Cytoadhesins of Mycoplasma hominis.

The mechanism of Mycoplasma hominis adherence to host cells of the urogenital tract was investigated with monoclonal antibodies (MAbs) directed against antigenic surface-localized polypeptides P50, P60, P80, and P100 of cytoadherent M. hominis FBG. A cell enzyme-linked immunosorbent assay was established allowing quantification of cytoadherent mycoplasmas detected by one of the following MAbs: four MAbs directed against P100 (molecular weight, about 100,000), three MAbs against P80, one MAb against P60, and three MAbs against P50. MAb binding to one of the surface proteins resulted in a decrease of mycoplasmal adherence to HeLa cells. To exclude the thesis that this is caused by nonspecific blocking of adherence, P100 and P50 were purified by affinity chromatography and tested instead of intact mycoplasmas in the cell enzyme-linked immunosorbent assay for cytoadherence. Both proteins bound to the surface of the eukaryotic cells. MAb binding to single epitopes of these proteins resulted in inhibition of protein adherence. These experiments strongly suggest that of the four surface-localized proteins at least P100 and P50 are adhesins of M. hominis FBG.     

Characterisation and epitope analysis of monoclonal antibodies to virions of clover yellow vein and Johnsongrass mosaic potyviruses

Summary Mouse monoclonal antibodies (MAbs) against the Australian B strain of clover yellow vein (C1YVV-B) and the JG strain of Johnsongrass mosaic (JGMV) potyviruses were produced, characterised and the epitopes with which they reacted were deduced. Using intact particles of C1YVV a total of ten MAbs were obtained which reacted strongly with C1YVV-B in an enzyme-linked immunosorbent assay and Western blots. Four of these MAbs (1, 2, 4, and 13) were found to be ClYVV-specific, as they reacted with all five C1YVV strains from Australia and the U.S.A. but not with 11 strains of bean yellow mosaic (BYMV), pea mosaic (PMV), and white lupin mosaic (WLMV) viruses which, together with C1YVV, form the BYMV subgroup of potyviruses. These MAbs failed to react with eight other potyvirus species, including six which infect legumes like the viruses in the BYMV subgroup. The C1YVV MAb 10 was found to be BYMV subgroup-specific. It reacted strongly with 15 of the 16 strains of viruses in the subgroup and gave no reaction with eight other potyviruses. The other five C1YVV MAbs reacted with varying degrees of specificity with the BYMV subgroup viruses and also with other potyviruses. Eight of the C1YVV MAbs (1, 2, 4, 5, 13, 17, 21, and 22) reacted with the intact coat proteins only and not with the truncated (minus amino terminus) coat protein of C1YVV suggesting that the epitopes for these MAbs are located in the surface-exposed, amino-terminal region of the C1YVV coat protein. Comparison of published coat protein sequences of BYMV and C1YVV isolates indicated that the epitopes for the four ClYVV-specific MAbs may be in the amino-terminal region spanning amino acid residues 18 to 30, whereas those for the other four MAbs may be located in the first 17 amino-terminal amino acid residue region. The epitopes that reacted with BYMV subgroup-specific MAb 10 and MAb 30 which reacted with 20 of the 24 potyvirus isolates, are probably located in the core region of C1YVV coat protein as these MAbs reacted with the intact as well as truncated coat protein of C1YVV. Analysis, in Western blot immunoassay, of 17 MAbs raised against virions of JGMV revealed that only two MAbs (1–25 and 4–30) were JGMV-specific, whereas others displayed varying degrees of specificity to different potyviruses. When these MAbs were screened against the intact and truncated (minus 67 amino-terminal amino acid residues) coat proteins of JGMV, the two JGMV-specific MAbs reacted only with the intact coat protein, whereas the other MAbs reacted with the intact as well as with truncated coat proteins, in Western blots. These results suggest that the epitopes for the two JGMV-specific MAbs are located in the surface-exposed amino-terminal 67 amino acid residue region and those for the cross-reactive MAbs are contained in the conserved core region of the JGMV coat protein. Screening of potyvirus MAbs against intact and truncated coat proteins thus appears to be a simple procedure to select virus-specific MAbs to potyviruses.     

Monoclonal antibody E8-18 identifies an integral membrane surface protein unique to Mycoplasma capricolum subsp. capripneumoniae.

