Antigen cocktails and uses thereof

Abstract

The present invention relates to combinations or mixtures of antigens which may be used in the detection of IgM and/or IgG antibodies to Toxoplasma gondii. Furthermore, the present invention also relates to methods of using these combinations of antigens, antibodies raised against these combinations of antigens or against the novel P29 antigen thereof, as well as kits and vaccines containing the antigens present in the combinations.

Description

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates to combinations or mixtures of antigens which may be used in the detection of IgM or IgG antibodies to Toxoplasma gondii . Furthermore, the present invention also relates to methods of using these combinations of antigens, antibodies raised against these combinations of antigens or against the novel P29 antigen thereof, as well as kits and vaccine containing the antigens present in the combinations.

2. Background Information

Toxoplasma gondii is an obligate intracellular parasite which is classified among the Coccidia. This parasite has relatively broad host range infecting both mammals and birds. The organism is ubiquitous in nature and exists in three forms: tachyzoite, cyst, and oocyst (Remington, J. S., McLeod, R., Desmonds, G., Infectious Diseases of the Fetus and Newborn Infant (J. S. Remington and J. O. Klein, Eds.), pp. 140-267, Saunders, Philadelphia (1995)). Tachyzoites, found during acute infection, are the invasive form capable of invading all nucleated mammalian cells. After the acute stage of infection, tissue cysts called bradyzoites are formed within host cells and persist within the host organism for the life of the host. Cysts are important in transmission of infection, especially in humans, as the ingestion of raw or undercooked meat can result in the ingestion of bradyzoites which can infect the individual resulting in an acute infection. Oocysts represent a stage of sexual reproduction which occurs only in the intestinal lining of the cat family from which they are excreted in the feces.

A T. gondii infection acquired through contaminated meat or cat feces in a healthy adult is often asymptomatic. In pregnant women and immunosuppressed patients, the clinical outcome can be very serious. An acute infection with T. gondii acquired during pregnancy, especially during the first trimester, can result in intrauterine transmission to the unborn fetus resulting in severe fetal and neonatal complications, including mental retardation and fetal death. Recrudesence of a previous T. gondii infection or an acute infection in an immunosuppressed individual can be pathogenic. Toxoplasmic encephalitis is a major cause of morbidity and mortality in AIDS patients. Toxoplasma infection has also been shown to be a significant cause of chorioretinitis in children and adults.

Diagnosis of infection with T. gondii may be established by the isolation of T. gondii from blood or body fluids, demonstration of the presence of the organism in the placenta or tissues of the fetus, demonstration of the presence of antigen by detection of specific nucleic acid sequences (e.g., DNA probes), or detection of T. gondii specific immunoglobulins synthesized by the host in response to infection using serologic tests.

The detection of T. gondii specific antibodies and determination of antibody titer are important tools used in the diagnosis of toxoplasmosis. The most widely used serologic tests for the diagnosis of toxoplasmosis are the Sabin-Feldman dye test (Sabin, A. B. and Feldman, H. A. (1948) Science 108, 660-663), the indirect hemagglutination (IHA) test (Jacobs, L. and Lunde, M. (1957) J. Parasitol. 43, 308-314), the IFA test (Walton, B. C. et al. (1966) Am. J. Trop. Med. Hyg. 15, 149-152), the agglutination test (Fondation Mérieux, Sérologie de I' Infection Toxoplasmique en Particulier à Son Début: Méthodes et Interprétation des Résultants, Lyon, 182 pp. (1975)) and the ELISA (Naot, Y. and Remington, J. S. (1980) J. Infect. Dis. 142, 757-766). The ELISA test is one the easiest tests to perform, and many automated serologic tests for the detection of Toxoplasma specific IgM and IgG are commercially available.

The current tests for the detection of IgM and IgG antibodies in infected individuals can vary widely in their ability to detect serum antibody. Hence, there is significant inter-assay variation seen among the commercially available kits. The differences observed between the different commercial kits are caused primarily by the preparation of the antigen used for the serologic test. Most kits use either whole or sonicated tachyzoites grown in tissue culture or in mice which contain a high proportion of extra-parasitic material, for example, mammalian cells, tissue culture components, etc. Due to the lack of a purified, standardized antigen or standard method for preparing the tachyzoite antigen, it is not surprising that inter-assay variability exists resulting in different assays having different performance characteristics in terms of assay sensitivity and specificity.

Given the limitations of serologic tests employing the tachyzoite antigen, purified recombinant antigens obtained by molecular biology are an attractive alternative in that they can be purified and standardized. In the literature, a number of Toxo genes have been cloned and expressed in a suitable host to produce immunoreactive, recombinant Toxo antigens. For example, the Toxo P22 (SAG2), P24 (GRA1), P25, P28 (GRA2), P30 (SAG1), P35, P41 (GRA4), P54 (ROP2), P66 (ROP1), and the Toxo P68 antigens have been described (Prince et al. (1990) Mol. Biochem. Parasitol 43, 97-106; Cesbron-Delauw et al. (1989) Proc. Nat. Acad. Sci. 86, 7537-7541; Johnson et al. (1991) Gene 99, 127-132; Prince et al. (1989) Mol. Biochem. Parasitol. 34, 3-13; Burg et al. (1988) J. Immunol. 141, 3584-3591; Knapp et al. (1989) EPA 431541A2; Mevelec et al. (1992) Mol. Biochem. Parasitol. 56, 227-238; Saavedra et al. (1991) J. Immunol. 147, 1975-1982).

It is plausible that no single Toxo antigen can replace the tachyzoite in an immunoassay for the detection of Toxo-specific immunoglobulins. This may be for several reasons. First, the antibodies produced during infection vary with the stage of infection, i.e., the antibodies produced by an infected individual vary over time reacting with different epitopes. Secondly, the epitopes present in a recombinant antigen may be different or less reactive than native antigen prepared from the tachyzoite depending on the host used for expression and the purification scheme employed. Thirdly, different recombinant antigens may be needed to detect the different classes of immunoglobulins produced in response to an infection, e.g., IgM, IgG, IgA, IgE.

In order to overcome the limitations of the tachyzoite antigen in terms of assay specificity and sensitivity, a search was begun for novel Toxo antigens which could be used in combination with known existing antigens in order to configure new assays for the detection of Toxo-specific immunoglobulins.

SUMMARY OF THE INVENTION

The present invention includes a composition comprising Toxoplasma gondii antigens P29, P30 and P35 as well as a composition comprising Toxoplasma gondii antigens P29, P35 and 66. These compositions may be used as diagnositic reagents, and the antigens within these compositions may be produced either recombinantly or synthetically.

Additionally, the present invention includes an isolated nucleic acid sequence represented by SEQ ID NO: 26 and a purified polypeptide having the amino acid sequence represented by SEQ ID NO: 27. The present invention also includes a polyclonal or monoclonal antibody directed against the purified polypeptide.

The present invention also encompasses a method for detecting the presence of IgM antibodies to Toxoplasma gondii in a test sample. This method comprises the steps of: a) contacting the test sample suspected of containing the IgM antibodies with a composition comprising P29, P35 and P66; and b) detecting the presence of the IgM antibodies.

Furthermore, the present invention includes an additional method for detecting the presence of IgM antibodies to Toxoplasma gondii in a test sample. This method comprises the steps of: a) contacting the test sample suspected of containing the IgM antibodies with a composition comprising antigen P29, P35 and P66 for a time and under conditions sufficient for the formation of IgM antibody/antigen complexes; b) adding a conjugate to the resulting IgM antibody/antigen complexes for a time and under conditions sufficient to allow the conjugate to bind to the bound antibody, wherein the conjugate comprises an antibody attached to a signal generating compound capable of generating a detectable signal; and c) detecting the presence of IgM antibodies which may be present in the test sample by detecting a signal generated by the signal generating compound.

Moreover, the present invention also includes a method for detecting the presence of IgG antibodies to Toxoplasma gondii in a test sample. This method comprises the steps of: a) contacting the test sample suspected of containing the IgG antibodies with a composition comprising P29, P30 and P35; and b) detecting the presence of the IgG antibodies.

Additionally, the present invention encompasses another method for detecting the presence of IgG antibodies to Toxoplasma gondii in a test sample. This method comprising the steps of: a) contacting said test sample suspected of containing the IgG antibodies with a composition comprising antigen P29, P30 and P35 for a time and under conditions sufficient for formation of IgG antibody/antigen complexes; b) adding a conjugate to resulting IgG antibody/antigen complexes for a time and under conditions sufficient to allow the conjugate to bind to bound antibody, wherein the conjugate comprises an antibody attached to a signal generating compound capable of generating a detectable signal; and c) detecting IgG antibodies which may be present in said test sample by detecting a signal generated by said signal generating compound.

Additionally, the present invention includes another method for detecting the presence of IgM antibodies to Toxoplasma gondii in a test sample. This method comprises the steps of: a) contacting the test sample suspected of containing the IgM antibodies with anti-antibody specific for the IgM antibodies for a time and under conditions sufficient to allow for formation of anti-antibody/IgM antibody complexes; b) adding a conjugate to resulting anti-antibody/IgM antibody complexes for a time and under conditions sufficient to allow the conjugate to bind to bound antibody, wherein the conjugate comprises P29, P35 and P66, each attached to a signal generating compound capable of generating a detectable signal; and c) detecting IgM antibodies which may be present in the test sample by detecting a signal generated by the signal generating compound.

Another method for detecting the presence of IgG antibodies to Toxoplasma gondii in a test sample, encompassed by the present invention, comprises the steps of: a) contacting the test sample suspected of containing the IgG antibodies with anti-antibody specific for the IgG antibodies for a time and under conditions sufficient to allow for formation of anti-antibody/IgG antibody complexes; b) adding a conjugate to resulting anti-antibody/IgG antibody complexes for a time and under conditions sufficient to allow the conjugate to bind to bound antibody, wherein the conjugate comprises P29, P30 and P35, each attached to a signal generating compound capable of generating a detectable signal; and c) detecting IgG antibodies which may be present in the test sample by detecting a signal generated by the signal generating compound.

Also, the present invention includes a vaccine comprising: 1) Toxoplasma gondii antigens P29, P30 and P35 and 2) a pharmaceutically acceptable adjuvant as well as a vaccine comprising: 1) Toxoplasma gondii antigens P29, P35 and P66 and 2) a pharmaceutically acceptable adjuvant.

Additionally, the present invention includes a kit for determining the presence of IgM antibodies to Toxoplasma gondii in a test sample comprising: a) a composition comprising Toxoplasma gondii antigens P29, P35 and P66 and b) a conjugate comprising an antibody attached to a signal generating compound capable of generating a detectable signal.

The present invention also includes a kit for determining the presence of IgG antibodies to Toxoplasma gondii in a test sample comprising: a) a composition comprising Toxoplasma gondii antigens P29, P30 and P35 and b) a conjugate comprising an antibody attached to a signal generating compound capable of generating a detectable signal.

An additional kit for determining the presence of IgM antibodies to Toxoplasma gondii in a test sample, encompassed by the present invention, comprises: a) an anti-antibody specific for IgM antibody and b) a composition comprising Toxoplasma gondii antigens P29, P35 and P66.

The present invention also includes a kit for determining the presence of IgM antibodies to Toxoplasma gondii in a test sample comprising: a) an anti-antibody specific for IgM antibody and b) a conjugate comprising: 1) Toxoplasma gondii antigens P29, P35 and P66, each attached to 2) a signal generating compound capable of generating a detectable signal.

Additionally, the present invention includes a kit for determining the presence of IgG antibodies to Toxoplasma gondii in a test sample comprising: a) an anti-antibody specific for IgG antibody and b) a composition comprising Toxoplasma gondii antigens P29, P30 and P35.

The present invention also includes an additional kit for determining the presence of antibodies to Toxoplasma gondii in a test sample comprising: a) an anti-antibody specific for IgG antibody and b) a conjugate comprising: 1) Toxoplasma gondii antigens P29, P30 and P35, each attached to 2) a signal generating compound capable of generating a detectable signal.

Additionally, the present invention includes a method for detecting the presence of IgM antibodies to Toxoplasma gondii in a test sample comprising the steps of: (a) contacting the test sample suspected of containing IgM antibodies with anti-antibody specific for the IgM antibodies for a time and under conditions sufficient to allow for formation of anti-antibody IgM complexes; (b) adding antigen to resulting anti-antibody/IgM complexes for a time and under conditions sufficient to allow the antigen to bind to bound IgM antibody, the antigen comprising a mixture of P29, P35 and P66; and (c) adding a conjugate to resulting anti-antibody/IgM/antigen complexes, the conjugate comprising a composition comprising monoclonal or polyclonal antibody attached to a signal generating compound capable of generating a detectable signal; and (d) detecting IgM antibodies which may be present in the test sample by detecting a signal generated by the signal generating compound.

The present invention also includes a method for detecting the presence of IgG antibodies to Toxoplasma gondii in a test sample comprising the steps of: (a) contacting the test sample suspected of containing IgG antibodies with anti-antibody specific for said IgG antibodies for a time and under conditions sufficient to allow for formation of anti-antibody IgG complexes; (b) adding antigen to resulting anti-antibody/IgG complexes for a time and under conditions sufficient to allow said antigen to bind to bound IgG antibody, the antigen comprising a mixture of P29, P30 and P35; and (c) adding a conjugate to resulting anti-antibody/IgG/antigen complexes, the conjugate comprising a composition comprising monoclonal or polyclonal antibody attached to a signal generating compound capable of generating a detectable signal; and (d) detecting IgG antibodies which may be present in the test sample by detecting a signal generated by the signal generating compound.

A further method for detecting the presence of IgM and IgG antibodies to Toxoplasma gondii in a test sample, included within the present invention, comprises the steps of: a) contacting the test sample suspected of containing the IgM and IgG antibodies with a composition comprising antigen P29, P30, P35 and P66 for a time and under conditions sufficient for the formation of IgM antibody/antigen complexes and IgG antibody/antigen complexes; b) adding a conjugate to the resulting IgM antibody/antigen complexes and IgG antibody/antigen complexes for a time and under conditions sufficient to allow the conjugate to bind to the bound IgM and IgG antibody, wherein said conjugate comprises an antibody attached to a signal generating compound capable of generating a detectable signal; and c) detecting the presence of IgM and IgG antibodies which may be present in the test sample by detecting a signal generated by the signal generating compound.

The present invention also includes method for detecting the presence of IgM and IgG antibodies to Toxoplasma gondii in a test sample comprising the steps of: a) contacting the test sample suspected of containing the IgM and IgG antibodies with anti-antibody specific for said IgM antibodies and the IgG antibodies for a time and under conditions sufficient to allow for formation of anti-antibody/IgM antibody complexes and anti-antibody/IgG antibody complexes; b) adding a conjugate to resulting anti-antibody/IgM antibody complexes and resulting anti-antibody/IgG antibody complexes for a time and under conditions sufficient to allow the conjugate to bind to bound antibody, wherein the conjugate comprises P29, P30, P35 and P66, each attached to a signal generating compound capable of generating a detectable signal; and c) detecting IgM and IgG antibodies which may be present in the test sample by detecting a signal generated by the signal generating compound.

The present invention also includes a method for detecting the presence of IgM and IgG antibodies to Toxoplasma gondii in a test sample comprising the steps of: (a) contacting the test sample suspected of containing IgM and IgG antibodies with anti-antibody specific for the IgM antibodies and with anti-antibody specific for the IgG antibodies for a time and under conditions sufficient to allow for formation of anti-antibody/IgM complexes and anti-antibody/IgG complexes; (b) adding antigen to resulting anti-antibody/IgM complexes and resulting anti-antibody/IgG complexes for a time and under conditions sufficient to allow the antigen to bind to bound IgM antibody and bound IgG antibody, the antigen comprising a mixture of P29, P30, P35 and P66; and (c) adding a conjugate to resulting anti-antibody/IgM/antigen complexes and anti-antibody/IgG/antigen complexes, the conjugate comprising a composition comprising monoclonal or polyclonal antibody attached to a signal generating compound capable of generating a detectable signal; and (d) detecting IgM and IgG antibodies which may be present in the test sample by detecting a signal generated by the signal generating compound.

Additionally, the present invention encompasses a method of producing monoclonal antibodies comprising the steps of:

a) injecting a non-human mammal with an antigen;

b) administering a composition comprising antibiotics to the non-human mammal;

c) fusing spleen cells of the non-human mammal with myeloma cells in order to generate hybridomas; and

d) culturing the hybridomas under sufficient time and conditions such that the hybridomas produce monoclonal antibodies.

The antigen utilized may be derived from, for example, T. gondii.

All U.S. patents and publications referred to herein are hereby incorporated in their entirety by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C represent the DNA sequence [SEQ ID NO: 23] of nucleotides 1-1268 and the corresponding amino acid sequence [SEQ ID NO: 24] of plasmid pGM613.

FIG. 2 represents the DNA sequence [SEQ ID NO: 25] of nucleotides 1-477 of plasmid pTXG1-2.

FIGS. 3A-3D represent the composite DNA sequence [SEQ ID NO: 26] of nucleotides 1-1648 and the corresponding amino acid sequence [SEQ ID NO: 27] for the P29 gene.

FIG. 4 is a schematic representation of (A) the construction of plasmid pEE2; (B) the nucleotide sequence [SEQ ID NO: 28] and the corresponding amino acid sequence [SEQ ID NO: 49] of the polylinker to be removed from pEE1 by digestion with BglII; and (C) the nucleotide sequence [SEQ ID NO: 29] and the corresponding amino acid sequence [SEQ ID NO: 50] of the synthetic DNA to be introduced into the BglII site of pEE1 to generate plasmid pEE2.

FIG. 5 is a schematic representation of (A) the construction of plasmid pEE3; and (B) the nucleotide sequence [SEQ ID NO: 32] and the corresponding amino acid sequence [SEQ ID NO: 51] of the synthetic DNA polylinker to be introduced into the StuI/MluI sites of pEE2 to generate plasmid pEE3.

FIG. 6 is a schematic representation of the construction of plasmid pToxo-P29.

FIGS. 7A-7J illustrates the DNA sequence [SEQ ID NO: 37] of nucleotides 1-4775 and the corresponding amino acid sequence [SEQ ID NO: 52] of the CKS-P29-CKS fusion protein of plasmid pToxo-P29.

FIG. 8 is a schematic representation of the construction of plasmid pToxo-P30.

FIGS. 9A-9J represent the DNA sequence [SEQ ID NO: 40] of nucleotides 1-4910 and the corresponding amino acid sequence [SEQ ID NO: 53] of the CKS-P30-CKS fusion protein of plasmid pToxo-P30.

FIG. 10 is a schematic representation of the construction of plasmid pToxo-P35S.

FIGS. 11A-11I illustrate the DNA sequence [SEQ ID NO: 45] of nucleotides 1-4451 and the corresponding amino acid sequence [SEQ ID NO: 54] of the CKS-P35-CKS fusion protein of plasmid pToxo-P35S.

FIG. 12 is a schematic representation of the construction of plasmid pToxo-P66g.

FIGS. 13A-13K represent the DNA sequence [SEQ ID NO: 48] of nucleotides 1-5258 and the corresponding amino acid sequence [SEQ ID NO: 55] of the CKS-P66-CKS fusion protein of plasmid pToxo-P66g.

DETAILED DESCRIPTION OF THE INVENTION

The difficulties of known assays for the detection of IgG and IgM antibodies to T. gondii have been described, in detail, above. Thus, there was a need to discover immunoassays which could accurately detect the presence of such antibodies in positive serum, thereby eliminating the problem of false negative or false positive tests. The present invention provides such needed immunoassays and, in particular, combinations of antigens which accurately detect the presence of IgG or IgM antibodies in human sera.

In particular, the present invention includes a novel antigen which, for purposes of the present invention, is referred to as P29. The nucleotide sequence of the gene encoding this antigen is shown in FIG. 3 and is represented by SEQ ID NO. 26. The amino acid sequence of this antigen is shown in FIG. 3 and is represented by SEQ ID NO. 27.

P29, a dense granule protein, when used in combination with other known antigens, may accurately detect the presence of IgG or IgM in human sera. In particular, P29, when used in combination with other known antigens, may replace the tachyzoite previously used in assays for T. gondii antibodies.

Furthermore, the present invention also includes a polyclonal or monoclonal antibody raised against P29. Such an antibody may be used, for example, in an immunoassay, a vaccine, a kit, or for research purposes.

The present invention also encompasses a composition or mixture comprising the following three antigens: P29, P30 and P35. This combination or mixture of antigens may be utilized for the detection of IgG in IgG-positive sera (i.e., as a diagnostic reagent). Furthermore, the antigens may be produced either recombinantly or synthetically. Additionally, the present invention also includes a composition comprising antibodies raised against these antigens.

The present invention also includes a composition or mixture comprising the following three antigens: P29, P35 and P66. This combination or mixture of antigens may be used for the detection of IgM in IgM-positive sera (i.e., as a diagnostic reagent), and the antigens may be produced either recombinantly or synthetically. Furthermore, the present invention also includes a composition comprising antibodies raised against these antigens.

If, in fact, one wishes to measure both the titer of IgM and IgG in an individual, then a composition or mixture of antigens P29, P30, P35 and P66 may be utilized in an immunoassay. Such a combination of antigens is also included within the scope of the present invention.

The present invention also includes methods of detecting IgM and/or IgG using the combinations of antigens described above. More specifically, there are two basic types of assays, competitive and non-competitive (e.g., immunometric and sandwich). In both assays, antibody or antigen reagents are covalently or non-covalently attached to the solid phase. Linking agents for covalent attachment are known and may be part of the solid phase or derivatized to it prior to coating. Examples of solid phases used in immunoassays are porous and non-porous materials, latex particles, magnetic particles, microparticles, beads, membranes, microtiter wells and plastic tubes. The choice of solid phase material and method of labeling the antigen or antibody reagent are determined based upon desired assay format performance characteristics. For some immunoassays, no label is required. For example, if the antigen is on a detectable particle such as a red blood cell, reactivity can be established based upon agglutination. Alternatively, an antigen-antibody reaction may result in a visible change (e.g., radial immunodiffusion). In most cases, one of the antibody or antigen reagents used in an immunoassay is attached to a signal generating compound or “label”. This signal generating compound or “label” is in itself detectable or may be reacted with one or more additional compounds to generate a detectable product. Examples of such signal generating compounds include chromogens, radioisotopes (e.g., 125I, 131I, 32P, 3H, 35S, and 14C), fluorescent compounds (e.g., fluorescein, rhodamine), chemiluminescent compounds, particles (visible or fluorescent), nucleic acids, complexing agents, or catalysts such as enzymes (e.g., alkaline phosphatase, acid phosphatase, horseradish peroxidase, beta-galactosidase, and ribonuclease). In the case of enzyme use, addition of chromo-, fluoro-, or lumo-genic substrate results in generation of a detectable signal. Other detection systems such as time-resolved fluorescence, internal-reflection fluorescence, amplification (e.g., polymerase chain reaction) and Raman spectroscopy are also useful.

