Patent Application
20120208176
The present invention relates to a new method to determine the presence of antibodies to a pathogen in a serological sample using a new detection reagent which comprises at least two and preferably at least four copies of an antigen from the pathogen and a detectable marker. The present invention also relates to a new detection reagent which consists of at least two and preferably at least four antigenic peptides in a complex with a detectable marker via interacting portions upon both the antigenic peptides and detectable marker.
In order to best treat a disease or illness afflicting a patient it is necessary to determine as soon as possible the causative agent of the disease. In the case of illnesses caused by infective agents such as viruses, bacteria or protozoa a further problem is the identification of the particular infective agent, as in general the infection of a patient gives rise to certain common symptoms.
Febrile illnesses are characterised by a period of fever which can last a short time, continue throughout the infection or arise intermittently whilst the infection persists. Within the group of febrile illnesses are a number of ‘low’ risk illnesses, that is illnesses which generally do not have any long term effects upon the patients health, such as the common cold, measles and chicken pox; and ‘high’ risk diseases, that is diseases which often lead to patient mortality or severe symptoms which prevent the patient from recovering to their original level of health. ‘High’ risk febrile diseases include severe acute respiratory syndrome (SARS), typhoid fever as well as the group of diseases caused by flaviviruses such as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS).
This commonality of early symptoms can make the accurate diagnosis of the agent of disease very difficult if not impossible using directly measurable or observable symptoms. The resulting delay in diagnosis can in turn retard the deployment of an effective treatment regime for the patients' disease and lead to a reduction in the speed and in some cases chances of the patient recovering.
It is also currently difficult to make an early and definitive diagnosis using more advanced techniques such as a serological analysis of reacting antibodies for the pathogen or the direct detection of the pathogen by for instance RT-PCR. The reasons for this vary between pathogens but in general most existing serological techniques exhibit non-specific cross reactions between antibodies with non-target antigens or limitations in terms of when a PCR assay can lead to a meaningful result. Further problems include the inability using current assays to differentiate between a primary and secondary (or further) infection by the same pathogen and also the time necessary to perform the assay. The determination of whether an infection is the first or a subsequent re-infection can be of critical importance as certain pathogens are known to induce more pathogenic reactions upon subsequent infection due in part to the altered nature of the patients' immune response.
To illustrate the problems detailed above the important group of disease causing viruses, flaviviruses, will be discussed in detail together with an analysis of their epidemiology and the current problems with making an early diagnosis so that an appropriate treatment regime can be deployed.
Flaviviruses are enveloped RNA viruses. They encode three structural proteins, the core protein C, the premembrane/membrane protein prM/M and the envelope glycoprotein gpE, and seven non-structural (NS) proteins. The structures of the whole virus and of gpE have been solved for several flaviviruses by electron cryomicroscopy and X-ray crystallography. Ninety dimers of gpE cover the surface of the virus. Each monomer comprises three ectodomains, ED1 to ED3, and a transmembrane segment. ED1 and ED2 are interwoven. ED2 contains the dimerization interface and the peptide of fusion with the cellular membrane. ED3 is continuous and comprises residues 296-400 of gpE. Its fold is compact, immunoglobulin-like and stabilized by a disulfide bond (Mukhopadhyay et al. 2005).
Many flaviviruses are responsible for widespread emerging or reemerging diseases, e.g. the four serotypes of dengue virus (DENV1 to DENV4), the West-Nile virus (WNV), the yellow fever virus (YFV), and the Japanese encephalitis virus (JEV). Infections with flaviviruses cause three main clinical syndromes in humans: systemic febrile illness, hemorrhagic fever and meningoencephalitis. The illness generally follows a biphasic course with a mild febrile phase, often going unrecognized, followed by defervescence and an apparent relapse about five to seven days after the onset of symptoms, and then by a more severe illness in a few percent of cases (Gould and Solomon 2008). These syndromes are shared with other diseases, including measles, typhoid fever, leptospiroses and severe acute respiratory syndrome (SARS) and their co-occurence in the same area makes a specific diagnosis difficult (Potts and Rothman 2008).
The ‘high’ risk illnesses that are caused by DENV, include the dengue hemorrhagic fever DHF and shock syndrome DSS (WHO 1997). The high mortality associated with DHF and DSS (40 to 50% for untreated patients) can be minimized through timely medical care and fluid replacement, which depend on accurate diagnosis (Igarashi 1997). Infections by DENY result in the production of neutralizing antibodies and lifelong immunity against the homologous DENV serotype but there is no cross-immunity against the heterologous serotypes. Moreover, secondary infection is suspected to be a risk factor for DHF and DSS (Halstead 1988; Mathew and Rothman 2008).
Flavivirus and in particular DENV infections can be diagnosed during the acute phase of the disease by virus isolation, RT-PCR of viral RNA, or immunochemical detection of the viral NS1 protein in serum samples (Kuno 2003; Shu and Huang 2004; Dauphin and Zientara 2007). However, these methods can only be used during the viremic phase of infection, i.e. 4-6 days after the onset of symptoms and they no longer work after defervescence (Yamada et al. 1999; de Oliveira Poersch et al. 2005; Dussart et al. 2006). Moreover, these techniques cannot distinguish between primary and secondary infections (Schilling et al. 2004).
The kinetics of the immune response to a flaviviral infection have been well characterized for dengue. In primary infections, immunoglobulin M (IgM) antibodies that are directed against DENV, appear as early as 4-6 days after the onset of symptoms, peak at about 2 weeks, and wane thereafter. IgG antibodies appear shortly afterwards and persist for several years. In secondary infections, IgMs appear earlier than in primary infections, often with lower titers, whereas IgGs appear with high titers either before or along with IgMs (Innis et al. 1989; Kuno 2003; Schilling et al. 2004; Shu and Huang 2004; Teles et al. 2005; Dauphin and Zientara 2007).
Cross-reactive antibodies are produced during an infection with a flavivirus. They react with the infecting virus but also with other flaviviruses, Flaviviridae or even unrelated infectious agents. These cross-reactive antibodies give rise to false positives in serological tests, e.g. enzyme-linked immunosorbent assays (ELISA) and complicate their interpretation. For example, cross-reactions arise between sera from patients infected with DENV or vaccinated against YFV and JEV, when whole virus antigens are used in the tests. They also arise between sera from patients with flaviviral diseases and those from patients with malaria, typhoid, leptospirosis or autoimmune diseases (Schwartz et al. 2000; Takasaki et al. 2002; Kuno 2003; Mongkolsapaya et al. 2003; AnandaRao et al. 2005; Holmes et al. 2005).
Generally, IgMs are less cross-reactive than IgG but they have a lower affinity for their antigen than IgGs since they do not undergo somatic hypermutagenesis (Westaway et al. 1975; Makino et al. 1994; Martin et al. 2002; Kuno 2003; Dauphin and Zientara 2007). In IgM-specific indirect ELISA, see below, the IgM signal can be quenched by the high amount of non-specific IgG present in the serum. In IgM-antibody specific capture ELISA (MAC-ELISA) see below, the IgMs of the serum are first captured by anti-IgM antibodies. MAC-ELISAs are therefore more sensitive and specific than IgM-specific indirect ELISAs and preferred over them (Kuno 2003).
