Patent Application
20210293807
The official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named “80859-US-REG-ORG-P-1_SeqList_ST25.txt”, originally created on Sep. 8, 2015, and having a size of 48 kilobytes and is filed concurrently with the specification. The sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety.
The invention relates generally to immunoassays, and more particularly to monoclonal antibodies and immunoassays for the differential detection and quantitation of wild-type crystal proteins from Bacillus thuringiensis and hybrid crystal proteins in complex biological samples.
Bacillus thuringiensis is a ubiquitous gram-positive, spore-forming bacterium that forms a crystalline protein inclusion during the stationary phase of its growth cycle. The crystal proteins (Cry proteins) are toxic to a number of plant pests, including many insects in the orders Lepidoptera and Coleoptera. Prior to 1990, the major Cry protein classes were defined by their spectrum of activity with the Cry1 proteins active against Lepidoptera (moths and butterflies), Cry2 proteins active against both Lepidoptera and Diptera (flies and mosquitoes), Cry3 proteins active against Coleoptera (beetles) and Cry4 proteins active against Diptera (Hofte and Whitely, 1989, Microbiol. Rev. 53:242 255). Subsequent to the Hofte and Whitely nomenclature scheme, a different nomenclature was developed which systematically classifies the Cry proteins based on amino acid sequence homology rather than insect target specificities (Crickmore et al. 1998, Microbiol. Mol. Biol. Rev. 62:807 813).
Most Cry proteins active against lepidopteran or coleopteran insects are formed in the crystalline matrix as 130-140 kDa or 60-70 kDa protoxins, respectively. In lepidopteran insects, the alkaline pH of the gut solubilizes the crystal and then gut proteases process the 130-140 kDa protoxin to toxic proteins of approximately 60-70 kDa. In coleopteran insects, the 60-70 kDa protoxins are processed to 55-67 kDa toxins. Examples of lepidopteran-active Cry proteins include Cry1A, Cry1B, Cry1C, Cry1D, Cry1E, Cry1F and Cry9. Examples of coleopteran-active Cry proteins include, Cry3A, Cry3B, Cry3C, Cry8, the binary Cry23-Cry37 and the binary Cry34-Cry35. Processing of the Cry protein protoxin to a toxin has been reported to proceed by removal of both N- and C-terminal amino acids with the exact location of processing being dependent on the specific Cry protein and the specific insect gut fluids involved (Ogiwara et al., 1992. J. Invert. Pathol. 60:121-126). The proteolytic activation of a Cry protoxin can play a significant role in determining its specificity.
All or parts of certain wild-type Cry proteins have been used to engineer hybrid Cry proteins in attempts to create insecticidal proteins with improved specific activity or broader spectrum of insecticidal activity. Targeted engineering was made more possible by solving the three dimensional structure of Cry3A by Li el al. (1991, Nature 353:815-821). Based on this work, it has been determined that Cry proteins in general have three structural domains: the N-terminal domain I, from residues 1-290, consists of 7 alpha helices, domain II, from residues 291-500, contains three beta-sheets and the C-terminal domain III, from residues 501-644, is a beta-sandwich. Based on this structure, a hypothesis has been formulated regarding the structure/function relationship of the Cry proteins. It is generally thought that domain I is primarily responsible for pore formation in the insect gut membrane (Gazit and Shai, 1993, Appl. Environ. Microbiol. 57:2816 2820), domain II is primarily responsible for interaction with the gut receptor (Ge el al., 1991, J. Biol. Chem. 32:3429 3436) and that domain III is most likely involved with protein stability (Li et al. 1991, supra) as well as having a regulatory impact on ion channel activity (Chen et al., 1993, PNAS 90:9041 9045).
Many successful attempts to create hybrid Cry proteins have been disclosed in the literature. For example, the silk moth (Bombyx mori) specificity domain from a Cry1Aa protein was moved to a Cry1Ac protein, thus imparting a new insecticidal activity to the resulting Cry1Aa-Cry1Ac chimeric protein (Ge et al. 1989, PNAS 86: 4037 4041). Thompson et al. 1996 and 1997 (U.S. Pat. Nos. 5,527,883 and 5,593,881) replaced the protoxin tail region of a wild-type Cry1F protein and Cry1C protein with the protoxin tail region of a Cry1Ab protein to make a Cry1F-Cry1Ab hybrid Cry protein and a Cry1C-Cry1Ab hybrid Cry protein, both having improved expression in certain expression host cells. Bosch et al. 1998 (U.S. Pat. No. 5,736,131), created new lepidopteran-active proteins by substituting domain III of a Cry1Ea protein and a Cry1Ab protein with domain III of Cry1Ca protein thus producing a Cry1E-Cry1C hybrid Cry protein called G27 and a Cry1Ab-Cry1C hybrid Cry protein called H04, both of which have a broader spectrum of lepidopteran activity than the wild-type Cry protein parent molecules. Malvar et al. 2001 (U.S. Pat. No. 6,242,241) combined domain I of a Cry1Ac protein with domains II and III and the protoxin tail of a Cry1F protein to create a Cry1Ac-Cry1F hybrid Cry protein with broader insecticidal activity than the parental wild-type Cry proteins. Bogdanova et al. 2011 (U.S. Pat. No. 8,034,997) combined domains I and II of a Cry1Ab protein with domain III of a Cry1Fa protein and added a Cry1Ac protein protoxin tail to create a new lepidopteran-active hybrid Cry protein called Cry1A.105. And, Hart et al. 2012 (U.S. Pat. No. 8,309,516) combined domains I and II of a modified Cry3A protein with domain III of a Cry1Ab protein and added a portion of a Cry1Ab protein protoxin tail to create a coleopteran-active hybrid Cry protein called FR8a (also called eCry3.1Ab). Most of the reported hybrid Cry proteins to date have used all or parts of the same classes of wild-type Cry proteins, such as Cry1Aa, Cry1Ab, Cry1Ac, Cry1C, Cry1F and Cry3A.
Several wild-type Cry proteins, for example Cry1Ab, Cry1Ac, Cry1C, Cry1F, Cry2A, Cry2Ba, Cry3A, Cry3B, Cry9C, and Cry34-Cry35 have been expressed in transgenic crop plants, including corn, cotton, rice and soybean, some of which have been exploited commercially to control certain lepidopteran and coleopteran insect pests since as early as 1996. More recently, transgenic crop products containing hybrid Cry proteins, for example Cry1A.105 (Cry1Ab-Cry1F-Cry1Ac) and eCry3.1Ab (mCry3A-Cry3A-Cry1Ab), have been introduced commercially.
Immunoassay is the current preferred method in the agricultural industry for detection and quantification of Cry proteins introduced through genetic modification of plants. The crucial component of an immunoassay is an antibody with specificity for the target molecule (antigen). Immunoassays can be highly specific and samples often need only a simple preparation before being analyzed. Moreover, immunoassays can be used qualitatively or quantitatively over a wide range of concentrations. Typically, immunoassays require separate tests for each Cry protein of interest.
The antibodies can be polyclonal, raised in animals, or monoclonal, produced by cell cultures. Commercially available polyclonal antiserum is often produced in rabbits, goats or sheep. Monoclonal antibodies offer some advantages over polyclonal antibodies because they express uniform affinity and specificity against a single epitope or antigenic determinant and can be produced in vast quantities. Both polyclonal and monoclonal antibodies may require further purification steps to enhance the sensitivity and reduce backgrounds in assays.
Making a valid identification of a product containing a Cry protein or quantitating a Cry protein in a commercial product depends on the accuracy of the immunoassay. Development of a successful immunoassay depends on certain characteristics of the antigen used for development of the antibody, i.e. size, hydrophobicity and the tertiary structure of the antigen. The specificity of the antibodies must be checked carefully to elucidate any cross-reactivity with similar substances, which might cause false positive results. A current problem in the industry is that many of the antibodies in commercially available tests kits do not differentiate between various products or wild-type Cry proteins, making differential product identification and quantitation difficult or impossible.
With many current commercial transgenic crop products using one or more of the same wild-type Cry proteins, for example Cry1Ab, Cry1F and Cry3, and with the introduction of crops expressing hybrid Cry proteins made of whole or parts of the same wild-type Cry proteins that are already in transgenic crop products, there is a continuing need to develop new and improved immunoassays to be able to distinguish a wild-type Cry protein from a hybrid Cry protein containing all or portions of that same wild-type Cry protein when they are together in complex biological samples, such as samples from transgenic plants.