There are two general formats commonly used to monitor specific antibody titer and type in humans: (1) antigen is presented on a solid phase, as described above, the human biological fluid containing the specific antibodies is allowed to react with the antigen, and then antibody bound to antigen is detected with an anti-human antibody coupled to a signal generating compound and (2) an anti-human antibody is bound to the solid phase, the human biological fluid containing specific antibodies is allowed to react with the bound antibody, and then antigen attached to a signal generating compound is added to detect specific antibody present in the fluid sample. In both formats, the anti-human antibody reagent may recognize all antibody classes, or alternatively, be specific for a particular class or subclass of antibody, depending upon the intended purpose of the assay. These assays formats as well as other known formats are intended to be within the scope of the present invention and are well known to those of ordinary skill in the art.

In particular, two illustrative examples of an immunometric antibody-capture based immunoassay are the Imx Toxo IgM and Toxo IgG antibody assays manufactured by Abbott Laboratories (Abbott Park, Ill.). Both assays are automated Microparticle Enzyme Immunoasssays (MEIA) which measure antibodies to Toxoplasma gondii ( T. gondii ) in human serum or plasma (Safford et al., J. Clin. Pathol . 44:238-242 (1991)). One assay quantitatively measures IgM antibodies, indicative of recent exposure or acute infection, and the other assay quantitatively measures IgG, indicative of chronic or past infection. These assays use microparticles coated with T. gondii antigens as the solid phase. In particular, specimen is added to the coated microparticles to allow antibodies specific for T. gondii to bind. Subsequently, an alkaline phosphatase conjugated anti-human IgM (or anti-human IgG) is added that specifically binds to IgM (or IgG) class antibodies complexed to the T. gondii antigens. Following addition of a suitable substrate (e.g., 4-methyumbelliferyl phosphate), the rate of enzyme-catalyzed turnover is monitored based upon fluorescence.

The mixture of P29, P30 and P35 may be used in the IgG Abbott immunoassay, and the mixture of P29, P35 and P66 may be utilized in the IgM Abbott immunoassay. Additionally, A mixture of P29, P30, P35, and P66 may be utilized in either assay, if desired. Furthermore, it must be noted that other non-Abbott assays or platforms may also be utilized, with each of the combinations of antigens (i.e., 3 or 4 antigens), for purposes of the present invention.

Thus, the present invention includes a method of detecting IgM antibodies in a test sample comprising the steps of: (a) contacting the test sample suspected of containing the IgM antibodies with P29, P35 and P66; (b) detecting the presence of IgM antibodies present in the test sample. More specifically, the present invention includes a method of detecting IgM antibodies in a test sample comprising the steps of: (a) contacting the test sample suspected of containing the IgM antibodies with P29, P35 and P66 for a time and under conditions sufficient to allow the formation of IgM antibody/antigen complexes; (b) adding a conjugate to the resulting IgM antibody/antigen complexes for a time and under conditions sufficient to allow the conjugate to bind to the bound antibody, the conjugate comprising an antibody (directed against the IgM) attached to a signal generating compound capable of generating a detectable signal; (c) detecting the presence of the IgM antibody which may be present in the test sample by detecting the signal generated by the signal generating compound. A control or calibrator may also be used which binds to the antigens. Furthermore, the method may also comprise the use of P30 in addition P29, P35 and P66.

In each of the above assays, IgG may be detected by substituting the P29, P35 and P66 mixture with a P29, P30 and P35 mixture. Additionally, the antibody in the conjugate will be directed against IgG rather than IgM. Additionally, if one wishes to detect both IgM and IgG antibodies, P29, P30, P35 and P66 may be utilized in the immunoassay. Furthermore, if desired, one may also add P66 to the assay, even if detection of antibodies to only IgG is required.

Additionally, the present invention also includes a method for detecting the presence of IgM which may be present in a test sample. This method comprises the steps of: (a) contacting the test sample suspected of containing IgM antibodies with anti-antibody specific for the IgM, for a time and under conditions sufficient to allow for formation of anti-antibody/IgM complexes and (b) detecting the presence of IgM which may be present in the test sample. (Such anti-antibodies are commercially available and may be created, for example, by immunizing a mammal with purified mu-chain of the antibody.)

More specifically, this method may comprise the steps of: (a) contacting the test sample suspected of containing the IgM antibodies with anti-antibody specific for the IgM, under time and conditions sufficient to allow the formation of anti-antibody/IgM complexes; (b) adding a conjugate to the resulting anti-antibody/IgM complexes for a time and under conditions sufficient to allow the conjugate to bind to the bound antibody, the conjugate comprising P29, P35 and P66, each being attached to a signal generating compound capable of generating a detectable signal; and (c) detecting the presence of the IgM antibodies which may be present in the test sample by detecting the signal generated by the signal generating compound. A control or calibrator may be used which comprises antibody to the anti-antibody. Furthermore, the conjugate may also comprise P30, if desired.

In each of the above assays, IgG may be detected by substituting the P29, P35 and P66 mixture with a P29, P30 and P35 mixture. Also, anti-antibody specific for IgG will be used. Additionally, if one wishes to detect both IgM and IgG antibodies, P29, P30, P35 and P66 may be utilized in the immunoassay. Moreover, even if one wishes to detect IgG only, P66 may also be added to the assay, if desired.

The present invention also encompasses a third method for detecting the presence of IgM in a test sample. This method comprises the steps of: (a) contacting the test sample suspected of containing IgM antibodies with anti-antibody specific for the IgM, under time and conditions sufficient to allow the formation of anti-antibody IgM compelxes; (b) adding antigen to the resulting anti-antibody/IgM complexes for a time and under conditions sufficient to allow the antigen to bind to the bound IgM antibody, the antigen comprising a mixture of P29, P35 and P66; and (c) adding a conjugate to the resulting anti-antibody/IgM/antigen complexes, the conjugate comprising a composition comprising monoclonal or polyclonal antibody attached to a signal generating compound capable of detecting a detectable signal, the monoclonal or polyclonal antibody being directed against the antigen; and (d) detecting the presence of the IgM antibodies which may be present in the test sample by detecting the signal generated by the signal generating compound. Again, a control or calibrator may be used which comprises antibody to the anti-antibody. The antigen mixture may further comprise P30, if desired.

In this method, IgG may be detected by substituting the P29, P35 and P66 mixture with a P29, P30 and P35 mixture and utilizing anti-antibody specific for IgG. However, if one wishes to detect both IgM and IgG antibodies, P29, P30, P35 and P66 may be utilized in the immunoassay. Even if one wishes to detect IgG alone, the assay may further comprise the use of P66.

It should also be noted that all of the above methods may be used to detect IgA antibodies (with an alpha-specific conjugate) and/or IgE antibodies (with an epsilon-specific conjugate) should such detection be desired.

Additionally, the present invention also includes a vaccine comprising a mixture of P29, P30 and P35 antigens and a pharmaceutically acceptable adjuvant. Such a vaccine may be administered if one desires to raise IgG antibodies in a mammal. The present invention also includes a vaccine comprising a mixture of P29, P35 and P66 antigens and a pharmaceutically acceptable adjuvant (e.g., Freund's adjuvant or Phosphate Buffered Saline). Such a vaccine may be administered if one desires to raise IgM antibodies in a mammal. Additionally, the present invention also includes a vaccine comprising a mixture of P29, P30, P35 and P66 antigens as well as a pharmaceutically acceptable adjuvant. This vaccine should be administered if one desires to raise both IgM and IgG antibodies in a mammal.

Kits are also included within the scope of the present invention. More specifically, the present invention includes kits for determining the presence of IgG and/or IgM. In particular, a kit for determining the presence of IgM in a test sample comprises a) a mixture of P29, P35 and P66; and b) a conjugate comprising an antibody (directed against IgM) attached to a signal generating compound capable of generating a detectable signal. The kit may also contain a control or calibrator which comprises a reagent which binds to P29, P35 and P66.

Again, if one desires to detect IgG, rather than IgM, the kit will comprise a mixture of P29, P30 and P35, rather than P29, P35 and P66, as well as an antibody directed against IgG. If one wishes to detect both IgM and IgG, the kit will comprise P29, P30, P35 and P66.

The present invention also includes another type of kit for detecting IgM and/or IgG in a test sample. If utilized for detecting the presence of IgM, the kit may comprise a) an anti-antibody specific for IgM, and b) a mixture of antigens P29, P35 and P66. A control or calibrator comprising a reagent which binds to P29, P35 and P66 may also be included. More specifically, the kit may comprise a) an anti-antibody specific for IgM, and b) a conjugate comprising P29, P35 and P66, the conjugate being attached to a signal generating compound capable of generating a detectable signal. Again, the kit may also comprise a control of calibrator comprising a reagent which binds to P29, P35 and P66.

Additionally, if one desires to detect IgG, rather than IgM, the kit will comprise a mixture of P29, P30 and P35, rather than P29, P35 and P66, as well as anti-antibody specific for IgG. If one wishes to detect both IgM and IgG, the kit may comprise P29, P30, P35 and P66.

The present invention may be illustrated by the use of the following non-limiting examples:

EXAMPLE 1

General Methodology

Materials and Sources

Restriction enzymes, T4 DNA ligase, calf intestinal alkaline phosphatase (CIAP), polynucleotide kinase, and the Klenow fragment of DNA Polymerase I were purchased from New England Biolabs, Inc. (Beverly, Mass.) or from Boehringer Mannheim Corp. (Indianapolis, Ind.). DNaseI and aprotinin were purchased from Boehringer Mannheim Corp.

DNA and protein molecular weight standards, Daiichi pre-cast gradient polyacrylamide gels were obtained from Integrated Separation Systems, Inc. (Natick, Mass.).

Isopropyl-β-D-thiogalactoside (IPTG), TRITON X-100, 4-chloro-1-naphthol, and sodium dodecyl sulfate (SDS) were purchased from BioRad Laboratories (Richmond, Calif.).

Plasma from patients with an acute Toxoplasma infection was obtained from Antibody Systems, Inc., Bedford, Tex.

Horseradish peroxidase (HRPO)-labelled antibodies were purchased from Kirkegaard & Perry Laboratories, Inc. (Gaithersburg, Md.).

EPICURIAN Coli™ XL-1 BLUE (recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F′ proAB lacI q ZDM15 Tn10 (Tet r )]) supercompetent E. coli cells, a DNA isolation kit, a RNA isolation kit, a ZAP™-cDNA Gigapack II Gold Cloning kit, a picoBLUE Immunoscreening kit, and Duralose-UV™ membranes, and a ZAP™-cDNA Synthesis kit were obtained from Stratagene Cloning Systems, Inc. (La Jolla, Calif.).

A GeneAmp™ reagent kit and AmpliTaq™ DNA Polymerase were purchased from Perkin-Elmer Cetus (Norwalk, Conn.). Deoxynucleotide triphosphates used in general procedures were from the GeneAmp™ reagent kit.

Supported nitrocellulose membrane was purchased from Schleicher & Schuell (Keene, N.H.).

A nucleotide kit for DNA sequencing with Sequenase™ and 7-deaza-dGTP and Sequenase™ version 2.0 DNA Polymerase were obtained from U.S. Biochemical Corp. (Cleveland, Ohio).

A Multiprime DNA labelling kit, alpha- 32 P-dCTP, and a- 32 P-dATP were purchased from Amersham Corp. (Arlington Heights, Ill.).

A PolyA + mRNA purification kit was purchased from Pharmacia LKB Biotechnology, Inc. (Piscataway, N.J.).

Polygard Cartridge filters, pore size 10 u, were purchased from Millipore Corp., Bedford, Mass.

Luria Broth plates with ampicillin (LBamp plates) were purchased from Micro Diagnostics, Inc. (Lombard, Ill.).

OPTI-MEM™ Medium, Iscove's Modified Dulbecco's Media, Hank's Balanced Salt Solution, fetal calf serum, phosphate-buffered saline, competent E. coli DH5-alpha (F −Ø 80dlacZDM15 D(lacZYA-argF)U169 deoR recA1 endA1 phoA hsdR17(r k − , m k + ) supE44 l − thi-1 grA96 relA1), and ultraPURE agarose were purchased from GIBCO BRL, Inc. (Grand Island, N.Y.).

Bacto-Tryptone, Bacto-Yeast Extract, and Bacto-Agar were obtained from Difco Laboratories (Detroit, Mich.).

NZY Broth was purchased from Becton Dickinson Microbiology Systems (Cockeysville, Md.).

Salmon sperm DNA, lysozyme, ampicillin, N-lauroyl sarcosine, thimerosal, buffers, casein acid hydrolysate, TWEEN 20™ (polyoxyethylenesorbitan monolaurate), diethylpyrocarbonate (DEPC), phenylmethylsulfonylfluoride (PMSF), bovine serum albumin (BSA), urea, glycerol, EDTA, sodium deoxycholate, pyrimethamine, sulfamethoxazole, mouse monoclonal antibody isotyping kits, and inorganic salts were purchased from Sigma Chemical Co. (Saint Louis, Mo.).

OPD (0-phenylenediamine dihydrochloride) and PBS (phosphate buffered saline) was purchased from Abbott Laboratories (Abbott Park, Ill.).

Hydrogen Peroxide (H 2O 2 ) was purchased from Mallinkrodt (Paris, Ky.).

Methanol was purchased from EM Science (Gibbstown, N.J.).

Microtiter MAXISORPIplates were purchased from NUNC, Inc. (Naperville, Ill.).

Media, Buffers and General Reagents

“Superbroth II” contained 11.25 g/L tryptone, 22.5 g/L yeast extract, 11.4 g/L potassium phosphate dibasic, 1.7 g/L potassium phosphate monobasic, 10 mL/L glycerol, adjusted pH to 7.2 with sodium hydroxide.

“Tris-buffered saline” or “TBS” consisted of 20 mM Tris, 500 mM NaCl at pH 7.5.

“Tris-buffered saline TWEEN 20™” or “TBST” consisted of TBS plus 0.05% TWEEN 20.

“Rubazyme specimen dilution buffer” or “Rubazyme SDB” consisted of 100 mM Tris at pH 7.5 with 135 mM NaCl, 10 mM EDTA, 0.2% TWEEN 20™, 0.01% thimerosal and 4% bovine calf serum.

“Rubazyme conjugate diluent dilution buffer” consisted of 100 mM Trisat pH 7.5 with 135 mM NaCl, 0.01% thimerosal and 10% bovine calf serum.

“Membrane blocking solution” consisted of 1% BSA, 1% casein acid hydrolysate, 0.05% TWEEN 20 in TBS.

“TE buffer” consisted of 10 mM Tris and 1 mM EDTA at pH 8.0.

“TEM lysis buffer” consisted of 50 mM Tris, 10 mM EDTA and 20 mM magnesium chloride at pH 8.5.

“PTE buffer” consisted of 50 mM Tris and 10 mM EDTA at pH 8.5.

Parasite, Cell, and Mouse Lines

The RH strain of T. gondii (ATCC 50174) and the HeLa S3 cell line (ATCC CCL 2.2) were obtained from the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209. The TS4 strain of T. gondii was also available from the American Type Culture Collection and from other sources. The Swiss mouse strain CD1 was obtained from Charles River Laboratories, Wilmington, Mass. Parasites were maintained by serial passage in the peritoneal cavity of Swiss mice. Tachyzoites were collected from the peritoneal cavity and used to inoculate a primary suspension culture of HeLa S3 cells. This infected suspension culture was grown for 2-4 days at 37° C. in Iscove's Modified Dulbecco's Media and then used to inoculate a secondary suspension culture of uninfected HeLa S3 cells. This secondary infected suspension culture was grown for 2-4 days at 37° C. in OPTI-MEM Reduced Serum Medium and used as a source of tachyzoites for screening monoclonal antibodies and for the preparation of DNA, RNA, and total tachyzoite protein.

General Methods

All enzyme digestions of DNA were performed according to suppliers' instructions. At least 5 units of enzyme were used per microgram of DNA, and sufficient incubation time was allowed for complete digestion of DNA. Supplier protocols were followed for the various kits used in manipulation of DNA and RNA, for polymerase chain reaction (PCR) DNA synthesis and for DNA sequencing. Standard procedures were used for Western and Southern Blots, partial restriction enzyme digestion of Toxoplasma genomic DNA with Sau 3AI, construction of a Toxoplasma genomic library, miniprep and large scale preparation of plasmid DNA from E. coli , preparation of phage lysate DNA from E. coli cells infected with phage lambda, preparation of E. coli lysates for the absorption of anti- E. coli antibodies, phenol-chloroform extraction and ethanol precipitation of DNA, restriction analysis of DNA on agarose gels, purification of DNA fragments from agarose gels, filling the recessed 3′ termini created by digestion with restriction enzymes using the Klenow fragment of DNA Polymerase I, and ligation of DNA fragments with T4 DNA ligase. (Maniatis et al., Molecular Cloning: A Laboratory Manual , 2nd ed. (Cold Spring Harbor Laboratory Press, New York, 1989)).

DNA fragments for cloning into plasmids that were generated by PCR amplification, were extracted with phenol-chloroform and precipitated with ethanol prior to restriction enzyme digestion of the PCR reaction mixture. Oligonucleotides for PCR and DNA sequencing were synthesized on an Applied Biosystems Oligonucleotide Synthesizer, model 380B or 394, per the manufacturer's protocol.

Mouse monoclonal antibody directed against the CKS protein was obtained by immunization of mice with purified rpHCV-23 (CKS-BCD), described in International Application No. W093/04088 by Dailey et al. The proteins used for immunization were approximately 90% pure as determined by SDS-PAGE. The procedure for the immunization of mice, cell fusion, screening and cloning of monoclonal antibodies, and characterization of monoclonal antibodies were as described in Published International Application No. W092/08738 by Mehta et al.

EXAMPLE 2

Isolation of Toxoplasma DNA, RNA, Protein and Synthesis of cDNA

A 10L secondary suspension culture of HeLa cells infected with the RH strain of T. gondii was grown to a tachyzoite density of approximately 1×10 7 per ml and filtered through a 10 m Millipore Polygard cartridge filter to remove HeLa cells from the tachyzoites. The tachyzoite filtrate obtained contained less than 1% HeLa cells. The tachyzoites were then concentrated by centrifugation, washed and resuspended in 1× Hank's Buffer. The tachyzoite concentrate was then pipetted dropwise into liquid nitrogen, and the frozen tachyzoite pellets were recovered and stored at −80° C. until further use. The tachyzoite pellets were converted to tachyzoite powder by grinding the pellets to a fine powder using a mortar and pestle chilled with dry ice and liquid nitrogen. The tachyzoite powder was subsequently used for the isolation of tachyzoite nucleic acid and protein as described below.

Step A: Isolation of Toxoplasma DNA

Total Toxoplasma DNA was isolated from the tachyzoite powder using the Stratagene DNA extraction kit. The tachyzoite powder was dissolved in Solution 2, and total DNA was isolated following the kit's protocol. After ethanol precipitation and resuspension of the DNA in TE buffer, undissolved DNA and contaminating polysaccharides were removed by centrifugation at 200,000×g for 1 hr.

Step B: Isolation of Toxoplasma RNA

Total Toxoplasma RNA was isolated from the tachyzoite powder using the Stratagene RNA isolation kit. The tachyzoite powder was dissolved in Solution D, and total RNA was isolated following the kit's protocol. After ethanol precipitation and resuspension of the RNA in DEPC-treated water, polyA+RNA was selected with an oligo-dT column using a Pharmacia mRNA isolation kit. The purified mRNA was concentrated by ethanol precipitation and stored in DEPC-treated water at −80° C. until further use.

Step C: Isolation of Total Toxoplasma Protein

Total Toxoplasma protein was isolated from the tachyzoite powder by dissolving the powder in SDS-PAGE loading buffer and boiling the sample for 5 min. The protein preparation was stored at −20° C. until further use.

Step D: Synthesis of Toxoplasma cDNA

Purified Toxoplasma mRNA was used as a template for the synthesis of cDNA using the Stratagene ZAP-cDNA Synthesis kit. The first strand was synthesized using Moloney-Murine Leukemia Virus Reverse Transcriptase and a 50 mer primer which included an Xho I restriction enzyme site and an poly-dT tract. The reaction mix included the analog 5-methyl dCTP to protect the cDNA from restriction enzymes used in subsequent cloning steps. The second strand was synthesized using RNase H and DNA polymerase I. The cDNA was then ethanol precipitated and resuspended in water and stored at −20° C. until further use as a template for PCR amplification and for construction of a Toxoplasma cDNA library.

EXAMPLE 3

Cloning Strategy for Genes Encoding Toxoplasma Antigens

The immune response that is generated by human patients with Toxoplasmosis is targeted against several T. gondii proteins and varies by individual and by the disease stage. Hence, a Toxoplasma immunoassay which is composed entirely of purified protein antigens will require more than one protein serological target to accurately detect serum antibody to T. gondii in a population of Toxoplasma infected individuals. In order to identify additional Toxoplasma antigens which are relevant for human diagnostic testing, a two-tiered cloning strategy for genes encoding Toxoplasma antigens was undertaken. The first-tier consisted of cloning known genes encoding Toxoplasma antigens, by using the published DNA sequences for these genes. The second-tier consisted of cloning novel, previously undescribed genes encoding Toxoplasma antigens, by using pooled human plasma from patients with toxoplasmosis to screen a Toxoplasma cDNA library. The genes cloned in the first tier were then used as DNA probes to screen the genes cloned in the second tier for uniqueness.