Current diagnostic assays for flavivirus infections utilize either ELISA or dipstick formats for the identification of flavivirus infections.
The immunosorbent assays (USA) for the detection of viral anti-bodies in the serum of patients belong to two main types: indirect ISA and antibody-specific capture ISA.
Support-virAg::Serum::@Ig-Reporter (1)
Support-@IgX::Serum::virAg::@virAg-Reporter (2)
Depending on the Ig binding protein (@IgX), one can speak of IgM, IgG or IgA antibody capture immunosorbent assays (MAC-ELISA, GAC-ELISA or AAC-ELISA).
The immunosorbent assays for IgM antibodies are among the most useful serologic procedures for determining recent infections by flaviviruses, since these IgM molecules appear early in infection, rise rapidly in the course of the disease, and are usually less cross-reactive with other viruses than IgG antibodies (Kuno, 2003). IgM molecules can be detected as soon as the 5th day after infection but their affinity for a monomeric antigen is generally lower than that of other immunoglobulin molecule types.
The MAC-ELISA is preferred over the IgM-specific indirect ELISA because the IgG antibodies from previous infections by related viruses can have a suppressive effect on the sensitivity of the latter assay (Vorndam and Kuno, 1997). It is recommended by WHO for the serological diagnosis of several flaviviral infections and in particular dengue (WHO, 1997).
The MAC-ELISA has the following advantages: If paired serum samples are available, a rising, stable or falling titer in IgM can indicate the time of infection. The ratio of IgM to IgG antibody in parallel MAC- and GAC-ELISA on a single sample can be used to differentiate primary from secondary infections since the IgM/IgG ratio is higher than one in the former case and lower in the latter (Innis et al., 1989). It can detect anti-flaviviral IgM in the cerebrospinal fluid and saliva (Kao et al., 2005; Teles et al., 2005). IgA specific ELISAs have also been developed. The IgA response develops after the IgM response but before the IgG one. The IgA/IgM ratio in parallel MAC- and AAC-ELISAs can indicate whether the infection is recent or dates from a few months, for DENY and WNV (Prince and Lape-Nixon, 2005; Talarmin et al., 1998).
The specificity of the immunosorbent assays comes mainly from the interaction between the serum under analysis and the antigen and thus depends on the nature of the antigen preparation. However, it may also come from the nature of the reporter molecule.
Another IgM capture assay described in Morinet et al., 1990, concerns an improved IgM capture assay for the detection of anti-IgM antibodies directed against the B19 Human Parvovirus. This method uses single copies of a biotinylated recombinant α-peptide from the B19 capsid which are complexed with an avidin-peroxidase.
ViljA et al., 1985 also described an assay to detect anti-Hepatitis B (HepB) core antigen IgM antibodies using an antibody capture assay, based upon a biotinylated recombinant HepB antigen which is mixed with the captured antibody and this first immunocomplex is subsequently mixed with an avidin-peroxidase. In this assay the HepB antigen was not presented in a multivalent form and the biotinylation of the HepB antigen was performed at random. This assay therefore has the same problems of a lower detection threshold as the other prior art assays discussed herein.
Other types of ISA exist such as sandwich ELISA (R. J. Kerschbaumer et al., 1996) whose format is the following:
Support-@GST::GST-(3D6epitope)scFv3D6-PhoA (3)
where @GST represents antibodies directed against the Glutathione-S-Transferase (GST); GST-(3D6 epitope), a hybrid protein between GST and an epitope of antibody 3D6; and scFv3D6-PhoA, a hybrid protein between a single-chain variable fragment (scFv) of antibody 3D6 and alkaline phosphatase.
Sandwich ELISA assays are used to detect the presence of an antigen in the serum of patients, but not antibodies directed against an infectious agent as in the current invention. Moreover such methods require the isolation and characterization of at least two non-competing antibodies to be used in the assay.
Another type of ISA is a reverse ELISA (D. Ludolfs et al., 2007) whose format is the following:
Support-RF::serum::rED3-HRP (4)
where the Rhumatoid Factor (RF) is an autoimmune antibody that recognizes the Fc fragment of the IgG immunoglobulins; and rED3-HRP is a chemical conjugate between Horseradish Peroxydase (HRP) and a recombinant domain 3 (rED3) of the envelope protein E from the West-Nile virus. HRP is a monomeric protein, whereas alkaline phosphatase is dimeric, and the rED3-HRP hybrid protein was obtained by chemical coupling of the two partners, rED3 and HRP.
Until recently, the antigens used for ISA were mainly extracts of suckling mouse brains (SMB) or cell cultures, infected by the virus under consideration. These are being progressively replaced with recombinant prM/gpE virus like particles (VLP), where prM and gpE are the precursor of the membrane protein and envelope glycoprotein of the virus or with a recombinant extracellular domain (sE) of gpE. The non-structural protein NS1 has also been used as an antigen in both IgG-specific indirect ELISA and MAC-ELISA. NS1 can differentiate between primary and secondary infections and correctly identify the serotype of the infecting DENV in the sera of patients with primary infection (Shu et al., 2004; Shu et al., 2003; Shu et al., 2002).
Previously (Bedouelle et al., 2008, WO2008/152528), the inventors have created a new class of reagent for use in ELISA method to detect antiflavivirus antibodies. This reagent consisted of a hexahistidine, the ED3 domain from a flavivirus and alkaline phosphatase from E. coli (H6-ED3-PhoA) in a single peptide. This reagent is an obligate dimer. In this way, the inventors generated a first class of reagents that can be used to detect lower affinity antibodies through an avidity effect and in particular IgM antibodies.
These dimeric reagents (H6-ED3-PhoA)2 validated the concept of using an antigenic viral domain, and in particular the ED3 domain from flaviviruses, in a bivalent state, to detect specific low affinity IgMs in the serum of patients by MAC-ELISA.
Serious problems still exist however with the detection of Flaviviruses and other pathogens using serological assays even using the new class of reagent developed by the inventors and these include the specificity of the assay. Although the dimeric reagents were shown to have much improved performance with respect to prior art techniques and in particular allowed the differentiation of DENV serotypes 1, 2 and 3 they were still unable to distinguish DENV4. In addition due to the levels of affinity between the bivalent reagent and its antibody targets a minimum of 3 hours was required for the chromogenic reaction (Bedouelle et al., 2008, WO2008/152528). These limitations also make it difficult to determine whether an infection is a primary or secondary infection by the pathogen. Finally the use of the E. coli PhoA limits these reagents as many immunoenzymatic diagnostic assays are based on horseradish peroxidase (HRP) rather than PhoA because the former has a higher activity and is smaller, more stable, and cheaper to produce than the latter. The inventors used the PhoA enzyme as a marker as it can be easily expressed as a single peptide in combination with the selected antigen. It is not possible to prepare a HRP/ED3 single antigenic peptide as ED3 requires specific conditions so as to allow the formation of an essential disulfide bond which in turn would render the HRP enzyme inactive. Such specific expression requirements for antigens are common and this limits the types of antigens which can be used with the most common detectable marker HRP to those antigens which have no such expression requirements. In addition the fusion of an antigen and a marker at the genetic level can lead to unwanted structural/chemical alterations of the antigen or marker which in turn can lead to an imperfect detection reagent either with altered antibody binding properties or marker activity.