Monoclonal antibody (MAb) E8-18 reacted with four isolates of Mycoplasma capricolum subsp. capripneumoniae in Western blots identifying an epitope on a 24 kDa antigen (p24). MAb E8-18 did not react with 11 isolates belonging to four other Mycoplasma species or subspecies closely related to M. capricolum subsp. capripneumoniae. A combination of trypsin treatment of intact organisms and detergent-phase partitioning revealed p24 to be an integral M. capricolum subsp. capripneumoniae surface membrane protein.     

Inhibitory activity on bacterial motility and in vivo protective activity of human monoclonal antibodies against flagella of Pseudomonas aeruginosa.

Three stable hybridoma cell lines, IN-2A8, IN-5D6, and ZI-3A8, that secrete human monoclonal antibodies (MAbs) specific for b-type flagella of Pseudomonas aeruginosa were established by fusing peripheral blood lymphocytes from healthy volunteers with murine myeloma P3X63-Ag8.653 cells. The immunoglobulin M MAbs reacted specifically with flagellin (Mr, 52,000) by Western blotting (immunoblotting) analysis and bound specifically to clinical isolates belonging to Homma serotypes A, B, H, I, and M at frequencies of 58, 50, 46, 30, and 35%, respectively, but did not bind to any serotype E or G isolates. Overall, the MAbs bound to 31% of the clinical isolates. MAb IN-2A8 strongly protected burned mice challenged with P. aeruginosa bearing b-type flagella from death following parenteral administration of 0.1 microgram per mouse. This MAb also inhibited P. aeruginosa colony spreading in soft agar at a concentration of more than 1 microgram/ml but only slightly enhanced opsonophagocytosis by human polymorphonuclear leukocytes. A line of evidence suggests that the potent in vivo activity of MAb IN-2A8 in the burned-mouse model is likely to be caused by its inhibition of bacterial motility after binding to flagella.     

Immunochemical characterization of variable epitopes of outer membrane protein P2 of nontypeable Haemophilus influenzae.

Monoclonal antibodies (MAbs) were elicited to the nontypeable Haemophilus influenzae variants d1 to d4, which differ in the outer membrane protein P2 to analyze the immunological properties of the variable parts of this protein. Five MAbs reacted in a whole-cell enzyme-linked immunosorbent assay (ELISA) only with the homologous strain and in some cases with its variants, but not with 69 unrelated nonencapsulated H. influenzae isolates; nine MAbs also reacted with some other H. influenzae isolates, and four MAbs showed broad cross-reactivity. All of the MAbs reacted with purified protein P2 in ELISAs and immunoblotting. The five MAbs which reacted with the homologous strain d3 and not with the variants d1, d2, and d4 promoted complement-dependent bactericidal activity against strain d3. These and four other MAbs reacted with the intact bacteria of strain d3 in immunogold electron microscopy, indicating that they were directed against surface-exposed epitopes of outer membrane protein P2. A mutant of strain d3 was isolated as a survivor from bacterial killing by complement and MAb 30DA5. This mutant had an altered P2 protein on sodium dodecyl sulfate-polyacrylamide gels and had lost its reactivity with all of the five H. influenzae d3-specific MAbs but not with the other MAbs. From these results, we conclude that the variable parts of outer membrane protein P2 of nonencapsulated H. influenzae from the sputum of patients with chronic obstructive pulmonary disease are immunogenic and mostly surface exposed. Only strain-specific MAbs promoted complement-dependent killing of the bacteria, which was abolished in a spontaneous mutant with an altered P2 protein.     

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.     

Protection against infection with Neisseria meningitidis group B serotype 2b by passive immunization with serotype-specific monoclonal antibody