Step A: Cloning of Toxoplasma Genes Encoding Known Toxoplasma Antigens

The CKS expression vector pJO200 described in U.S. patent application Ser. No. 08/742,619 of Maine and Chovan allows the fusion of recombinant proteins to the CMP-KDO synthetase (CKS) protein. The DNA gene sequence which encodes for the structural protein CKS (also known as the kdsB gene) is published in Goldman et al., J. Biol. Chem. 261:15831 (1986). The amino acid sequence of CKS includes 248 amino acid (aa) residues and is described in Goldman et al., supra. The pJO200 vector contained DNA encoding the sequence of the first 240 amino acids from the original kdsB gene followed by an additional 20 amino acids encoded for by the polylinker DNA sequence, for a total of 260 amino acids.

Oligonucleotide primers for use in the PCR amplification of known genes encoding Toxoplasma antigens were designed based on published DNA sequences. Each pair of PCR primers were “tailed” with additional DNA sequences to include restriction enzyme sites for subsequent cloning into the pJO200 CKS expression vector. PCR amplification of each Toxoplasma gene with the appropriate primers was carried out using the GeneAmp reagent kit and AmpliTaq DNA Polymerase purchased from Perkin-Elmer Cetus, Norwalk, Conn., following the kit's protocol. Approximately 20 ng of Toxoplasma cDNA prepared in Example 2D or 20 ng of Toxoplasma genomic DNA prepared in Example 2A (for P66 genomic clone only) was used in each reaction. The amplification cycles were 1 cycle of 95° C. for 120 sec., followed by 35 cycles of 95° C. for 60 sec., 55° C. for 60 sec., 72° C. for 120 sec., followed by 1 cycle at 72° C. for 300 sec., followed by a soak cycle at 4° C. The PCR products obtained from the amplification reaction were then digested with the appropriate restriction enzymes, purified on agarose gels, ligated into the pJO200 vector cut with the appropriate restriction enzymes and transformed into the Epicurean Coli XL-1 Blue Supercompetent E. coli cells following the kit protocol. Correct clones were confirmed by DNA sequence analysis of the cloned Toxoplasma DNA. The DNA sequences of the oligonucleotide primers used for the PCR amplification of the following Toxoplasma genes are shown below and how they were cloned into the pJO200 CKS vector:

Toxo P22 (SAG2) Gene

(Prince et al. (1990) Mol. Biochem. Parasitol 43, 97-106)

Sense Primer [SEQ ID NO:1]:

5′-CGCAGAATTCGATGTCCACCACCGAGACGCCAGCGCCCATTGA-3′ (EcoRI site is underlined)

Antisense Primer [SEQ ID NO:2]:

5′-CCCGGGATCCTTACACAAACGTGATCAACAAACCTGCGAGACC-3′ (BamH-I site is underlined)

Region Cloned: Nucleotides 260-739 of the Toxo P22 gene cloned into the EcoRI/BamH-I sites of pJO200 to yield plasmid pJO200-P22.

Toxo P24 (GRA1) Gene

(Cesbron-Delauw et al. (1989) Proc. Nat. Acad. Sci. 86, 7537-7541)

Sense Primer [SEQ ID NO:3]:

5′-GGCCGAATTCGATGGCCGAAGGCGGCGACAACCAGT-3′ (EcoRI site is underlined)

Antisense Primer [SEQ ID NO:4]:

5′-GCCCGGATCCTTACTCTCTCTCTCCTGTTAGGAACCCA-3′ (BamH-I site is underlined)

Region Cloned: Nucleotides 685-1183 of the Toxo P24 gene cloned into the EcoRI/BamH-I sites of pJO200 to yield plasmid pJO200-P24.

Toxo P25 Gene

(Johnson et al. (1991) Gene 99, 127-132) Sense Primer [SEQ ID NO:5]:

5′-GGCGAATTCGATGCAAGAGGAAATCAAAGAAGGGGTGGA-3′ (EcoRI site is underlined)

Antisense Primer [SEQ ID NO:6]:

5′-CGCACTCTAGATCACCTCGGAGTCGAGCCCAAC-3′ (XbaI site is underlined)

Region Cloned: Nucleotides 7-288 of the Toxo P25 gene cloned into the EcoRI/XbaI sites of pJO200 to yield plasmid pJO200-P25.

Toxo P28 (GRA2) Gene

(Prince et al. (1989) Mol. Biochem. Parasitol. 34, 3-13)

Sense Primer [SEQ ID NO:7]:

51′-GGCGAATTCGATGAGCGGTAAACCTCTTGATGAG-3′ (EcoRI site is underlined)

Antisense Primer [SEQ ID NO:8]:

5′-CGCTAGGATCCTTACTGCGAAAAGTCTGGGAC-3′ (BamH-I site is underlined)

Region Cloned: Nucleotides 489-924 of the Toxo P28 gene cloned into the EcoRI/BamH-I sites of pJO200 to yield plasmid pJO200-P28.

Toxo P30 (SAGI) Gene

(Burg et al. (1988) J. Immunol. 141, 3584-3591)

Sense Primer [SEQ ID NO:9]:

5′-GGCGAATTCGATGCTTGTTGCCAATCAAGTTGTCACC-3′ (EcoRI site is underlined)

Antisense Primer [SEQ ID NO:10]:

5′-CGCTAGGATCCTCACGCGACACAAGCTGCGA-3′ (BamH-I site is underlined)

Region Cloned: Nucleotides 464-1318 of the Toxo P30 gene cloned into the EcoRI/BamH-I sites of pJO200 to yield plasmid pJO200-P30.

Toxo P35 Gene

(Knapp et al. (1989) EPA 431541A2)

Sense Primer [SEQ ID NO:11]:

51′-GACGGCGAATTCGATGAACGGTCCTTTGAGTTATC-3′ (EcoRI site is underlined)

Antisense Primer [SEQ ID NO:12]:

5′-CGCTAGGATCCTTAATTCTGCGTCGTTACGGT-3′ (BamH-I site is underlined)

Region Cloned: Nucleotides 91-822 of the Toxo P35 gene cloned into the EcoRI/BamH-I sites of pJO200 to yield plasmid pJO200-P35.

Toxo P35 Gene Subclone#1 (1-135aa)

(Knapp et al. (1989) EPO 431541A2)

Sense Primer [SEQ ID NO:13]:

5′-GACGGCGAATTCGATGAACGGTCCTTTGAGTTATC-3′ (EcoRI site is underlined)

Antisense Primer [SEQ ID NO:14]:

5′-CGCTAGGATCCTCAATGGTGAACTGCCGGTATCTCC-3′ (BamH-I site is underlined) Region Cloned: Nucleotides 91-495 of the Toxo P35 gene cloned into the EcoRI/BamH-I sites of pJO200 to yield plasmid pJO200-P35S.

Toxo P41 (GRA4) Gene

(Mevelec et al. (1992) Mol. Biochem. Parasitol. 56, 227-238)

Sense Primer [SEQ ID NO:15]:

5′-GGCGAATTCGATGGGTGAGTGCAGCTTTGGTTCT-3′ (EcoRI site is underlined)

Antisense Primer [SEQ ID NO:16]:

5′-CGCACTCTAGATCACTCTTTGCGCATTCTTTCCA-3′ (XbaI site is underlined)

Region Cloned: Nucleotides 133-1107 of the Toxo P41 gene cloned into EcoRI/XbaI sites of pJO200 to yield plasmid pJO200-P41.

Toxo P54 (ROP2) Gene

(Saavedra et al. (1991) J. Immunol. 147, 1975-1982)

Sense Primer [SEQ ID NO:17]:

5′-GCCTGAATTCGATGCACGTACAGCAAGGCGCTGGCGTTGT-3′ (EcoRI site is underlined)

Antisense Primer [SEQ ID NO:18]:

51′-CGCTAGGATCCTCAGAAGTCTCCATGGCTTGCAATGGGAGGA-3′ (Cloned as a blunt end)

Region Cloned: Nucleotides 85-1620 of the Toxo P54 gene cloned into the EcoRI/SmaI sites of pJO200 to yield plasmid pJO200-P54.

Toxo P66 (ROP1) Gene

(Knapp et al. (1989) EPA 431541A2)

(Ossorio et al. (1992) Mol. Biochem. Parasitol. 50, 1-15.

Sense Primer [SEQ ID NO:19]:

51′-GGCGAATTCGATGAGCCACAATGGAGTCCCCGCTTATCCA-3′ (EcoRI site is underlined)

Antisense Primer [SEQ ID NO:20]:

5′-CGCTAGGATCCTTATTGCGATCCATCATCCTGCTCTCTTC-3′ (BamH-I site is underlined)

Region Cloned: Nucleotides 122-1330 of the Toxo P66 gene cloned into the EcoRI/BamH-I sites of pJO200 to yield plasmid pJO200-P66 using Toxoplasma cDNA as template. Nucleotides 122-1330 of the Toxo P66 gene cloned into the EcoRI/BamH-I sites of pJO200 to yield plasmid pJO200-P66g using Toxoplasma genomic DNA as template.

Toxo P68 Gene

(Knapp et al. (1989) EPA 431541A2)

Sense Primer [SEQ ID NO:21]:

5′-ACCCGAATTCGATGACAGCAACCGTAGGATTGAGCCAA-3′ (EcoRI site is underlined)

Antisense Primer [SEQ ID NO:22]:

5′-CGCTGGATCCTCAAGCTGCCTGTTCCGCTAAGAT-3′ (BamH-I site is underlined)

Region Cloned: Nucleotides 294-1580 of the Toxo P68 gene cloned into the EcoRI/BamH-I sites of pJO200 to yield plasmid pJO200-P68.

Step B: Construction and Immunoscreening of a Toxoplasma cDNA Library

A Toxoplasma cDNA library was constructed in the UNIZAP XR vector using the Stratagene ZAP-cDNA Synthesis kit and ZAP-cDNA Gigapack II Gold Cloning kit. The cDNA produced in Example 2D was further processed using the kit protocols as briefly outlined below. The cDNA ends were blunted with T4 DNA polymerase, and EcoRI restriction site adapters were ligated to the blunt-ended cDNA. The RI adaptors ligated to the CDNA were then kinased with T4 Polynucleotide Kinase. The cDNA was digested with the restriction enzymes EcoRI and XhoI and then ligated to the phage lambda UNIZAP XR vector arms. The cDNA is cloned unidirectionally into this vector, resulting in the 5′ end of the cDNA located downstream of the lacZ gene. If the coding sequence of the cDNA is in frame with the lacZ gene, a lacZ-Toxo fusion protein will be expressed. The UNIZAP XR-Toxo cDNA ligation mixture was packaged into phage in vitro, and a primary Toxoplasma cDNA phage library was obtained with 660,000 members. This library was amplified and checked for the size and frequency of the cloned cDNA inserts by converting a dozen random phage clones to E. coli phagemid (plasmid) clones using the Stratagene in vivo subcloning protocol from the ZAP-cDNA Synthesis kit. This procedure excises the cloned cDNA insert and the pBLUESCRIPT plasmid from the phage resulting in a pBLUESCRIPT plasmid clone containing the cloned cDNA. Miniprep DNA was made from the phagemid clones and analyzed with restriction enzymes on DNA agarose gels. Greater than 90% of the phagemid clones contained insert DNA with an average size of 0.8 Kb. This library was used for immunological screening with pooled plasma obtained from patients with Toxoplasmosis as described below.

Plasmas obtained from individuals in the acute phase of Toxoplasmosis infection were pooled. Samples used for this pool were tested by the Abbott IMx Toxo IgM and Toxo IgG immunoassays (Abbott Laboratories, Abbott Park, Ill.), and only samples that contained IgM antibodies and no detectable levels of IgG antibody were pooled. Prior to immunoscreening, the pooled plasma was treated to remove E. coli cross-reactive antibodies. The procedure followed was a modification of the protocol described in the Stratagene picoBLUE immunoscreening kit. Pooled plasma was initially diluted 1:5 in Rubazyme specimen dilution buffer and E. coli cross-reactive antibodies were removed by incubating the diluted pool plasma with several nitrocellulose filters coated with E. coli lysate as described in the kit protocol. After absorption of E. coli antibodies, the plasma pool was stored at 4° C. until further use.

The Toxoplasma cDNA library was immunologically screened following a modification of the Stratagene picoBLUE Immunoscreening kit protocol. Briefly, recombinant phage absorbed to the XL-1 Blue strain of E. coli were plated onto pre-warmed 150 mm NZY plates at a density of 20,000 phage per plate and incubated for 3.5 hrs. at 42° C. Duralose UV membranes pretreated with 10 mM IPTG and dried were then overlayed on each plate and incubated for an additional 4 hrs. at 37° C. The filters were oriented by piercing them with an 18 gauge needle, removed from the plate and washed 3× with TBST buffer at room temperature, 10 min. per wash. The filters were then washed once for 10 min. with TBS buffer at room temperature and blocked overnight at 4° C. in membrane blocking solution. The next day the filters were incubated for 2 hrs. at room temperature with the acute phase plasma pool (at 1:40 dilution in Rubazyme SDB). The filters were then washed 2× with TBST for 10 min. per wash and once with TBS for 10 min. and then incubated for 1 hr. at room temperature with goat anti-Human IgM (H+L) horseradish peroxidase-labelled antibody. The filters were washed again as before and developed for 10 min. in HRP color development solution. The filters were then extensively washed with tap water to stop the color development reaction, and plaques which gave a strong blue color were subsequently plaque purified twice and retested for immunoreactivity against the appropriate pool of plasma. Approximately 130,000 plaques were screened with the pooled acute phase plasma with the isolation of 4 positive clones. These phage clones were converted to plasmid clones using the Stratagene in vivo subcloning protocol from the ZAP-cDNA Synthesis Kit and further characterized as described below.

Step C: Characterization of the Immunopositive Clones Isolated With the Acute Phase Plasma Pool

The 4 immunopositive clones isolated with the acute phase plasma pool were designated pGM610, pGM611, pGM612, and pGM613 and were analyzed with restriction enzymes on DNA agarose gels. Clones pGM610 and pGM612 contained a 1.1 Kb insert of DNA, clone pGM611 contained a 0.7 Kb insert of DNA, and clone pGM613 contained a 1.3 Kb insert of DNA. The cDNA inserts contained in these clones were removed from the pBLUESCRIPT vector by restriction enzyme digestion and purified on DNA agarose gels. These 4 purified cDNA inserts were individually labelled with alpha- 32 P-dCTP using the Multiprime DNA labelling kit and protocol from Amersham for hybridization to colony filters and genomic Toxoplasma DNA. Filters for colony hybridization were prepared by gridding E. coli clones containing the cloned Toxoplasma genes described in Examples 3A and 3B onto Duralose UV membranes overlaid on LBamp plates. These plates- were grown overnite at 37° C., and the next day the E. coli colonies were lysed with alkali and prepared for DNA colony hybridization as described in GENERAL METHODS. After hybridization and washing, the hybridization signal was visualized by autoradiography with the result that all 4 immunopositive clones were homologous to one another and are non-homologous to the other 10 genes tested (see Example 3A). In order to determine the homology between the immunopositive clones and between Toxoplasma genomic DNA, the following Southern blot experiment was performed as described in GENERAL METHODS. Toxoplasma genomic DNA and two of the immunopositive clones were digested with restriction enzymes, run on DNA agarose gels, transferred to nitrocellulose and probed with purified radioactively-labelled cDNA inserts from clones pGM611 and pGM613. After hybridization and washing, the hybridization signal was visualized by autoradiography with the result that both clones were homologous to one another and all hybridized to the genomic blot of Toxoplasma DNA. Therefore, these 4 immunopositive clones contained the same Toxoplasma gene encoding a novel antigen which was designated P novel2 .

EXAMPLE 4

Construction of CKS-P novel2 Expression Vector Based on pJO200

The gene encoding the P novel2 antigen was subcloned into the pJO200 vector in order to produce adequate levels of fusion protein for further analysis. Since the reading frame of the lacZ gene in the pBLUESCRIPT vector and the reading frame of the CKS gene in the pJO200 vector are the same, presence of the EcoRI site at the juncture of the CKS and Toxoplasma genes ensured that the Toxoplasma gene was fused translationally in frame with the CKS gene. In order to remove the cDNA insert from the pBLUESCRIPT vector and subclone it into the pJO200 vector, the following digests were performed:

The CKS expression vector pJO200 described in Example 3A was digested with EcoRI and SmaI and the vector backbone was purified on an agarose gel in preparation for subcloning. Plasmid DNA from the largest P novel2 clone pGM613 was digested with Asp718 and then treated with the Klenow fragment of DNA Polymerase I to render the ends blunt-ended. Subsequently, the DNA was extracted and then digested with EcoRI, and the 1.3Kb EcoRI/Asp718 (Klenow) DNA fragment from pGM613 was purified on an agarose gel and ligated to pJO200/EcoRI/SmaI overnight at 16° C.

The next day, the ligation mixture was transformed into competent XL-1 Blue cells. Miniprep DNA was prepared from the transformants and screened for the presence of the 1.3 Kb DNA fragment inserted at the EcoRI/SmaI sites of pJO200. The correct CKS-P novel2 clone identified by restriction analysis was designated pJO200-P novel2 .

EXAMPLE 5

Expression of Recombinant Toxo Antigens and CKS in E. coli

Step A: Expression of cloned genes in E. coli

Bacterial clones pJO200-P22, pJO200-P24, pJO200-P25, pJO200-P28, pJO200-P30, pJO200-P35S, pJO200-P41, pJO200-66g, pJO200-68 and pJO200-P novel2 expressing the CKS fusion proteins rpJO200-P22, rpJO200-P24, rpJO200-P25, rpJO200-P28, rpJO200-P30, rpJO200-P35S, rpJO200-P41, rpJO 200-66 g, rPJ 0200-68 and rpJO200-P novel2 of Examples 3 and 4 and the control bacterial strain expressing unfused CKS were grown in “SUPERBROTH II” media containing 100 ug/ml ampicillin to log phase, and the synthesis of the CKS-Toxo fusion protein and unfused CKS was induced by the addition of IPTG as previously described (Robinson et al. (1993) J. Clin. Micro. 31, 629-635). After 4 hours post-induction, the cells were harvested, and the cell pellets were stored at −80° C. until protein purification occurred.

Step B: Purification of Recombinant Toxo Antigens and CKS Protein

Insoluble recombinant antigens rpJO200-P22, rpJO200-P25, rpJO200-P30, rpJO200-P35S, rpJO200-P41, rpJO200-66g, and rpJO200-P novel2 were purified after lysis from cell paste by a combination of detergent washes followed by solubilization in 8M urea (Robinson et al. (1993) J. Clin. Micro. 31, 629-635). After solubilization was complete, these proteins were filtered through a 0.2 u filter and further purified by chromatography on SEPHACRYL S-300 columns. The appropriate column fractions were pooled for each protein and stored at 2-8° C. for evaluation by microtiter ELISA. Soluble rpJO200-P24, rpJO200-P28, rpJO200-P68, and unfused CKS proteins were purified after cell lysis by ammonium sulfate precipitation followed by ion-exchange chromatography. The appropriate column fractions were pooled for each protein, dialyzed against the appropriate buffer, and stored at 2-8° C. for evaluation by microtiter ELISA.

EXAMPLE 6

Evaluation of Human Sera with the Recombinant Toxo Antigens in Microtiter ELISA

Step A: Human Sera for Testing

The tests used for determining the presence of IgG and IgM antibody in sera were the Abbott Toxo-G and Toxo-M MEIA assays, respectively. Twenty-four Toxo IgG positive sera, eighteen Toxo IgM positive sera, and nineteen sera negative for Toxo IgG and IgM antibody were evaluated using the recombinant Toxo antigens in Microtiter ELISA.

Step B: Evaluation of Human Sera in the Recombinant Toxo Antigen Microtiter ELISA

Purified recombinant Toxo antigens (Example 5B) were individually diluted to 5.0 ug per ml in PBS, and 0.1 ml of each antigen was added to separate wells of microtiter Maxisorp plates. Control wells for each sera were coated with E. coli lysate at 5.0 ug per ml. Plates were incubated at 37° C. for 1 hr and stored overnight at 4° C. The next day, the plates were washed three times with distilled water and blocked for 2 hrs at 37° C. with 0.2 ml of blocking solution (3% fish gelatin, 10% fetal calf serum in PBS, 0.22 u). The plates were then washed three times with distilled water and ready for incubation with serum. Each serum specimen was tested in duplicate with each antigen at a 1:200 dilution into Rubazyme SDB containing 2% E. coli lysate. After adding 0.1 ml of diluted specimen to each well, the plates were incubated for 1 hr. at 37° C. The plates were then washed three times with PBS-Tween and three times with distilled water. Bound human IgG and IgM were detected by using goat anti-human IgG-HRPO and IgM-HRPO conjugates, respectively, diluted 1:1,000 in Rubazyme conjugate diluent buffer and filtered. After addition of 0.1 ml of the appropriate diluted conjugate, the plates were incubated for 1 hr. at 37° C. and washed three times with PBS-Tween and three times with distilled water. The OPD color development reagent was prepared per manufacturer's directions and 0.1 ml was added to each well. After 2 minutes, the color development reaction was stopped by adding 0.1 ml of 1N sulfuric acid, and the plate was read in a microtiter plate reader. The net OD was obtained by subtracting the OD for the E. coli lysate-control from that of the test with each recombinant antigen. The cut-off for these assays was between 2 to 3 standard deviations from the mean of the negative population for each antigen.

The results of the evaluation of human sera in the recombinant microtiter ELISA are shown in Table 1 for detection of Toxoplasma-specific IgG antibody and in Table 2 for detection of Toxoplasma-specific IgM antibody. The performance of each antigen was ranked in decreasing order of the antigen with the largest number of positive specimen results per total number of positive (IgM or IgG) specimens tested.