Seeing the problems associated with prior art methods and materials in diagnosing early the disease causing agent of febrile disease the inventors have developed a new set of materials and method for this purpose.
In accordance with a first aspect of the present invention, there is provided a method for the detection of antibodies against at least one pathogenic micro organism in a human or animal host, characterized in that it comprises:
a). contacting a sample from said host with a solid support coated on at least one side with an Immunoglobulin (Ig) binding protein which is directed against a specific class of Ig molecules of the species of said host, and
b). incubating the immunocomplex formed in step a). with a detection reagent comprising a complex between at least two and preferably at least four copies of an antigen from said at least one pathogenic micro organism each of which comprises a first multimerization domain and a detectable marker which comprises a second multimerization domain, wherein said complex is formed via the interaction of said first and said second multimerization domains,
c). determining the presence of said detection reagent in association with said immunocomplex formed in step a).
The Inventors have found that using a multivalent detection reagent and in particular a tetravalent detection reagent, the problems of specificity and efficiency of detection are overcome. The inventors have used again the ED3 domain from Flavivirus to validate this new method. The inventors have also found that using a detection reagent which consists of a multivalent antigen complexed with a marker, difficult to express antigens can be combined with markers that can not be expressed in the same peptide with a marker.
In the present patent application a complex between the antigenic peptide and a marker is a molecular structure in which the components therein are linked by non-covalent bonds.
In the present patent application a multivalent detection reagent consists of at least two antigens and preferably at least four antigens.
In the present patent application an antigen is a substance that prompts the generation of antibodies and can cause an immune response. In particular an antigen may be in the form of a peptide this being an antigenic peptide. Alternatively an antigen may be a lipid, saccharide or other molecule and/or a combination of any of these for instance a glycolipid or glycoprotein.
More generally an antigen is any molecule recognized by the immune system and in particular an antigen is any molecule or molecular fragment that can be bound by a major histocompatibility complex (MHC) and so presented.
In accordance with the present invention therefore the exact nature of the antigen is irrelevant as any given antigen would be expected to behave in an identical fashion to an antigen from any other antigenic component of the same organism or other pathogenic virus, bacteria or eukaryote. The nature of the antigen is not an essential aspect of the present invention, it is instead the finding of the inventors that by presenting an antigen in a multiplexed form, non-covalently bound to a marker which allows the routine determination of the IgG and IgM reactivity of a serum sample, that this allows the routine differentiation of a primary or non-primary infections of the pathogen from which the antigen comes and whether the infection is recent.
In the present patent application a copy of an antigen may comprise a number of epitopes each of which may be targeted by one or more antibodies.
In the present patent application the antigen can each comprise a first multimerization or alternatively groups of for instance two or more antigens may comprise a first multimerization domain.
In particular the Ig binding protein is selected in the group consisting of anti-IgM, anti-IgG and anti-IgA antibodies.
In particular the antigen is from a pathogenic micro organism which causes a febrile illness and in particular is from an organism selected from the group consisting of: a pathogenic virus, selected from the family Adenoviruses, Herpesviruses, Poxviruses, Parvoviruses, Reoviruses, Picornaviruses, Togaviruses, Orthomyxoviruses, Rhabdoviruses, Retroviruses, Hepadnaviruses; a pathogenic bacteria, selected from the Genus Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia, Clostridium, Corynebacterium, Enterococcus, Escherichia, Francisella, Haemophilus, Helicobacter, Legionella, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema, Vibrio, Yersinia; a pathogenic protozoa from the phylum Apicomplexa, Sarcomastigophora, Microspora, Ciliophora; in particular said antigen is the ED3 domain polypeptide of a flavivirus and is selected from the group consisting of: a yellow fever virus ED3 domain polypeptide, a West Nile virus ED3 domain polypeptide, a Dengue virus ED3 domain polypeptide, a St Louis encephalitis virus ED3 domain polypeptide, a Murray Valley encephalitis virus ED3 domain polypeptide and a Japanese encephalitis virus ED3 domain polypeptide.
This new method using multivalent antigen detection reagents can be used to detect a number of pathogenic micro organisms of the various types listed above.
In particular the detectable marker is selected from the group consisting of: an enzyme which catalyses the production of a detectable product, a fluorescent, radioactive or metal detectable tag, in particular wherein said detectable marker is selected from the group horseradish peroxidase (HRP), alkaline phosphatase (PhoA), a fluorophore or colloidal gold.
In particular the detection reagent also comprises a polypeptide tag selected in the group consisting of HIS, c-MYC, HA, VSV-G, HSV, V5 and FLAG.
These polypeptide tags can be used to identify the detection reagent in situ, for instance as an antibody mediated detection marker or as a site to attach a fluorophore and/or can be used to affinity purify the detection reagent.
In particular the method comprises detecting antibodies against a panel of detection reagents each comprising a different antigen from the same pathogenic micro organism or a group of different pathogenic micro organisms, wherein the immunocomplex formed in step a). are incubated with said panel of detection reagents and the presence of said panel of detection reagents in association with said immunocomplex formed in step a). is detected.
The present method therefore can be used to detect either simultaneously using a panel of reagents comprising antigens from different pathogens upon a panel of bound serum antibodies or alternatively sequentially upon the same bound serum antibody sample the presence or absence of several pathogens and/or antigens from the same pathogen.
In particular the method may also comprise the formation of two sets of immunocomplexes as formed in step a)., a first immunocomplex consisting of a first solid surface coated in IgG binding proteins bound to IgG molecules in said sample and a second immunocomplex consisting of a second solid surface coated in IgM binding proteins bound to IgM molecules in said sample; following which;
said first and said second immunocomplexes are incubated with a detection reagent comprising a at least two and preferably at least four copies of an antigen from said at least one pathogenic micro organism each of which comprises a first multimerization domain and a detectable marker which comprises a second multimerization domain, wherein said complex is formed via the interaction of said first and said second multimerization domains, and
the amount of said detection reagent in association with said first and said second immunocomplexes is determined quantitatively and compared.
This comparison of the levels of reacting IgM and IgG antibodies allows an investigator to determine whether an infection is a primary or secondary infection with the pathogen, as in a primary infection the level of IgM would be expected to be much elevated in comparison to IgG whereas for secondary (and subsequent) infections the levels of IgM/IgG would be comparable or show more IgG than IgM.