Hybridomas derived from mice immunized with Neisseria meningitidis serogroup B serotype 2b (B,2b) outer membrane preparations produced monoclonal antibodies (MAbs) specific for major outer membrane proteins of classes 1, 2, and 5. The MAbs were examined by enzyme-linked immunosorbent assay against a selected panel of seven strains of N. meningitidis (B,2b) of different sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns, a serotype 2a, and a nontypable strain. The five MAbs selected were all bactericidal and of different immunoglobulin subclasses. None of the MAbs reacted with other bacterial strains in a dot-enzyme immunoassay. The corresponding antigenic determinant for each MAb was localized on a specific outer membrane protein by immunoblotting of sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of major outer membrane proteins. MAbs M5-11 and M5-30 bound to the class 2 protein and were serotype 2b specific. MAb M2-20 bound to the class 1 protein, and MAbs M5-16 and M5-19 bound to the class 5 protein. A mouse model of infection was established whereby a local infection progressed to lethal bacteremia over 3 days, and 50% of the animals were killed with an intraperitoneal injection of 10 meningococci plus 4% mucin and 1.6% hemoglobin. The ability of the MAbs to provide passive protection against experimental infection with N. meningitidis (B,2b) was examined. Both serotype-specific MAbs M5-11 and M5-30 were highly protective even though they were of different immunoglobulin subclasses. The class 5-specific MAb offered no protection, while the class 1-specific MAb gave limited protection. It may therefore be possible to provide protection against serotype 2b infection by using as vaccine the class 2 serotype-specific surface-exposed outer membrane protein epitopes defined by MAb M5-11 or M5-30.     

Monoclonal antibodies to hog thyroglobulin recognizing disulfide-dependent conformational structures

Monoclonal antibodies (MAbs) to hog thyroglobulin (Tg) were obtained by fusion of myeloma cells with B-lymphocytes from mice immunized with the protein. The five MAbs were classified into three groups which recognized different determinants; (1) MAb 4, (2) MAbs 6 and 10, and (3) MAbs 16 and 18. MAb 16 was IgG2b and the others IgG1. The immunoreactivity of Tg to the five MAbs was lost after reduction of the protein under denaturing conditions but remained after limited digestion by proteolytic enzymes. Tryptic fragments were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electrophoretically blotted and stained by a peroxidase-linked second antibody. Various sized tryptic fragments reacted similarly with the three MAbs which had different determinant specificities. All the MAb-reactable fragments in the tryptic digest completely lost their immunoreactivity after reduction of their disulfide linkages.     

Characterization of a tick isolate of Borrelia burgdorferi that possesses a major low-molecular-weight surface protein.

An unusual strain of Borrelia burgdorferi (DN 127 cl 9-2) that was isolated from an Ixodes pacificus tick did not react with monoclonal antibodies (MAbs) to OspA and OspB surface proteins, which are found in most U.S. strains. The strain exhibited an abundant protein with an apparent molecular weight of 25,000 (25K protein). A MAb, 86 DN-1, that was prepared to the 25K protein was used in studies on the effect of proteases on the intact spirochetes, immune electron microscopy, and Western blot (immunoblot) analyses; the results indicated that the low-molecular-weight protein was an apparent surface protein that was loosely attached to the spirochete. Five tick isolates from California possessed low-molecular-weight proteins in the 20,000- to 25,000-molecular-weight range that reacted with the 86 DN-1 MAb. The 25K protein of DN 127 cl 9-2 was unaffected by prolonged in vitro passage of cultures in BSK II medium, while the low-molecular-weight proteins of the other strains of B. burgdorferi from California either decreased in quantity or became undetectable on long-term in vitro passage.     

Monoclonal antibodies directed against conserved epitopes on the nucleocapsid protein and the major envelope glycoprotein of equine arteritis virus

We recently developed a highly effective immunization procedure for the generation of monoclonal antibodies (MAbs) directed against the porcine reproductive and respiratory syndrome virus (E. Weiland, M. Wieczorek-Krohmer, D. Kohl, K. K. Conzelmann, and F. Weiland, Vet. Microbiol. 66:171-186, 1999). The same method was used to produce a panel of 16 MAbs specific for the equine arteritis virus (EAV). Ten MAbs were directed against the EAV nucleocapsid (N) protein, and five MAbs recognized the major viral envelope glycoprotein (G(L)). Two of the EAV G(L)-specific MAbs and one antibody of unknown specificity neutralized virus infectivity. A comparison of the reactivities of the MAbs with 1 U.S. and 22 newly obtained European field isolates of EAV demonstrated that all N-specific MAbs, the three nonneutralizing anti-G(L) MAbs, and the weakest neutralizing MAb (MAb E7/d15-c9) recognized conserved epitopes. In contrast, the two MAbs with the highest neutralization titers bound to 17 of 23 (MAb E6/A3) and 10 of 23 (MAb E7/d15-c1) of the field isolates. Ten of the virus isolates reacted with only one of these two MAbs, indicating that they recognized different epitopes. The G(L)-specific MAbs and the strongly neutralizing MAb of unknown specificity (MAb E6/A3) were used for the selection of neutralization-resistant (NR) virus variants. The observation that the E6/A3-specific NR virus variants were neutralized by MAb E7/d15-c1 and that MAb E6/A3 blocked the infectivity of the E7/d15-c1-specific NR escape mutant confirmed that these antibodies reacted with distinct antigenic sites. Immunoelectron microscopy revealed for the first time that the antigenic determinants recognized by the anti-G(L) MAbs were localized on the virion surface. Surprisingly, although the immunofluorescence signal obtained with the neutralizing antibodies was relatively weak, they mediated binding of about three times as much gold granules to the viral envelope than the nonneutralizing anti-G(L) MAbs.     