TABLE 1
Relative rank of Antigen Performance in
Microtiter IgG ELISA
	Immunoreactivity
	IgG −	IgG +
	# Pos Results/Total # IgG−	# Pos Results/Total # IgG+
Antigen	Specimens Tested	Specimens Tested
P68	1/19	16/24
P35S	1/19	14/24
P24	0/19	14/24
P30	2/18	13/24
Pnove12 (P29)	1/19	13/24
P22	0/19	13/24
P30	2/18	13/24
P41	0/19	10/24
P25	1/19	10/24
P28	1/19	10/24
P66	2/19	9/24

TABLE 2
Relative rank of Antigen Performance in Microtiter
IgM ELISA
	Immunoreactivity
	IgM −	IgM +
	# Pos Results/Total # IgM−	# Pos Results/Total # IgM+
Antigen	Specimens	Specimens
P66	1/18	17/18
P35 (1-135)	0/18	15/18
Pnove12 (P29)	0/19	10/18
P68	0/19	5/18
P22	0/19	5/18
P28	1/18	4/18
P41	0/18	3/18
P25	0/19	3/18
P30	1/18	2/18
P24	1/19	0/18

As can be seen from Table 1, there was no single recombinant Toxo antigen capable of detecting as positive all 24 IgG positive specimens. Hence, an immunoassay employing some combination of the antigens listed in Table 1 is required to detect all the IgG positive specimens.

As can be seen from Table 2, there was no single recombinant Toxo antigen capable of detecting as positive all 18 IgM positive specimens. Hence, an immunoassay employing some combination of the antigens listed in Table 2 is required to detect all the IgM positive specimens.

EXAMPLE 7

Generation of a Monoclonal Antibody Reactive With CKS-P novel2 Antigen

Step A: Immune Response Study in Mice and Generation of Hybridomas

Animals, including mice, rats, hamsters, rabbits, goats and sheep may be infected with a lethal dose of tachyzoites, rescued from death with drug therapy and later used for hybridoma development. There are two hydbridoma development advantages for using this process that otherwise would not be possible. The first advantage is that time is allowed for a diverse repertoire of antibodies to be generated against native T. gondii (or Borrelia burgdorferi , Schistosoma sp., for example, Schistosoma treponema , or sporozoans other than T. gondii , for example, members of the genus Plasmodium (e.g., P. vivax and P. falciparum ) and other possible members of the genus Toxoplasma)), and the second advantage is that the rescue allows time for affinity maturation of the immune response.

In the present experiment, Swiss mice were infected intraperitonally with 2.5×10 7 tachyozoites of T. gondii strain TS4. Five days later mice were treated orally with 10 mg pyrimethamine and 200 mg sulfamethoxazole per kg daily for 10 days. (This technique can be repeated every 6-8 weeks if desired.) After 12 additional weeks, these mice were injected intravenously with 1.2×10 7 sonicated tachyzoites 3 days prior to fusion to minimize the biohazardous status. One hundred percent of the mice survived (providing evidence of a humane method). Resulting hybrids from the PEG mediated fusion of splenocytes and the SP2/0 myeloma were screened on the sonicated tachyzoites and CKS-P novel2 antigen (Kohler, G. and Milstein, C. (1975) Nature 256, 495-497; Kohler, G. and Milstein, C. (1976) Eur. J. Immunol. 6, 511-519; Goding, J. (1986) Monoclonal Antibodies: Principles and Practice. 2nd Ed. Academic Press London).

It should also be noted that monoclonal antibodies may be produced by immunizing mice by intraperitoneal infection with T. gondii (Mineo et al. (1993) J. Immunol. 150, 3951-3964; Handman et al. (1980) J. Immunol. 124, 2578-2583; Grimwood and Smith (1992) Exp. Parasitol. 74, 106-111) or with fractions of T. gondii (Prince et al. (1990) Mol. Biochem. Parasitol. 43, 97-106). Fusion of spleen cells and myeloma cells may then be carried out directly, subsequent to immunization, without a drug therapy step (see, e.g., Kohler and Milstein, supra (1975)).

Step B: Screening and Isolation of a Monoclonal Antibody to rpCKS-P novel2

Bacterial clone pJO200-P novel2 expressing the CKS-P novel2 fusion protein of Example 4 (rpJO200-P novel2 ) and the control bacterial strain expressing unfused CKS were grown in Superbroth II media containing 100 ug/ml ampicillin to log phase, and the synthesis of the CKS-Toxo fusion protein and unfused CKS was induced by the addition of IPTG as previously described in Example 5A. In preparation for screening hybridoma fluids obtained in Example 7A, cell pellets were thawed, resuspended in 10 ml of PBS and sonicated for 0.5 min in an icewater bath. The antigen preparation was diluted 1:40 in 0.05 M sodium carbonate-bicarbonate, pH 9.6, containing 15 mM sodium azide after which 0.1 ml of this suspension was placed in wells of NUNC MAXISORB microtiter plates. When tachyzoites were tested, 3×10 6 sonicated tachyzoites were added to wells. Plates were incubated at 37° C. for 1 hr, stored 1 to 3 days at 4° C., and washed three times with distilled water. Hybridoma fluids obtained in Example 7A were diluted 1:10 in Rubazyme SDB. The remainder of the ELISA was performed as described above in Example 6B except bound antibody was detected by mixture of horseradish peroxidase-conjugated goat anti-mouse IgG and IgM, each diluted to 1.0 ug per ml in Rubazyme conjugate diluent buffer.

Positive hybridoma clones were cloned by limiting dilution, and hybridoma fluid was retested by microtiter ELISA containing rpJO200-P novel2 , unfused CKS, and sonicated tachyzoites. One highly reactive monoclonal antibody clone was isolated which was designated Toxo Mab 5-241-178, which reacted very strongly with sonicated tachyzoites and rpJO200-P novel2 but showed no reactivity to unfused CKS. This hybridoma clone was found to produce IgG type antibodies as determined using a mouse monoclonal antibody isotyping kit from Sigma.

Step C: Identification of the P novel2 Gene Encoding the Toxoplasma P29 Antigen Using Toxo Mab 5-241-178

Total Toxoplasma protein prepared as described in Example 2C was loaded onto an 4-20% gradient Daiichi SDS-PAGE gel along with protein standard molecular weight markers, and transferred to nitrocellulose as described in General Methods. The Western blot was probed with the Toxo Mab 5-241-178 antibody, and the blot was visualized with a goat anti-mouse IgG-HRPO conjugate followed by BioRad Color Development Reagent (4-chloro-1-naphthol and hydrogen peroxide) per manufacturer's directions. A single protein band of 29,000 molecular weight from the Toxoplasma protein prepared from tachyzoites was immunoreactive with the Toxo Mab 5-241-178 indicating that the P novel2 gene cloned in plasmid pGM613 (Example 3C) and pJO200-P novel2 (Example 4) encodes the P29 antigen of Toxoplasma.

EXAMPLE 8

DNA Sequence of Clone pGM613 and Deduced Amino Acid Sequence

The 1.3 Kb EcoRI/XhoI insert of Toxoplasma cDNA contained in pGM613 was sequenced as described in General Methods. The DNA sequence (1268 bp) [SEQ ID NO:23] and the deduced amino acid sequence (228 aa) [SEQ ID NO:24] in-frame with the lacZ gene are shown in FIG. 1 . The open reading frame (nucleotide position 2 to 685) present in this sequence can code for a protein of approximately 25,000 molecular weight. The first ATG present in the DNA sequence is located at nucleotide position 80 and is not surrounded by sequences fulfilling the criteria for initiation of translation (Kozak, M. (1986) Cell 44, 283-292) and is probably not the initiator methionine residue. Hence, it is likely that the insert of Toxoplasma cDNA present in clone pGM613 is not full-length.

Genebank's non-redundant protein, DNA, and dbEST/dbSTS sequences (tags) database and the Derwent DNA and protein patent databases were searched for homology to the DNA sequence and the deduced amino acid sequence of clone pGM613. Homology of DNA sequence and the deduced amino acid sequence was found between a portion of the pGM613 clone (nucleotide positions 461-684, amino acid residues 153-228) and the F29 clone of Knapp et al. contained in European Patent Application 0431541A2. In addition, homology between the DNA sequence of pGM613 and several T. gondii expressed sequence tags of unknown function isolated by Wan, K. -L. et al. (1996) Molec. and Biochem. Parasitol. 75, 179-186 was also found.

EXAMPLE 9

Isolation and Characterization of a Genomic Clone Containing the P29 Gene and Generation of a Composite DNA Sequence

Since the cDNA insert of pGM613 encoding the P29 antigen of Toxoplasma appeared to be less than full-length, portion of the pGM613 cDNA sequence was used as a probe to isolate a genomic clone of the P29 antigen with the goal of cloning the remaining 5′ end of the gene.

Step A: Construction of a Toxoplasma Genomic DNA Library in pJO200

A Toxoplasma genomic DNA library was constructed in the pJO200 vector as follows. Toxoplasma genomic DNA prepared in Example 2A was treated by a partial digestion with the restriction enzyme Sau 3AI as described in General Methods. The partially digested genomic DNA was subsequently electrophoresed on a 0.7% agarose gel with molecular weight standards and the 6-15 Kb molecular weight range of the DNA was isolated, purified, and extracted as described in General Methods. In preparation for ligation with the genomic DNA, plasmid pJO200 was digested with BamH-I followed by dephosphorylation with the CIAP enzyme. The resulting vector backbone was extracted and then ligated overnight at 16° C. with the Sau 3AI digested DNA. The ligation mixture was transformed the next day into competent XL-1 Blue cells, and the resulting transformants were pooled resulting in a primary Toxoplasma genomic library containing 80,000 members.

Step B: Screening Toxoplasma Genomic Library With P29 5′ Gene Probe

In order to isolate the 5′ end of the P29 gene from the genomic library, a portion of the 5′ end of the cDNA clone present in pGM613 was selected as a probe. This portion of the cDNA was then used to probe the Toxoplasma genomic library prepared in Example 9A for genomic clones homologous to the 5′ end of the cDNA.

Plasmid pGM613 was digested with SacII and HindIII, and the 326 bp SacII/HindIII fragment containing the 5′ end of the CDNA insert in pGM613 (nucleotide positions 55-380, see FIG. 1 ) was gel purified. This gene fragment was radioactively labelled and used to probe the Toxoplasma genomic library by colony hybridization as described in General Methods. Positive clones obtained by hybridization were colony purified and retested. One positive clone designated pTXG1-2 containing a 6.5 Kb insert of DNA was further characterized as described below.

Step C: DNA Sequence of Genomic Clone pTXG1-2 and Composite DNA Sequence for the P29 Gene and the Deduced Amino Acid Sequence

The 5′ end of the P29 gene contained in clone pTXG-12 was sequenced as described in General Methods using DNA primers complementary to the 5′ end of the cDNA contained in clone pGM613. The DNA sequence obtained for clone pTXG1-2 [SEQ ID NO:25] is shown in FIG. 2 . An alignment of the DNA sequences for genomic clone pTXG-1 and the CDNA clone pGM613 was then performed resulting in the composite DNA sequence [SEQ ID NO:26] and deduced amino acid sequence [SEQ ID NO:27] for the P29 gene as shown in FIG. 3 . The composite DNA sequence is derived from the genomic sequence of clone pTXG-1 ( FIG. 2 , [SEQ ID NO:25]) and the cDNA sequence of pGM613 ( FIG. 1 , [SEQ ID NO:23]) as shown below in Table 3.

TABLE 3
Source of Sequence for the Composite DNA Sequence for
the P29 Gene
	Nucleotide	Nucleotide	Nucleotide
	Position	Position	Position
	Composite	Genomic	cDNA
	Sequence	Sequence	Sequence
	1-419	1-419	None
	420-477	420-477	40-97
	478-1648	None	98-1268

The only good candidate for the initiator methionine residue for the start of translation of the P29 gene is the first methionine shown on FIG. 3 starting at nucleotide position 358. This is the only methionine in-frame with the reading frame present in the cDNA clone pGM613. If the same reading frame is examined further upstream of the methionine at position 358, no further methionine residues are found before an in-frame UAA stop codon present at position 325. The methionine at nucleotide position 358 is surrounded by sequences fulfilling the criteria for initiation of translation (Kozak, M. (1986) Cell 44, 283-292) and is followed by amino acid residues that constitute a signal peptide (von Heijne, G. (1986) Nucleic Acids Res. 14, 4683-4690).

EXAMPLE 10

Construction of an Improved CKS Epitope-Embedding Vector pEE3

The CKS epitope-embedding expression vector pEE1 described in U.S. patent application Ser. No. 08/742,619 of Maine and Chovan allows for the embedded fusion of recombinant proteins to the CMP-KDO synthetase (CKS) protein. In order to facilitate the cloning of the P29 gene into the CKS epitope-embedding vector, the pEE1 vector was modified in two steps. First, an obsolete polylinker near the 3′ end of the CKS gene in the pEE1 vector was removed generating an intermediate vector pEE2. Secondly, a new polylinker was introduced into the coding region of CKS, thus permitting the embedding of genes using a variety of restriction sites (StuI, EcoRI, SacI, BamH-I, PstI, MluI) into the CKS gene.

Step A: Construction of pEE2

The plasmid pEE2, a derivative of the CKS expression vector pEE1 (FIG. 4 A), was constructed by digesting pEE1 with the Bgl II restriction enzyme and removing a polylinker located at the 3′ end of the CKS gene which had the sequence (5′-3′) [SEQ ID NO:28] ( FIG. 4B ) and the deduced amino acid sequence [SEQ ID NO:49]

AGATCTCGACCCGTCGACGAATTCGAGCTCGGTACCCGGGGATCCTCTAGAC AspLeuAspProSerThrAsnSerSerSerValProGlyAspProLeuAsp

TGCAGGCATGCTAAGTAAGTAGATCT CysArgHisAlaLys

and replacing it with the following sequence (5′-3′) [SEQ ID NO:29] (see FIG. 4C ) and the deduced amino acid sequence [SEQ ID NO:50]

AGATCTCGACCCATCTACCAATTCGTCTTCTGTTCCGGGTGATCCGCTAGAC AspLeuAspProserThrAsnserserservalProGlyAspProLeuAsp

TGCCGTCACGCTAAGTAAGTAGATCT CysArgHisAlaLys.

As shown in FIGS. 4B and 4C , this sequence replacement removes the restriction sites SalI, EcoRI, SacI, KpnI, SmaI, BamH-I, XbaI, PstI, and SphI, thus enabling the use of these sites in a new polylinker to be embedded later within the CKS gene further upstream (Example 10B).

Plasmid pEE1 was digested with Bgl II and then treated with the CIAP enzyme to remove the 5′ phosphate groups to prevent self-ligation. The pEE1/Bgl II dephoshorylated vector backbone was then purified on an agarose gel. Two oligonucleotides shown below (5′-3′) were synthesized for ligation into the pEE1/Bgl II backbone.

[SEQ ID NO:30]
CCTGAAGATCTCGACCCATCTACCAATTCGTCTTCTGTTCCGGGTGATCC
GCTAGACTGCCGTCACGCTAAGTAAGTAGATCTTGACT
[SEQ ID NO:31]
AGTCAAGATCTACTTACTTAGCGTGACGGCAGTCTAGCGGATCACCCGGA
ACAGAAGACGAATTGGTAGATGGGTCGAGATCTTCAGG

These oligonucleotides were mixed together, heated to 85° C. and then allowed to cool gradually overnight to 4° C. to permit annealing of the oligonucleotides. The annealed oligonucleotides were then digested with the Bgl II enzyme, extracted, and then ligated to the pEE1/Bgl II backbone overnight at 16° C. The ligation mixture was transformed the next day into competent XL-1 Blue cells. Miniprep DNA was prepared from the transformants and screened for the presence of the new sequence by restriction enzyme analysis. Putative correct clones were then sequenced to verify the correct sequence in the proper orientation. Plasmid pEE2 was isolated which contains the new sequence [SEQ ID NO:29] at the Bgl II site.

Step B: Construction of pEE3

The plasmid pEE3, a derivative of the CKS expression vector pEE2 (FIG. 5 A), was constructed by digesting pEE2 with StuI and MluI and cloning in a new polylinker with the following sequence (5′-3′) [SEQ ID NO:32](see FIG. 5B ) and deduced amino acid sequence [SEQ ID NO:51]

AGGCCTGAATTCGAGCTCTGGGATCCGTCTGCAGACGCGT GlyLeuAsnSerSerSerGlyIleArgLeuGlnThrArg

which contains the restriction sites StuI, EcoRI, SacI, BamH-I, PstI, and MluI.

Plasmid pEE2 was digested with StuI and MluI, and the vector backbone was purified on an agarose gel. Two oligonucleotides shown below (5′-3′) were synthesized for ligation into the pEE2/StuI/MluI 30 backbone.

[SEQ ID NO:33]
CCTGAATTCGAGCTCTGGGATCCGTCTGCAGA
[SEQ ID NO:34]
CGCGTCTGCAGACGGATCCCAGAGCTCGAATTCAGG

These oligonucleotides were mixed together, heated to 80° C. for 10 minutes and then allowed to cool gradually overnight to 4° C. to permit annealing of the oligonucleotides. The annealed oligonucleotides were then ligated to the pEE2/StuI/MluI backbone overnight at 16° C. The ligation mixture was transformed the next day into competent XL-1 Blue cells. Miniprep DNA was prepared from the transformants and screened for the presence of the new sequence by restriction enzyme analysis. Putative correct clones were then sequenced to verify the correct sequence. Plasmid pEE3 was isolated which contains the new sequence [SEQ ID NO:32] at the StuI/MluI sites.

EXAMPLE 11

Construction of CKS-Toxo Ag-CKS Epitope-Embedding Expression Vectors

The CKS expression vectors pJO200, pEE1, and pEE3 were utilized for the construction of four CKS-Toxo Ag-CKS gene fusion constructs using the Toxo P29, P30, P35, and P66 genes.

Step A: Construction of pToxo-P29: CKS-P29(1-236aa)-CKS

The plasmid pToxo-P29, a derivative of plasmid pEE3 (FIG. 6 ), was constructed by cloning a DNA fragment containing Toxo P29, obtained by PCR amplification of Toxo P29 DNA contained in plasmid pTXG1-2 (Example 9C), into the EcoRI/BamH-I sites of pEE3. Plasmid pToxo-P29 was deposited with the ATCC 10801 University Boulevard, Manassas, Va. 20110-2209, under the terms of the Budapest Treaty on May 19, 1998, and was accorded Accession No. ATCC 98758.

Large scale plasmid DNAs (pTXG1-2 and pEE3) were isolated by general methods. Plasmid pEE3 was digested with EcoRI and BamH-I, and the vector backbone, pEE3/EcoRI/BamH-I, was purified on an agarose gel. A sense primer, starting at nucleotide 358 of the P29 gene ( FIG. 3 ) containing an EcoRI site, and an antisense primer containing a BamH-I site, starting at nucleotide 1065 of the P29 gene, were synthesized as shown below:

Sense Primer [SEQ ID NO:35]

5′-ACTTAGAATTCGATGGCCCGACACGCAATTTTTTCC-3′ (EcoRI site is underlined)

Antisense Primer [SEQ ID NO:36]

5′-ACATGGATCCGCTGGCGGGCATCCTCCCCATCTTC-3′ (BamH-I site is underlined)

The sense and antisense primers were added to a PCR reaction mixture containing plasmid pTXG1-2. After PCR amplification, the reaction mixture was digested with EcoRI and BamH-I, and the 708 base pair DNA fragment containing P29 was purified on an agarose gel. The purified 708 base pair DNA fragment was ligated to pEE3/EcoRI/BamH-I overnight at 16° C. The ligation mixture was transformed the next day into competent XL-1 Blue cells. Miniprep DNA was prepared from the transformants and screened for the presence of the P29 DNA sequence by restriction enzyme analysis. Plasmid pToxo-P29 contained the P29 gene embedded at the EcoRI/BamH-I sites of pEE3. This CKS-ToxoP29-CKS fusion construct was designated:

“CKS (1-171aa)-N-S-ToxoP29(1-236aa)-R-I -R-L-Q-T-R-CKS(171-260aa)”

where N, S, R, I, R, L, Q, T, R are the asparagine, serine, arginine, isoleucine, arginine, leucine, glutamine, threonine, and arginine residues, respectively, encoded by the polylinker DNA sequence of the vector. The complete DNA sequence [SEQ ID NO:37] of plasmid pToxo-P29 and the corresponding amino acid sequence [SEQ ID NO:52] of the CKS-P29-CKS fusion protein is shown in FIG. 7 , wherein amino acid residues 174-409 of SEQ ID NO:52 correspond to the full length amino acid sequence of the P29 antigen of Toxoplasma gondii.

Step B: Construction of pToxo-P30:CKS-P30(1-236aa)-CKS

The plasmid pToxo-P30, a derivative of plasmid pEE1 (FIG. 8 ), was constructed by cloning a DNA fragment containing Toxo P30, obtained by PCR amplification of Toxo P30 DNA contained in plasmid pJO200-P30 (Example 3A), into the StuI/MluI sites of pEE1. Plasmid pToxo-P30 was deposited with the ATCC 10801 University Boulevard, Manassas, Va. 20110-2209, under the terms of the Budapest Treaty on May 19, 1998, and was accorded Acession No. ATCC 98761.

Large scale plasmid DNAs (pJO200-P30 and pEE1) were isolated by general methods. Plasmid pEE1 was digested with StuI and MluI, and the vector backbone, pEE1/StuI/MluI, was purifed on an agarose gel. A sense primer, starting at nucleotide 464 of the P30 gene containing an StuI site, and an antisense primer containing a MluI site, starting at nucleotide 1318 of the P30 gene (Burg et al. (1988) J. Immunol. 141, 3584-3591) were synthesized as shown below:

Sense Primer [SEQ ID NO:38]

5′-TCCTAGGCCTTAATTCGATGCTTGTTGCCAATCAAG- 3′ (StuI site is underlined)

Antisense Primer [SEQ ID NO:39]

5′-ACATACGCGTCGCGACACAAGCTGCGATAGAG-3′ (MluI site is underlined)

The sense and antisense primers were added to a PCR reaction mixture containing plasmid pJO200-P30. After PCR amplification, the reaction mixture was digested with StuI and MluI, and the 855 base pair DNA fragment containing P30 was purified on an agarose gel. The purified 855 base pair DNA fragment was ligated to pEE1/StuI/MluI overnight at 16° C. The ligation mixture was transformed the next day into competent XL-1 Blue cells. Miniprep DNA was prepared from the transformants and screened for the presence of the P30 DNA sequence by restriction enzyme analysis. Plasmid pToxo-P30 contained the P30 gene embedded at the StuI/MluI sites of pEE1. This CKS-ToxoP30-CKS fusion construct was designated:

“CKS(1-171aa)-N-S-M-ToxoP30(5-289aa)-T-R-CKS(171-260aa)”

where N, S, M, T, R are the asparagine, serine, methionine, threonine, and arginine residues, respectively, encoded by the synthetic DNA sequence of the vector. The complete DNA sequence [SEQ ID NO:40] of plasmid pToxo-P30 is shown in FIG. 9 and the corresponding amino acid sequence [SEQ ID NO:53] of the CKS-P30-CKS fusion protein are shown in FIG. 9 , wherein amino acid residues 175-459 of SEQ ID NO:53 correspond to amino acids 5-289 of the P30 antigen of Toxoplasma gondii.