Alternatively the method may comprise the formation of two sets of immunocomplexes as formed in step a)., a first immunocomplex consisting of a first solid surface coated in IgA binding proteins bound to IgA molecules in said sample and a second immunocomplex consisting of a second solid surface coated in IgM binding proteins bound to IgM molecules in said sample; following which;
said first and said second immunocomplexes are incubated with a detection reagent comprising at least two and preferably at least four copies of an antigen from said at least one pathogenic micro organism each of which comprises a first multimerization domain and a detectable marker which comprises a second multimerization domain, wherein said complex is formed via the interaction of said first and said second multimerization domains, and
the amount of said detection reagent in association with said first and said second immunocomplexes is determined quantitatively and compared.
IgA forms an essential component of mucosal immunity and is produced at higher levels in the muscosal linings than any other antibody type. For pathogenic organisms whose route of entry and/or site of pathology is the mucosal linings therefore a comparison and IgA and IgM antibody levels, together with or in replacement of a comparison of IgG and IgM levels, allows a worker to even more clearly elucidate whether an infection is a primary infection or not.
In accordance with a second aspect of the present invention there is provided a detection reagent, comprising:
an antigenic peptide comprising at least one antigen from a pathogenic micro organism and a first multimerization domain;
a detectable marker and a second multimerization domain;
wherein said detection reagent comprises at least two and preferably at least four antigenic peptides in complex with said detectable marker via the interaction of said first and said second multimerization domains.
In the present patent application the first and second multimerization domains may be any peptide, saccharide or other chemical moieties which cause a first molecule comprising the first multimerization domain to form a complex with a second molecule comprising the second multimerization domain. In particular the first and second multimerization domains are naturally occurring or adapted systems which involve the recognition and binding of one molecule to another such as the recognition and binding of avidin to biotin or any of the various known variants of this system. Alternatively the first and second multimerization domains may be an epitope and an antibody or a fragment thereof which recognises and binds to the epitope. The multimerization domains may also be protein domains which in vivo cause protein-protein multimerization such as tubulin dimerisation domains, actin recognition and binding domains or any other protein-protein binding domain. The first and second multimerization domains can also consist of reactive chemical groups such as thiol groups, maleimide groups or other suitable bond forming chemical groups. The function of the first and second multimerization domains is therefore to create a stable molecular complex between several copies of an antigenic peptide and a detectable marker or alternatively, several different antigenic peptides and a detectable marker.
In particular the first and second multimerization domains could be a coiled coil domain (Blondel & Bedouelle, 1991; Müller et al., 2000); a four helix-bundle (Pack & Plückthun, 1992); the pentamerization domain of the E. coli verotoxin (Zhang et al., 2004).
In another embodiment of the present invention the antigenic peptide may comprise a single polypeptide chain, comprising several copies of the same antigenic peptide, such an arrangement could be constructed at the genetic level using known molecular biology techniques.
This new class of reagent shows vastly improved performance over existing reagents for use in ELISA and in particular MAC-ELISA assays. In addition the fact that the antigenic peptide is in complex with a separate detectable marker allows the combination of antigens with markers that were not previously possible. For instance certain antigens comprise disulfide bonds or other complex chemical structures which require specific conditions to form. ED3 is an example of such an antigen. To produce this antigen or an antigenic peptide comprising this antigen or a fragment thereof, it is necessary either for the protein to be exported into the periplasmic space or alternatively produced as inclusion bodies in the cytoplasm and refolded in vitro so as to provide the required conditions for the formation of the disulfide bond. It is therefore difficult to produce a unified construct comprising the antigenic and marker functional portions. Further it is difficult to generate a multi-valent antigen construct wherein each of the antigens is presented correctly so that they can interact with bound serum antibodies. The inventors have overcome these problems by generating two separate components, an antigenic peptide and a detectable marker each of which comprises a multimerization domain and via this multimerization domain several copies of the antigenic protein form a molecular complex with the detectable marker. This separation of the antigenic and detectable portions of the final detection reagent and the defined means by which the two components can polymerise with each other ensures that the important structure of the antigen is maintained in the final detection reagent and also the relative quantities of antigenic peptide:detectable marker can be adjusted by modifying the multimerization domains used.
In the present patent application by molecular complex it is intended to mean a non-covalent molecular assembly between the antigenic peptide and detectable marker.
Detection reagents consisting of antigenic peptides covalently attached to the detectable marker are also encompassed by the present invention.
In addition to the use of this reagent in MAC-ELISA it can also be used as an adsorbed monovalent antigen in an IgG-specific indirect ELISA.
In particular the antigenic peptide and/or detectable marker may comprise an affinity tag.
In particular the antigenic peptide comprises a hexahistidine affinity tag, a flaviviral ED3 domain, selected from the group consisting of: a yellow fever virus ED3 domain polypeptide, a West Nile virus ED3 domain polypeptide, a Dengue virus ED3 domain polypeptide, a St Louis encephalitis virus ED3 domain polypeptide, a Murray Valley encephalitis virus ED3 domain polypeptide and a Japanese encephalitis virus ED3 domain polypeptide and wherein the first multimerization domain comprises a biotinylation site or avidin binding peptide for instance Strep-tag™ (Schmidt et al 2007); and
said detectable marker comprises a Horseradish Peroxidase enzyme and a second multimerization domain which is selected from the group consisting of avidin, strepavidin, neutravidin or a functional derivative thereof.
The inventors have therefore extended the concept of simplified MAC-ELISA to include the use of HRP. As explained above the extension of the inventors prior work to include HRP could not be implemented simply by creating hybrid proteins between the ED3 domain and HRP at the genetic level because HRP is a monomeric and cytoplasmic protein whereas ED3 carries an essential disulfide bond that cannot form in the reducing environment of the cytoplasm. To solve this problem, the Inventors constructed recombinant plasmids that directed the expression of bt-ED3-H6 hybrids between (i) an AviTag peptide, which is biotinylated in vivo and that is labelled “bt” in this patent application; (ii) the ED3 domain from the flavivirus under consideration; and (iii) an hexahistidine for its immobilisation on a nickel ion column and purification by affinity chromatography. These hybrids were produced in the cytoplasm of E. coli, refolded in vitro, purified and then associated with a (SA)4-HRP conjugate between the tetramer of streptavidin (SA) from Streptomyces avidinii and HRP.
The (bt-ED3-H6:SA)4-HRP complexes displayed the ED3 domain in a form that was tetravalent and strongly associated with HRP. The complex corresponding to serotype 1 of dengue virus was produced and evaluated with success in a MAC-ELISA of serums from human patients that had been infected by one of the four serotypes of DENV.
HRP is a preferred marker as many substrates exist for HRP, giving chromogenic, fluorescent, or luminescent products. All of these could be used to generate a measurable signal in a MAC-ELISA performed with the (bt-ED3-H6:SA)4-HRP complexes.