Production and characterization of two monoclonal antibodies to human pancreatic trypsin 1

Two monoclonal antibodies (MAb) G6 and A8 directed against human trypsin 1 have been produced by hybridization of myeloma cells with spleen cells of OF1 immunized mice. Antibodies were screened by radioimmunoassay. Monoclonal antibodies were purified by affinity chromatography on Protein A-Sepharose, and we found that MAb G6 was of the IgG2b class and MAb A8 of the IgG2a class. Both MAbs had a high affinity for trypsin 1 with the respective affinity constants equal to 1.3 x 10(8) l/mol for G6 and 3.2 x 10(7) l/mol for A8. Epitope specificity was studied by western blotting, using human trypsinogens 1 and 2. Both MAbs gave a positive reaction with native trypsinogen 1 and no reaction with the same protein after reduction. Only MAb G6 reacted with trypsinogen 2 in the native form. Its affinity for trypsin 2 was found similar to that for trypsin 1 with a constant equal to 2.7 x 10(7) l/mol. Both antibodies appeared directed against conformational and not sequential epitopes.     

A murine monoclonal antibody defines a unique epitope shared by Klebsiella lipopolysaccharides.

A hybridoma secreting a monoclonal antibody (MAb) directed against Klebsiella lipopolysaccharide (LPS) was derived from spleen cells of mice immunized a smooth, nonencapsulated Klebsiella strain (Friedländer 201; serogroup O1). The MAb, called V/9-5 (immunoglobulin G2a), cross-reacted with LPS preparations produced from reference strains for the Klebsiella O serogroups O1, O2ab, O2ac, O3, O4, O5, and O12. Furthermore, the MAb reacted with LPSs from serogroup reference strains O6/O8, O9, and O11, which are regarded as being identical to O1, O2, and O4, respectively. When testing the supernatant of clinically isolated Klebsiella strains by means of an inhibition enzyme-linked immunosorbent assay, we found that 86 (92.4%) of 93 Klebsiella pneumoniae subsp. pneumoniae isolates and 24 (96.0%) of 25 K. oxytoca isolates harbored the cross-reactive epitope. By contrast, two laboratory strains of K. pneumoniae subsp. rhinoscleromatis did not react with MAb V/9-5. The MAb proved to be specific for the genus Klebsiella, since it did not react with any of a total of 73 strains belonging to other gram-negative bacterial genera. In conjunction with other LPS-specific MAbs, MAb V/9-5 might become a useful reagent for rapid identification of klebsiellae in clinical specimens. Furthermore, the epitope recognized by MAb V/9-5 might serve as a target epitope for the production of human MAbs for immunotherapeutic purposes.     

Identification and characterization of outer membrane proteins of Pasteurella multocida serotype D by using monoclonal antibodies.

Monoclonal antibodies (MAbs) against Pasteurella multocida serotype D were obtained by fusion of spleen cells from BALB/c mice immunized with outer membrane proteins (OMPs) with SP2/0-Ag 14 murine myeloma cells. Desirable MAbs were selected by enzyme-linked immunosorbent assay (ELISA) with OMP as the antigen. MAbs MT1 and MT2 identified two different proteins (H [heavy] and W [weak]), each with a molecular mass of 32 kDa, in Western blots (immunoblots). Treatment of the OMPs with proteolytic enzymes and sodium periodate indicated that the binding sites of MAbs MT1 and MT2 are of protein and glycoprotein natures, respectively. The epitopes reactive with MAbs were surface exposed, as visualized by immunoelectron microscopy. Among field isolates of P. multocida serotype D, two distinct OMP patterns were recognized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and these patterns were designated types I and II. In both the ELISA and the Western blot, MAb MT1 recognized only type I isolates, whereas MAb MT2 recognized both type I and II isolates. Neither MAb MT1 nor MAb MT2 reacted with either reference strains of capsular serotypes A, B, E, and F or field isolates of capsular serotype A of P. multocida. This is the first report of MAbs identifying the serotype D-specific OMP of P. multocida.     