Step C: Construction of pToxo-P35S:CKS-P35(1-135aa)-CKS

The plasmid pToxo-P35S, a derivative of plasmid pJO200 (FIG. 10 ), was constructed by cloning a DNA fragment containing Toxo P35, obtained by PCR amplification of Toxo P35 DNA contained in plasmid pJO200-P35 (Example 3A), into the StuI site of pJO200. Plasmid pToxo-P35S was deposited with the ATCC 10802 University Boulevard, Manassas, Va. 20110-2209, under the terms of the Budapest Treaty on May 19, 1998, and was accorded Accession No. ATCC 98759.

Large scale plasmid DNAs (pJO200-P35 and pJO200) were isolated by general methods. Plasmid pJO200 was digested with StuI and BamH-I, and the vector backbone, pJO200/StuI/BamH-I, was purified on an agarose gel. A sense primer, starting at nucleotide 91 of the P35 gene containing an StuI site, and an antisense primer containing a MluI site, starting at nucleotide 495 of the P35 gene (Knapp et al., 1989 (EPA 431541A2)) were synthesized as shown below:

Sense Primer [SEQ ID NO:41]

5′-GAGCAGAAGGCCTTATGAACGGTCCTTTGAGTTATCATCC-3′ (StuI site is underlined)

Antisense Primer [SEQ ID NO:42]

5′-TTCGCTCACGCGTATGGTGAACTGCCGGTATCT-3′ (MluI site is underlined)

The sense and antisense primers were added to a PCR reaction mixture containing plasmid pJO200-P35. After PCR amplification, the reaction mixture was digested with StuI and MluI, and the 405 base pair DNA fragment containing P35 was purified on an agarose gel. A sense primer, starting at nucleotide 640 of pJO200 containing an MluI site, and an antisense primer starting at nucleotide 905 of pJO200 were synthesized as shown below:

Sense Primer [SEQ ID NO:43]

5′-GACGGAGACGCGTCTTGAACCGTTGGCGATAACT-3′ (MluI site is underlined)

Antisense Primer [SEQ ID NO:44]

5′-GCATGCCTGCAGTCTAGAGGA-3′

The sense and antisense primers were added to a PCR reaction mixture containing plasmid pJO200. After PCR amplification, the reaction mixture was digested with MluI and BamH-I, and the 266 base pair DNA fragment containing P35 was purified on an agarose gel.

The purified 405 base pair DNA fragment containing the P35 gene and the purified 266 base pair DNA fragment containing the 3′ end of the CKS gene, were ligated to pJO200/StuI/BamH-I overnight at 16° C. The ligation mixture was transformed the next day into competent XL-1 Blue cells. Miniprep DNA was prepared from the transformants and screened for the presence of the P35 DNA sequence by restriction enzyme analysis. Plasmid pToxo-P35S contained the P35 gene embedded at the StuI/MluI sites of pJO200. This CKS-ToxoP35-CKS fusion construct was designated:

“CKS(1-171aa)-ToxoP35(1-135aa)-T-R-CKS(171-260aa)”

where T and R are the threonine and arginine residues, respectively, encoded by the synthetic DNA sequence of the vector. The complete DNA sequence [SEQ ID NO:45] of plasmid pToxo-P35S and the corresponding amino acid sequence [SEQ ID NO:54] of the CKS-P35-CKS fusion protein are shown in FIG. 11 , wherein amino acid residues 172-306 of SEQ ID NO:54 correspond to amino acids 1-135 of the P35 antigen of Toxoplasma gondii.

Step D: Construction of pToxo-P66g:

CKS-P66(26-428aa)-CKS

The plasmid pToxo-66g, a derivative of plasmid pEE1 (FIG. 12 ), was constructed by cloning a DNA fragment containing Toxo P66, obtained by PCR amplification of Toxo P66 DNA contained in plasmid pJO200-P66g (Example 3A), into the StuI/MluI sites of pEE1. Plasmid pToxo-P66g was deposited with the ATCC 10801 University Boulevard, Manassas, Va. 20110-2209, under the terms of the Budapest Treaty on May 19, 1998, and was accorded Accession No. ATCC 98760.

Large scale plasmid DNAs (pJO200-P66g and pEE1) were isolated by general methods. Plasmid pEE1 was digested with StuI and MluI, and the vector backbone, pEE1/StuI/MluI, was purified on an agarose gel. A sense primer, starting at nucleotide 122 of the P30 gene containing an StuI site, and an antisense primer containing a MluI site, starting at nucleotide 1330 of the P66 gene (Knapp et al., supra (1989)) were synthesized as shown below:

Sense Primer [SEQ ID NO:46]

5′-ATATTAGGCCTTATGAGCCACAATGGAGTCCCCGCTTATCC-3′ (StuI site is underlined)

Antisense Primer [SEQ ID NO:47]

30 5′-CAGTGTACGCGTTTGCGATCCATCATCCTGCTCTCTTC-3′ (MluI site is underlined)

The sense and antisense primers were added to a PCR reaction mixture containing plasmid pJO200-P66g. After PCR amplification, the reaction mixture was digested with StuI and MiuI, and the 1209 base pair DNA fragment containing P66 was purified on an agarose gel. The purified 1209 base pair DNA fragment was ligated to pEE1/StuI/MluI overnight at 16° C. The ligation mixture was transformed the next day into competent XL-1 Blue cells. Miniprep DNA was prepared from the transformants and screened for the presence of the P66 DNA sequence by restriction enzyme analysis. Plasmid pToxo-P66g contained the P66 gene embedded at the StuI/MluI sites of pEE1. This CKS-ToxoP66-CKS fusion construct was designated:

“CKS (1-171aa)-M-ToxoP66(26-428aa)-T-R-CKS (171-260aa)”

where M, T, and R are the methionine, threonine and arginine residues, respectively, encoded by the synthetic DNA sequence of the vector. The complete DNA sequence [SEQ ID NO:48] of plasmid pToxo-P66g and the corresponding amino acid sequence [SEQ ID NO:55] of the CKS-P66-CKS fusion protein are shown in FIG. 13 , wherein amino acid residues 173-575 of SEQ ID NO:55 correspond to amino acids 26-428 of the P66 antigen of Toxoplasma gondii.

EXAMPLE 12

Development of a Toxo Recombinant Antigen Cocktail for the Detection of Toxoplasma-Specific IgG and IgM

The results in Tables 1 and 2 of Example 6B indicated that more than one recombinant antigen would be required to detect Toxoplasma-specific IgG and IgM in order to replace the tachyzoite in an immunoassay. Additional sera were sourced from patients with an acute or chronic Toxolasmosis and tested with the individual antigens coated in separate wells listed in Tables 1 and 2 using the IgG or IgM Microtiter ELISA described in Example 6B. These results indicated that a cocktail of recombinant antigens necessary and sufficient to replace the tachyzoite in an immunoassay should be composed of the following Toxo antigens:

Toxo IgG Immunoassay: P29+P30+P35

Toxo IgM Immunoassay: P29+P35+P66

In order to demonstrate the diagnostic utility of the Toxo recombinant antigens in the proposed above combinations in an immunoassay, i.e. the coating of the Toxo antigens P29, P30, and P35 in a single microtiter plate well (Microtiter format) or other solid phase, e.g. microparticles (MEIA format), to detect Toxoplasma-specific IgG antibodies and the coating of the Toxo antigens P29, P35, and P66 in a single microtiter plate well (Microtiter format) or other solid phase, e.g. microparticles (MEIA format), to detect Toxoplasma-specific IgM antibodies, the following experiments were performed:

Step A: Expression of cloned genes in E. coli

Bacterial clones pToxo-P29, pToxo-P30, pToxo-P35S, and pToxo-P66g expressing the CKS fusion proteins rpToxo-P29, rpToxo-P30, rpToxo-P35S, and rpToxo-P66g, respectively, were grown in SUPERBROTH II media containing 100 ug/ml ampicillin to log phase, and the synthesis of the CKS-Toxo fusion protein was induced by the addition of IPTG as previously described (Robinson et al. (1993) J. Clin. Micro. 31, 629-635). After 4 hours post-induction, the cells were harvested, and the cell pellets were stored at −80° C. until protein purification.

Step B: Purification of Recombinant Toxo Antigens

Insoluble recombinant antigens rpToxo-P29, rpToxo-P30, rpToxo-P35S, and rpToxo-P66g were purified after lysis from cell paste by a combination of detergent washes followed by solubilization in 8M urea (Robinson et al., supra (1993)). After solubilization was complete, these proteins were filtered through a 0.2 m filter and either stored at 2-8° C. (w/urea) or dialyzed against 50 mM Tris, pH 8.5 and then stored at 2-8° C. (w/o urea).

Step C: Human Sera for Testing

Four groups of serum specimens from a French population were evaluated for the presence of Toxoplasma-specific IgG and IgM antibodies using the Microtiter ELISA. These serum specimens collectively cover the entire span of Toxoplasma infection from early seroconversion (acute toxoplasmosis) to convalesence (latent infection, chronic toxoplasmosis) and represent the types of specimens normally encountered in routine Toxoplasma serology.

Group 1: Negative Serum Specimens

This group contained 200 serum specimens negative for Toxoplasma IgG and IgM antibodies as determined by the Abbott IMx Toxo IgG and IgM immunoassays.

Group 2: “Ancienne” Serum Specimens

This group contained 100 serum specimens negative for Toxoplasma IgM antibodies and positive for Toxoplasma IgG antibodies by the Abbott IMx Toxo IgG and IgM immunoassays. These specimens were negative for Toxoplasma IgA antibodies as determined by an immunocapture assay using a suspension of tachyzoites (IC-A) (Pinon, J. M. (1986) Diag. Immunol. 4:223-227).

Group 3: “Evolutive” Serum Specimens

This group contained 99 serum specimens positive for Toxoplasma IgG antibodies by a high sensitivity direct agglutination assay (HSDA) (Desmonts, G. and Remington, J. S. (1980) J. Clin. Micro. 11:562-568) and positive for Toxoplasma IgM and IgA antibodies using a specific immunocapture assay (IC-M, IC-A).

Group 4: “Precoce” Serum Specimens

This group contained 66 specimens sourced from individuals with evidence of a early seroconversion of Toxoplasma-specific antibodies (absence or early manifestation of IgG antibodies and positive for IgM and IgA antibodies using a specific immunocapture assay (IC-M, IC-A)).

Step D: Evaluation of Human Sera in the Recombinant Toxo Antigen Microtiter ELISA

Purified recombinant Toxo antigens (Example 12B) were coated onto the wells of the microtiter plate as follows:

For the IgG microtiter ELISA, the three Toxo antigens rpToxo-P29, rpToxo-P30, and rpToxo-P35S (w or w/o urea) were diluted together into PBS to a final concentration of 5 ug/ml for each antigen, and plates were coated and processed as described in Example 6B using a goat anti-human IgG-HRPO conjugate to detect bound human IgG. All three Toxo antigens were coated together into the same microtiter well to detect Toxoplasma-specific IgG. For the IgM microtiter ELISA, the three Toxo antigens rpToxo-P29, rpToxo-P35S, and rpToxo-P66g (w or w/o urea) were diluted together into PBS to a final concentration of 5 mg/ml for each antigen, and plates were coated and processed as described in Example 6B using a goat anti-human IgM-HRPO conjugate to detect bound human IgM. All three Toxo antigens were coated together into the same microtiter well to detect Toxoplasma-specific IgM. The cut-off for these assays was between 2 to 3 standard deviations from the mean of the negative population.

Step E: Results of the Evaluation of Human Sera in the Recombinant Toxo Antigen (P29+P30+P35) IgG Microtiter ELISA

The serum specimens from Groups 1-4 (Example 12C) were tested for the presence of Toxoplasma-specific IgG using the recombinant Toxo antigen IgG microtiter ELISA (rpToxo-P29 (P29)+rpToxo-P30 (P30)+rpToxo-P35S (P35)). The results from this evaluation are presented in Tables 4-8.

TABLE 4
Evaluation of Group 1 Negative Serum Specimens by
Toxo IgG Microtiter ELISA

TABLE 4
Evaluation of Group 1 Negative Serum Specimens by
Toxo IgG Microtiter ELISA

TABLE 4
Evaluation of Group 1 Negative Serum Specimens by
Toxo IgG Microtiter ELISA

TABLE 7
Evaluation of Group 4 “Précoce” Serum Specimens by
Toxo IgG Microtiter ELISA

TABLE 7
Evaluation of Group 4 “Précoce” Serum Specimens by
Toxo IgG Microtiter ELISA

As can be seen from Tables 4-8, the Toxo IgG microtiter ELISA is both a sensitive and specific assay for the detection of Toxoplasma-specific IgG as demonstrated by the overall high relative diagnostic specificity (95.7%) and sensitivity (98.4%) (Table 8) of the assay. The Toxo recombinant antigen cocktail comprised of the Toxo antigens P29, P30 and P35, in combination with the Toxo IgG assay, is both necessary and sufficient to replace the tachyzoite for the detection of Toxoplasma-specific IgG antibody.

Furthermore, there are several advantages of the recombinant antigen cocktail over the tachyzoite antigen for use in detection of IgG antibodies. First, the antigens are purified, and the amount of each antigen loaded into the immunoassay can be accurately determined and standardized, e.g., protein concentration. This minimizes interlot differences commonly observed in kits manufactured with different tachyzoite antigen lots. Hence, different lots of kits manufactured with different antigen cocktail lots will be very consistent from lot to lot. Secondly, mouse monoclonal antibodies to the individual recombinant Toxo antigens are used to monitor coating of the proteins to the solid phase. This further ensures that each lot produced is consistent. Third, the true clinical sensitivity of the assay using the purified antigens will be higher by virtue of the fact of the higher specific activity of the purified antigens. Finally, kits manufactured with the antigen cocktail are more stable during storage over time, and the performance of the assay using these antigens remains consistent over the shelf life of the assay. Kits manufactured with the tachyzoite antigen are not as stable and their performance may vary over time.

Additionally, there are many advantages of using a cocktail over using a single antigen alone. For example, an immune response to infection varies by individual. Some individuals produce antibodies to P35 and not to P66, whereas some individuals produce antibodies to P66 and not to P35. Thus, the antigen cocktail of the present invention will detect both groups of individuals.

Moreover, immune responses vary with time. For example. One individual may produce antibodies against P35 first and then later produce antibodies to only P66. Thus, the present cocktail will detect both types of “positive” individuals.

Furthermore, individuals may be infected with different Toxo serotypes, strains or isolates. Thus, the immune response may be such that multiple antigens are needed to detect the presence of all antibodies being produced. Again, the present cocktail allows for such detection.

Also, it is known from previous Western Blot experiments with tachyzoite proteins that the immune response to Toxoplasma is directed against several antigens. Once again, the present antigen cocktail will allow for the detection of all antibodies produced in response to these antigens.

Step F: Results of the Evaluation of Human Sera in the Recombinant Toxo Antigen (P29+P35+P66) IgM Microtiter ELISA

The serum specimens from Groups 1-4 (Example 12C) were tested for the presence of Toxoplasma-specific IgM using the recombinant Toxo antigen IgM microtiter ELISA (rpToxo-P29 (P29)+rpToxo-P35S (P35)+rpToxo-P66g (P66)). The results from this evaluation are presented in Tables 9-13.

TABLE 9
Evaluation of Group 1 Negative Serum Specimens by
Toxo IgM Microtiter ELISA

TABLE 10
Evaluation of Group 2 “Ancienne” Serum Specimens by
Toxo IgM Microtiter ELISA

TABLE 10
Evaluation of Group 2 “Ancienne” Serum Specimens by
Toxo IgM Microtiter ELISA

TABLE 10
Evaluation of Group 2 “Ancienne” Serum Specimens by
Toxo IgM Microtiter ELISA

TABLE 13
Summary of Evaluation of Groups 1-4 Serum Specimens
by Toxo IgM Microtiter ELISA

As can be seen from Tables 9-13, the Toxo IgM microtiter ELISA is a specific assay for the detection of Toxoplasma-specific IgM as demonstrated by the overall high relative diagnostic specificity (94.9.%) (Table 13) of the assay. However, the assay appeared to be relatively insensitive to detection of Toxoplasma-specific IgM present in serum specimens from Group 3 “évolutive” (relative diagnostic sensitivity=70%, Table 11) but sensitive to detection of Toxoplasma-specific IgM present in serum specimens from Group 4 “précoce” (relative diagnostic sensitivity=96.7%, Table 12). These data suggest that the Toxo IgM microtiter ELISA may be more sensitive to the detection of Toxoplasma-specific IgM indicative of an acute or recent infection than the IC-M immunocapture assay used as the reference assay.

Further resolution testing was performed with the Abbott IMx Toxo IgM assay and a Toxo IgG avidity assay on the 30 discordant specimens listed in Table 11 that were positive for IgM antibody using the IC-M immunocapture assay and negative for IgM antibody by the Toxo IgM microtiter ELISA. Of the 30 specimens that were false negative by the Toxo IgM microtiter assay, 11 were resolved true negative by the Abbott IMx Toxo IgM assay. Furthermore, all 11 specimens contained Toxoplasma IgG with elevated avidity, representative of a past infection. Of the remaining 19 specimens that were false negative by the Toxo IgM microtiter assay, an additional 11 specimens corresponded to Toxoplasma infections which probably occurred greater than 6 months ago, as demonstrated by the presence of Toxoplasma-specific IgG high avidity antibodies. In addition, one specimen was from a patient with reactivation of toxoplasmosis where normally Toxo IgM antibodies are absent (an IC-M and Abbott IMx Toxo IgM false positive), and one specimen was from a patient with congenital toxoplasmosis. Therefore, after resolution by the Abbott IMx Toxo IgM assay followed by consideration of the Toxo IgG avidity data and clinical history of the specimens, of the 32 specimens false negative by the microtiter IgM assay, 11 were resolved true negative, 13 specimens (from congenitally infected patients) were removed from the calculation of relative diagnostic specificity and sensitivity, and 6 specimens remained false negative. The resolved data and recalculated sensitivity and specificity for the Toxo IgM microtiter assay are shown in Tables 14 and 15.

TABLE 14
Evaluation of Group 3 “Évolutive” Serum Specimens by
Toxo IgM Microtiter ELISA
After Resolution of Discordant Specimens

TABLE 14
Evaluation of Group 3 “Évolutive” Serum Specimens by
Toxo IgM Microtiter ELISA
After Resolution of Discordant Specimens

As can be seen from Tables 14 and 15 after resolution of discordant specimens, the Toxo IgM microtiter ELISA configured with the antigen cocktail is both a sensitive and specific assay for the detection of Toxoplasma-specific IgM as demonstrated by the overall high relative diagnostic specificity (95.0%) and sensitivity (93.8%) (Table 15) of the assay. The Toxo recombinant antigen cocktail comprised of the Toxo antigens P29, P35, and P66 is both necessary and sufficient to replace the tachyzoite for the detection of Toxoplasma-specific IgM indicative of a recent toxoplasmosis.

Furthermore, there are several advantages of this recombinant antigen cocktail over the tachyzoite antigen for use in detection of antibodies to IgM. First, the antigens are purified and the amount of each antigen loaded into the immunoassay can be accurately determined and standardized, e.g., protein concentration. This minimizes interlot differences commonly observed in kits manufactured with different tachyzoite antigen lots. Hence, different lots of kits manufactured with different antigen cocktail lots will be very consistent from lot to lot. Secondly, mouse monoclonal antibodies to the individual recombinant Toxo antigens are used to monitor coating of the proteins to the solid phase. This further ensures that each lot produced is consistent. Third, the true clinical sensitivity of the assay using the purified antigens will be higher by virtue of the fact of the higher specific activity of the purified antigens. Fourth, an IgM assay with the antigen cocktail will preferentially detect IgM antibodies produced in response to a recent infection. This can be seen in Tables 11 and 14 where specimens with high avidity IgG antibodies (indicative of a past or chronic infection) were negative for Toxo-specific IgM using the antigen cocktail in a microtiter ELISA. Finally, kits manufactured with the antigen cocktail are more stable during storage over time, and the performance of the assay using these antigens remains consistent over the shelf life of the assay. Kits manufactured with the tachyzoite antigen are not as stable, and their performance may vary over time.

Additionally, there are many advantages of using a cocktail over using a single antigen alone. For example, an immune response to infection varies by individual. Some individuals produce antibodies to P35 and not to P30 whereas some individuals produce antibodies to P30 and not to P35. Thus, the antigen cocktail of the present invention will detect both groups of individuals.

Also, immune responses vary with time. For example, one individual may produce antibodies against P35 first and then later produce antibodies to only P30. Thus, the present cocktail will detect both types of “positive” individuals.

Furthermore, individuals may be infected with different Toxo serotypes, strains or isolates. Thus, the immune response may be such that multiple antigens are needed to detect the presence of all antibodies being produced. Again, the present cocktail allows for such detection.

Also, it is knownn from previous Western Blot experiments with tachyzoite proteins that the immune response to Toxoplasma is directed against several antigens. Once again, the present antigen cocktail will allow for the detection of all antibodies produced in response to these antigens.

