Anti-idiotypic antibodies to bovine herpesvirus-1 inhibit virus infection in cell cultures

Summary A panel of murine monoclonal antibodies (MAbs) to bovine herpesvirus-1 (BHV-1) was prepared. Three of them were neutralizing MAbs and reacted against 130/75/50 kDa, 77 kDa, or 97 kDa glycoproteins (gp). A fourth non-neutralizing MAb recognized the 97 kDa gp. Competition radioimmunoassay demonstrated that each of the four MAbs reacted against a different virus epitope. Anti-idiotypic antibodies (anti-id) to the four MAbs were produced in rabbits and purified by sequential immunoaffinity chromatography. Each anti-id inhibited the binding of its respective MAb to BHV-1 in competitive ELISA and blocked BHV-1 neutralizing activity of the MAb. This inhibition suggested that the anti-ids were specific for the antigen binding site of the MAbs. Treatment of MDBK cells with anti-ids inhibited BHV-1 infection, which suggested that the anti-ids block a cellular component essential for virus infection. Absence of significant cross-reactivity among the anti-ids for heterologous MAbs indicated that they recognized unique determinants on the antigen binding site of the homologous MAb.     

Rapid production of monoclonal antibodies to T lymphocyte functional antigens

Brief immunization of rats with mouse lymphoid cells was combined with the rat/mouse hybridoma technology and functional hybridoma screening to yield a rapid method for the production of monoclonal antibodies (MAb) against functionally important T lymphocyte cell surface antigens. Two protocols were used. In one, rats were immunized once with mouse thymocytes followed by fusion and screening of the hybridomas for interference with the thymocyte co-stimulator (interleukin 1) assay. The resultant hybridomas included producers of MAbs against the L3T4-antigen (inhibitory), the Ly-1-antigen (stimulatory), and the Thy-1-antigen (inhibitory?). In the second protocol, rats were immunized twice with a T cell hybridoma. The resultant hybridomas were screened for inhibition of polyclonal T cell activation, induced by an anti-Thy-1 (MAb G7). A panel of MAbs against the Thy-1 antigen with different reactivity profiles was generated by this procedure. Most of the MAbs were of the IgM class. Short-term immunization may lead to less selection of response to highly immunogenic determinants than a protocol involving several boosters. Thus, this alternative may be useful for producing MAbs against rare or weakly immunogenic cell surface molecules, as suggested by the ease with which we were able to make MAbs against the L3T4-molecule.     

Anti-idiotypic antibodies against Puumala virus glycoprotein-specific monoclonal antibodies inhibit virus infection in cell cultures

Summary Rabbit anti-idiotypic antibodies (anti-ids) were generated against three bank vole and one human monoclonal antibody (MAb) specific for the two envelope glycoproteins of Puumala virus (G 1 and G 2). The anti-ids were purified by sequential immunoaffinity chromatography. Each anti-id inhibited the antigen binding of its respective MAb in a competitive ELISA. This inhibition, and the absence of cross-reactivity among the anti-ids for heterologous MAbs, showed that they all were specific for unique determinants on the antigen binding site of the homologous MAb. The anti-ids reacted with non-infected Vero E 6 cells when examined by immunofluorescence and ELISA, indicating the presence of antibodies that mimic epitopes on the virus. Preincubation of Vero E 6 cells with two of the anti-ids produced against neutralizing MAbs inhibited Puumala virus infection, suggesting that these two anti-ids blocked a cellular component involved in virus infection.     

Preparation and characterization of anti-sulphamethoxazole (SMX) monoclonal antibody

Anti-sulphamethoxazole (SMX) monoclonal antibody (MAb) has a significant role in monitoring of SMX residue levels in edible animal products. In this study, SMX was diazotizated and coupled to human serum albumin to form an antigen for animal immunization. Anti-SMX hybridomas were prepared by cell fusion using an enzyme-linked immunosorbent assay (ELISA) method to screen for positive hybridomas. A monoclonal antibody (MAb) against SMX was secreted by the hybridomas and its specificity was characterized by ELISA, SDS-PAGE and Western-blot. The results showed that the SMX-MAb was of the IgG1 type and its molecular weight was 162 000 Daltons. The affinity constant of MAb for the conjugated antigen was 2.3×10⁸ l/mol. The detection limit of 50% inhibition rate in the range 10 pg/mL to 10 ng/mL was 0.82 ng/mL and the recoveries of SMX were above 90%. There was no cross-reaction between MAb and SMX analogues.     