Antigenic evidence of prevalence and diversity of Mycobacterium tuberculosis arabinomannan

Arabinomannan (AM) is a polysaccharide of the mycobacterial capsule. The capsular polysaccharides of various microorganisms are diverse, and this diversity is important for classification of organisms into serotypes and vaccine development. In the present study we examined the prevalence and diversity of AM among Mycobacterium tuberculosis strains using four AM-binding monoclonal antibodies (MAbs). One of these MAbs, MAb 9d8, is known to bind to AM specifically. By whole-cell enzyme-linked immunosorbent assay (ELISA), the AM recognized by MAb 9d8 was detected on the surfaces of 9 of 11 strains, while 2 strains showed no reactivity with MAb 9d8. However, the AM recognized by MAb 9d8 was found in the culture supernatants of all 11 M. tuberculosis strains tested, as demonstrated by capture ELISA. Other AM-binding MAbs reacted both with the surfaces and with the culture supernatants of all 11 strains. Mice immunized with an experimental AM-recombinant Pseudomonas aeruginosa exoprotein A (rEPA) conjugate vaccine had an increased antibody response to AM and a moderate reduction in the numbers of CFU in their organs 7 days after challenge. Our results indicate that AM was detected in all M. tuberculosis strains tested, with differences in epitope distributions of certain strains. In addition, our results suggest that an experimental AM-rEPA vaccine has a moderate effect on the numbers of CFU in organs early after infection.     

Extensive antigenic diversity among human parainfluenza type 2 virus isolates and immunological relationships among paramyxoviruses revealed by monoclonal antibodies

A panel of 128 monoclonal antibodies (MAbs) directed against hemagglutinin-neuraminidase (HN), fusion (F), matrix (M), and polymerase (P) proteins, and nucleoprotein (NP) of the Toshiba strain of human parainfluenza type 2 virus (PIV2) was prepared to examine the antigenic relationships among clinical isolates of PIV2 and among paramyxoviruses by indirect enzyme-linked immunosorbent assays. The HN proteins of 18 clinical isolates of PIV2 showed extensive antigenic diversity: 23 of 33 anti-HN MAbs showed no or limited reactivity to many isolates, while other structural proteins were antigenically well conserved. Some anti-HN MAbs recognizing conserved epitopes of the isolates exhibited two types of neutralizing activity, that is, these antibodies inhibited viral infectivity through attachment inhibition or fusion inhibition. This result also showed the presence of a potential third function of the HN protein which might affect the fusing activity of the F protein besides the hemagglutinating and neuraminidase activities. Many of the anti-NP and anti-P MAbs reacted with simian virus 41 (SV41) and simian virus 5 (SV5), whereas a few reacted with mumps virus or PIV4. Two of 6 anti-F MAbs reacted with SV41. None of the 128 MAbs showed reactivity with PIV1, PIV3, Newcastle disease virus (NDV), and measles virus. This result confirmed antigenic proximity of SV5 and SV41 to PIV2 and revealed comparatively restricted immunological relatedness among PIV2, PIV4, and mumps virus.     

Production and characterization of murine monoclonal antibodies to Histoplasma capsulatum yeast cell antigens

Four monoclonal antibodies (MAbs) were produced by immunizing mice with a disrupted yeast cell homogenate of Histoplasma capsulatum. MAbs 1 and 2 reacted only with the yeast cell antigens of H. capsulatum and Blastomyces dermatitidis, whereas MAbs 3 and 4 showed broader cross-reactivity. MAb 3 cross-reacted with B. dermatitidis, Paracoccidioides brasiliensis, Sporothrix schenckii, and Candida albicans, and MAb 4 cross-reacted with B. dermatitidis, C. albicans, Coccidioides immitis, Aspergillus fumigatus, and Mycobacterium tuberculosis. All four MAbs exhibited unique specificity when reacted with three different strains of H. capsulatum (G217B, A811, and P-IN). MAb 1 belonged to the IgG2b subclass, MAb 3 belonged to the IgG1 subclass, and MAbs 2 and 4 belonged to the IgG3 subclass. MAbs 1, 2, and 3 formed bands in the Western immunoblot assay; the two dominant distinct bands had apparent molecular masses of 72 and 62 kilodaltons.     