55 1 43 DNA Toxoplasma gondii 1cgcagaattc gatgtccacc accgagacgc cagcgcccat tga 43 2 43 DNA Toxoplasma gondii 2cccgggatcc ttacacaaac gtgatcaaca aacctgcgag acc 43 3 36 DNA Toxoplasma gondii 3ggccgaattc gatggccgaa ggcggcgaca accagt 36 4 38 DNA Toxoplasma gondii 4gcccggatcc ttactctctc tctcctgtta ggaaccca 38 5 39 DNA Toxoplasma gondii 5ggcgaattcg atgcaagagg aaatcaaaga aggggtgga 39 6 33 DNA Toxoplasma gondii 6cgcactctag atcacctcgg agtcgagccc aac 33 7 34 DNA Toxoplasma gondii 7ggcgaattcg atgagcggta aacctcttga tgag 34 8 32 DNA Toxoplasma gondii 8cgctaggatc cttactgcga aaagtctggg ac 32 9 37 DNA Toxoplasma gondii 9ggcgaattcg atgcttgttg ccaatcaagt tgtcacc 37 10 31 DNA Toxoplasma gondii 10cgctaggatc ctcacgcgac acaagctgcg a 31 11 35 DNA Toxoplasma gondii 11gacggcgaat tcgatgaacg gtcctttgag ttatc 35 12 32 DNA Toxoplasma gondii 12cgctaggatc cttaattctg cgtcgttacg gt 32 13 35 DNA Toxoplasma gondii 13gacggcgaat tcgatgaacg gtcctttgag ttatc 35 14 36 DNA Toxoplasma gondii 14cgctaggatc ctcaatggtg aactgccggt atctcc 36 15 34 DNA Toxoplasma gondii 15ggcgaattcg atgggtgagt gcagctttgg ttct 34 16 34 DNA Toxoplasma gondii 16cgcactctag atcactcttt gcgcattctt tcca 34 17 40 DNA Toxoplasma gondii 17gcctgaattc gatgcacgta cagcaaggcg ctggcgttgt 40 18 42 DNA Toxoplasma gondii 18cgctaggatc ctcagaagtc tccatggctt gcaatgggag ga 42 19 40 DNA Toxoplasma gondii 19ggcgaattcg atgagccaca atggagtccc cgcttatcca 40 20 40 DNA Toxoplasma gondii 20cgctaggatc cttattgcga tccatcatcc tgctctcttc 40 21 38 DNA Toxoplasma gondii 21acccgaattc gatgacagca accgtaggat tgagccaa 38 22 34 DNA Toxoplasma gondii 22cgctggatcc tcaagctgcc tgttccgcta agat 34 23 1268 DNA Toxoplasma gondii 23gaattcggca cgaggcgaac tggggcaaag ccgccgccac cagttcgcta ccgcggccac 60cgcgtcagat gacgaactga tgagtcgaat ccgaaattct gactttttcg atggtcaagc 120acccgttgac agtctcagac cgacgaacgc cggtgtcgac tcgaaaggga ccgacgatca 180cctcaccacc agcatggata aggcatctgt agagagtcag cttccgagaa gagagccatt 240ggagacggag ccagatgaac aagaagaagt tcatttcagg aagcgaggcg tccgttccga 300cgctgaagtg actgacgaca acatctacga ggagcacact gatcgtaagg tggttccgag 360gaagtcggag ggcaagcgaa gcttcaaaga cttgctgaag aagctcgcgc tgccggctgt 420tggtatgggt gcatcgtatt ttgccgctga tagacttgtg ccggaactaa cagaggagca 480acagagaggc gacgaacccc taaccaccgg ccagaatgtg ggcactgtgt taggcttcgc 540agcgcttgct gctgccgcag cgttccttgg catgggtctc acgaggacgt accgacattt 600ttccccacgc aaaaacagat cacggcagcc tgcactcgag caagaggtgc ctgaatcagg 660cgaagatggg gaggatgccc gccagtagga tatgggggct aataaaagtg agtaggagct 720cgaggacagt gtcccgaacg cgcctgagag gcagacagac acagaagagt gaagaaaaac 780aacatggtat tacgtgcggt gagtgtttgc tgtcacgtgt tttttgcgcc acaaagacag 840cttgtgttgt atgcatggga tcgacagttc atggacggcg ctacccagag aggcggcatt 900tgcgtacacc gtgggtcgtc atgagtaccg ggacatcgtg ttcgtgttta tttgttcatg 960tcgaagtgca ctaagacacg agacgaaagg gtggttccgc ccctggcagc atcacgtagt 1020ggtttctttg tcgagaacag cggcagtccg aggccacttg agacaggatg tttgagtgta 1080tacagacaac gtggtcacag catgaggcaa agctgtctaa gcagccattt gcgcgagcga 1140agtcatccat gccgactgtg tgagcctctt tcgtcacttt gaatgagaca gaaactaaga 1200ctcgcagcag gtctgaatat tgcgaataat ctacttttaa aaccaaaaaa aaaaaaaaaa 1260aactcgag 1268 24 228 PRT Toxoplasma gondii 24Asn Ser Ala Arg Gly Glu Leu Gly Gln Ser Arg Arg His Gln Phe Ala 1 5 10 15Thr Ala Ala Thr Ala Ser Asp Asp Glu Leu Met Ser Arg Ile Arg Asn 20 25 30Ser Asp Phe Phe Asp Gly Gln Ala Pro Val Asp Ser Leu Arg Pro Thr 35 40 45Asn Ala Gly Val Asp Ser Lys Gly Thr Asp Asp His Leu Thr Thr Ser 50 55 60Met Asp Lys Ala Ser Val Glu Ser Gln Leu Pro Arg Arg Glu Pro Leu65 70 75 80Glu Thr Glu Pro Asp Glu Gln Glu Glu Val His Phe Arg Lys Arg Gly 85 90 95Val Arg Ser Asp Ala Glu Val Thr Asp Asp Asn Ile Tyr Glu Glu His 100 105 110Thr Asp Arg Lys Val Val Pro Arg Lys Ser Glu Gly Lys Arg Ser Phe 115 120 125Lys Asp Leu Leu Lys Lys Leu Ala Leu Pro Ala Val Gly Met Gly Ala 130 135 140Ser Tyr Phe Ala Ala Asp Arg Leu Val Pro Glu Leu Thr Glu Glu Gln145 150 155 160Gln Arg Gly Asp Glu Pro Leu Thr Thr Gly Gln Asn Val Gly Thr Val 165 170 175Leu Gly Phe Ala Ala Leu Ala Ala Ala Ala Ala Phe Leu Gly Met Gly 180 185 190Leu Thr Arg Thr Tyr Arg His Phe Ser Pro Arg Lys Asn Arg Ser Arg 195 200 205Gln Pro Ala Leu Glu Gln Glu Val Pro Glu Ser Gly Glu Asp Gly Glu 210 215 220Asp Ala Arg Gln225 25 477 DNA Toxoplasma gondii 25agaccccgcc accgcccgtg acgaaccacg aaccgcggcg aacggcgagc tcaccgggtt 60ttcagagacg cgcgagatcc ctgatttcgt ttaccattga cgcccgccgc cgtcgacgtc 120tttggaacgt gtttcacgtt tgagttgcac tgttactttc ttcggattac attcttccac 180taaaagctgg ttttgtccag tatccattcg tcgctaccgt tgcgcagtca cgttgaattt 240tgcagcggca aaacatcttg tgtaaaattc gagttttgtt gatgattgaa gtaccctata 300ttggggcttg ctaacgtttt gtattaaaag ggattactgc ggcgtctcat ttccaaaatg 360gcccgacacg caattttttc cgcgctttgt gttttaggcc tggtggcggc ggctttgccc 420cagttcgcta ccgcggccac cgcgtcagat gacgaactga tgagtcgaat ccgaaat 477 26 1648 DNA Toxoplasma gondii 26agaccccgcc accgcccgtg acgaaccacg aaccgcggcg aacggcgagc tcaccgggtt 60ttcagagacg cgcgagatcc ctgatttcgt ttaccattga cgcccgccgc cgtcgacgtc 120tttggaacgt gtttcacgtt tgagttgcac tgttactttc ttcggattac attcttccac 180taaaagctgg ttttgtccag tatccattcg tcgctaccgt tgcgcagtca cgttgaattt 240tgcagcggca aaacatcttg tgtaaaattc gagttttgtt gatgattgaa gtaccctata 300ttggggcttg ctaacgtttt gtattaaaag ggattactgc ggcgtctcat ttccaaaatg 360gcccgacacg caattttttc cgcgctttgt gttttaggcc tggtggcggc ggctttgccc 420cagttcgcta ccgcggccac cgcgtcagat gacgaactga tgagtcgaat ccgaaattct 480gactttttcg atggtcaagc acccgttgac agtctcagac cgacgaacgc cggtgtcgac 540tcgaaaggga ccgacgatca cctcaccacc agcatggata aggcatctgt agagagtcag 600cttccgagaa gagagccatt ggagacggag ccagatgaac aagaagaagt tcatttcagg 660aagcgaggcg tccgttccga cgctgaagtg actgacgaca acatctacga ggagcacact 720gatcgtaagg tggttccgag gaagtcggag ggcaagcgaa gcttcaaaga cttgctgaag 780aagctcgcgc tgccggctgt tggtatgggt gcatcgtatt ttgccgctga tagacttgtg 840ccggaactaa cagaggagca acagagaggc gacgaacccc taaccaccgg ccagaatgtg 900ggcactgtgt taggcttcgc agcgcttgct gctgccgcag cgttccttgg catgggtctc 960acgaggacgt accgacattt ttccccacgc aaaaacagat cacggcagcc tgcactcgag 1020caagaggtgc ctgaatcagg cgaagatggg gaggatgccc gccagtagga tatgggggct 1080aataaaagtg agtaggagct cgaggacagt gtcccgaacg cgcctgagag gcagacagac 1140acagaagagt gaagaaaaac aacatggtat tacgtgcggt gagtgtttgc tgtcacgtgt 1200tttttgcgcc acaaagacag cttgtgttgt atgcatggga tcgacagttc atggacggcg 1260ctacccagag aggcggcatt tgcgtacacc gtgggtcgtc atgagtaccg ggacatcgtg 1320ttcgtgttta tttgttcatg tcgaagtgca ctaagacacg agacgaaagg gtggttccgc 1380ccctggcagc atcacgtagt ggtttctttg tcgagaacag cggcagtccg aggccacttg 1440agacaggatg tttgagtgta tacagacaac gtggtcacag catgaggcaa agctgtctaa 1500gcagccattt gcgcgagcga agtcatccat gccgactgtg tgagcctctt tcgtcacttt 1560gaatgagaca gaaactaaga ctcgcagcag gtctgaatat tgcgaataat ctacttttaa 1620aaccaaaaaa aaaaaaaaaa aactcgag 1648 27 236 PRT Toxoplasma gondii 27Met Ala Arg His Ala Ile Phe Ser Ala Leu Cys Val Leu Gly Leu Val 1 5 10 15Ala Ala Ala Leu Pro Gln Phe Ala Thr Ala Ala Thr Ala Ser Asp Asp 20 25 30Glu Leu Met Ser Arg Ile Arg Asn Ser Asp Phe Phe Asp Gly Gln Ala 35 40 45Pro Val Asp Ser Leu Arg Pro Thr Asn Ala Gly Val Asp Ser Lys Gly 50 55 60Thr Asp Asp His Leu Thr Thr Ser Met Asp Lys Ala Ser Val Glu Ser65 70 75 80Gln Leu Pro Arg Arg Glu Pro Leu Glu Thr Glu Pro Asp Glu Gln Glu 85 90 95Glu Val His Phe Arg Lys Arg Gly Val Arg Ser Asp Ala Glu Val Thr 100 105 110Asp Asp Asn Ile Tyr Glu Glu His Thr Asp Arg Lys Val Val Pro Arg 115 120 125Lys Ser Glu Gly Lys Arg Ser Phe Lys Asp Leu Leu Lys Lys Leu Ala 130 135 140Leu Pro Ala Val Gly Met Gly Ala Ser Tyr Phe Ala Ala Asp Arg Leu145 150 155 160Val Pro Glu Leu Thr Glu Glu Gln Gln Arg Gly Asp Glu Pro Leu Thr 165 170 175Thr Gly Gln Asn Val Gly Thr Val Leu Gly Phe Ala Ala Leu Ala Ala 180 185 190Ala Ala Ala Phe Leu Gly Met Gly Leu Thr Arg Thr Tyr Arg His Phe 195 200 205Ser Pro Arg Lys Asn Arg Ser Arg Gln Pro Ala Leu Glu Gln Glu Val 210 215 220Pro Glu Ser Gly Glu Asp Gly Glu Asp Ala Arg Gln225 230 235 28 78 DNA Toxoplasma gondii 28agatctcgac ccgtcgacga attcgagctc ggtacccggg gatcctctag actgcaggca 60tgctaagtaa gtagatct 78 29 78 DNA Toxoplasma gondii 29agatctcgac ccatctacca attcgtcttc tgttccgggt gatccgctag actgccgtca 60cgctaagtaa gtagatct 78 30 88 DNA Toxoplasma gondii 30cctgaagatc tcgacccatc taccaattcg tcttctgttc cgggtgatcc gctagactgc 60cgtcacgcta agtaagtaga tcttgact 88 31 88 DNA Toxoplasma gondii 31agtcaagatc tacttactta gcgtgacggc agtctagcgg atcacccgga acagaagacg 60aattggtaga tgggtcgaga tcttcagg 88 32 40 DNA Toxoplasma gondii 32aggcctgaat tcgagctctg ggatccgtct gcagacgcgt 40 33 32 DNA Toxoplasma gondii 33cctgaattcg agctctggga tccgtctgca ga 32 34 36 DNA Toxoplasma gondii 34cgcgtctgca gacggatccc agagctcgaa ttcagg 36 35 36 DNA Toxoplasma gondii 35acttagaatt cgatggcccg acacgcaatt ttttcc 36 36 35 DNA Toxoplasma gondii 36acatggatcc gctggcgggc atcctcccca tcttc 35 37 4775 DNA Toxoplasma gondii 37gaattaattc ccattaatgt gagttagctc actcattagg caccccaggc tttacacttt 60atgttccggc tcgtattttg tgtggaattg tgagcggata acaattgggc atccagtaag 120gaggtttaaa tgagttttgt ggtcattatt cccgcgcgct acgcgacgtc gcgtctgccc 180ggtaaaccat tggttgatat taacggcaaa cccatgattg ttcatgttct tgaacgcgcg 240cgtgaatcag gtgccgagcg catcatcgtg gcaaccgatc atgaggatgt tgcccgcgcc 300gttgaagccg ctggcggtga agtatgtatg acgcgcgccg atcatcagtc aggaacagaa 360cgtctggcgg aagttgtcga aaaatgcgca ttcagcgacg acacggtgat cgttaatgtg 420cagggtgatg aaccgatgat ccctgcgaca atcattcgtc aggttgctga taacctcgct 480cagcgtcagg tgggtatgac gactctggcg gtgccaatcc acaatgcgga agaagcgttt 540aacccgaatg cggtgaaagt ggttctcgac gctgaagggt atgcactgta cttctctcgc 600gccaccattc cttgggatcg tgatcgtttt gcagaaggcc tgaattcgat ggcccgacac 660gcaatttttt ccgcgctttg tgttttaggc ctggtggcgg cggctttgcc ccagttcgct 720accgcggcca ccgcgtcaga tgacgaactg atgagtcgaa tccgaaattc tgactttttc 780gatggtcaag cacccgttga cagtctcaga ccgacgaacg ccggtgtcga ctcgaaaggg 840accgacgatc acctcaccac cagcatggat aaggcatctg tagagagtca gcttccgaga 900agagagccat tggagacgga gccagatgaa caagaagaag ttcatttcag gaagcgaggc 960gtccgttccg acgctgaagt gactgacgac aacatctacg aggagcacac tgatcgtaag 1020gtggttccga ggaagtcgga gggcaagcga agcttcaaag acttgctgaa gaagctcgcg 1080ctgccggctg ttggtatggg tgcatcgtat tttgccgctg atagacttgt gccggaacta 1140acagaggagc aacagagagg cgacgaaccc ctaaccaccg gccagaatgt gggcactgtg 1200ttaggcttcg cagcgcttgc tgctgccgca gcgttccttg gcatgggtct cacgaggacg 1260taccgacatt tttccccacg caaaaacaga tcacggcagc ctgcactcga gcaagaggtg 1320cctgaatcag gcgaagatgg ggaggatgcc cgccagcgga tccgtctgca gacgcgtctt 1380gaaaccgttg gcgataactt cctgcgtcat cttggtattt atggctaccg tgcaggcttt 1440atccgtcgtt acgtcaactg gcagccaagt ccgttagaac acatcgaaat gttagagcag 1500cttcgtgttc tgtggtacgg cgaaaaaatc catgttgctg ttgctcagga agttcctggc 1560acaggtgtgg atacccctga agatctcgac ccatctacca attcgtcttc tgttccgggt 1620gatccgctag actgccgtca cgctaagtaa gtagatcttg agcgcgttcg cgctgaaatg 1680cgctaatttc acttcacgac acttcagcca attttgggag gagtgtcgta ccgttacgat 1740tttcctcaat ttttcttttc aacaattgat ctcattcagg tgacatcttt tatattggcg 1800ctcattatga aagcagtagc ttttatgagg gtaatctgaa tggaacagct gcgtgccgaa 1860ttaagccatt tactgggcga aaaactcagt cgtattgagt gcgtcaatga aaaagcggat 1920acggcgttgt gggctttgta tgacagccag ggaaacccaa tgccgttaat ggcaagaagc 1980ttagcccgcc taatgagcgg gctttttttt cgacgcgagg ctggatggcc ttccccatta 2040tgattcttct cgcttccggc ggcatcggga tgcccgcgtt gcaggccatg ctgtccaggc 2100aggtagatga cgaccatcag ggacagcttc aaggatcgct cgcggctctt accagcctaa 2160cttcgatcac tggaccgctg atcgtcacgg cgatttatgc cgcctcggcg agcacatgga 2220acgggttggc atggattgta ggcgccgccc tataccttgt ctgcctcccc gcgttgcgtc 2280gcggtgcatg gagccgggcc acctcgacct gaatggaagc cggcggcacc tcgctaacgg 2340attcaccact ccaagaattg gagccaatca attcttgcgg agaactgtga atgcgcaaac 2400caacccttgg cagaacatat ccatcgcgtc cgccatctcc agcagccgca cgcggcgcat 2460ctcgggcagc gttgggtcct ggccacgggt gcgcatgatc gtgctcctgt cgttgaggac 2520ccggctaggc tggcggggtt gccttactgg ttagcagaat gaatcaccga tacgcgagcg 2580aacgtgaagc gactgctgct gcaaaacgtc tgcgacctga gcaacaacat gaatggtctt 2640cggtttccgt gtttcgtaaa gtctggaaac gcggaagtca gcgccctgca ccattatgtt 2700ccggatctgc atcgcaggat gctgctggct accctgtgga acacctacat ctgtattaac 2760gaagcgcttc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc 2820gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg 2880caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt 2940tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa 3000gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct 3060ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc 3120cttcgggaag cgtggcgctt tctcaatgct cacgctgtag gtatctcagt tcggtgtagg 3180tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct 3240tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag 3300cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga 3360agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga 3420agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg 3480gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag 3540aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag 3600ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat 3660gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct 3720taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac 3780tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa 3840tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg 3900gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt 3960gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca 4020ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt 4080cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct 4140tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg 4200cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg 4260agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg 4320cgtcaacacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa 4380aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt 4440aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt 4500gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt 4560gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca 4620tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat 4680ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata 4740aaaataggcg tatcacgagg ccctttcgtc ttcaa 4775 38 36 DNA Toxoplasma gondii 38tcctaggcct taattcgatg cttgttgcca atcaag 36 39 32 DNA Toxoplasma gondii 39acatacgcgt cgcgacacaa gctgcgatag ag 32 40 4910 DNA Toxoplasma gondii 40gaattaattc ccattaatgt gagttagctc actcattagg caccccaggc tttacacttt 60atgttccggc tcgtattttg tgtggaattg tgagcggata acaattgggc atccagtaag 120gaggtttaaa tgagttttgt ggtcattatt cccgcgcgct acgcgacgtc gcgtctgccc 180ggtaaaccat tggttgatat taacggcaaa cccatgattg ttcatgttct tgaacgcgcg 240cgtgaatcag gtgccgagcg catcatcgtg gcaaccgatc atgaggatgt tgcccgcgcc 300gttgaagccg ctggcggtga agtatgtatg acgcgcgccg atcatcagtc aggaacagaa 360cgtctggcgg aagttgtcga aaaatgcgca ttcagcgacg acacggtgat cgttaatgtg 420cagggtgatg aaccgatgat ccctgcgaca atcattcgtc aggttgctga taacctcgct 480cagcgtcagg tgggtatgac gactctggcg gtgccaatcc acaatgcgga agaagcgttt 540aacccgaatg cggtgaaagt ggttctcgac gctgaagggt atgcactgta cttctctcgc 600gccaccattc cttgggatcg tgatcgtttt gcagaaggcc ttaattcgat gcttgttgcc 660aatcaagttg tcacctgccc agataaaaaa tcgacagccg cggtcattct cacaccgacg 720gagaaccact tcactctcaa gtgccctaaa acagcgctca cagagcctcc cactcttgcg 780tactcaccca acaggcaaat ctgcccagcg ggtactacaa gtagctgtac atcaaaggct 840gtaacattga gctccttgat tcctgaagca gaagatagct ggtggacggg ggattctgct 900agtctcgaca cggcaggcat caaactcaca gttccaatcg agaagttccc cgtgacaacg 960cagacgtttg tggtcggttg catcaaggga gacgacgcac agagttgtat ggtcacggtg 1020acagtacaag ccagagcctc atcggtcgtc aataatgtcg caaggtgctc ctacggtgca 1080gacagcactc ttggtcctgt caagttgtct gcggaaggac ccactacaat gaccctcgtg 1140tgcgggaaag atggagtcaa agttcctcaa gacaacaatc agtactgttc cgggacgacg 1200ctgactggtt gcaacgagaa atcgttcaaa gatattttgc caaaattaac tgagaacccg 1260tggcagggta acgcttcgag tgataagggt gccacgctaa cgatcaagaa ggaagcattt 1320ccagccgagt caaaaagcgt cattattgga tgcacagggg gatcgcctga gaagcatcac 1380tgtaccgtga aactggagtt tgccggggct gcagggtcag caaaatcggc tgcgggaaca 1440gccagtcacg tttccatttt tgccatggtg atcggactta ttggctctat cgcagcttgt 1500gtcgcgacgc gtcttgaaac cgttggcgat aacttcctgc gtcatcttgg tatttatggc 1560taccgtgcag gctttatccg tcgttacgtc aactggcagc caagtccgtt agaacacatc 1620gaaatgttag agcagcttcg tgttctgtgg tacggcgaaa aaatccatgt tgctgttgct 1680caggaagttc ctggcacagg tgtggatacc cctgaagatc tcgacccgtc gacgaattcg 1740agctcggtac ccggggatcc tctagactgc aggcatgcta agtaagtaga tcttgagcgc 1800gttcgcgctg aaatgcgcta atttcacttc acgacacttc agccaatttt gggaggagtg 1860tcgtaccgtt acgattttcc tcaatttttc ttttcaacaa ttgatctcat tcaggtgaca 1920tcttttatat tggcgctcat tatgaaagca gtagctttta tgagggtaat ctgaatggaa 1980cagctgcgtg ccgaattaag ccatttactg ggcgaaaaac tcagtcgtat tgagtgcgtc 2040aatgaaaaag cggatacggc gttgtgggct ttgtatgaca gccagggaaa cccaatgccg 2100ttaatggcaa gaagcttagc ccgcctaatg agcgggcttt tttttcgacg cgaggctgga 2160tggccttccc cattatgatt cttctcgctt ccggcggcat cgggatgccc gcgttgcagg 2220ccatgctgtc caggcaggta gatgacgacc atcagggaca gcttcaagga tcgctcgcgg 2280ctcttaccag cctaacttcg atcactggac cgctgatcgt cacggcgatt tatgccgcct 2340cggcgagcac atggaacggg ttggcatgga ttgtaggcgc cgccctatac cttgtctgcc 2400tccccgcgtt gcgtcgcggt gcatggagcc gggccacctc gacctgaatg gaagccggcg 2460gcacctcgct aacggattca ccactccaag aattggagcc aatcaattct tgcggagaac 2520tgtgaatgcg caaaccaacc cttggcagaa catatccatc gcgtccgcca tctccagcag 2580ccgcacgcgg cgcatctcgg gcagcgttgg gtcctggcca cgggtgcgca tgatcgtgct 2640cctgtcgttg aggacccggc taggctggcg gggttgcctt actggttagc agaatgaatc 2700accgatacgc gagcgaacgt gaagcgactg ctgctgcaaa acgtctgcga cctgagcaac 2760aacatgaatg gtcttcggtt tccgtgtttc gtaaagtctg gaaacgcgga agtcagcgcc 2820ctgcaccatt atgttccgga tctgcatcgc aggatgctgc tggctaccct gtggaacacc 2880tacatctgta ttaacgaagc gcttcttccg cttcctcgct cactgactcg ctgcgctcgg 2940tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag 3000aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc 3060gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca 3120aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt 3180ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc 3240tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc 3300tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 3360ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 3420tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 3480ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta 3540tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 3600aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa 3660aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg 3720aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc 3780ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg 3840acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat 3900ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg 3960gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa 4020taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca 4080tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc 4140gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt 4200cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa 4260aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat 4320cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct 4380tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga 4440gttgctcttg cccggcgtca acacgggata ataccgcgcc acatagcaga actttaaaag 4500tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga 4560gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca 4620ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg 4680cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc 4740agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag 4800gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca 4860tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtcttcaa 4910 41 40 DNA Toxoplasma gondii 41gagcagaagg ccttatgaac ggtcctttga gttatcatcc 40 42 33 DNA Toxoplasma gondii 42ttcgctcacg cgtatggtga actgccggta tct 33 43 34 DNA Toxoplasma gondii 43gacggagacg cgtcttgaac cgttggcgat aact 34 44 21 DNA Toxoplasma gondii 44gcatgcctgc agtctagagg a 21 45 4451 DNA Toxoplasma gondii 45gaattaattc ccattaatgt gagttagctc actcattagg caccccaggc tttacacttt 60atgttccggc tcgtattttg tgtggaattg tgagcggata acaattgggc atccagtaag 120gaggtttaaa tgagttttgt ggtcattatt cccgcgcgct acgcgtcgac gcgtctgccc 180ggtaaaccat tggttgatat taacggcaaa cccatgattg ttcatgttct tgaacgcgcg 240cgtgaatcag gtgccgagcg catcatcgtg gcaaccgatc atgaggatgt tgcccgcgcc 300gttgaagccg ctggcggtga agtatgtatg acgcgcgccg atcatcagtc aggaacagaa 360cgtctggcgg aagttgtcga aaaatgcgca ttcagcgacg acacggtgat cgttaatgtg 420cagggtgatg aaccgatgat ccctgcgaca atcattcgtc aggttgctga taacctcgct 480cagcgtcagg tgggtatgac gactctggcg gtgccaatcc acaatgcgga agaagcgttt 540aacccgaatg cggtgaaagt ggttctcgac gctgaagggt atgcactgta cttctctcgc 600gccaccattc cttgggatcg tgatcgtttt gcagaaggcc ttatgaacgg tcctttgagt 