The present invention also relates to multivalent antigen complexes linked via streptavidin or via other means, with other enzymes (e.g. alkaline phosphatases, luciferases), fluorescent proteins, fluorophores, radioactive tracers, etc, and used in all the applications where multivalency is necessary for an assay or brings additional sensitivity, whether in vivo, ex vivo or in vitro.
In particular the peptide sequence of the antigenic peptide is selected from the group consisting of: SEQ ID NO: 1; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10.
According to another aspect of the present invention there is provided a nucleic acid encoding the antigenic peptide or detectable marker according the second aspect of the present invention.
According to a further aspect of the present invention there is provided a method of preparing a detection reagent according to the present invention, wherein it comprises the steps:
a). preparing an expression vector containing a sequence encoding said antigenic peptide according to the present invention;
b). transforming an appropriate E. coli strain, with said expression vector and culturing said transformed strains;
c). purifying said antigenic peptide from the periplasmic extract by affinity purification for said affinity tag;
d). mixing said purified antigenic peptide with said detectable marker under conditions which permit the formation of a complex between said purified antigenic peptide and said detectable marker via said first and said second multimerization domains.
The preparation of recombinant antigens, produced in E. coli, offers many advantages over the more traditional and empirical methods, e.g. the preparation of brain extracts from suckling mice or of supernatants from cell cultures infected with the virus. The antigenic peptide component of detection reagents according to the present invention can be produced in E. coli and purified with low levels of security and expertise, in contrast to the more traditional preparations of antigens, which must be performed in high level security laboratories.
In addition wherein the antigenic peptide comprises a biotinylation site, biotinylation of this site can be performed in vivo and this is considered preferable to enzymatic biotinylation in vitro, which involves additional method steps. The inventors also consider in vivo biotinylation preferable to an in vitro chemical biotinylation, which generally occurs at random sites of the target polypeptides, e.g. the amine groups, results in an inhomogeneous population of conjugated molecules and may mask the epitopes of the antigenic peptide.
According to a further aspect of the present invention there is provided a kit for diagnosing and/or screening for anti-flavivirus antibodies in a subject comprising:
In particular the kit comprises an Ig binding protein selected from the group consisting of anti-IgM, anti-IgG and anti-IgA, and said detection reagent comprises a hexahistidine tag, a flavivirus ED3 domain and the Horseradish Peroxidase enzyme.
According to a further aspect of the present invention there is provided a method for the detection of antibodies against at least one pathogenic micro organism in a human or animal host, characterized in that it comprises:
a). contacting a sample from said host with a solid support coated on at least one side with an Immunoglobulin (Ig) binding protein which is directed against a specific class of Ig molecules of the species of said host, and
b). incubating the immunocomplex formed in step a). with a detection reagent comprising a complex between at least four copies of an antigen from said at least one pathogenic micro organism and a detectable marker,
c). determining the presence of said detection reagent in association with said immunocomplex formed in step a).
For a better understanding of the invention and to show how the same may be carried into effect, there will now be shown by way of example only, specific embodiments, methods and processes according to the present invention with reference to the accompanying drawings in which:
There will now be described by way of example a specific mode contemplated by the Inventors. In the following description numerous specific details are set forth in order to provide a thorough understanding. It will be apparent however, to one skilled in the art, that the present invention may be practiced without limitation to these specific details. In other instances, well known methods and structures have not been described so as not to unnecessarily obscure the description.
Abbreviations
gpE, envelope glycoprotein E; sE, extracellular domain of gpE; ED3, domain 3 of gpE; ED3.DEN1, ED3 from serotype DENV1 of dengue virus; ELISA, enzyme-linked immunosorbent assay; MAC-ELISA, IgM antibody specific capture ELISA; VLP, virus like particle; RT-PCR, a combination of reverse transcription and polymerase chain reactions; bt, biotinylated AviTag peptide; SA, streptavidin monomer; (SA)4, SA tetramer; HRP, horse radish peroxidase.
Media, Buffers and Kits
The culture media LB and 2-YT (Invitrogen) have been described (Sambrook and Russell 2001). Ampicillin was used at 200 μg/mL, kanamycin at 50 μg/mL, tetracycline at 15 μg/mL and chloramphenicol at 10 μg/mL. LB medium with ampicillin was used for all the genetic constructions. PBS (phosphate buffered saline), Tween 20, the streptavidin-alkaline phosphatase conjugate, the streptavidin-horseradish peroxidase conjugate, the (anti-human IgG)-peroxidase conjugate, oxidized L-glutathione, reduced L-glutathione and The 3,3′,5,5′-tetramethylbenzidine (TMB) liquid substrate system were purchased from Sigma-Aldrich; bovine serum albumin (BSA) from Roche; low-fat dry milk from Regilait. Buffer A contained 20 mM Tris-HCl, pH 8.0, 500 mM NaCl; buffer B, 100 mM NaH2PO4, 10 mM Tris-HCl, 8 M urea, 10 mM 2-mercaptoethanol, pH 8.0; buffer C, 20 mM Tris-HCl, pH 8.0, 100 mM NaCl; buffer D, 1 mM reduced L-glutathione, 0.5 mM oxidized L-glutathione in buffer C; buffer E, 0.1% Tween 20 in PBS; buffer F, 1% low-fat dried milk in buffer E; buffer G, 0.5% Tween 20 and 5% low-fat dried milk in PBS.
Bacterial, Plasmid and Viral Strains
The E. coli strains XL1-Blue (Bullock et al. 1987), NEB-Express (F−, ion, ompT; New England Biolabs) and AVB99 (pBirAcm in XL1-Blue; Avidity); and plasmids pET20b+ (Novagen), pLB11 (Lisova et al. 2007) and pAT224 (GenBank accession no. AY327139) (Binz et al. 2004) have been described. Plasmid pBirAcm carries the birA gene under control of the ptac promoter, and the lacIq gene, both from E. coli. pLB11 is a derivative of pET20b+ and codes for a hybrid ED3.DEN1-H6 between the ED3 domain (residues 296-400) of DENV1 (strain FGA/89, GenBank accession No AF226687) and a hexahistidine. pAT224 codes for a hybrid AviTag-MalE-H6 between the AviTag peptide, the MalE protein of E. coli and a hexahistidine, under control of the T5 promoter and lacO operator; it also carries the lacIq gene. Strain NZ2 was constructed by introducing the pBirAcm plasmid of AVB99 into NEB-Express by transformation.
Antibodies and Antiserums
Human sera were from the collection of the National Reference Center (NRC) for Arboviruses, Institut Pasteur de la Guyane, French Guiana. They were collected from patients who displayed clinical symptoms of dengue (temperature of ≧38.5° C., headache, myalgia and/or arthralgia), associated or not with rash and minor hemorrhagic manifestations, in the context of the NRC activity. Six serum samples were tested: two from patients infected with DENV 1, one with DENV2, one with DENV3, one with DENV4, and one from an individual who was not infected by DENV. The samples were collected during the early convalescent phase of the disease, i.e. 5-6 days or later after fever onset. They were characterized by standard diagnostic methods, i.e. MAC-ELISA and antigen capture IgG indirect ELISA, using mouse-brain extracts as antigens (Dussart et al. 2008). The DENV serotype that was responsible for the disease, was identified by RT-PCR as described (Lanciotti et al. 1992). This identification was performed on serum samples that were collected from the patients in the acute phase of the disease. Microtiter plates coated with antibodies directed against human immunoglobulin u-chains (Platelia™ IgM microplates) were purchased from Bio-Rad.