Use of monoclonal antibodies in an ELISA for the diagnosis of bovine leukaemia virus infection.

An ELISA diagnostic test for detection of bovine leukaemia virus (BLV) infected animals was developed. The test is based on the use of a mixture of monoclonal antibodies (MAbs) against envelope glycoprotein and against viral structural protein p24. The sensitivity and specificity of the test were found to be dependent on the relative proportions of MAbs of the appropriate epitope specificity. Polystyrene microtitre plates, wells or sticks were firstly coated with a mixture of purified MAbs and then non-purified viral antigens were adsorbed from tissue culture fluid obtained from BLV-producing cells. The optimal conditions for adsorption of MAbs and viral antigens as well as for the ELISA procedure were established. The test is more sensitive and cheaper (no need for virus antigen purification) than the routinely used ELISA using purified virus antigens. The assay is highly specific, rapid, practical and could be easily automated. It is suitable for the detection of BLV-antibodies in blood serum or milk in the large-scale screening programs for BLV-infected animals.     

Induction of monoclonal antibody with predefined ELNKWA epitope specificity by epitope vaccine

Since the hybridoma technique to produce monoclonal antibodies (MAbs) was discovered, thousands of MAbs with predefined protein specificity have been produced, and a natural or recombinant protein as antigen is necessary for inducing MAbs in the conventional hybridoma technique. To induce epitope-specific MAbs, we suggest an epitope vaccine as a new technique to induce MAbs with predefined epitope specificity. ELDKWA was identified as an important neutralizing epitope on HIV-1 gp41. The MAb 2F5, recognizing ELDKWA epitope, has shown broad neutralizing activity to many HIV strains, including primary isolates, but the mutant in ELNKWA epitope results in escape 2F5-based neutralization. To produce MAbs recognizing this mutated epitope for consideration of passive immunotherapy against the mutant bearing the ELNKWA epitope, MAbs with predefined ELNKWA epitope specificity were induced by synthetic epitope-peptide instead of a natural or recombinant gp41 bearing this epitope. Three MAbs were identified to recognize ELNKWA epitope on the synthetic epitope-peptide, and interestingly could bind the recombinant gp41 with ELDKWA epitope in an ELISA assay and immunoblotting analysis.     

Genus-specific epitope on the 60-kilodalton Legionella heat shock protein recognized by a monoclonal antibody.

A monoclonal antibody (MAb) immunoglobulin G2a (2125) was produced against a 60-kDa Legionella heat shock protein (HSP), recognizing a unique epitope common to all species of the genus Legionella. The antibody reacted in the immunoblot with 59 Legionella species and serogroups that were tested and showed no cross-reactivity with other bacteria, including Acinetobacter spp., Bordetella spp., Pseudomonas spp., Mycobacterium spp., and Escherichia coli. Two other MAbs (2122 and 2130) reacted with the 60-kDa Legionella protein as well but showed different cross-reactivities with other gram-negative bacteria in the same molecular mass range. The genus-specific MAb 2125 as well as the cross-reacting MAbs 2122 and 2130 were shown to be reactive with the expressed protein of the cloned gene of the 60-kDa HSP of Legionella micdadei and Legionella pneumophila. These antibodies demonstrate that Legionella-specific and nonspecific epitopes are present on this protein. A sandwich enzyme-linked immunosorbent assay (ELISA) in which the genus-specific MAb is used both as a capture antibody and as a biotinylated second antibody has been established. With this test it is possible to detect Legionella whole cells, sonicated cells, and cell fractions containing the 60-kDa HSP. The main part of the 60-kDa HSP is found in the cytoplasmic fraction. The sandwich ELISA can be used to demonstrate the increased expression of the 60-kDa protein in Legionella cells following heat shock as well as marked differences in the detection of the 60-kDa HSP on whole cells of different Legionella strains. The high specificity and sensitivity of the sandwich ELISA for sonicated cells might be very useful to screen on a genus level for Legionella cells or the 60-kDa antigen in environmental isolates or body fluids of patients.     