660tatcatccaa gcagttacgg agcgtcgtat ccgaatccga gtaatcctct gcatggaatg 720cccaagccag agaacccggt gagaccgcct cctcccggtt tccatccaag cgttattccc 780aatcccccgt acccgctggg cactccagcg agcatgccac agccagaggt tccgccactt 840cagcatcccc cgccaacggg ttcccctccc gcggccgctc cccagcctcc atatccagtg 900ggtactccag taatgccaca gccagagata ccgcctgttc atcggccgcc gcctccgggt 960ttccgtcccg aagtggctcc cgtgcccccg tatccagtgg gcactccaac gggcatgccc 1020cagccggaga taccggcagt tcaccatacg cgtcttgaaa ccgttggcga taacttcctg 1080cgtcatcttg gtatttatgg ctaccgtgca ggctttatcc gtcgttacgt caactggcag 1140ccaagtccgt tagaacacat cgaaatgtta gagcagcttc gtgttctgtg gtacggcgaa 1200aaaatccatg ttgctgttgc tcaggaagtt cctggcacag gtgtggatac ccctgaagat 1260ctcgacccgt cgacgaattc gagctcggta cccggggatc ctctagactg caggcatgct 1320aagtaagtag atcttgagcg cgttcgcgct gaaatgcgct aatttcactt cacgacactt 1380cagccaattt tgggaggagt gtcgtaccgt tacgattttc ctcaattttt cttttcaaca 1440attgatctca ttcaggtgac atcttttata ttggcgctca ttatgaaagc agtagctttt 1500atgagggtaa tctgaatgga acagctgcgt gccgaattaa gccatttact gggcgaaaaa 1560ctcagtcgta ttgagtgcgt caatgaaaaa gcggatacgg cgttgtgggc tttgtatgac 1620agccagggaa acccaatgcc gttaatggca agaagcttag cccgcctaat gagcgggctt 1680ttttttcgac gcgaggctgg atggccttcc ccattatgat tcttctcgct tccggcggca 1740tcgggatgcc cgcgttgcag gccatgctgt ccaggcaggt agatgacgac catcagggac 1800agcttcaagg atcgctcgcg gctcttacca gcctaacttc gatcactgga ccgctgatcg 1860tcacggcgat ttatgccgcc tcggcgagca catggaacgg gttggcatgg attgtaggcg 1920ccgccctata ccttgtctgc ctccccgcgt tgcgtcgcgg tgcatggagc cgggccacct 1980cgacctgaat ggaagccggc ggcacctcgc taacggattc accactccaa gaattggagc 2040caatcaattc ttgcggagaa ctgtgaatgc gcaaaccaac ccttggcaga acatatccat 2100cgcgtccgcc atctccagca gccgcacgcg gcgcatctcg ggcagcgttg ggtcctggcc 2160acgggtgcgc atgatcgtgc tcctgtcgtt gaggacccgg ctaggctggc ggggttgcct 2220tactggttag cagaatgaat caccgatacg cgagcgaacg tgaagcgact gctgctgcaa 2280aacgtctgcg acctgagcaa caacatgaat ggtcttcggt ttccgtgttt cgtaaagtct 2340ggaaacgcgg aagtcagcgc cctgcaccat tatgttccgg atctgcatcg caggatgctg 2400ctggctaccc tgtggaacac ctacatctgt attaacgaag cgcttcttcc gcttcctcgc 2460tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg 2520cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag 2580gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 2640gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 2700gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 2760ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 2820aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 2880tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 2940ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 3000gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 3060ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 3120ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca 3180agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 3240ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa 3300aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta 3360tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag 3420cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga 3480tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac 3540cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc 3600ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta 3660gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac 3720gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat 3780gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa 3840gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg 3900tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag 3960aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aacacgggat aataccgcgc 4020cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct 4080caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat 4140cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg 4200ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc 4260aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta 4320tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg 4380tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct 4440ttcgtcttca a 4451 46 41 DNA Toxoplasma gondii 46atattaggcc ttatgagcca caatggagtc cccgcttatc c 41 47 38 DNA Toxoplasma gondii 47cagtgtacgc gtttgcgatc catcatcctg ctctcttc 38 48 5258 DNA Toxoplasma gondii 48gaattaattc ccattaatgt gagttagctc actcattagg caccccaggc tttacacttt 60atgttccggc tcgtattttg tgtggaattg tgagcggata acaattgggc atccagtaag 120gaggtttaaa tgagttttgt ggtcattatt cccgcgcgct acgcgacgtc gcgtctgccc 180ggtaaaccat tggttgatat taacggcaaa cccatgattg ttcatgttct tgaacgcgcg 240cgtgaatcag gtgccgagcg catcatcgtg gcaaccgatc atgaggatgt tgcccgcgcc 300gttgaagccg ctggcggtga agtatgtatg acgcgcgccg atcatcagtc aggaacagaa 360cgtctggcgg aagttgtcga aaaatgcgca ttcagcgacg acacggtgat cgttaatgtg 420cagggtgatg aaccgatgat ccctgcgaca atcattcgtc aggttgctga taacctcgct 480cagcgtcagg tgggtatgac gactctggcg gtgccaatcc acaatgcgga agaagcgttt 540aacccgaatg cggtgaaagt ggttctcgac gctgaagggt atgcactgta cttctctcgc 600gccaccattc cttgggatcg tgatcgtttt gcagaaggcc ttatgagcca caatggagtc 660cccgcttatc catcgtatgc acaggtatcg ctctcttcca acggcgagcc acggcacagg 720ggcatacgcg gcagcttcct catgtccgta aagccacacg caaacgctga tgacttcgcc 780tccgacgaca actacgaacc gctgccgagt ttcgtggaag ctcctgtcag aggcccggac 840caagtccctg ccagaggaga agctgctctt gtcacagagg agactccagc gcaacagccg 900gcggtggctc taggcagtgc agaaggggag gggacctcca ctactgaatc cgcctccgaa 960aattctgaag atgatgacac gtttcacgat gccctccaag agcttccaga ggatggcctc 1020gaagtgcgcc caccaaatgc acaggagctg cccccaccaa atgtacagga gctgccccca 1080ccaaatgtac aggagctgcc cccaccaact gaacaggagc tgcccccacc aactgaacag 1140gagctgcccc caccaactga acaggagctg cccccaccaa ctgaacagga gctaccccca 1200tcaactgaac aggagctgcc cccaccagtg ggcgaaggtc aacgtctgca agtccctggg 1260gaacatgggc cacaggggcc cccatacgat gatcagcagc tgcttttaga gcctacggaa 1320gagcaacagg agggccctca ggagccgctg ccaccgccgc cgcccccgac tcggggcgaa 1380caacccgaag gacagcagcc gcagggacca gttcgtcaaa atttttttcg tcgggcgttg 1440ggggccgcaa gaagccgatt cggaggtgca cgacgccatg tcagtggggt gttccgaaga 1500gtcagaggtg gtttgaaccg tatagtaggt ggagtgagga gtggtttcag gcgtgcaaga 1560gaaggtgtcg ttgggggagt ccgtcgttta acaagtggtg ccagtctggg tctccgtcgt 1620gtaggagaag gtttacgtag gagtttctat cgtgtaagag gagctgtcag tagcggtcgt 1680aggcgtgcag cagatggtgc cagcaatgta agagaaagat tcgttgccgc aggcgggaga 1740gtcagagacg ctttcggcgc gggattgacg cgcctccgca ggcgcggcag aactaatggc 1800gaggagggca ggcccctact gggcgaagga agagagcagg atgatggatc gcaaacgcgt 1860cttgaaaccg ttggcgataa cttcctgcgt catcttggta tttatggcta ccgtgcaggc 1920tttatccgtc gttacgtcaa ctggcagcca agtccgttag aacacatcga aatgttagag 1980cagcttcgtg ttctgtggta cggcgaaaaa atccatgttg ctgttgctca ggaagttcct 2040ggcacaggtg tggatacccc tgaagatctc gacccgtcga cgaattcgag ctcggtaccc 2100ggggatcctc tagactgcag gcatgctaag taagtagatc ttgagcgcgt tcgcgctgaa 2160atgcgctaat ttcacttcac gacacttcag ccaattttgg gaggagtgtc gtaccgttac 2220gattttcctc aatttttctt ttcaacaatt gatctcattc aggtgacatc ttttatattg 2280gcgctcatta tgaaagcagt agcttttatg agggtaatct gaatggaaca gctgcgtgcc 2340gaattaagcc atttactggg cgaaaaactc agtcgtattg agtgcgtcaa tgaaaaagcg 2400gatacggcgt tgtgggcttt gtatgacagc cagggaaacc caatgccgtt aatggcaaga 2460agcttagccc gcctaatgag cgggcttttt tttcgacgcg aggctggatg gccttcccca 2520ttatgattct tctcgcttcc ggcggcatcg ggatgcccgc gttgcaggcc atgctgtcca 2580ggcaggtaga tgacgaccat cagggacagc ttcaaggatc gctcgcggct cttaccagcc 2640taacttcgat cactggaccg ctgatcgtca cggcgattta tgccgcctcg gcgagcacat 2700ggaacgggtt ggcatggatt gtaggcgccg ccctatacct tgtctgcctc cccgcgttgc 2760gtcgcggtgc atggagccgg gccacctcga cctgaatgga agccggcggc acctcgctaa 2820cggattcacc actccaagaa ttggagccaa tcaattcttg cggagaactg tgaatgcgca 2880aaccaaccct tggcagaaca tatccatcgc gtccgccatc tccagcagcc gcacgcggcg 2940catctcgggc agcgttgggt cctggccacg ggtgcgcatg atcgtgctcc tgtcgttgag 3000gacccggcta ggctggcggg gttgccttac tggttagcag aatgaatcac cgatacgcga 3060gcgaacgtga agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa catgaatggt 3120cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag tcagcgccct gcaccattat 3180gttccggatc tgcatcgcag gatgctgctg gctaccctgt ggaacaccta catctgtatt 3240aacgaagcgc ttcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc 3300ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata 3360acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg 3420cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct 3480caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa 3540gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc 3600tcccttcggg aagcgtggcg ctttctcaat gctcacgctg taggtatctc agttcggtgt 3660aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg 3720ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg 3780cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct 3840tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc 3900tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg 3960ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc 4020aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt 4080aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa 4140aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat 4200gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct 4260gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg 4320caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag 4380ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta 4440attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg 4500ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg 4560gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct 4620ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta 4680tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg 4740gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc 4800cggcgtcaac acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg 4860gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga 4920tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg 4980ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat 5040gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc 5100tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca 5160catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct 5220ataaaaatag gcgtatcacg aggccctttc gtcttcaa 5258 49 22 PRT Toxoplasma gondii 49Asp Leu Asp Pro Ser Thr Asn Ser Ser Ser Val Pro Gly Asp Pro Leu 1 5 10 15Asp Cys Arg His Ala Lys 20 50 22 PRT Toxoplasma gondii 50Asp Leu Asp Pro Ser Thr Asn Ser Ser Ser Val Pro Gly Asp Pro Leu 1 5 10 15Asp Cys Arg His Ala Lys 20 51 13 PRT Toxoplasma gondii 51Gly Leu Asn Ser Ser Ser Gly Ile Arg Leu Gln Thr Arg 1 5 10 52 506 PRT Toxoplasma gondii 52Met Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Thr Ser Arg Leu 1 5 10 15Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His 20 25 30Val Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala 35 40 45Thr Asp His Glu Asp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu 50 55 60Val Cys Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala65 70 75 80Glu Val Val Glu Lys Cys Ala Phe Ser Asp Asp Thr Val Ile Val Asn 85 90 95Val Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val 100 105 110Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Met Thr Thr Leu Ala Val 115 120 125Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val 130 135 140Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile145 150 155 160Pro Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Asn Ser Met Ala Arg 165 170 175His Ala Ile Phe Ser Ala Leu Cys Val Leu Gly Leu Val Ala Ala Ala 180 185 190Leu Pro Gln Phe Ala Thr Ala Ala Thr Ala Ser Asp Asp Glu Leu Met 195 200 205Ser Arg Ile Arg Asn Ser Asp Phe Phe Asp Gly Gln Ala Pro Val Asp 210 215 220Ser Leu Arg Pro Thr Asn Ala Gly Val Asp Ser Lys Gly Thr Asp Asp225 230 235 240His Leu Thr Thr Ser Met Asp Lys Ala Ser Val Glu Ser Gln Leu Pro 245 250 255Arg Arg Glu Pro Leu Glu Thr Glu Pro Asp Glu Gln Glu Glu Val His 260 265 270Phe Arg Lys Arg Gly Val Arg Ser Asp Ala Glu Val Thr Asp Asp Asn 275 280 285Ile Tyr Glu Glu His Thr Asp Arg Lys Val Val Pro Arg Lys Ser Glu 290 295 300Gly Lys Arg Ser Phe Lys Asp Leu Leu Lys Lys Leu Ala Leu Pro Ala305 310 315 320Val Gly Met Gly Ala Ser Tyr Phe Ala Ala Asp Arg Leu Val Pro Glu 325 330 335Leu Thr Glu Glu Gln Gln Arg Gly Asp Glu Pro Leu Thr Thr Gly Gln 340 345 350Asn Val Gly Thr Val Leu Gly Phe Ala Ala Leu Ala Ala Ala Ala Ala 355 360 365Phe Leu Gly Met Gly Leu Thr Arg Thr Tyr Arg His Phe Ser Pro Arg 370 375 380Lys Asn Arg Ser Arg Gln Pro Ala Leu Glu Gln Glu Val Pro Glu Ser385 390 395 400Gly Glu Asp Gly Glu Asp Ala Arg Gln Arg Ile Arg Leu Gln Thr Arg 405 410 415Leu Glu Thr Val Gly Asp Asn Phe Leu Arg His Leu Gly Ile Tyr Gly 420 425 430Tyr Arg Ala Gly Phe Ile Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro 435 440 445Leu Glu His Ile Glu Met Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly 450 455 460Glu Lys Ile His Val Ala Val Ala Gln Glu Val Pro Gly Thr Gly Val465 470 475 480Asp Thr Pro Glu Asp Leu Asp Pro Ser Thr Asn Ser Ser Ser Val Pro 485 490 495Gly Asp Pro Leu Asp Cys Arg His Ala Lys 500 505 53 551 PRT Toxoplasma gondii 53Met Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Thr Ser Arg Leu 1 5 10 15Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His 20 25 30Val Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala 35 40 45Thr Asp His Glu Asp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu 50 55 60Val Cys Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala65 70 75 80Glu Val Val Glu Lys Cys Ala Phe Ser Asp Asp Thr Val Ile Val Asn 85 90 95Val Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val 100 105 110Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Met Thr Thr Leu Ala Val 115 120 125Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val 130 135 140Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile145 150 155 160Pro Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Asn Ser Met Leu Val 165 170 175Ala Asn Gln Val Val Thr Cys Pro Asp Lys Lys Ser Thr Ala Ala Val 180 185 190Ile Leu Thr Pro Thr Glu Asn His Phe Thr Leu Lys Cys Pro Lys Thr 195 200 205Ala Leu Thr Glu Pro Pro Thr Leu Ala Tyr Ser Pro Asn Arg Gln Ile 210 215 220Cys Pro Ala Gly Thr Thr Ser Ser Cys Thr Ser Lys Ala Val Thr Leu225 230 235 240Ser Ser Leu Ile Pro Glu Ala Glu Asp Ser Trp Trp Thr Gly Asp Ser 245 250 255Ala Ser Leu Asp Thr Ala Gly Ile Lys Leu Thr Val Pro Ile Glu Lys 260 265 270Phe Pro Val Thr Thr Gln Thr Phe Val Val Gly Cys Ile Lys Gly Asp 275 280 285Asp Ala Gln Ser Cys Met Val Thr Val Thr Val Gln Ala Arg Ala Ser 290 295 300Ser Val Val Asn Asn Val Ala Arg Cys Ser Tyr Gly Ala Asp Ser Thr305 310 315 320Leu Gly Pro Val Lys Leu Ser Ala Glu Gly Pro Thr Thr Met Thr Leu 325 330 335Val Cys Gly Lys Asp Gly Val Lys Val Pro Gln Asp Asn Asn Gln Tyr 340 345 350Cys Ser Gly Thr Thr Leu Thr Gly Cys Asn Glu Lys Ser Phe Lys Asp 355 360 365Ile Leu Pro Lys Leu Thr Glu Asn Pro Trp Gln Gly Asn Ala Ser Ser 370 375 380Asp Lys Gly Ala Thr Leu Thr Ile Lys Lys Glu Ala Phe Pro Ala Glu385 390 395 400Ser Lys Ser Val Ile Ile Gly Cys Thr Gly Gly Ser Pro Glu Lys His 405 410 415His Cys Thr Val Lys Leu Glu Phe Ala Gly Ala Ala Gly Ser Ala Lys 420 425 430Ser Ala Ala Gly Thr Ala Ser His Val Ser Ile Phe Ala Met Val Ile 435 440 445Gly Leu Ile Gly Ser Ile Ala Ala Cys Val Ala Thr Arg Leu Glu Thr 450 455 460Val Gly Asp Asn Phe Leu Arg His Leu Gly Ile Tyr Gly Tyr Arg Ala465 470 475 480Gly Phe Ile Arg Arg Tyr Val Asn Trp Gln Pro Ser Pro Leu Glu His 485 490 495Ile Glu Met Leu Glu Gln Leu Arg Val Leu Trp Tyr Gly Glu Lys Ile 500 505 510His Val Ala Val Ala Gln Glu Val Pro Gly Thr Gly Val Asp Thr Pro 515 520 525Glu Asp Leu Asp Pro Ser Thr Asn Ser Ser Ser Val Pro Gly Asp Pro 530 535 540Leu Asp Cys Arg His Ala Lys545 550 54 398 PRT Toxoplasma gondii 54Met Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Ser Thr Arg Leu 1 5 10 15Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His 20 25 30Val Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala 35 40 45Thr Asp His Glu Asp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu 50 55 60Val Cys Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala65 70 75 80Glu Val Val Glu Lys Cys Ala Phe Ser Asp Asp Thr Val Ile Val Asn 85 90 95Val Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val 100 105 110Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Met Thr Thr Leu Ala Val 115 120 125Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val 130 135 140Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile145 150 155 160Pro Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Met Asn Gly Pro Leu 165 170 175Ser Tyr His Pro Ser Ser Tyr Gly Ala Ser Tyr Pro Asn Pro Ser Asn 180 185 190Pro Leu His Gly Met Pro Lys Pro Glu Asn Pro Val Arg Pro Pro Pro 195 200 205Pro Gly Phe His Pro Ser Val Ile Pro Asn Pro Pro Tyr Pro Leu Gly 210 215 220Thr Pro Ala Ser Met Pro Gln Pro Glu Val Pro Pro Leu Gln His Pro225 230 235 240Pro Pro Thr Gly Ser Pro Pro Ala Ala Ala Pro Gln Pro Pro Tyr Pro 245 250 255Val Gly Thr Pro Val Met Pro Gln Pro Glu Ile Pro Pro Val His Arg 260 265 270Pro Pro Pro Pro Gly Phe Arg Pro Glu Val Ala Pro Val Pro Pro Tyr 275 280 285Pro Val Gly Thr Pro Thr Gly Met Pro Gln Pro Glu Ile Pro Ala Val 290 295 300His His Thr Arg Leu Glu Thr Val Gly Asp Asn Phe Leu Arg His Leu305 310 315 320Gly Ile Tyr Gly Tyr Arg Ala Gly Phe Ile Arg Arg Tyr Val Asn Trp 325 330 335Gln Pro Ser Pro Leu Glu His Ile Glu Met Leu Glu Gln Leu Arg Val 340 345 350Leu Trp Tyr Gly Glu Lys Ile His Val Ala Val Ala Gln Glu Val Pro 355 360 365Gly Thr Gly Val Asp Thr Pro Glu Asp Leu Asp Pro Ser Thr Asn Ser 370 375 380Ser Ser Val Pro Gly Asp Pro Leu Asp Cys Arg His Ala Lys385 390 395 55 667 PRT Toxoplasma gondii 55Met Ser Phe Val Val Ile Ile Pro Ala Arg Tyr Ala Thr Ser Arg Leu 1 5 10 15Pro Gly Lys Pro Leu Val Asp Ile Asn Gly Lys Pro Met Ile Val His 20 25 30Val Leu Glu Arg Ala Arg Glu Ser Gly Ala Glu Arg Ile Ile Val Ala 35 40 45Thr Asp His Glu Asp Val Ala Arg Ala Val Glu Ala Ala Gly Gly Glu 50 55 60Val Cys Met Thr Arg Ala Asp His Gln Ser Gly Thr Glu Arg Leu Ala65 70 75 80Glu Val Val Glu Lys Cys Ala Phe Ser Asp Asp Thr Val Ile Val Asn 85 90 95Val Gln Gly Asp Glu Pro Met Ile Pro Ala Thr Ile Ile Arg Gln Val 100 105 110Ala Asp Asn Leu Ala Gln Arg Gln Val Gly Met Thr Thr Leu Ala Val 115 120 125Pro Ile His Asn Ala Glu Glu Ala Phe Asn Pro Asn Ala Val Lys Val 130 135 140Val Leu Asp Ala Glu Gly Tyr Ala Leu Tyr Phe Ser Arg Ala Thr Ile145 150 155 160Pro Trp Asp Arg Asp Arg Phe Ala Glu Gly Leu Met Ser His Asn Gly 165 170 175Val Pro Ala Tyr Pro Ser Tyr Ala Gln Val Ser Leu Ser Ser Asn Gly 180 185 190Glu Pro Arg His Arg Gly Ile Arg Gly Ser Phe Leu Met Ser Val Lys 195 200 205Pro His Ala Asn Ala Asp Asp Phe Ala Ser Asp Asp Asn Tyr Glu Pro 210 215 220Leu Pro Ser Phe Val Glu Ala Pro Val Arg Gly Pro Asp Gln Val Pro225 230 235 240Ala Arg Gly Glu Ala Ala Leu Val Thr Glu Glu Thr Pro Ala Gln Gln 245 250 255Pro Ala Val Ala Leu Gly Ser Ala Glu Gly Glu Gly Thr Ser Thr Thr 260 265 270Glu Ser Ala Ser Glu Asn Ser Glu Asp Asp Asp Thr Phe His Asp Ala 275 280 285Leu Gln Glu Leu Pro Glu Asp Gly Leu Glu Val Arg Pro Pro Asn Ala 290 295 300Gln Glu Leu Pro Pro Pro Asn Val Gln Glu Leu Pro Pro Pro Asn Val305 310 315 320Gln Glu Leu Pro Pro Pro Thr Glu Gln Glu Leu Pro Pro Pro Thr Glu 325 330 335Gln Glu Leu Pro Pro Pro Thr Glu Gln Glu Leu Pro Pro Pro Thr Glu 340 345 350Gln Glu Leu Pro Pro Ser Thr Glu Gln Glu Leu Pro Pro Pro Val Gly 355 360 365Glu Gly Gln Arg Leu Gln Val Pro Gly Glu His Gly Pro Gln Gly Pro 370 375 380Pro Tyr Asp Asp Gln Gln Leu Leu Leu Glu Pro Thr Glu Glu Gln Gln385 390 395 400Glu Gly Pro Gln Glu Pro Leu Pro Pro Pro Pro Pro Pro Thr Arg Gly 405 410 415Glu Gln Pro Glu Gly Gln Gln Pro Gln Gly Pro Val Arg Gln Asn Phe 420 425 430Phe Arg Arg Ala Leu Gly Ala Ala Arg Ser Arg Phe Gly Gly Ala Arg 435 440 445Arg His Val Ser Gly Val Phe Arg Arg Val Arg Gly Gly Leu Asn Arg 450 455 460Ile Val Gly Gly Val Arg Ser Gly Phe Arg Arg Ala Arg Glu Gly Val465 470 475 480Val Gly Gly Val Arg Arg Leu Thr Ser Gly Ala Ser Leu Gly Leu Arg 485 490 495Arg Val Gly Glu Gly Leu Arg Arg Ser Phe Tyr Arg Val Arg Gly Ala 500 505 510Val Ser Ser Gly Arg Arg Arg Ala Ala Asp Gly Ala Ser Asn Val Arg 515 520 525Glu Arg Phe Val Ala Ala Gly Gly Arg Val Arg Asp Ala Phe Gly Ala 530 535 540Gly Leu Thr Arg Leu Arg Arg Arg Gly Arg Thr Asn Gly Glu Glu Gly545 550 555 560Arg Pro Leu Leu Gly Glu Gly Arg Glu Gln Asp Asp Gly Ser Gln Thr 565 570 575Arg Leu Glu Thr Val Gly Asp Asn Phe Leu Arg His Leu Gly Ile Tyr 580 585 590Gly Tyr Arg Ala Gly Phe Ile Arg Arg Tyr Val Asn Trp Gln Pro Ser 595 600 605Pro Leu Glu His Ile Glu Met Leu Glu Gln Leu Arg Val Leu Trp Tyr 610 615 620Gly Glu Lys Ile His Val Ala Val Ala Gln Glu Val Pro Gly Thr Gly625 630 635 640Val Asp Thr Pro Glu Asp Leu Asp Pro Ser Thr Asn Ser Ser Ser Val 645 650 655Pro Gly Asp Pro Leu Asp Cys Arg His Ala Lys 660 665