The present invention addresses the need for new and improved immunoassays by providing compositions useful in specific detection and differentiation of certain wild-type Cry proteins and engineered hybrid Cry proteins comprising all or part of the wild-type Cry protein amino acid sequence in complex biological samples, including transgenic plant samples. The invention also relates to methods, assays and kits to specifically detect and differentiate wild-type Cry proteins from engineered hybrid Cry proteins comprising all or part of the wild-type Cry protein amino acid sequence in biological samples comprising the wild-type Cry protein and the hybrid Cry protein.
According to one aspect, the invention provides a composition comprising a first antibody and a second antibody that function together to specifically detect or quantitate a Cry1Ab protein in an immunoassay of a biological sample comprising the Cry1Ab protein and a hybrid Cry protein having at least a contiguous 27% of the Cry1Ab protein's amino acid sequence, wherein the first antibody and the second antibody individually are capable of binding to both the wild-type Cry1Ab protein and the hybrid Cry protein.
According to another aspect, the invention also provides a pair of purified antibodies comprising a first antibody and a second antibody that function together in an immunoassay to specifically detect or quantitate a Cry1Ab protein in a biological sample comprising the Cry1Ab protein and a hybrid Cry protein having at least a contiguous 27% of the Cry1Ab protein's amino acid sequence, wherein the first antibody and the second antibody individually are capable of binding to both the Cry1Ab protein and the hybrid Cry protein.
According to yet another aspect of this invention, a diagnostic kit is provided for detecting a Cry1Ab protein in a biological sample which comprises the Cry1Ab protein and a hybrid Cry protein having at least a contiguous 27% of the Cry1Ab protein's amino acid sequence, wherein the kit comprises a first antibody and a second antibody that function together to specifically detect or quantitate the Cry1Ab protein and that individually are capable of binding both the Cry1Ab protein and the hybrid Cry protein.
In certain embodiments of the above described aspects of the invention, the immunoassay is a enzyme-linked immunosorbent assay (ELISA), preferably a sandwich ELISA. In other embodiments of these aspects of the invention, the first antibody is the coating antibody and the second antibody is the detecting antibody. In still other embodiments, the first and the second antibodies are monoclonal. In yet other embodiments, the first or coating antibody is the monoclonal antibody 87AB1.1, which is available from Romer Labs, Inc (Union, Mo.). In still other embodiments, the second or detecting antibody is the monoclonal antibody H04MAb70 produced by the hybridoma cell line H04MAb70 deposited as ATCC Accession No. PTA-122984. In yet other embodiments, the coating antibody is 87AB1.1 and the detecting antibody is H04MAb70.
In other embodiments of the above described aspects of the invention, the Cry1Ab protein comprises an amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:2 or SEQ ID N:6. In other embodiments, the hybrid Cry protein is eCry3.1Ab, the amino acid sequence of which is set forth in SEQ ID NO:3, H04, the amino acid sequence of which is set forth in SEQ ID NO:4 or Cry1A.105, the amino acid sequence of which is set forth in SEQ ID NO:5.
In other embodiments of the above described aspects of the invention, the biological sample is a transgenic plant sample. In other embodiments, the transgenic plant is a transgenic corn plant comprising a transgenic event selected from the group consisting of event Bt11, which comprises the Cry1Ab protein of SEQ ID NO:1, event 5307, which comprises the eCry3.1Ab hybrid Cry protein of SEQ ID NO:3, event MON89034, which comprises the Cry1A.105 hybrid Cry protein of SEQ ID NO:5 and event MON810, which comprises the Cry1Ab protein of SEQ ID NO:6.
According to another aspect of the invention, there is provided a hybridoma cell line, designated H04MAb70, deposited as ATCC Accession No. PTA-122984, and a hybridoma cell line designated MAb58, deposited as ATCC Accession No. PTA-122985. In certain embodiments of this aspect of the invention, there is provided a monoclonal antibody designated H04MAb70 produced by the H04MAb hybridoma cell line that binds to both a wild-type Cry1Ab protein and a hybrid Cry protein. In certain other embodiments of this aspect of the invention, there is provided a monoclonal antibody designated MAb58 produced by the hybridoma cell line MAb58 that binds to a hybrid Cry protein.
According to another aspect of the invention there is provided immunoassay methods to specifically detect or quantitate a Cry1Ab protein in a biological sample comprising the Cry1Ab protein and a hybrid Cry protein having at least a contiguous 27% of the Cry1Ab protein's amino acid sequence. Such methods comprise (a) obtaining a biological sample comprising the Cry1Ab protein and the hybrid Cry protein; and (b) performing an immunoassay on the biological sample, wherein the immunoassay comprises use of a first antibody and a second antibody that individually are capable of binding to the Cry1Ab protein and the hybrid Cry protein but that function together in the immunoassay to specifically detect or quantitate the Cry1Ab protein and not the hybrid Cry protein, resulting in the specific detection or quantitation of the Cry1Ab protein.
In some embodiments of this aspect, the immunoassay is an enzyme-linked immunosorbent assay (ELISA). In other embodiments, the first antibody is a coating antibody and the second antibody is a detecting antibody. In further embodiments, the coating antibody and the detecting antibody are monoclonal. In still other embodiments, the coating antibody is the monoclonal antibody 87AB1.1 available from Romer Labs, Inc. (Union, Mo.). In yet other embodiments, the detecting antibody is the monoclonal antibody H04MAb70 produced by the hybridoma cell line deposited as ATCC Accession No. PTA-122984. In other embodiments, the coating antibody is 87AB1.1 and the detecting antibody is H04MAb70.
In some embodiments of this aspect of the invention, the Cry1Ab protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:6. In other embodiments, the hybrid Cry protein is selected from the group consisting of eCry3.1Ab (SEQ ID NO:3), Cry1A.105 (SEQ ID NO:4) and H04 (SEQ ID NO:5). In still other embodiments, the Cry1Ab protein comprises the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:6 and the hybrid Cry protein is eCry3.1Ab (SEQ ID NO:3) or Cry1A.105 (SEQ ID NO:4) or H04 (SEQ ID NO:5).
In some embodiments of this aspect of the invention, the biological sample tested in the immunoassay is a transgenic plant sample. In other embodiments, the transgenic plant sample is from a transgenic corn plant. In other embodiments, the transgenic corn plant comprises a transgenic corn event selected from the group consisting of event Bt11, event 5307, event MON89034 and event MON810. In still other embodiments, the transgenic corn plant comprises event Bt11, event 5307 and event MON89034. In yet further embodiments, the biological sample comprises a Cry1Ab protein from event Bt11 and an eCry3.1Ab hybrid Cry protein from event 5307 and optionally a Cry1A.105 hybrid Cry protein from event MON89034. In still further embodiments, the biological sample comprises a Cry1Ab protein from event MON810 and a Cry1A.105 hybrid Cry protein from event MON89034.
In another aspect of the invention there is provided immunoassay methods for detecting or quantitating both a Cry1Ab protein and a hybrid Cry protein having at least a contiguous 27% of a Cry1Ab protein's amino acid sequence in a biological sample comprising the hybrid Cry protein and the Cry1Ab protein. Such immunoassay methods comprise: (a) obtaining a biological sample comprising the hybrid Cry protein and the Cry1Ab protein; (b) contacting the biological sample with a first antibody under conditions effective to allow the formation of a Cry1Ab-first antibody complex but not a hybrid Cry protein-first antibody complex, resulting in a biological sample depleted of the Cry1Ab protein; (c) removing the Cry1Ab-depleted biological sample from contact with the first antibody; (d) contacting the Cry1Ab-depleted biological sample with a second antibody under conditions effective to allow the formation of a hybrid Cry protein-second antibody complex; and (e) detecting or quantitating the hybrid Cry protein in the hybrid Cry protein-second antibody complex.