Monoclonal antibody characterization of Jamestown Canyon (California serogroup) virus topotypes isolated in Canada.

Jamestown Canyon (JC) virus of the California (CAL) serogroup has been isolated in 12 American states and 6 Canadian provinces. A study was undertaken to produce monoclonal antibodies (MAbs) to JC virus and to use these MAbs to assay for possible heterogeneity among naturally occurring JC topotypes in Canada. MAbs were produced to the prototype strain of JC virus using BALB/c mice. Twenty-seven secreting MAbs were obtained and three of these MAbs were propagated and studied. All three MAbs, M1 (IgG1), M2 (IgG2b), and M3 (IgG2a), were reactive by immunofluorescent antibody assay against JC-infected vero cells and by ELISA against JC antigen. MAb M2 reacted with all members of the Melao complex, MAb M1 reacted only with Keystone virus, while MAb M3 exhibited no reactivity with other CAL serogroup viruses. Only MAb M3 possessed neutralization and hemagglutination inhibition activities against JC virus. The MAbs were also tested by ELISA and for neutralizing activity against 13 JC topotypes isolated in 5 provinces from Newfoundland to Saskatchewan. ELISA confirmed closer identity of the Canadian topotypes to JC as opposed to the closely related South River virus. The MAbs verified all Canadian topotypes to be JC virus but revealed different patterns of reactivity between these topotypes and prototype JC virus.     

A convenient method for epitope competition analysis of two monoclonal antibodies for their antigen binding

Choosing optimum pair of capturing antibody and detecting antibody when developing monoclonal antibody (MAb)-based, sandwich enzyme-linked immunosorbent assays is a time-consuming process requiring the coupling of individual antibodies to an enzyme like horseradish peroxidase or alkaline phosphatase. The MAbs required for the two-site sandwich ELISA should bind to distinct epitopes of the antigen, and their binding should not be mutually exclusive. To determine if two monoclonal antibodies would occupy distinct sites of their antigen in binding, and enzyme-linked immunosorbent assay was devised, which is easy-to-use and does not require any coupling of monoclonal antibodies to enzymes. Microplate wells are coated with rabbit polyclonal antibodies raised against the same antigen of MAbs. After blocking, a limited amount of the antigen is added for incubation with the rabbit antibodies. Mouse monoclonal antibody 1 (Mab 1) is added to saturation. A serial dilution of MAb 2 (for analysis) or MAb 1 (for control) is added subsequently. An enzyme-labeled, goat anti-mouse secondary antibody and its substrates are added for color development. Thus, the epitope competition of two MAbs for their antigen binding is easily determined by the measurement and comparison of color development between the two MAb additions.     

单克隆抗体相对亲和力和特异性的测定

在单克隆抗体的制备中,亲和力和特异性的测定是非常重要的。实验证明单抗的特异性不是绝对的,我们也发现在免疫酶组化染色过程中,当单抗浓度达到一定水平时会出现交叉反应,即抗3型(或7型)腺病毒单抗与7型(或3型)腺病毒出现反应。因此有必要测定单抗特异性,以便筛选高特异性的单抗。     

Avidity, potency, and cross-reactivity of monoclonal antibodies to pneumococcal capsular polysaccharide serotype 6B