Claims

1. A method for detecting the presence of IgM antibodies to Toxoplasma gondii (T. gondii) in a test sample comprising the steps of: a) contacting said test sample suspected of containing said IgM antibodies with a composition comprising a recombinant P29 antigen of T. gondii comprising SEQ ID NO:27, a recombinant fusion protein comprising a portion of the P35 antigen of T. gondii, said portion of said P35 antigen corresponding to amino acids 172-306 of SEQ ID NO:54, and a recombinant fusion protein comprising a portion of the P66 antigen of T. gondii, said portion of said P66 antigen corresponding to amino acids 173-575 of SEQ ID NO:55; and b) detecting the presence of said IgM antibodies.

2. A method for detecting the presence of IgM antibodies to Toxoplasma gondii in a test sample comprising the steps of: a) contacting said test sample suspected of containing said IgM antibodies with a composition comprising a recombinant P29 antigen of T. gondii comprising SEQ ID NO:27, a recombinant fusion protein comprising a portion of the P35 antigen of T. gondii, said portion of said P35 antigen corresponding to amino acids 172-306 of SEQ ID NO:54, and a recombinant fusion protein comprising a portion of the P66 antigen of T. gondii, said portion of said P66 antigen corresponding to amino acids 173-575 of SEQ ID NO:55, for a time and under conditions sufficient for the formation of IgM antibody/antigen complexes; b) adding a conjugate to the resulting IgM antibody/antigen complexes for a time and under conditions sufficient to allow said conjugate to bind to the bound antibody, wherein said conjugate comprises an anti-IgM antibody attached to a signal-generating compound capable of generating a detectable signal; and c) detecting the presence of IgM antibodies which may be present in said test sample by detecting a signal generated by said signal-generating compound.

3. The method according to claim 2 wherein said composition further comprises a fusion protein comprising a portion of the P30 antigen of T. gondii, said portion of said P30 antigen corresponding to amino acids 175-459 of SEQ ID NO:53.

4. A method for detecting the presence of IgG antibodies to Toxoplasma gondii in a test sample comprising the steps of: a) contacting said test sample suspected of containing said IgG antibodies with a composition comprising a recombinant P29 antigen of T. gondii comprising SEQ ID NO:27, a recombinant fusion protein comprising a portion of the P30 antigen of T. gondii, said portion of said P30 antigen corresponding to amino acids 175-459 of SEQ ID NO:53, and a recombinant fusion protein comprising a portion of the P35 antigen of T. gondii, said portion of said P35 antigen corresponding to amino acids 172-306 of SEQ ID NO:54; and b) detecting the presence of said IgG antibodies.

5. A method for detecting the presence of IgG antibodies to Toxoplasma gondii in a test sample comprising the steps of: a) contacting said test sample suspected of containing said IgG antibodies with a composition comprising a recombinant P29 antigen of T. gondii comprising SEQ ID NO:27, a recombinant fusion protein comprising a portion of the P30 antigen of T. gondii, said portion of said P30 antigen corresponding to amino acids 175-459 of SEQ ID NO:53, and a recombinant fusion protein comprising a portion of the P35 antigen of T. gondii, said portion of said P35 antigen corresponding to amino acids 172-306 of SEQ ID NO:54, for a time and under conditions sufficient for formation of IgG antibody/antigen complexes; b) adding a conjugate to resulting IgG antibody/antigen complexes for a time and under conditions sufficient to allow said conjugate to bind to bound antibody, wherein said conjugate comprises an anti-IgG antibody attached to a signal-generating compound capable of generating a detectable signal; and c) detecting IgG antibodies which may be present in said test sample by detecting a signal generated by said signal-generating compound.

6. The method according to claim 5 wherein said composition further comprises a recombinant fusion protein comprising a portion of the P66 antigen of T. gondii, said portion of said P66 antigen corresponding to amino acids 173-575 of SEQ ID NO:55.

7. A method for detecting the presence of IgM antibodies to Toxoplasma gondii in a test sample comprising the steps of: a) contacting said test sample suspected of containing said IgM antibodies with anti-antibody specific for said IgM antibodies for a time and under conditions sufficient to allow for formation of anti-antibody/IgM antibody complexes; b) adding a conjugate to resulting anti-antibody/IgM antibody complexes for a time and under conditions sufficient to allow said conjugate to bind to bound antibody, wherein said conjugate comprises a composition comprising a recombinant P29 antigen of T. gondii comprising SEQ ID NO:27, a recombinant fusion protein comprising a portion of the P35 antigen of T. gondii, said portion of said P35 antigen corresponding to amino acids 172-306 of SEQ ID NO:54, and a recombinant fusion protein comprising a portion of the P66 antigen of T. gondii, said portion of said P66 antigen corresponding to amino acids 173-575 of SEQ ID NO:55, each attached to a signal-generating compound capable of generating a detectable signal; and c) detecting IgM antibodies which may be present in said test sample by detecting a signal generated by said signal-generating compound.

8. The method according to claim 7 wherein said composition further comprises a recombinant fusion protein comprising a portion of the P30 antigen of T. gondii, said portion of said P30 antigen corresponding to amino acids 175-459 of SEQ ID NO:53.

9. A method for detecting the presence of IgG antibodies to Toxoplasma gondii in a test sample comprising the steps of: a) contacting said test sample suspected of containing said IgG antibodies with anti-antibody specific for said IgG antibodies for a time and under conditions sufficient to allow for formation of anti-antibody/IgG antibody complexes; b) adding a conjugate to resulting anti-antibody/IgG antibody complexes for a time and under conditions sufficient to allow said conjugate to bind to bound antibody, wherein said conjugate comprises a composition comprising a recombinant P29 antigen of T. gondii comprising SEQ ID NO:27, a recombinant fusion protein comprising a portion of the P30 antigen of T. gondii, said portion of said P30 antigen corresponding to amino acids 175-459 of SEQ ID NO:53, and a recombinant fusion protein comprising a portion of the P35 antigen of T. gondii, said portion of said P35 antigen corresponding to amino acids 172-306 of SEQ ID NO:54, each attached to a signal-generating compound capable of generating a detectable signal; and c) detecting IgG antibodies which may be present in said test sample by detecting a signal generated by said signal-generating compounds.

10. The method according to claim 9 wherein said composition further comprises a recombinant fusion protein comprising a portion of the P66 antigen of T. gondii, said portion of said P66 antigen corresponding to amino acids 173-575 of SEQ ID NO:55.

11. A method for detecting the presence of IgM antibodies to Toxoplasma gondii in a test sample comprising the steps of: (a) contacting said test sample suspected of containing IgM antibodies with anti-antibody specific for said IgM antibodies for a time and under conditions sufficient to allow for formation of anti-antibody IgM complexes; (b) adding an antigen to resulting anti-antibody/IgM complexes for a time and under conditions sufficient to allow said antigen to bind to bound IgM antibody, said antigen comprising a mixture of a recombinant P29 antigen of T. gondii comprising SEQ ID NO:27, a recombinant fusion protein comprising a portion of the P35 antigen of T. gondii, said portion of said P35 antigen corresponding to amino acids 172-306 of SEQ ID NO:54, and a recombinant fusion protein comprising a portion of the P66 antigen of T. gondii, said portion of said P66 antigen corresponding to amino acids 173-575 of SEQ ID NO:55; and (c) adding a conjugate to resulting anti-antibody/IgM/antigen complexes, said conjugate comprising a composition comprising monoclonal or polyclonal anti-IgM antibody attached to a signal-generating compound capable of generating a detectable signal; and (d) detecting IgM antibodies which may be present in said test sample by detecting a signal generated by said signal-generating compound.

12. The method according to claim 11 wherein said mixture further comprises a recombinant fusion protein comprising a portion of the P30 antigen of T. gondii, said portion of said P30 antigen corresponding to amino acids 175-459 of SEQ ID NO:53.

13. A method for detecting the presence of IgG antibodies to Toxoplasma gondii in a test sample comprising the steps of: (a) contacting said test sample suspected of containing IgG antibodies with anti-antibody specific for said IgG antibodies for a time and under conditions sufficient to allow for formation of anti-antibody IgG complexes; (b) adding an antigen to resulting anti-antibody/IgG complexes for a time and under conditions sufficient to allow said antigen to bind to bound IgG antibody, said antigen comprising a mixture a recombinant P29 antigen of T. gondii comprising SEQ ID NO:27, a recombinant fusion protein comprising a portion of the P30 antigen of T. gondii, said portion of said P30 antigen corresponding to amino acids 175-459 of SEQ ID NO:53, and a recombinant fusion protein comprising a portion of the P35 antigen of T. gondii, said portion of said P35 antigen corresponding to amino acids 172-306 of SEQ ID NO:54; (c) adding a conjugate to resulting anti-antibody/IgG/antigen complexes, said conjugate comprising a composition comprising monoclonal or polyclonal IgG antibody attached to a signal-generating compound capable of generating a detectable signal; and (d) detecting IgG antibodies which may be present in said test sample by-detecting a signal generated by said signal-generating compound.

14. The method according to claim 13 wherein said mixture further comprises a recombinant fusion protein comprising a portion of the P66 antigen of T. gondii, said portion of said P66 antigen corresponding to amino acids 173-575 of SEQ ID NO:55.

15. A method for detecting the presence of IgM and IgG antibodies to Toxoplasma gondii in a test sample comprising the steps of: a) contacting said test sample suspected of containing said IgM and IgG antibodies with a composition comprising a recombinant P29 antigen of T. gondii comprising SEQ ID NO:27, a recombinant fusion protein comprising a portion of the P30 antigen of T. gondii, said portion of said P30 antigen corresponding to amino acids 175-459 of SEQ ID NO:53, a recombinant fusion protein comprising a portion of the P35 antigen of T. gondii, said portion of said P35 antigen corresponding to amino acids 172-306 of SEQ ID NO:54, and a recombinant fusion protein comprising a portion of the P66 antigen of T. gondii, said portion of said P66 antigen corresponding to amino acids 173-575 of SEQ ID NO:55, for a time and under conditions sufficient for the formation of IgM antibody/antigen complexes and IgG antibody/antigen complexes; b) adding a first conjugate to resulting IgM antibody/antigen complexes and a second conjugate to resulting IgG antibody/antigen complexes for a time and under conditions sufficient to allow said first and second conjugates to bind to the bound IgM and IgG antibody, respectively, wherein said first conjugate comprises an anti-IgM antibody attached to a signal- generating compound capable of generating a detectable signal and said second conjugate comprises an anti-IgG antibody attached to a signal-generating compound capable of generating a detectable signal; and c) detecting the presence of IgM and IgG antibodies which may be present in said test sample by detecting a signal generated by said signal-generating compounds.

16. A method for detecting the presence of IgM and IgG antibodies to Toxoplasma gondii in a test sample comprising the steps of: a) contacting said test sample suspected of containing said IgM and IgG antibodies with anti-antibody specific for said IgM antibodies and said IgG antibodies for a time and under conditions sufficient to allow for formation of anti-antibody/IgM antibody complexes and anti-antibody/IgG antibody complexes; b) adding a conjugate to resulting anti-antibody/IgM antibody complexes and resulting anti-antibody/IgG antibody complexes for a time and under conditions sufficient to allow said conjugate to bind to bound antibody, wherein said conjugate comprises a recombinant P29 antigen of T. gondii comprising SEQ ID NO:27, a recombinant fusion protein comprising a portion of the P30 antigen of T. gondii, said portion of said P30 antigen corresponding to amino acids 175-459 of SEQ ID NO:53, a recombinant fusion protein comprising a portion of the P35 antigen of T. gondii, said portion of said P35 antigen corresponding to amino acids 172-306 of SEQ ID NO:54, and a recombinant fusion protein comprising a portion of the P66 antigen of T. gondii, said portion of said P66 antigen corresponding to amino acids 173-575 of SEQ ID NO:55, each attached to a signal-generating compound capable of generating a detectable signal; and c) detecting IgM and IgG antibodies which may be present in said test sample by detecting a signal generated by said signal-generating compounds.

17. A method for detecting the presence of IgM and IgG antibodies to Toxoplasma gondii in a test sample comprising the steps of: (a) contacting said test sample suspected of containing IgM and IgG antibodies with anti-antibody specific for said IgM antibodies and with anti-antibody specific for said IgG antibodies for a time and under conditions sufficient to allow for formation of anti-antibody/IgM complexes and anti-antibody/IgG complexes; (b) adding an antigen to resulting anti-antibody/IgM complexes and resulting anti-antibody/IgG complexes for a time and under conditions sufficient to allow said antigen to bind to bound IgM and IgG antibody, respectively, said antigen comprising a mixture of a recombinant P29 antigen of T. gondii comprising SEQ ID NO:27, a recombinant fusion protein comprising a portion of the P30 antigen of T. gondii, said portion of said P30 antigen corresponding to amino acids 175-459 of SEQ ID NO:53, a recombinant fusion protein comprising a portion of the P35 antigen of T. gondii, said portion of said P35 antigen corresponding to amino acids 172-306 of SEQ ID NO:54, and a recombinant fusion protein comprising a portion of the P66 antigen of T. gondii, said portion of said P66 antigen corresponding to amino acids 173-575 of SEQ ID NO:55; and (c) adding a first conjugate to resulting anti-antibody/IgM/antigen complexes and a second conjugate to resulting anti-antibody/IgG/antigen complexes, said first conjugate comprising a composition comprising an anti-IgM monoclonal or polyclonal antibody attached to a signal-generating compound capable of generating a detectable signal and said second conjugate comprising a composition comprising an anti-IgG monoclonal or polyclonal antibody attached to a signal-generating compound capable of generating a detectable signal; and (d) detecting IgM and IgG antibodies which may be present in said test sample by detecting a signal generated by said signal-generating compounds.