In some embodiments of this aspect, the immunoassay is an enzyme-linked immunosorbent assay, wherein the solid surface is a well in a microtiter dish. In other embodiments, the hybrid Cry protein is eCry3.1Ab (SEQ ID NO:3) or Cry1A.105 (SEQ ID NO:4). In other embodiments, the first antibody is 87AB1.1 which is available from Romer Labs, Inc. (Union, Mo.). In other embodiments, the second antibody is the monoclonal antibody MAb58 produced by the hybridoma cell line deposited as ATCC Accession No. PTA-122985. In yet other embodiments of this aspect, the detecting of step (d) is carried out using a composition comprising a monoclonal antibody capable of binding to the Cry1Ab protein and the hybrid Cry protein. In still further embodiments, the monoclonal antibody is H04MAb70. In yet other embodiments of this aspect, the detecting step (e) is carried out using a composition comprising a monoclonal antibody or a polyclonal antibody capable of binding to the hybrid Cry protein and the Cry1Ab protein. In further embodiments, the monoclonal antibody is H04MAb70 or the polyclonal antibody is PAb713 available from Romer Labs, Inc. (Union, Mo.).
According to yet another aspect of the invention there is provided immunoassay methods to detect or quantitate a hybrid Cry protein having at least a contiguous 27% of a Cry1Ab protein's amino acid sequence in a biological sample comprising the hybrid Cry protein and the Cry1Ab protein. Such methods comprise: (a) obtaining a biological sample comprising the hybrid Cry protein and the Cry1Ab protein; (b) contacting the biological sample with a 87AB1.1 antibody under conditions effective to allow the formation of a Cry1Ab-87AB1.1 complex but not a hybrid Cry protein-87AB1.1 complex, resulting in a biological sample depleted of the Cry1Ab protein; (c) removing the Cry1Ab-depleted biological sample from contact with the 87AB1.1 antibody; (d) contacting the Cry1Ab-depleted biological sample with a second antibody under conditions effective to allow the formation of a hybrid Cry protein-second antibody complex; and (e) detecting or quantitating the hybrid Cry protein in the hybrid Cry protein-second antibody complex.
In some embodiments of this aspect, the hybrid Cry protein is eCry3.1Ab (SEQ ID NO:3) or Cry1A.105 (SEQ ID NO:4).
In other embodiments, the biological sample is from a transgenic plant. In some other embodiments, the transgenic plant is a transgenic corn plant. In still other embodiments, the transgenic corn plant comprises a transgenic corn event selected from the group consisting of event Bt11, event 5307, event MON89034 and event MON810. In yet other embodiments, the transgenic corn plant comprises the transgenic corn event Bt11, event 5307 and event MON89034. In other embodiments, the transgenic corn plant comprises the transgenic event MON810 and the transgenic event MON89034. In other embodiments, the biological sample comprises a Cry1Ab protein from event Bt11 and an eCry3.1Ab hybrid Cry protein from event 5307 and optionally, a Cry1A.105 hybrid Cry protein from event MON89034. In still further embodiments, the biological sample comprises a Cry1Ab protein from event MON810 and a Cry1A.105 hybrid Cry protein from event MON89034.
Various objects, features, aspects, and advantages of the present invention will become more apparent from the following detailed description of preferred embodiments of the invention, along with the accompanying drawings and sequence listing.
SEQ ID NO:1 is an amino acid sequence of a wild-type Cry1Ab protein expressed in the transgenic corn event B tn.
SEQ ID NO:2 is an amino acid sequence of a wild-type Cry1Ab protein used to make the MAb58 monoclonal antibody.
SEQ ID NO:3 is an amino acid sequence of a eCry3.1Ab hybrid Cry protein expressed in the transgenic corn event 5307.
SEQ ID NO:4 is an amino acid sequence of a Cry1A.105 hybrid Cry protein expressed in the transgenic corn event MON89034.
SEQ ID NO:5 is an amino acid sequence of a H04 hybrid Cry used to make the H04MAb70 monoclonal antibody.
SEQ ID NO:6 is an amino acid sequence of a wild-type Cry1Ab protein expressed in the transgenic corn event MON810.
Hybridoma cell line H04MAb70 and Hybridoma cell line MAb58 were deposited with the American Type Culture Collection, 10801 University Blvd., Manassas, Va., on Mar. 30, 2016 and have the following accession numbers: HO4Mab70: PTA-122984 and Mab58: PTA-122985. Both deposits were made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. All restrictions on the availability of the deposited material will be irrevocably removed upon granting of the patent.
This description is not intended to be a detailed catalog of all the different ways in which the invention may be implemented, or all the features that may be added to the instant invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. Thus, the invention contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure, which do not depart from the instant invention. Hence, the following descriptions are intended to illustrate some particular embodiments of the invention, and not to exhaustively specify all permutations, combinations and variations thereof.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.
As used herein and in the appended claims, the singular forms “a,” “and,” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to “a plant” is a reference to one or more plants and includes equivalents thereof known to those skilled in the art, and so forth. As used herein, the word “or” means any one member of a particular list and also includes any combination of members of that list (i.e., includes also “and”).
The term “about” is used herein to mean approximately, roughly, around, or in the region of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20 percent, preferably 10 percent up or down (higher or lower). With regard to a temperature the term “about” means±1° C., preferably ±0.5° C. Where the term “about” is used in the context of this invention (e.g., in combinations with temperature or molecular weight values) the exact value (i.e., without “about”) is preferred.
The term “antibody”, as used herein, broadly refers to any immunoglobulin (Ig) molecule comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art. Non-limiting embodiments of which are discussed below. In a full-length antibody, each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.
The term “antigen” as used herein means a protein used to trigger an immune response in an organism for the production of an antibody. Antigens according to the present invention include wild-type Cry1Ab proteins and hybrid Cry proteins.
The terms “comprises” and/or “comprising,” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.
As used herein, the transitional phrase “consisting essentially of” (and grammatical variants) means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim” and those that do not materially alter the basic and novel characteristic(s)” of the claimed invention. Thus, the term “consisting essentially of” when used in a claim of this invention is not intended to be interpreted to be equivalent to “comprising.”
The term “contacting” refers to a combining action that brings an antibody of the invention into contact with the biological sample and more particularly to a combining action which brings the antibody into contact with the Cry1Ab protein or hybrid Cry protein in a manner that a binding interaction will occur between the antibody and the Cry1Ab protein present in the biological sample.
The term “cross-reactivity” refers to the ability of an antibody to bind a target protein other than that against which it was raised.
The term “depleted” as used herein means that all or a significant portion of a target protein has been removed from a complex biological sample by contacting the biological sample with an antibody that binds to the target protein. A biological sample that has been “depleted” of the target protein has 100% of the target protein removed, or at least 95% of the target protein removed, or at least 90%, or at least 85%, or at least 80% of the target protein removed. Thus, for example, a biological sample of the invention comprising Cry1Ab, eCry3.1Ab and Cry1A.105 that has been depleted of Cry1Ab, has at least 80% to 100% of the Cry1Ab protein removed.
“Detecting antibodies” and “labeled antibodies/labeling antibody” refer to antibodies that are capable of being discovered. The detecting antibody may be directly or indirectly (e.g. through another antibody) conjugated to a detectable signal or to a signal-generating moiety. The signal can be radioactive (e.g. radioactive iodine, tritium, carbon, sulfur or the like), colorimetric, fluorescent signal and the like. Signal-generating moieties that act on signal-generating substrates include, but are not limited to, horseradish peroxidase (HRP) [suitable substrates include 3,3′,5,5′-tetramethylbenzidine (TMB); OPD; 2,2′-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid diammonium salt]; alkaline phosphatase [suitable substrates include p-nitrophenyl phosphate disodium salt]; and .beta.-galactosidase [suitable substrates include O-nitrophenyl-beta-D-galactopyranoside]. The signal may be amplified by using an Avidin-Biotin conjugation system.
As used herein the term transgenic “event” refers to a recombinant plant produced by transformation and regeneration of a single plant cell with heterologous DNA, for example, an expression cassette that includes a gene of interest. The term “event” refers to the original transformant and/or progeny of the transformant that include the heterologous DNA. The term “event” also refers to progeny produced by a sexual outcross between the transformant and another corn line. Even after repeated backcrossing to a recurrent parent, the inserted DNA and the flanking DNA from the transformed parent is present in the progeny of the cross at the same chromosomal location. Normally, transformation of plant tissue produces multiple events, each of which represent insertion of a DNA construct into a different location in the genome of a plant cell. Based on the expression of the transgene or other desirable characteristics, a particular event is selected. Non-limiting examples of such transgenic events include “event Bt11” (also Bt11 event or just Bt11), “event 5307” (also 5307 event or just 5307), “event MON810” (also MON810 event or just MON810) and “event MON89034” (also MON89034 event or just MON89034. Thus for example, the term “event Bt11”, “Bt11 event” or “Bt11” as used herein means the original Bt11 transformant and/or progeny of the Bt11 transformant, including any plant derived therefrom.