Many pneumococcal capsular polysaccharides (PSs) are similar in structure, and a pneumococcal antibody often binds to all of the PSs with a similar structure. Yet, these cross-reactive antibodies may bind to the structurally related pneumococcal capsular PSs with an avidity too low to be effective. If memory B cells producing such weakly cross-reactive antibodies are elicited with pneumococcal conjugate vaccines, the memory cells for low-avidity antibodies could compromise the subsequent immune responses to the cross-reactive PS (original antigenic sin). To investigate these issues, we produced 14 hybridomas secreting monoclonal antibodies (MAbs) to the capsular PS of Streptococcus pneumoniae serotype 6B by immunizing BALB/c mice with antigens containing 6B PS and studied their epitope, avidity, in vitro opsonizing capacity, in vivo protective capacity, and "antigen binding titer" by enzyme-linked immunosorbent assay (ELISA) of 6A and 6B capsular PSs. Six MAbs bound to the non-cross-reactive 6B-specific epitope, and seven MAbs bound to the cross-reactive epitope present in both 6A and 6B PSs One MAb (Hyp6BM6) revealed a novel epitope. This epitope was found on 6A PS in solution, but not on 6A PS adsorbed onto the plastic surface of the ELISA plates. The avidity of the MAb for 6A or 6B PS ranged from 7.8 x 10(6) M(-1) to 4.1 x 10(11) M(-1). No MAbs were weakly cross-reactive, since none of the cross-reactive MAbs showed any tendency toward having less avidity to 6A PS (the cross-reactive PS) than to 6B PS. Avidity influenced the results of several antibody assays. When all of the hybridomas were examined, avidity strongly correlated with the titer of a unit amount of MAb to bind antigen-coated ELISA plates (r = 0.91) or to opsonize pneumococci in vitro (r = -0.85). Because both assay results are avidity dependent, the ELISA and the opsonization assay results were strongly correlated (r = 0.91), regardless of avidity. Avidity also correlated with the potency of a MAb to passively protect mice against pneumococcal infections. When only the immunoglobulin G hybridomas were examined, little increase in opsonizing capacity and in vivo protective potency was observed above 10(9) M(-1). Taken together, an ELISA measuring antigen binding titer may be an adequate measure of the protective immunity induced with pneumococcal vaccines, and the absence of a partially cross-reactive MAb suggests that antigenic sin may not be significant in responses to vaccines against the S. pneumoniae 6B serotype.     

Monoclonal antibodies against Blo t 13, a recombinant allergen from Blomia tropicalis

BACKGROUND: The recombinant allergen of Blomia tropicalis, rBlo t 13, shows 11% of IgE reactivity to sera from allergic patients. This allergen belongs to the fatty acid-binding protein family and its natural equivalent remains to be isolated. Monoclonal antibodies (MAbs) are important tools for specific determination and isolation of natural allergens as well as for characterization of recombinant proteins. METHODS: Mice were immunized with partially purified preparation of rBlo t 13 allergen expressed in the yeast Pichia pastoris. Spleen cells were fused with myeloma cells using polyethylene glycol. Hybridoma screening was performed using a direct ELISA with recombinant allergen. MAb specificity to rBlo t 13 was tested by immunoblotting. Topography of binding sites and binding of MAb to native allergen was studied by ELISA. Reactivity of MAb against allergenic extract of B. tropicalis and Dermatophagoides siboney was analyzed by ELISA inhibition. In addition, the reactivity of MAbs against rBlot 13 from Escherichia coli and P. pastoris expression was compared. RESULTS: Two MAbs, 5G3 and 3G4 with IgG1 isotype, were generated. These MAbs specifically recognized the 16-kD band, which corresponds to the molecular weight shown by rBlo t 13 on SDS-PAGE. In ELISA, the binding of 5G3 MAb to B. tropicalis and D. siboney extracts was inhibited by rBlo t 13. Both MAbs showed the highest reactivity when the allergen was expressed in P. pastoris. CONCLUSION: Two MAbs specific for Blo t 13 were obtained. These MAbs recognized the same or close epitopes on the rBlo t 13 molecule. The occurrence of homologous allergens to Blo t 13 in D. siboney is suggested by the ELISA inhibition assay.     