Production of a monoclonal antibody specific to the El Amar strain of plum pox virus

Plum pox virus (PPV) isolates are grouped into three clusters differentiated by biological, serological, molecular and epidemiological characteristics: Marcus (M), Dideron (D) and Cherry (C). The El Amar (EA) isolate that does not fit any of the above groups is also known. Monoclonal antibodies (MAbs) that specifically recognize M, D, and C strains of PPV are already available. To complete the set of PPV strain-specific serological reagents, MAbs against the EA isolate were raised by immunizing BALB/c mice and fusing their spleen cells with NS0/1 myeloma cells. After a preliminary characterization by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), 1 of 13 selected MAbs proved to be EA strain-specific. This MAb (EA24) reacted equally well with a homologous antigen and several PPV isolates from Egyptian apricot trees, supporting the hypothesis of an additional specific PPV group. MAb EA24 did not react either with about a hundred PPV isolates belonging to the D and M groups or with PPV-SwC and PPV-SoC isolates belonging to the C group. The strain specificity of MAb EA24 was confirmed by Western blot analysis and immunoelectron microscopy. We conclude that there is now available a set of MAbs which are highly specific to the four currently known groups of PPV strains.     

Production and characterization of monoclonal antibodies to type 8 lipooligosaccharide of Neisseria meningitidis.

Eight monoclonal antibodies (MAbs) to lipooligosaccharides (LOSs) of Neisseria meningitidis were produced by immunizing mice with purified LOS from group A meningococcal strain A1. The specificities of the MAbs were examined by enzyme-linked immunosorbent assay (ELISA), immunodot assay, and ELISA inhibition by using the homologous A1 LOS, 12 immunotype LOSs of N. meningitidis (L1 through L12), and LOSs or lipopolysaccharides from other gram-negative bacteria. Two of the MAbs, 4385G7 (immunoglobulin G2b [IgG2b]) and 4387A5 (IgG2a), had the strongest reactivities with the homologous A1 LOS, moderate reactivities with the M978 (L8) LOS, but no reactivity with other LOSs. The other six MAbs (4 IgM and 2 IgG3) reacted with the A1 LOS and with several or many of the 12 LOSs. ELISA inhibition at 50% showed that the inhibitory activities of the LOSs from strains A1 and BB431 (a group B strain) to the specific MAb 4387A5 were about 10 to 20 times greater than that of the M978 (L8) LOS. When compared with MAb 2-1-L8 (L8) by Western blot (immunoblot) analysis and ELISA inhibition, the two specific MAbs recognized a different epitope in the 3.6-kDa LOSs of strains A1 and BB431. We propose that the new epitope is L8a, since the MAbs also reacted with the M978 (L8) LOS. The expression of the L8a epitope in the A1 LOS requires a few monosaccharide residues in its oligosaccharide moiety, and the fatty acid residues in its lipid A moiety also play a role. In a whole-cell ELISA, the two specific MAbs bound specifically to the homologous strain A1 and the L8 prototype strain M978 but not to any other LOS prototype strains. These results suggest that the two specific MAbs can be used for LOS typing of N. meningitidis.     

Identification of Helicobacter pylori by immunological dot blot method based on reaction of a species-specific monoclonal antibody with a surface-exposed protein.

Monoclonal antibodies (MAbs) against membrane preparations of Helicobacter pylori were produced. One MAb was found to be specific for H. pylori, because it did not react with a number of other bacterial species, including Helicobacter felis and Campylobacter jejuni. This MAb reacted with a 30-kDa protein found in outer membrane preparations of H. pylori. The protein was also detected on the cell surface on intact bacteria when analyzed by immunoelectron microscopy. To facilitate the identification of H. pylori isolates after culturing of biopsies, an immunodot blot assay based on the reaction of this MAb was developed. This assay was found to be highly specific for H. pylori. Sixty-six clinical isolates typed as H. pylori by conventional biochemical tests were found to be positive, whereas no other bacterial species tested gave a positive result. By this method, reliable and rapid identification of H. pylori could be accomplished.     

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.