Construction of the AviTag-ED3-H6 Hybrid Gene
The AviTag-ED3.DEN1-H6 hybrid gene (SEQ ID NO: 1), coding for a hybrid protein between the AviTag, the ED3.DEN1 domain and a hexahistidine, was constructed as follows. Plasmids pLB11 and pAT224 were digested with the restriction enzymes NcoI and B1pI. The products of digestion were purified with a PCR purification kit (Qiagen) to remove the B1pI enzyme, which is heat stable, ligated and then introduced into XL1-Blue by transformation. The recombinant plasmids were identified by digestion with the restriction enzyme NdeI and the sequence of the hybrid gene checked by DNA sequencing. The resulting plasmid, pNZ21, coded for the AviTag-ED3.DEN1-H6 hybrid protein (SEQ ID NO: 2), under control of the T5 promoter and lacO operator.
Production and Purification of the bt-ED3-H6 Hybrid
The bt-ED3-H6 hybrid was expressed from plasmid pNZ21 in strain NZ2. A pre-culture of the producing strain was obtained by inoculation of 2×YT broth, containing ampicillin and chloramphenicol, with an isolated colony and overnight incubation at 30° C. The production was obtained by dilution of the pre-culture in one volume of 2×YT broth, containing ampicillin, to obtain an initial absorbance A600nm=0.1, growth at 30° C. until A600nm=1.0, induction of the ptac and T5 promoters with 1.0 mM IPTG, addition of 50 mM biotin, and further incubation for 4 h at the same temperature. The subsequent steps were performed at 4° C. The culture was cooled on ice and then centrifuged 10 min at 5000 rpm to pellet the bacteria. The bacterial pellet was resuspended in 1/20th volume of 1 mg/mL hen egg white lysozyme, 5 mM imidazole in buffer A and the cells were broken open by sonication. The insoluble materials were pelleted by centrifugation for 20 min at 10,000 g and the supernatant discarded.
The subsequent steps were performed at room temperature to avoid crystallisation of urea. The insolubles were resuspended in 1/20th volume of buffer B. The suspension was mildly agitated until it became translucent, and then centrifuged 20 min at 10,000 g to remove the cell debris. The supernatant contained the solubilized proteins. The bt-ED3-H6 hybrid was separated from the other solubilized proteins by affinity chromatography on a column of NiNTA resin (2-3 mL/L of culture, Qiagen), and refolded in the column before elution. The column was equilibrated with 6 volumes of buffer B, loaded with the preparation of solubilized proteins, and then washed with 20 volumes of buffer C to remove urea and the unbound proteins.
The subsequent steps were performed at 4° C. The column was equilibrated with 5 volumes of buffer D, its outlet was capped, 1 volume of buffer D was added and the reaction of refolding was left to run overnight. The column was washed with 5 volumes of buffer A to remove glutathione and then with 5 mM imidazole in buffer A to remove the weakly bound proteins. bt-ED3-H6 was then eluted by a step gradient of imidazole in buffer A. The fractions of purifications were analyzed by SDS-PAGE (12% acrylamide, NuPage Novex system, Invitrogen) in reducing conditions. The purest fractions were pooled, snap-frozen and kept at −80° C. They were homogeneous at >98%.
Proteins and their Complexes
The molecular mass MM and extinction coefficient ε280nm of the proteins were calculated from their amino acid sequences with the subroutine Pepstats of the software suite EMBOSS (Rice et al. 2000). The MM value of the biotin moiety is equal to 226 (Beckett et al. 1999). The MM value of the bt-ED3-H6 hybrid was equal to 14989 and its ε280nm value to 15220 M−1 cm−1. The MM value of the streptavidine monomer from Streptomyces avidinii (GenBank accession No CAA00084) was equal to 16622, that of horse radish peroxidase to 44000 (Sigma-Aldrich) and therefore that of the (SA)4-HRP conjugate to 110488 (Sigma-Aldrich), corresponding to an average MM=27622 for each monomer of the conjugate.
Therefore, a mixture of 1.00 μg of (SA)4 and 0.90 μg of bt-ED3-H6 is equimolar, i.e. the number of molecules of bt-ED3-H6 is equal to the number of binding sites for biotin. Likewise, a mixture of 1.00 μg of the conjugate (SA)4-HRP and 0.54 μg of bt-ED3-H6 is equimolar.
The (bt-ED3-H6:SA)4-HRP complex was prepared by mixing the bt-ED3-H6 hybrid and (SA)4-HRP conjugate in a 12:1 molar ratio (i.e. 3 moles of bt-ED3-H6 for 1 mole of SA monomer; see Results). More precisely, a preparation of complex was formed extemporaneously by mixing 13.1 μL of bt-ED3.DEN1-H6 hybrid (purified preparation at 0.87 mg/mL) and 7 μL of (SA)4-HRP conjugate (stock solution at 1 mg/mL in PBS), followed by incubation for 1 hour at room temperature. The concentration of complex was therefore equal to 3.15 μM in the resulting preparation. Two additional mixes were prepared for control experiments. Mix-1 was prepared by mixing 13.1 μL of the purified preparation of bt-ED3.DEN1-H6 and 7 PBS; Mix-2, by mixing 13.1 μL of PBS and 7 μL of the stock solution of (SA)4-HRP.
MAC-ELISA
The IgM antibody specific capture ELISA (MAC-ELISA) was performed using microtiter plates coated with anti-human IgM and a volume of 100 μL/well. The human serum samples were diluted 100-fold in buffer F, unless otherwise indicated. The preparation of (bt-ED3-H6:SA)4-HRP complex was diluted 10- to 1280-fold in buffer F. Wells from one column of the microplate were loaded with the diluted serums or buffer F for the blank. The plate was incubated for 1 h at 37° C. for the reaction of antibody capture. The wells were washed three times with buffer E and then loaded with the dilutions of complex, except for the blank well. The plate was incubated for 1 h at 37° C. for the binding reaction. The wells were washed three times with buffer E and the bound (bt-ED3-H6:SA)4-HRP molecules detected by addition of TMB. A620nm was measured after 5, 10, 15 and 20 min at 25° C. The reaction was stopped by addition of 50 μL of 1 N sulfuric acid and the ratio A450nm/A620nm measured. The signal of the serum was considered as significant if its value was at least twice that of the blank controls. This condition was used to define the end-point value of the serum concentration when the latter was varied.