Characterization and formulation of multiple epitope-specific neutralizing monoclonal antibodies for passive immunization against cryptosporidiosis

The coccidian parasite Cryptosporidium parvum causes diarrhea in humans, calves, and other mammals. Neither immunization nor parasite-specific pharmaceuticals that are consistently effective against this organism are available. While polyclonal antibodies against whole C. parvum reduce infection, their efficacy and predictability are suboptimal. We hypothesized that passive immunization against cryptosporidiosis could be improved by using neutralizing monoclonal antibodies (MAbs) targeting functionally defined antigens on the infective stages. We previously reported that the apical complex and surface-exposed zoite antigens CSL, GP25-200, and P23 are critical in the infection process and are therefore rational targets. In the present study, a panel of 126 MAbs generated against affinity-purified CSL, GP25-200, and P23 was characterized to identify the most efficacious neutralizing MAb formulation targeting each antigen. To identify neutralizing MAbs, sporozoite infectivity following exposure to individual MAbs was assessed by enzyme-linked immunosorbent assay. Of 126 MAbs evaluated, 47 had neutralizing activity. These were then evaluated individually in oocyst-challenged neonatal mice, and 14 MAbs having highly significant efficacy were identified for further testing in formulations. Epitope specificity assays were performed to determine if candidate MAbs recognized the same or different epitopes. Formulations of two or three neutralizing MAbs, each recognizing distinct epitopes, were then evaluated. A formulation of MAbs 3E2 (anti-CSL [alphaCSL]), 3H2 (alphaGP25-200), and 1E10 (alphaP23) provided highly significant additive efficacy over that of either individual MAbs or combinations of two MAbs and reduced intestinal infection by 86 to 93%. These findings indicate that polyvalent neutralizing MAb formulations targeting epitopes on defined antigens may provide optimal passive immunization against cryptosporidiosis.     

Monoclonal antibodies specific for immunorecessive epitopes of glucuronoxylomannan, the major capsular polysaccharide of Cryptococcus neoformans, reduce serotype bias in an immunoassay for cryptococcal antigen

Immunoassay for detection of glucuronoxylomannan (GXM), the major capsular polysaccharide of Cryptococcus neoformans, is an important tool for diagnosis of cryptococcosis. However, immunoassays that are based solely or in part on detection with polyclonal antibodies may show serotype bias in detection of GXM, particularly limited sensitivity for serotype C. In this study, we describe detection of GXM in an antigen capture sandwich enzyme-linked immunosorbent assay (ELISA) that used a cocktail of two monoclonal antibodies (MAbs). MAb F12D2 was previously produced by immunization with GXM that had been treated to remove O-acetyl groups, a major source of serotype specificity. MAb F12D2 has a high degree of reactivity with GXM of serotypes A, B, C, and D, but the reactivity with serotype D was less than was found with other MAbs. MAb 339 is highly reactive with GXM of serotypes A and D. Use of a combination of the two MAbs produced an immunoassay that had the best properties of both MAbs, including good reactivity with serotype C, which is an emerging threat in sub-Saharan Africa. These results suggest that next-generation immunoassays for diagnosis of cryptococcosis may be formulated by (i) use of immunization and hybridoma screening strategies that are designed to prospectively meet the needs of immunoassay performance and (ii) careful selection of MAbs that span the expected polysaccharide serotypes in the subject patient population.     

Production and characterisation of monoclonal antibodies to outer-membrane-protein antigens of Vibrio cholerae O1

Monoclonal antibodies (MAbs) were raised against the major, 46-48-Kda outer-membrane proteins of Vibrio cholerae O1. The hybridoma clones were screened by enzyme-linked immunosorbent assay (ELISA) with cell-surface proteins of V. cholerae O1 as the coating antigen. Four hybridomas, which secreted anti-V. cholerae cell-surface-protein antibodies, were subcloned by limiting dilution and obtained as ascites in vivo. A MAb of the IgG1 subclass was isolated in good yield from the murine ascites by affinity chromatography with recombinant protein G-Sepharose 4B. It gave positive reactions, as determined by ELISA, against cell-surface proteins prepared from both biotypes (classical and El Tor) and both serotypes (Ogawa and Inaba) of V. cholerae O1. The MAb did not have any reactivity towards V. cholerae lipopolysaccharide preparations. Immunoblotting studies were performed on cell-surface proteins separated by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (1D SDS-PAGE) and also by two-dimensional (2D) electrophoresis with iso-electric focusing in the first dimension followed by SDS-PAGE in the second dimension. When proteins were separated by 1D SDS-PAGE, only one band at 46-48 Kda reacted with the MAb. This protein appeared to consist of two narrowly-spaced and cross-reactive bands when a nitrocellulose blot, obtained by 2D SDS-PAGE, was exposed to the MAb.     