As used herein the term “hybrid Cry protein” is an engineered insecticidal protein that does not exist in nature and at least a portion of which comprises at least a contiguous 27% of a Cry1Ab protein's amino acid sequence. The 27% limitation is calculated by dividing the number of contiguous Cry1Ab amino acids in the hybrid Cry protein divided by the total number of amino acids in the hybrid Cry protein. For example, the hybrid Cry protein, eCry3.1Ab (SEQ ID NO:3) has 174 Cry1Ab amino acids (positions 480-653) and a total of 653 amino acids. Therefore, eCry3A.1Ab has at least a contiguous 27% of a Cry1Ab protein's amino acid sequence. Other examples of a hybrid Cry protein according to the present invention is represented by SEQ ID NO:4 and SEQ ID NO:5.
The term “isolated” nucleic acid molecule, polynucleotide or toxin is a nucleic acid molecule, polynucleotide or toxic protein that no longer exists in its natural environment. An isolated nucleic acid molecule, polynucleotide or toxin of the invention may exist in a purified form or may exist in a recombinant host such as in a transgenic bacterial cell or a transgenic plant.
The term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. The term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology. Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, it is preferred that monoclonal antibodies of the present disclosure be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporated by reference in their entireties). Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art. Briefly, mice can be immunized with the antigen of interest. In a preferred embodiment, the antigen is administered with an adjuvant to stimulate the immune response. Such adjuvants include complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptides) or ISCOM (immunostimulating complexes). Such adjuvants may protect the polypeptide from rapid dispersal by sequestering it in a local deposit, or they may contain substances that stimulate the host to secrete factors that are chemotactic for macrophages and other components of the immune system. Preferably, if a polypeptide is being administered, the immunization schedule will involve two or more administrations of the polypeptide, spread out over several weeks.
A “plant” is any plant at any stage of development, particularly a seed plant.
A “plant cell” is a structural and physiological unit of a plant, comprising a protoplast and a cell wall. The plant cell may be in the form of an isolated single cell or a cultured cell, or as a part of a higher organized unit such as, for example, plant tissue, a plant organ, or a whole plant.
“Plant cell culture” means cultures of plant units such as, for example, protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes and embryos at various stages of development.
“Plant material” refers to leaves, stems, roots, flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant.
A “plant organ” is a distinct and visibly structured and differentiated part of a plant such as a root, stem, leaf, flower bud, or embryo.
“Plant tissue” as used herein means a group of plant cells organized into a structural and functional unit. Any tissue of a plant in planta or in culture is included. This term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue culture and any groups of plant cells organized into structural and/or functional units. The use of this term in conjunction with, or in the absence of, any specific type of plant tissue as listed above or otherwise embraced by this definition is not intended to be exclusive of any other type of plant tissue
A “wild-type Cry1Ab protein” means a naturally occurring Cry1Ab protein or a
Cry1Ab protein with minimal amino acid additions or substitutions to a naturally occurring Cry1Ab amino acid and having the same or similar insecticidal activity or spectrum as the naturally occurring Cry1Ab protein. A “wild-type Cry1Ab protein” can be either a full-length protein or the truncated toxin portion thereof. For example, without limitation, wild-type Cry1Ab proteins according to the present invention include the Cry1Ab protein of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:6.
The present invention provides immunoassay methods and compositions and kits useful in carrying out the immunoassay methods that allow for the specific detection of a Cry1Ab protein in complex biological samples comprising the Cry1Ab protein and at least one hybrid Cry protein having a contiguous at least 27% of the Cry1Ab protein's amino acid sequence. The current state of the art is such that commercially available immunoassays are not useful in differentially detecting a Cry1Ab protein from a hybrid Cry protein engineered using a significant amount of the Cry1Ab protein's amino acid sequence when the two proteins are in the same biological sample because there is high cross-reactivity between the two types of proteins. For example, an antibody raised against a wild-type Cry1Ab for use in a Cry1Ab-detecting immunoassay cross reacts with a hybrid Cry protein having a significant number, e.g. at least 27%, of its amino acids derived from the wild-type Cry1Ab protein when the two proteins are in the same biological sample. Therefore, for example, the quantitation of the wild-type Cry1Ab in such a complex biological sample may be confounded by the presence of one or more hybrid Cry proteins. Furthermore, using detection of expressed proteins for identity preservation of commercial transgenic plant products comprising a wild-type Cry1Ab and one or more hybrid Cry proteins of the present invention is difficult because of cross-reactivity of antibodies to both the Cry1Ab proteins and the hybrid Cry proteins in the transgenic plant products. The methods and compositions disclosed herein provide a solution to these problems and rely on pairs of antibodies, e.g. a first antibody and a second antibody, that alone are capable of binding both a wild-type Cry1Ab protein and a hybrid Cry protein having at least a contiguous 27% of the wild-type Cry1Ab protein's amino acid sequence, but surprisingly function together in an immunoassay of the invention to specifically detect or quantitate the wild-type Cry1Ab protein.
Immunoassays can be carried out in many different formats, examples of which include an enzyme-linked immunosorbent assay (ELISA) and a dipstick format, which is also called a lateral flow stick. In ELISA, the protein antigen-antibody reaction takes place on a solid phase, typically in wells on microtiter plates. Antigen and this first antibody, also called the coating antibody, react and produce a stable complex, which can be visualized by addition of a second antibody, also called the detecting antibody, linked to an enzyme. Addition of a substrate for that enzyme results in a color formation, which can be measured photometrically or recognized by eye.
Dipstick formats (lateral flow sticks) typically use paper strips or plastic paddles as support for the capture antibody and this is then the reaction site. The strip/paddle is dipped in vials containing the different biological samples. Each dip is followed by a rinsing step. The final reaction includes a color change in the vial, where the strip/paddle is placed. Recent development of dipstick format has led to lateral flow techniques where reactants are transported through the channels of a membrane by capillary forces. One single step is enough for performing the assay, and controls for reagent performance are included. Antibodies specific to the foreign protein are coupled to a color reagent and incorporated into the lateral flow strip. When the lateral flow strip is placed in a small amount of a biological sample, for example an extract from plant tissue, that contains foreign protein, binding occurs between the coupled antibody and the protein. A sandwich is formed with some, but not all the antibody that is coupled to the color reagent. The membrane contains two capture zones, one captures the bound foreign protein and the other captures color reagent. These capture zones display a reddish color when the sandwich and/or non-reacted colored reagents are captured in the specific zones on the membrane. The presence of a single line (control line) on the membrane indicates a negative sample and the presence of two lines indicates a positive sample.
In certain embodiments, an antibody as disclosed herein has a detectable label. Detectable labels suitable for use in the detection antibodies of the present invention include any compound or composition having a moiety that is detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Such labels include, for example, an enzyme, oligonucleotide, nanoparticle chemiluminophore, fluorophore, fluorescence quencher, chemiluminescence quencher, or biotin. Thus for example, in an immunoassay employing an optical signal, the optical signal is measured as an analyte concentration dependent change in chemiluminescence, fluorescence, phosphorescence, electrochemiluminescence, ultraviolet absorption, visible absorption, infrared absorption, refraction, surface plasmon resonance. In an immunoassay employing an electrical signal, the electrical signal is measured as an analyte concentration dependent change in current, resistance, potential, mass to charge ratio, or ion count. In an immunoassay employing a change-of-state signal, the change of state signal is measured as an analyte concentration dependent change in size, solubility, mass, or resonance.
Useful labels according to the present disclosure include magnetic beads (e.g., Dynabeads™), fluorescent dyes (e.g., fluorescein, Texas Red, rhodamine, green fluorescent protein) and the like (see, e.g., Molecular Probes, Eugene, Oreg., USA), chemiluminescent compounds such as acridinium (e.g., acridinium-9-carboxamide), phenanthridinium, dioxetanes, luminol and the like, radiolabels (e.g., 3H, 125I, 35S, 14C, or 32P), catalysts such as enzymes (e.g., horse radish peroxidase, alkaline phosphatase, beta-galactosidase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold (e.g., gold particles in the 40-80 nm diameter size range scatter green light with high efficiency) or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. Patents teaching the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.