In a control experiments, the dilutions of the (bt-ED3.DEN1-H6:SA)4-HRP complex were replaced by those of Mix-1, containing only the bt-ED3.DEN1-H6 hybrid (see previous paragraph). The plate was incubated for 1 h at 37° C. for the binding reaction. The wells were washed three times with buffer E and then loaded with the corresponding dilutions of Mix-2, containing only the (SA)4-HRP conjugate. The plate was incubated for 1 h at 37° C. for the capture of the conjugate by the bound bt-ED3.DEN1-H6 hybrid. The wells were washed three times with buffer E and then the reaction with TMB was run as above.
IgG-Specific Indirect ELISA
The indirect ELISAs were performed in microtiter plates (Polysorb, Nunc) with volumes of 100 μL/well. The wells of a plate were sensitized with the bt-ED3.DEN1-H6 hybrid. The hybrid (0.3 μg/mL in PBS) was loaded in the wells and the plate incubated overnight at 4° C. The wells were washed three times with buffer E and then blocked with buffer G for 1 h at 37° C. The wells were washed as above. The positive and negative human serums were diluted 400- to 102400-fold in buffer F and then loaded in duplicate in the sample wells while buffer F alone was loaded in the blank wells. The plate was incubated for 1 h at 37° C. for the binding reaction to occur and then the wells were washed three times with buffer E. A dilution of (anti-human IgG)-HRP conjugate (1000-fold in buffer F) was added in the sample and blank wells. The plate was incubated for 1 h at 37° C. for the reaction of capture and then the wells were washed as above. The bound molecules of (anti-human Ig)-HRP were detected by addition of TMB. A620nm was measured after 5, 10 and 15 min at 25° C. The reaction was stopped by addition of 50 μL of 1 N sulfuric acid and A450nm/A620nm measured. The signal of the serum was considered as significant if its value was at least twice that of the blank control. This condition was used to define the end-point value of the serum concentration.
Materials and Buffers for Multimerisation Assays
Phosphate buffered saline (PBS), p-nitrophenyl phosphate (pNPP), streptavidin from Streptomyces avidinii and the conjugate between alkaline phosphatase and an anti-mouse IgG (Fc specific) antibody, produced in goat, were purchased from Sigma-Aldrich; bovine serum albumin (BSA) from Roche; and the PentaHis antibody from Qiagen. Buffer A was 1% BSA in PBS; buffer B, 0.05% Tween 20 in PBS.
ELISA Assay for the Formation of a Tetravalent Complex
The (SA)4 tetramer and increasing amounts of the bt-ED3-H6 hybrid were mixed in buffer A (110 μL final volume), and the mixes incubated for 1 h at room temperature to allow the formation of the complex. The reaction mixtures were then sequentially diluted 10-, 100- and 1000-fold in the same buffer. The proportion of free bt-ED3-H6 in the reaction mixtures was determined by an indirect ELISA in microtiter plates (Polysorb, Nunc) that had been sensitized with streptavidin. Briefly, streptavidin (1 μg/mL in PBS) was loaded in the wells (100 μL/well) and the plate incubated overnight at 4° C. The wells were emptied and then blocked with 3% BSA in PBS (200 μL/well) for 1 h at 37° C. The wells were washed three times with buffer B, three times with PBS alone, and then loaded with the dilutions of the binding mixtures. The plates were incubated 1 h at room temperature for the capture of the free bt-ED3-H6 molecules and then the wells were washed as above. The captured molecules of bt-ED3-H6 were detected by adding successively a PentaHis antibody, a conjugate between alkaline phosphatase and an anti-mouse IgG antibody, and p-nitrophenyl phosphate as a substrate, and then by measuring A405nm.
Native PAGE
The (SA)4 tetramer (2 μL at 1 mg/mL) and increasing amounts of the bt-ED3-H6 hybrid were mixed and incubated for 1 h at room temperature to allow the formation of the complex. The reaction mixtures were then analyzed by PAGE in native conditions, i.e. non-denaturing non-reducing conditions. We used the NativeMark unstained protein standard (Catalog No LC0725), NativePAGE Novex 4-16% Bis-Tris gels (1.0 mm, 10 wells, No BN1002Box), NativePAGE Sample buffer (4×, No BN2003) and NativePage Running buffer kit (No BN2007), all from Invitrogen.
Construction of the Biotinylated Hybrids bt-ED3-H6
The inventors constructed plasmid vectors that coded for the cytoplasmic expression of AviTag-ED3-H6 hybrids in E. coli, under control of the T5 promoter and lacO operator (Table 1).
These hybrid proteins comprised three segments from N— to C-terminus: (i) the AviTag peptide, which is specifically biotinylated by biotin-protein ligase BirA, product of the birA gene from E. coli (Barker and Campbell 1981); (ii) the ED3 domain from the envelope protein of a flavivirus, which contains one disulfide bond; (iii) a hexahistidine for the immobilisation and purification of the hybrid on a nickel ion column. Plasmid pNZ21, which corresponded to the ED3 domain from DENV1, was introduced into the E. coli strain NZ2 for expression of the hybrid protein bt-ED3.DEN1-H6. Strain NZ2 harboured plasmid pBirA, which carried the birA gene under control of the ptac promoter. Both pNZ21 and pBirA carried the lacIq gene and both expressions of BirA and bt-ED3-H6 were under control of the Lad repressor.
Production, In Vitro Refolding and Purification of the Bt-Ed3-H6 Hybrid
The producing bacteria were broken open by sonication and the biotinylated hybrid, bt-ED3-H6, was purified from the insoluble fraction. Briefly, the insoluble proteins were solubilized with 8 M urea and 2-mercaptoethanol to reduce the intermolecular disulfide-bonds. The solubilized proteins were loaded on a NiNTA column, the unbound proteins and 2-mercaptoethanol were washed out of the column, and then the bound proteins were refolded in the column in the presence of a mixture of reduced and oxidized glutathione to favour the formation of the correct intramolecular disulfide bond. Glutathione was washed out of the column and then the refolded proteins were eluted from the column by a step-gradient of imidazole. The purified bt-ED3-H6 hybrid was >98% pure. We obtained 7.1 mg of the purified, refolded, biotinylated bt-ED3.DEN1-H6 hybrid/L of culture in flask by this procedure. Efficient biotinylation was confirmed by ELISA and blotting with a streptavidin-alkaline phosphatase conjugate.
Formation of the Complex Between bt-ED3-H6 and the (SA)4-HRP conjugate
The (SA)4-HRP conjugate between streptavidin and horseradish peroxidase, contains four binding sites for biotin, one per SA monomer. It was essential that these four sites be occupied by the bt-ED3-H6 hybrid to generate a multivalency of the ED3 antigen and an avidity effect. We therefore run the binding reaction between bt-ED3-H6 and (SA)4-HRP in a molar ratio of 12:1, which corresponded to three molecules of bt-ED3-H6 per SA monomer. This ratio was calculated under the assumption that each molecule of conjugate contained one molecule of HRP per SA tetramer. We tested different dilutions (10 to 1280-fold) of the complex preparation in MAC-ELISA of human serums (see below). We represent such a complex by (bt-ED3.DEN1-H6:SA)4-HRP, where “-” stands for a covalent bond and “:” for a non-covalent interaction.