Antigenic structure of the E2 glycoprotein from transmissible gastroenteritis coronavirus

The antigenic structure of transmissible gastroenteritis (TGE) virus E2 glycoprotein has been defined at three levels: antigenic sites, antigenic subsites and epitopes. Four antigenic sites (A, B, C and D) were defined by competitive radioimmunoassay (RIA) using monoclonal antibodies (MAbs) selected from 9 fusions. About 20% (197) of the hybridomas specific for TGE virus produced neutralizing MAbs specific for site A, which was one of the antigenically dominant determinants. Site A was differentiated in three antigenic subsites: a, b and c, by characterization of 11 MAb resistant (mar) mutants, that were defined by 8, 3, and 3 MAbs, respectively. These subsites were further subdivided in epitopes. A total of 11 epitopes were defined in E2 glycoprotein, eight of which were critical for virus neutralization. Neutralizing MAbs were obtained only when native virus was used to immunize mice, although to produce hybridomas mice immunizations were made with antigen in the native, denatured, or mixtures of native and denatured form. All neutralizing MAbs reacted to conformational epitopes. The antigenic structure of the E2-glycoprotein has been defined with murine MAbs, but the antigenic sites were relevant in the swine, the natural host of the virus, because porcine sera reacted against these sites. MAbs specific for TGE virus site C reacted to non-immune porcine sera. This reactivity was not directed against porcine immunoglobulins. These results indicated that TGE virus contains epitope(s) also present in some non-immunoglobulin component of porcine serum.     

Production and characterization of monoclonal antibodies to cell wall antigens of Aspergillus fumigatus

Two murine monoclonal antibodies (MAbs) against Aspergillus fumigatus were produced and characterized. Splenocytes from cell wall-immunized BALB/c mice were fused with SP2/0 myeloma cells. The hybridomas were screened with a cold alkali (CA) extract of mycelium containing protein, mannose, and galactose, and two MAbs of the immunoglobulin M class were purified from ascites fluid. MAbs 1 and 40 were characterized by double immunodiffusion against CA antigen, indirect enzyme immunoassay with mannans of Candida albicans serotypes A or B or Candida tropicalis, indirect immunofluorescence with C. albicans- or A. fumigatus-infected tissues, indirect immunofluorescence with smears of other pathogenic fungi, Western blotting (immunoblotting) with the lectin concanavalin A or BS-1 from the seeds of Bandeirea simplicifolia, and immunoelectron microscopy. MAb 1 did not cross-react with Candida mannan and recognized a periodate-sensitive, pronase- and heat-resistant epitope in CA antigen and three mannose- and galactose-containing components (80, 62, and 49 kilodaltons) of a mycelial homogenate. Immunoelectron microscopy demonstrated binding of MAb 1 to the inner cell wall and intracellular membranes of hyphae and conidia of A. fumigatus. Circulating antigen was detected in experimental invasive aspergillosis by inhibition enzyme immunoassay with MAb 1 and CA antigen. MAb 40 was a nonprecipitating antibody cross-reactive with Candida species, and competition for an epitope located diffusely in the cell wall of A. fumigatus hyphae was demonstrated by incubating MAb 40 with mannan of C. albicans serotype A. These results suggest that MAb 1 recognizes immunodominant oligogalactoside side chains of A. fumigatus galactomannan, while MAb 40 binds to mannopyranosyl side chains common to A. fumigatus galactomannan and C. albicans mannan.     