The label can be attached to each antibody prior to, or during, or after contact with the biological sample. So-called “direct labels” are detectable labels that are directly attached to or incorporated into the detection antibody prior to use in the assay. Direct labels can be attached to or incorporated into the antibody by any of a number of means well known to those of skill in the art. In contrast, so-called “indirect labels” typically bind to each antibody at some point during the assay. Often, the indirect label binds to a moiety that is attached to or incorporated into the detection agent prior to use. Thus, for example, each antibody can be biotinylated before use in an assay. During the assay, an avidin-conjugated fluorophore can bind the biotin-bearing detection agent, to provide a label that is easily detected.
In another example of indirect labeling, polypeptides capable of specifically binding immunoglobulin constant regions, such as polypeptide A or polypeptide G, can also be used as labels for detection antibodies. These polypeptides are normal constituents of the cell walls of streptococcal bacteria. They exhibit a strong non-immunogenic reactivity with immunoglobulin constant regions from a variety of species (see, generally Kronval, et al. (1973) J. Immunol., 111: 1401-1406, and Akerstrom (1985) J. Immunol., 135: 2589-2542). Such polypeptides can thus be labeled and added to the assay mixture, where they will bind to each antibody, as well as to the autoantibodies, labeling all and providing a composite signal attributable to analyte and autoantibody present in the sample.
Some labels useful in the present disclosure may require the use of an additional reagent(s) to produce a detectable signal. In an ELISA, for example, an enzyme label (e.g., beta-galactosidase) will require the addition of a substrate (e.g., X-gal) to produce a detectable signal. In immunoassays using an acridinium compound as the direct label, a basic solution and a source of hydrogen peroxide are added.
According to one embodiment, the invention encompasses a composition comprising a first antibody and a second antibody that function together to specifically detect or quantitate a Cry1Ab protein in an immunoassay of a biological sample comprising the Cry1Ab protein and a hybrid Cry protein having at least a contiguous 27% of the Cry1Ab protein's amino acid sequence, wherein the first antibody and the second antibody individually are capable of binding to both the wild-type Cry1Ab protein and the hybrid Cry protein.
In other embodiments of the invention, the immunoassay is an enzyme-linked immunosorbent assay (ELISA), preferably a sandwich ELISA. In still other embodiments, the first antibody is the coating antibody and the second antibody is the detecting antibody. In still other embodiments, the first and the second antibodies are monoclonal. In yet other embodiments, the first or coating antibody is the monoclonal antibody 87AB1.1 which is available from Romer Labs, Inc (Union, Mo.). In other embodiments, the second or detecting antibody is the monoclonal antibody H04MAb70 produced by the hybridoma cell line H04MAb70 deposited as ATCC Accession No. PTA-122984. In still other embodiments, the coating antibody is 87AB1.1 and the detecting antibody is H04MAb70.
In some embodiments of the invention, the Cry1Ab protein that is specifically detected in an immunoassay of the invention comprises an amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:2 or SEQ ID N:6. In other embodiments, the hybrid Cry protein is eCry3.1Ab, comprising an amino acid sequence set forth in SEQ ID NO:3, Cry1A.105, comprising an amino acid sequence set forth in SEQ ID NO:4, or Cry1A.105, comprising an amino acid sequence set forth in SEQ ID NO:5.
In other embodiments of the invention, the biological sample is a transgenic plant sample. In still other embodiments, the transgenic plant is a transgenic corn plant comprising a transgenic event selected from the group consisting of event Bt11, which expresses the Cry1Ab protein of SEQ ID NO:1, event 5307, which expresses the eCry3.1Ab hybrid Cry protein of SEQ ID NO:3, event MON89034, which expresses the Cry1A.105 hybrid Cry protein of SEQ ID NO:5 and event MON810, which expresses the Cry1Ab protein of SEQ ID NO:6. In other embodiments, the transgenic plant comprises event Bt11, event 5307 and event MON89034. Is still other embodiments, the transgenic plant comprises event MON810 and MON89034.
According to another embodiment, the invention encompasses a pair of purified antibodies comprising a first antibody and a second antibody that function together in an immunoassay to specifically detect or quantitate a Cry1Ab protein in a biological sample comprising the Cry1Ab protein and a hybrid Cry protein having at least a contiguous 27% of the Cry1Ab protein's amino acid sequence, wherein the first antibody and the second antibody individually are capable of binding to both the Cry1Ab protein and the hybrid Cry protein.
In other embodiments of the invention, the immunoassay is an enzyme-linked immunosorbent assay (ELISA), preferably a sandwich ELISA. In still other embodiments, the first antibody is the coating antibody and the second antibody is the detecting antibody. In still other embodiments, the first and the second antibodies are monoclonal. In yet other embodiments, the first or coating antibody is the monoclonal antibody 87AB1.1, which is available from Romer Labs, Inc (Union, Mo.). In other embodiments, the second or detecting antibody is the monoclonal antibody H04MAb70 produced by the hybridoma cell line H04MAb70 deposited as ATCC Accession No. PTA-122984. In still other embodiments, the coating antibody is 87AB1.1 and the detecting antibody is H04MAb70.
In some embodiments of the invention, the Cry1Ab protein that is specifically detected in an immunoassay of the invention comprises an amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:2 or SEQ ID N:6. In other embodiments, the hybrid Cry protein is eCry3.1Ab (SEQ ID NO:3), Cry1A.105 (SEQ ID NO:4) or Cry1A.105 (SEQ ID NO:5).
In other embodiments of the invention, the biological sample is a transgenic plant sample. In still other embodiments, the transgenic plant is a transgenic corn plant comprising a transgenic event selected from the group consisting of event Bt11, which expresses the Cry1Ab protein of SEQ ID NO:1, event 5307, which expresses the eCry3.1Ab hybrid Cry protein of SEQ ID NO:3, event MON89034, which expresses the Cry1A.105 hybrid Cry protein of SEQ ID NO:5 and event MON810, which expresses the Cry1Ab protein of SEQ ID NO:6. In other embodiments, the transgenic plant comprises event Bt11, event 5307 and event MON89034. Is still other embodiments, the transgenic plant comprises event MON810 and MON89034.
According to yet another embodiment, the invention encompasses a diagnostic kit for detecting a Cry1Ab protein in a biological sample which comprises the Cry1Ab protein and a hybrid Cry protein having at least a contiguous 27% of the Cry1Ab protein's amino acid sequence, wherein the kit comprises a first antibody and a second antibody that function together to specifically detect or quantitate the Cry1Ab protein and that individually are capable of binding both the Cry1Ab protein and the hybrid Cry protein.
In other embodiments of the invention, the immunoassay is an enzyme-linked immunosorbent assay (ELISA), preferably a sandwich ELISA. In still other embodiments, the first antibody is the coating antibody and the second antibody is the detecting antibody. In still other embodiments, the first and the second antibodies are monoclonal. In yet other embodiments, the first or coating antibody is the monoclonal antibody 87AB1.1, which is available from Romer Labs, Inc (Union, Mo.). In other embodiments, the second or detecting antibody is the monoclonal antibody H04MAb70 produced by the hybridoma cell line H04MAb70 deposited as ATCC Accession No. PTA-122984. In still other embodiments, the coating antibody is 87AB1.1 and the detecting antibody is H04MAb70.
In some embodiments of the invention, the Cry1Ab protein that is specifically detected in an immunoassay of the invention comprises an amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:2 or SEQ ID N:6. In other embodiments, the hybrid Cry protein is eCry3.1Ab (SEQ ID NO:3), Cry1A.105 (SEQ ID NO:4) or Cry1A.105 (SEQ ID NO:5).