Assay of Human Serums with a Simplified MAC-ELISA
We used the (bt-ED3.DEN1-H6:SA)4-HRP complex, corresponding to serotype DENV1 of the dengue virus, to detect IgMs, directed against DENV1, in the serum of patients who had been infected recently. We used microtiter-plates in the wells of which an antibody, directed against human IgMs, had been immobilized. This immobilized antibody was used to capture the IgMs of the human serum. The IgMs that were directed against the ED3.DEN1 domain, were detected with (bt-ED3.DEN1-H6:SA)4-HRP, through the binding of its ED3.DEN1 moiety and the enzymatic activity of its HRP moiety. We also tested the serums of patients who had been infected recently with the DENV2, -3 or -4 serotypes of the dengue virus, the serum of a patient who had not been infected with any DENV, and a blank sample in which both serum and complex were omitted (
We observed an important cross-reaction of the (bt-ED3.DEN1-H6:SA)4-HRP complex with all the samples, when it was diluted 10 to 160-fold. This cross-reaction totally disappeared for dilutions above 500-fold. A reaction time of 20 minutes was sufficient to develop the signal. At high dilution, the complex reacted very strongly with a first DENV1 serum and significantly with a second one. It reacted very weakly with the DENV2, -3 and -4 serums and with the negative serum (
We checked that the MAC-ELISA signal was an increasing and saturating function of the concentration in DENV 1-infected serum, and a constant and nil function of the concentration in non-infected serum, when the (bt-ED3.DEN1-H6:SA)4-HRP complex was used at a high dilution (1280-fold;
Monovalent Versus Multivalent Presentation of the Antigen
In the MAC-ELISA of the previous paragraph, the (bt-ED3.DEN1-H6:SA)4-HRP complex was pre-formed, it displayed the ED3 domain tetravalently, and its two components were captured in one step by the IgMs of the serum under assay. We also performed the capture of these two components in two steps to test the importance of the multivalency of the ED3 domain. In a first step, the bt-ED3.DEN1-H6 hybrid was presented in a free monovalent state to the IgMs that were bound to the plates. The unbound bt-ED3.DEN1-H6 molecules were subsequently washed off In a second step, the (SA)4-HRP conjugate was added for capture by the bound bt-ED3.DEN1-H6 hybrid. We found that the measured signal was similar for the infected serum, the non-infected serum and the blank control when the MAC-ELISA was performed in two steps, whereas it was 20-fold higher for the infected serum when the assay was done in one steps (
Use of a bt-ED3-H6 Hybrid in an IgG Specific Indirect ELISA
The ED3 domains can be used in an isolated format to detect IgG antibodies that are directed against the cognate flaviviruses in the serum of patients (see Introduction). In such assays, the ED3 domain is immobilized in the wells of a microtiter plate by passive adsorption and captures IgG antibodies in the serum under assay. The captured IgGs are then detected with a conjugate between a secondary antibody, directed against human IgGs, and an enzyme, usually HRP. We tested whether the bt-ED3-H6 hybrids could be used in such an indirect ELISA (
Establishing Optimum Ratio Between Antigen and Strepavidin Core
The new diagnostic assay according to the present invention depends crucially on the multivalent display of the antigenic domain in the final complex.
Two parallel methods to empirically determine the optimum ratio of the concentrations in the bt-ED3-H6 hybrid and (SA)4-HRP conjugate that should be used to obtain such a tetravalent complex. These methods also enabled the verification that the tetravalent complex is indeed formed. The methods were based either on an indirect ELISA or native PAGE.
The association between the bt-ED3-H6 hybrid and the tetramer (SA)4 of streptavidin (or a conjugate of this tetramer) occurs according to the reaction:
4×bt-ED3-H6+(SA)4→(bt-ED3-H6:SA)4
The reaction is practically irreversible because of the very strong affinity between biotin (bt) and streptavidin. The principle of the assay consisted in titrating bt-ED3-H6 with (SA)4 and detecting the appearance of free bt-ED3-H6, according to the reaction:
nbt-ED3-H6+(SA)4→(bt-ED3-H6:SA)4+(n−4) bt-ED3-H6
This assay enables one to empirically determine the amounts of the bt-ED3-H6 and (SA)4 preparations that must be mixed to obtain tetravalent molecular complexes. Two methods were used to detect the appearance of free bt-ED3-H6: (i) a polyacrylamide gel electrophoresis performed in native conditions (native PAGE); (ii) an indirect ELISA assay in which the free molecules of bt-ED3-H6 were captured by immobilized streptavidin, and detected with a murine anti-His5 monoclonal antibody.
In the set-up of the native PAGE assay, it was found that the saturated complex (bt-ED3-H6:SA)4 migrated faster than (SA)4 and slower than bt-ED3-H6. Free molecules of (SA)4 were no more detectable for molar ratios >2 between monomers of bt-ED3-H6 and monomers of SA. Free molecules of bt-ED3-H6 were not detectable for molar ratios <0.5. They were strongly present for ratios >2 (
The advantages of the (bt-ED3-H6:SA)4-HRP complexes over the (H6-ED3-PhoA)2 hybrids are important. As stated in the Introduction section, HRP is smaller, more stable and less expensive to produce than PhoA. It has a much higher catalytic turnover number that generates a strong signal in a short time span. Whereas the full-length H6-ED3-PhoA hybrid must be produced in E. coli, only the much smaller bt-ED3-H6 partner of the complex has to be produced in E. coli, the (SA)4-HRP partner being commercially available at a reasonable price. Whereas the hybrid must be exported into the periplasmic space of E. coli for its proper folding, the bt-ED3-H6 domain can be produced in the E. coli cytoplasm as inclusion bodies and then folded within the purification column, according to a simple protocol. As a result, the yields of production and purification are higher for the isolated bt-ED3-H6 domain (7.1 mg/L of culture; 470 nmoles/L of culture) than for the H6-ED3-PhoA hybrid (1.0 mg monomer/L; 16 nmoles/L), especially when calculated as moles/L of culture because the MM value of the isolated domain, 14989, is much smaller than that of the hybrid, 60631. Moreover, whereas the H6-ED3-PhoA hybrid is used at a monomer concentration of 0.066 μM in MAC-ELISA, the bt-ED3-H6 domain was used at a monomer concentration of 0.0025 μM, i.e. >25-fold lower for a 10-fold faster generation of the chromogenic signal.
Mathew, A., and Rothman, A. L. 2008. Understanding the contribution of cellular immunity to dengue disease pathogenesis. Immunol Rev 225: 300-313.
| Number | Date | Country | Kind |
|---|---|---|---|
| 09290560.3 | Jul 2009 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB10/53096 | 7/6/2010 | WO | 00 | 5/1/2012 |