Development and characterization of monoclonal antibodies and aptamers against major antigens of Mycobacterium avium subsp. paratuberculosis

Specific antibodies, available in unlimited quantities, have not been produced against Mycobacterium avium subsp. paratuberculosis, the bacterium that causes Johne's disease (JD). To fill this gap in JD research, monoclonal antibodies (MAbs) against M. avium subsp. paratuberculosis were produced from BALB/c mice immunized with a whole-cell extract of M. avium subsp. paratuberculosis. A total of 10 hybridomas producing MAbs to proteins ranging from 25 to 85 kDa were obtained. All MAbs showed some degree of cross-reactivity when they were analyzed against a panel of whole-cell protein lysates comprising seven different mycobacterial species. The MAbs were characterized by several methods, which included isotype analysis, specificity analysis, epitope analysis, reactivity in immunoblot assays, and electron microscopy. The identities of the antigens that bound to two selected MAbs were determined by screening an M. avium subsp. paratuberculosis lambda phage expression library. This approach revealed that MAb 9G10 detects MAP1643 (isocitrate lyase) and that MAb 11G4 detects MAP3840 (a 70-kDa heat shock protein), two proteins present in high relative abundance in M. avium subsp. paratuberculosis. The epitopes for MAb 11G4 were mapped to the N-terminal half of MAP3840, whereas MAb 9G10 bound to the C-terminal half of MAP1643. Aptamers, nucleic acids that bind to specific protein sequences, against the hypothetical protein encoded by MAP0105c were also generated and tested for their binding to M. avium subsp. paratuberculosis as well as other mycobacteria. These detection reagents may be beneficial in many JD research applications.     

Monoclonal antibodies to Marburg virus proteins and their immunochemical characteristics]

Hybridomas producing monoclonal antibodies (MAb) to Marburg virus proteins are prepared. Positive hybridomas were selected by solid-phase enzyme immunoassay (EIA) by specificity of their immunoglobulin reaction with Marburg virus antigen. Virus proteins reacting with MAbs were identified by immunoblotting. Out of 20 examined hybridoma antibodies, 5 reacted with protein VP35, 5 with VP40, 3 with NP, 1 with protein complex VP35-VP40, MAb 7H10 detected 2 proteins (VP40 and NP), and 5 MAbs did not bind virus proteins in this assay. Marburg virus antigen adsorbed on the surface of plates were detected by indirect EIA with biotin-treated MAbs (PEIA-MAb) and indirect EIA (IEIA-MAb). The sensitivity of both methods differed with different hybridoma antibodies and was the maximum with MAb 5F1 specific to Marburg virus nucleoprotein: 5-10 and 1-2 ng/ml for the direct and indirect methods, respectively. Purified MAbs 7C4, 7D8, and 5F1 were used as antigen captures in EIA for detecting immunoglobulins to Marburg virus in a serum from convalescent after Marburg fever. The results recommend the above MAbs for use in test systems for the diagnosis of the disease and detecting virus antigen.     

Detection and characterization of the coli surface antigen 6 of enterotoxigenic Escherichia coli strains by using monoclonal antibodies.

We describe, for the first time, the production of monoclonal antibodies (MAbs) against coli surface antigen 6 (CS6) of enterotoxigenic Escherichia coli (ETEC) and their use for characterization and diagnosis of CS6. Two MAbs, MAbs CS6-20:11:9 and CS6-2A:14, were produced by immunizing mice with purified CS6 or CS6-containing bacterial extracts. The MAb specificity was demonstrated by enzyme-linked immunosorbent assay (ELISA), immunoblotting, and immunoelectron microscopy, which showed that the MAbs bound to CS6-expressing bacteria as well as to purified CS6 and CS6 structural subunits but not to CS6-negative bacteria or other purified ETEC colonization factors. By using bacterial recombinants, i.e., strains with a complete CS6 operon or parts thereof, it was found that both MAbs were specific for CssB, one of the two structural subunits of CS6. Although the MAbs bound specifically to the entire surface of CS6-expressing bacteria, no structure of CS6 could be identified. The usefulness of the MAbs for the detection of CS6 was evaluated in an inhibition ELISA and in a dot blot test. Ninety-two ETEC strains with known colonization factors were analyzed, and all CS6-positive strains were identified by either assay with MAb CS6-2A:14, whereas MAb CS6-20:11:9 failed to identify two CS6-positive strains; in no instance was any CS6-negative strain identified by either of the MAbs. Parallel analyses of 48 strains with a gene probe specific for the other structural subunit of CS6, i.e., CssA, and the MAb-based assays gave identical results, suggesting the simultaneous presence of both subunits.