In other embodiments of the invention, the biological sample is a transgenic plant sample. In still other embodiments, the transgenic plant is a transgenic corn plant comprising a transgenic event selected from the group consisting of event Bt11, which expresses the Cry1Ab protein of SEQ ID NO:1, event 5307, which expresses the eCry3.1Ab hybrid Cry protein of SEQ ID NO:3, event MON89034, which expresses the Cry1A.105 hybrid Cry protein of SEQ ID NO:5 and event MON810, which expresses the Cry1Ab protein of SEQ ID NO:6. In yet further embodiments, the biological sample comprises a Cry1Ab protein from event Bt11 and an eCry3.1Ab hybrid Cry protein from event 5307 and optionally a Cry1A.105 hybrid Cry protein from event MON89034. In still further embodiments, the biological sample comprises a Cry1Ab protein from event MON810 and a Cry1A.105 hybrid Cry protein from event MON89034.
According to another embodiment, the invention encompasses a pair of purified antibodies comprising a first antibody and a second antibody that function together in an immunoassay to specifically detect or quantitate a Cry1Ab protein in a biological sample comprising the Cry1Ab protein and a hybrid Cry protein having at least a contiguous 27% of the Cry1Ab protein's amino acid sequence, wherein the first antibody and the second antibody individually are capable of binding to both the Cry1Ab protein and the hybrid Cry protein.
In other embodiments of the invention, the immunoassay is an enzyme-linked immunosorbent assay (ELISA), preferably a sandwich ELISA. In still other embodiments, the first antibody is the coating antibody and the second antibody is the detecting antibody. In still other embodiments, the first and the second antibodies are monoclonal. In yet other embodiments, the first or coating antibody is the monoclonal antibody 87AB1.1, which is available from Romer Labs, Inc (Union, Mo.). In other embodiments, the second or detecting antibody is the monoclonal antibody H04MAb70 produced by the hybridoma cell line H04MAb70 deposited as ATCC Accession No. PTA-122984. In still other embodiments, the coating antibody is 87AB1.1 and the detecting antibody is H04MAb70.
In some embodiments of the invention, the Cry1Ab protein that is specifically detected in an immunoassay of the invention comprises an amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:2 or SEQ ID N:6. In other embodiments, the hybrid Cry protein is eCry3.1Ab (SEQ ID NO:3), Cry1A.105 (SEQ ID NO:4) or Cry1A.105 (SEQ ID NO:5).
In other embodiments of the invention, the biological sample is a transgenic plant sample. In still other embodiments, the transgenic plant is a transgenic corn plant comprising a transgenic event selected from the group consisting of event Bt11, which expresses the Cry1Ab protein of SEQ ID NO:1, event 5307, which expresses the eCry3.1Ab hybrid Cry protein of SEQ ID NO:3, event MON89034, which expresses the Cry1A.105 hybrid Cry protein of SEQ ID NO:5 and event MON810, which expresses the Cry1Ab protein of SEQ ID NO:6. . In further embodiments, the biological sample comprises a Cry1Ab protein from event Bt11 and an eCry3.1Ab hybrid Cry protein from event 5307 and optionally a Cry1A.105 hybrid Cry protein from event MON89034. In still further embodiments, the biological sample comprises a Cry1Ab protein from event MON810 and a Cry1A.105 hybrid Cry protein from event MON89034.
According to another embodiment, the invention encompasses a hybridoma cell line, designated H04MAb70, deposited as ATCC Accession No. PTA-122984, and a hybridoma cell line designated MAb58, deposited as ATCC Accession No. PTA-122985. In other embodiments, the invention encompasses a monoclonal antibody designated HO4MAb70 produced by the HO4MAb hybridoma cell line that binds to both a wild-type Cry1Ab protein and a hybrid Cry protein. In certain other embodiments, the invention encompasses a monoclonal antibody designated MAb58 produced by the hybridoma cell line MAb58 that binds to a hybrid Cry protein.
According to another embodiment, the invention encompasses immunoassay methods to specifically detect or quantitate a Cry1Ab protein in a biological sample comprising the Cry1Ab protein and a hybrid Cry protein having at least a contiguous 27% of the Cry1Ab protein's amino acid sequence. Such methods comprise (a) obtaining a biological sample comprising the Cry1Ab protein and the hybrid Cry protein; and (b) performing an immunoassay on the biological sample, wherein the immunoassay comprises use of a first antibody and a second antibody that individually are capable of binding to the Cry1Ab protein and the hybrid Cry protein but that function together in the immunoassay to specifically detect or quantitate the Cry1Ab protein and not the hybrid Cry protein, resulting in the specific detection or quantitation of the Cry1Ab protein.
In some embodiments, the immunoassay is an enzyme-linked immunosorbent assay (ELISA). In other embodiments, the first antibody is a coating antibody and the second antibody is a detecting antibody. In further embodiments, the coating antibody and the detecting antibody are monoclonal. In still other embodiments, the coating antibody is the monoclonal antibody 87AB1.1 available from Romer Labs, Inc. (Union, Mo.). In yet other embodiments, the detecting antibody is the monoclonal antibody HO4MAb70 produced by the hybridoma cell line deposited as ATCC Accession No. PTA-122984. In other embodiments, the coating antibody is 87AB1.1 and the detecting antibody is HO4MAb70.
In some embodiments of the invention, the Cry1Ab protein is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:6. In other embodiments, the hybrid Cry protein is selected from the group consisting of eCry3.1Ab (SEQ ID NO:3), Cry1A.105 (SEQ ID NO:4) and H04 (SEQ ID NO:5). In still other embodiments, the Cry1Ab protein comprises an amino acid of SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:6 and the hybrid Cry protein is eCry3.1Ab (SEQ ID NO:3) or Cry1A.105 (SEQ ID NO:4) or H04 (SEQ ID NO:5). In other embodiments, the Cry1Ab protein comprises the amino acid sequence of SEQ ID NO:6 and the Cry1A.105 hybrid Cry protein comprises the amino acid of SEQ ID NO:4.
In some other embodiments of the invention, the biological sample tested in the immunoassay is a transgenic plant sample. In other embodiments, the transgenic plant sample is from a transgenic corn plant. In other embodiments, the transgenic corn plant comprises a transgenic corn event selected from the group consisting of event Bt11, event 5307, event MON89034 and event MON810. In still other embodiments, the transgenic corn plant comprises event Bt11, event 5307 and event MON89034. In yet other embodiments, the biological sample comprises a Cry1Ab protein from event Bt11 and an eCry3.1Ab hybrid Cry protein from event 5307 and optionally a Cry1A.105 hybrid Cry protein from event MON89034. In other embodiments, the biological sample comprises a Cry1Ab protein from event MON810 and a Cry1A.105 hybrid Cry protein from event MON89034.
In another embodiment, the invention encompasses an immunoassay method for detecting or quantitating both a Cry1Ab protein and a hybrid Cry protein having at least a contiguous 27% of a Cry1Ab protein's amino acid sequence in a biological sample comprising the hybrid Cry protein and the Cry1Ab protein, the immunoassay method comprising: (a) obtaining a biological sample comprising the hybrid Cry protein and the Cry1Ab protein; (b) contacting the biological sample with a first antibody under conditions effective to allow the formation of a Cry1Ab-first antibody complex but not a hybrid Cry protein-first antibody complex, resulting in a biological sample depleted of the Cry1Ab protein; (c) removing the Cry1Ab-depleted biological sample from contact with the first antibody; (d) contacting the Cry1Ab-depleted biological sample with a second antibody under conditions effective to allow the formation of a hybrid Cry protein-second antibody complex; and (e) detecting or quantitating the hybrid Cry protein in the hybrid Cry protein-second antibody complex.
In some embodiments, the immunoassay is an enzyme-linked immunosorbent assay, wherein the solid surface is plastic well in a microtiter dish. In some embodiments, the Cry1Ab protein is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:6. In other embodiments, the hybrid Cry protein is selected from the group consisting of eCry3.1Ab (SEQ ID NO:3), Cry1A.105 (SEQ ID NO:4) and H04 (SEQ ID NO:5). In still other embodiments, the Cry1Ab protein is SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:6 and the hybrid Cry protein is eCry3.1Ab (SEQ ID NO:3) or Cry1A.105 (SEQ ID NO:4) or H04 (SEQ ID NO:5). In other embodiments, the Cry1Ab protein comprises the amino acid sequence set forth in SEQ ID NO:6 and the Cry1A.105 hybrid Cry protein comprises the amino acid sequence set forth in SEQ ID NO:4. In still other embodiments, the Cry1Ab protein is SEQ ID NO:1 and the hybrid Cry protein is eCry3.1Ab (SEQ ID NO:3) or Cry1A.105 (SEQ ID NO:4).
In other embodiments, the first antibody is 87AB1.1 which is available from Romer Labs, Inc. (Union, Mo.). In still other embodiments, the second antibody is the monoclonal antibody MAb58 produced by the hybridoma cell line deposited as ATCC Accession No. PTA-122985. In yet other embodiments of this aspect, the detecting of step (d) is carried out using a composition comprising a monoclonal antibody capable of binding to the Cry1Ab protein and the hybrid Cry protein. In still further embodiments, the monoclonal antibody is HO4MAb70. In yet other embodiments of this aspect, the detecting step (e) is carried out using a composition comprising a monoclonal antibody or a polyclonal antibody capable of binding to the hybrid Cry protein and the Cry1Ab protein. In further embodiments, the monoclonal antibody is HO4MAb70 or the polyclonal antibody is R-PAb available from Romer Labs, Inc. (Union, Mo.).
According to yet another embodiment, the invention encompasses an immunoassay method to detect or quantitate a hybrid Cry protein having at least a contiguous 27% of a Cry1Ab protein's amino acid sequence in a biological sample comprising the hybrid Cry protein and the Cry1Ab protein. Such methods comprise: (a) obtaining a biological sample comprising the hybrid Cry protein and the Cry1Ab protein; (b) contacting the biological sample with a 87AB1.1 antibody under conditions effective to allow the formation of a Cry1Ab-87AB1.1 complex but not a hybrid Cry protein-87AB1.1 complex, resulting in a biological sample depleted of the Cry1Ab protein; (c) removing the Cry1Ab-depleted biological sample from contact with the 87AB1.1 antibody; (d) contacting the Cry1Ab-depleted biological sample with a second antibody under conditions effective to allow the formation of a hybrid Cry protein-second antibody complex; and (e) detecting or quantitating the hybrid Cry protein in the hybrid Cry protein-second antibody complex.
In some embodiments, the immunoassay is an enzyme-linked immunosorbent assay (ELISA). In some other embodiments of the invention, the Cry1Ab protein is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:6. In other embodiments, the hybrid Cry protein is selected from the group consisting of eCry3.1Ab (SEQ ID NO:3), Cry1A.105 (SEQ ID NO:4) and H04 (SEQ ID NO:5). In still other embodiments, the Cry1Ab protein is SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:6 and the hybrid Cry protein is eCry3.1Ab (SEQ ID NO:3) or Cry1A.105 (SEQ ID NO:4) or H04 (SEQ ID NO:5). In other embodiments, the Cry1Ab protein comprises the amino acid sequence set forth in SEQ ID NO:6 and the Cry1A.105 hybrid Cry protein comprises the amino acid sequence set forth in SEQ ID NO:4.
In some other embodiments of the invention, the biological sample tested in the immunoassay is a transgenic plant sample. In other embodiments, the transgenic plant sample is from a transgenic corn plant. In other embodiments, the transgenic corn plant comprises a transgenic corn event selected from the group consisting of event Bt11, event 5307, event MON89034 and event MON810. In still other embodiments, the transgenic corn plant comprises event Bt11, event 5307 and event MON89034. In yet other embodiments, the biological sample comprises a Cry1Ab protein from event Bt11 and an eCry3.1Ab hybrid Cry protein from event 5307 and optionally a Cry1A.105 hybrid Cry protein from event MON89034. In other embodiments, the biological sample comprises a Cry1Ab protein from event MON810 and a Cry1A.105 hybrid Cry protein from event MON89034.
The invention will be further described by reference to the following detailed examples. These examples are provided for the purposes of illustration only, and are not intended to be limiting unless otherwise specified. Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described by J. Sambrook, et al., Molecular Cloning: A Laboratory Manual, 3d Ed., Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press (2001); by T. J. Silhavy, M. L. Berman, and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984) and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, New York, John Wiley and Sons Inc., (1988), Reiter, et al., Methods in Arabidopsis Research, World Scientific Press (1992), and Schultz et al., Plant Molecular Biology Manual, Kluwer Academic Publishers (1998).
This example describes antibody embodiments of the invention and the identification of functional antibody pairs. Antibody pairs were tested for their ability to specifically detect a Cry1Ab protein (SEQ ID NO:1) when in the presence of an eCry3.1Ab protein (SEQ ID NO:3) or a Cry1A.105 protein (SEQ ID NO:4) or both, which are hybrid Cry proteins having at least a contiguous 27% of the Cry1Ab protein's amino acid sequence as shown in
Antibody pairs are tested in a standard sandwich ELISA. Wells of a 96-well plate are coated with a first antibody (coating antibody), typically by incubating over night at approximately 2°-8° C. Plates are then washed before adding a sample comprising a Cry1Ab protein, a Cry1A.105 protein and an eCry3.1Ab protein. Wells are then thoroughly mixed by moving the plate for approximately 20-30 sec, after which the plates are incubated at ambient temperature for approximately 1 to 2 hours. Optionally the plate may be shaken on an orbital shaker at approximately 200 rpm during the incubation period. After incubation, sample liquid is removed from the wells and the wells are washed completely 2-3× with a wash buffer. After washing, a second antibody (detection antibody) labelled with a detection enzyme is added to the wells. The plates are incubated at ambient temperature for another approximately 1 to 2 hours. After incubation the plates are washed completely with wash buffer. Approximately 100 μl of substrate is then added to each well and the plates incubated at ambient temperature for approximately 15-30 minutes. Plates are inspected for substrate color development and scored as positive, “+” or negative “−” for each of the three proteins of interest.
Surprisingly, the results, shown in Table 2, demonstrate that when individual antibodies that are capable of binding all three proteins, i.e. Cry1Ab, eCry3.1Ab and Cry1A.105, are used in pairs, the pairs function collectively to be selective for either Cry1Ab or a hybrid Cry protein. This is even more surprising for individual antibodies that are raised against a wild-type Cry1Ab protein like 87AB1.1 and MAb58 or for an antibody raised against a hybrid protein made up of almost 70% of wild-type Cry1Ab amino acid sequence like H04MAb70 (See
The results also surprisingly demonstrate that an immunoassay of a complex biological sample comprising Cry1Ab, Cry1A.105 and eCry3.1Ab using a pair of antibodies, where the first antibody is MAb58, a monoclonal antibody raised against a wild-type Cry1Ab protein, and the second antibody is H04MAb70, is selective for the Cry1A.105 protein. Thus, when used in combination, the functionality of the first antibody, MAb58, and the second antibody, H04MAb70, is markedly different than either antibody alone.
This example describes a procedure for specific detection and quantification of a
Cry1Ab protein in a complex biological sample, for example a transgenic plant sample, comprising the Cry1Ab protein and two hybrid Cry proteins having at least a contiguous 27% of the Cry1Ab protein's amino acid sequence by enzyme linked immunosorbent assay (ELISA).
A 96-well Nunc Maxisorp plate is coated with 100 μl per well of a solution comprising the monoclonal antibody 87AB1.1, available from Romer Labs, Inc (Union, Mo.). Plates are covered with a 96-well plate lid and incubated minimally overnight at approximately 2° C. to 8° C. If the plates are allowed to incubate longer than overnight, cover the plate with an adhesive plate sealer to prevent evaporation and contamination of the wells. Wash the plates at least three times with 1× phosphate buffered saline with Tween® 20 (PBST) wash buffer (§ 11.2.3) using approximately 300 μl/well. As a blocking step, add approximately 200 μl of Superblock® per well. Incubate the plates at room temperature while shaking at approximately 400 rpm for at least 30 minutes. Wash the plates at least three times with 1×PBST wash buffer using approximately 300 μl/well.
Dilutions: Sample extracts are diluted in 1× PBST (137 mM sodium chloride, 2.7 mM potassium chloride, 10 mM sodium phosphate dibasic, 1.8 mM potassium phosphate monobasic and 0.05% Tween® 20) with 1% bovine serum albumin (BSA) buffer. At least two dilutions of each sample extract should be analyzed to obtain multiple concentrations across the quantitative range of the standard curve. Sample extracts should be mixed thoroughly before use. Prepare a minimum volume of the initial dilution so that sufficient volume is available to create subsequent dilutions for accurate duplicate analysis (100 μl each). Increase the volume of the initial dilution appropriately if samples will be analyzed in triplicate. Mix dilutions by pipetting up and down several times. For example, a 1:20 dilution would be made up as follows: 760 μl of 1×PBST with 1% BSA buffer+40 μl of extract=800 μl (total volume). Subsequent 1:2 dilutions can be made from the first dilution. The minimum dilution factor for all matrices is located in Table 1.
Preparation of Standard Curve: Prepare the standard curve containing the following concentrations of Cry1Ab in 1×PBST with 1% BSA buffer: 12.5, 6.25, 3.13, 1.56, 0.781, 0.391, 0.195 and 0.098 ng/ml. See Table 3 for example dilution series. Solutions may be prepared in microtubes to facilitate efficient loading of ELISA plates with standard curve solution.
Loading the Plate: Sample extract dilutions should be prepared so that the plate can be loaded quickly and efficiently to minimize variation in incubation time between the first and last samples loaded. Place 100 μl of the standards in the wells in triplicate, then place 100 μl of samples and PACS into the wells in no less than duplicate. Cover the plate and incubate at room temperature for at least one hour while shaking at approximately 400 rpm.
Wash the plate at least five times with 1× PBST buffer with approximately 300 μl/well. Add 100 μl/well of the appropriately diluted H04Mab 70-HRP antibody. Cover the plate and incubate at room temperature for at least one hour while shaking at approximately 400 rpm. Wash the plate at least five times with 1×PBST buffer with approximately 300 μl/well. Add 100 μl/well of the TMB substrate solution (1 tablet TMB, 10 ml citrate phosphate buffer and 2 μl hydrogen peroxide) using an appropriate multichannel device to each well of the plate(s) and incubate the plate at room temperature, in the dark, while shaking at approximately 400 rpm until the appropriate color development has been achieved (approximately 15 minutes). Stop the reaction by adding 50 μl/well of 3 M sulfuric acid. Read the absorbance of the wells at a wavelength of 450 nm using a SPECTRAmax Pro plate reader. Other plate readers may be used if necessary.
Interpretation of Results: Data are analyzed using SoftMax® Pro software or equivalent.
Standard Curve: A 4-parameter logistic algorithm is used to fit the standard curve. For each standard curve dilution, the coefficient of variation (CV) of the optical density (OD) values must be ≤20%. A minimum of two replicates is required for analysis; one of the triplicate OD values may be omitted as needed to yield an acceptable CV value. For documentation purposes, this is indicated on the data printout by both drawing a single line through the omitted data point and writing the corrected mean in the “mean” column or by masking the well in the software program used for analysis. If the CV of the OD values of duplicate standards is >20%, then the ELISA must be repeated. The mean back calculated concentration (MBCC) value must be within 70%-120% of the nominal concentration for each standard point within the quantitative range of the assay. One of the triplicate OD values may be omitted as needed to yield an acceptable criterion, however a minimum of two replicates is required for analysis; See § 9.2.2 for documentation guidelines. The quantitative range of the Cry1Ab standard curve is 0.195 ng/ml to 12.5 ng/ml. The coefficient of determination (R2) must be ≥0.990.
Positive Assay Control Sample: For each PACS dilution, the CV between replicate concentrations must be ≤20%. To be a valid PACS dilution, a minimum of two replicates is required for analysis. If assayed in triplicate, one of the triplicate concentrations may be omitted as needed to yield an acceptable CV value. To calculate the mean PACS concentration, multiply the measured concentration (ng/ml) by the corresponding dilution factor to determine the calculated concentration for each dilution in μg/ml. Average all calculated concentrations that fall within the accurate range of the assay to determine the overall Cry1Ab PACS concentration in μg/ml. The CV of the calculated concentration among PACS dilutions must be ≤20%. If the CV of the calculated concentration among PACS dilutions is >20%, and/or the mean PACS concentration is outside the specification range, the ELISA plate data is invalidated and all samples on that plate will be reanalyzed in another ELISA session.
Test Samples: For each sample dilution, the CV between replicate concentrations must be ≤20%. To be a valid sample dilution, a minimum of two replicates is required for analysis. If assayed in triplicate, one of the triplicate concentrations may be omitted as needed to yield an acceptable CV value. The mean measured concentration obtained by ELISA for each sample dilution must lie within the quantitative range of the ELISA (Table 2). To calculate the sample concentration, multiply each measured sample protein concentration that is within the quantitative range of the ELISA by the corresponding dilution factor. Average the results from at least two sample dilutions to determine the overall sample concentration, unless the sample dilution at the minimum dilution factor for a given matrix is the only result within the quantitative range. The CV of the calculated concentration among sample dilutions must be ≤20%. If the CV among sample dilutions is >20% and more than two dilutions are used to calculate the concentration, one outlying dilution may be omitted and the mean concentration recalculated from the remaining dilutions.
Results: Samples of leaf, kernel and root tissue from a triple stack (three events) transgenic corn plant comprising the transgenic corn events Bt11 expressing Cry1Ab, 5307 expressing eCry3.1Ab and MON89034 expressing Cry1A.105 were tested using the immunoassay described above to quantitate specifically the Cry1Ab protein concentration in the samples. Four different samples for each tissue were prepared and 3 replicates from each sample were assayed. The concentration of Cry1Ab protein in transgenic corn comprising the Bt11 event alone has been reported to range from approximately 12-154 μg/g dry weight (DW) in leaves and approximately 9-22 μg/gDW in roots (September 2010. US Environmental Protection Agency Biopesticides Registration Action Document. Cry1Ab and Cry1F Bacillus thuringiensis (Bt) Corn Plant-Incorporated Protectants). Results of the immunoassay of a stacked product, shown in Table 4, demonstrate that the concentration of Cry1Ab in the stacked product is comparable to the Cry1Ab concentration in Bt11 alone, indicating that the immunoassay can specifically detect and quantitate Cry1Ab in the presence of one or more hybrid Cry proteins having at a contiguous 27% of the Cry1Ab protein's amino acid sequence.
Based on the discovery that certain antibody pairs have markedly different function than either antibody when used alone, a serial immunoassay was developed that allows for detection or quantitation of both a wild-type Cry1Ab and a hybrid Cry protein having a contiguous 27% of the Cry1Ab protein's amino acid sequence in the same immunoassay. Briefly the method comprises coating a first 96-well microtiter plate with the 87AB1.1 monoclonal antibody, which when used in a pair of antibodies selectively detects a wild-type Cry1Ab protein in a mixture of Cry1Ab and a hybrid Cry protein and a second 96-well microtiter plate with a MAb58 antibody. Contacting a biological sample from a transgenic plant comprising a Cry1Ab protein from event Bt11, a hybrid Cry protein, eCry3.1Ab, from event 5307 and a hybrid Cry protein, Cry1A.105, from event MON89034 with the 87AB1.1 antibody under conditions effective to allow the formation of a Cry1Ab-87AB1.1 complex but not a hybrid Cry protein-87AB1.1 complex as described above in Example 2. This results in a biological sample in the microtiter wells that is depleted of the Cry1Ab protein. Next remove the Cry1Ab-depleted biological sample from the first microtiter dish and transfer to the second microtiter dish, contacting the Cry1Ab-depleted biological sample with the MAb58 antibody under conditions effective to allow the formation of a Cry1A.105-MAb58 complex. Next add the appropriate dilution of a solution comprising a H04MAb70-HRP labelled antibody or a PAb713-HRP labelled antibody. Incubated under conditions effective to allow the formation of a Cry1A.105-H04MAb70 or Cry1A.105-PAb713 complex. Then add a substrate to detect or quantitate the Cry1A.105 protein by comparing to a Cry1A.105 standard curve.
Optionally, the Cry1Ab protein on the first microtiter plate can also be detected or quantitated in the same immunoassay by following the procedure described in Example 2.
It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims.
All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art that this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
This application is a divisional of co-pending U.S. application Ser. No. 15/755,721, filed Feb. 27, 2018, which is a 35 U.S.C. § 371 National Stage application of PCT Application No. PCT/US16/48580, filed Aug. 25, 2016, and published as WO2017/044310 on Mar. 16, 2017, which claims the benefit of U.S. Provisional Application No. 62/215,982, filed Sep. 9, 2015, all of which are incorporated by reference herein in their entirety.
