Monoclonal antibodies to a specific 54-kilodalton antigen of Nocardia spp.

Two monoclonal antibodies (MAbs) of the immunoglobulin G2A isotype, reacting with a Nocardia-specific 54-kDa antigen, were generated. As determined by Western blot (immunoblot), both MAbs reacted only with the 54-kDa band. As determined by indirect immunofluorescence or enzyme immunoassay with whole microorganisms, the MAbs did not react with Nocardia cells. One of the MAbs showed weak cross-reactivity with mycobacterial antigens, while the other showed no cross-reactivity.     

Surface antigen detected by a Schistosoma mansoni monoclonal antibody in worm extracts and kidney deposits of infected mice and hamsters.

Four monoclonal antibodies (MAbs) derived from a Schistosoma mansoni-infected mouse reacted against the tegument or the cell layer of the digestive tract of the adult worm. They also showed similar patterns of immunofluorescence staining when schistosomula were used as antigens. Two of the MAbs (4A10 and 4D3) recognized immune complexes deposited in the kidneys of infected mice and hamsters as detected by indirect and direct immunofluorescence reactions. When adsorbed to polystyrene beads, both MAbs allowed the quantitative detection of antigen by an enzyme immunoassay. 125I-labeled 4A10 binding to live schistosomula and corresponding inhibition assay ruled out the possibility that this binding could be through its Fc fragment. The same MAb detected an antigen migrating as an 80-kilodalton protein by Western blot analysis of soluble worm antigen preparation after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Use of MAb 4A10 may help to elucidate mechanisms of renal pathology and also be of value in the development of immunodiagnostic assays.     

Characterization of microneme antigens of Cryptosporidium parvum (Protozoa, Apicomplexa).

Two monoclonal antibodies (MAbs) raised against purified excysted oocysts and sporozoites of cryptosporidium parvum reacted in an immunofluorescence assay with antigens located at the anterior pole of the zoites. On Western blots of purified oocysts, these MAbs reacted with a series of bands between 210 and 40 kDa; several of these bands were recognized by both MAbs; others were specific. One MAb (TOU) did not react after periodic acid treatment and was therefore considered to recognize a carbohydrate epitope; as determined by immunoelectron microscopy, this MAb reacted on micronemes of sporozoites and merozoites and also with the peripheral cytoplasm and the parasitophorous vacuole of trophozoites and macrogametes. The other MAb (HAD) reacted with an epitope that was insensitive to periodate treatment but did not react in the immunoelectron microscopy assay. However, the similar labeling pattern obtained with the immunofluorescence assay with both MAbs and the fact that the two antibodies share common bands on Western immunoblots suggest that both MAbs react with molecules located in Cryptosporidium micronemes, one reacting with a glycannic epitope and the other reacting with a peptidic epitope.     

Production and characterization of monoclonal antibodies to porcine group C rotaviruses cross-reactive with group A rotaviruses.

Five monoclonal antibodies (MAbs) to porcine group (gp) C rotaviruses (Cowden and Ah strains) reactive with both gp A and C rotaviruses in cell culture immunofluorescence (CCIF) tests were produced and characterized. These MAbs reacted with three strains of gp A and two strains of gp C rotaviruses in a CCIF test and were classified into two groups based on their CCIF titers. The MAbs also reacted to various degrees with cell-culture-propagated porcine gp C rotavirus (Cowden) and bovine gp A rotavirus (NCDV) in an enzyme-linked immunosorbent assay by using the MAbs as capture antibodies. Fecal samples containing human, bovine, and porcine strains of gp A and C rotaviruses were positive when tested using one of the MAbs in this assay. The MAbs recognized VP6 of gp A rotavirus and the VP6 counterpart (41-kDa protein) of gp C rotavirus in a Western blot assay. Results of competitive binding assays on four MAbs indicated that gp A and gp C rotaviruses share three overlapping epitopes within a single antigenic domain. These results suggest that gp A and C rotaviruses share a common antigen located on the VP6 protein, which is recognized by certain MAbs in various serologic assays.     

Enhanced protection by use of a combination of anticapsule and antilipopolysaccharide monoclonal antibodies against lethal Escherichia coli O18K5 infection of mice.

To study antibody-mediated protection against Escherichia coli peritonitis in BALB/c mice, monoclonal antibodies (MAbs) were generated against the capsule (K5) and the lipopolysaccharide (O18) of E. coli. Flow cytometric analysis with two selected immunoglobulin M MAbs revealed that bacteria were antigenically heterogeneous. Arbitrarily, three subpopulations in E. coli O18K5 cultures could be distinguished by double immunofluorescence. A subpopulation bound only the anti-K5 MAb, and another subpopulation bound only the anti-O18 MAb. An intermediate subpopulation, however, bound both MAbs. In agreement with this result, combinations of both MAbs enhanced phagocytosis of fluorescein isothiocyanate-labeled bacteria by human polymorphonuclear leukocytes and mouse macrophage J774 cells as well. In protection experiments, combinations of both MAbs, preincubated with 3 50% lethal doses of E. coli O18K5, protected all mice upon intraperitoneal challenge. Relatively high doses of either MAb alone proved to be not fully protective in this infection model. Protection of mice by the combination of MAbs was associated with significantly lower (P < 0.02) tumor necrosis factor levels in serum 90 min after challenge compared with any other treatment group. Similarly, prophylactic administration of MAbs yielded significantly lower (P < 0.01) tumor necrosis factor levels in mice that received the combination of MAbs than in any other treatment group.     

Detection of enterovirus 70 with monoclonal antibodies.

To improve the ability to identify enterovirus-70 (EV-70) from patients with acute hemorrhagic conjunctivitis, we developed four monoclonal antibodies (MAbs) to EV-70. We reacted the four MAbs against nine previously characterized strains of EV-70 and heterologous viruses by virus neutralization, indirect immunofluorescence, and enzyme-linked immunosorbent assay (ELISA). Two of the MAbs neutralized all nine strains of EV-70 and none of the other enterovirus types tested. Two of the MAbs gave a positive reaction with all nine strains by indirect immunofluorescence, and three reacted with all nine strains by ELISA. None of the MAbs gave a positive reaction with heterologous viruses, including those associated with eye disease, by indirect immunofluorescence or ELISA. The two neutralizing MAbs failed to give a positive reaction with some of the strains of EV-70 by indirect immunofluorescence and ELISA, yet they neutralized these viruses. By ELISA with a polyclonal serum as capture antibody and a mixture of MAbs as detector antibody, we were able to detect from 10(2.2) to 10(5.8) 50% tissue culture infective doses of virus and to type lyophilized isolates of EV-70 sent from Taiwan from which we could not recover infectious virus. By choosing the appropriate MAb, or mixture of MAbs, we could construct a test which had the type specificity and strain sensitivity needed to type isolates of EV-70.     

Studies of murine malaria antigens using monoclonal antibodies

A panel of ten monoclonal antibodies made againstPlasmodium chabaudi andPlasmodium yoelii infected mouse erythrocytes were used for characterization of antigens present in murine malaria. Screening of the antibodies in ELISA with different fractions of infected erythrocytes revealed both species-specific and fraction-specific monoclonal antibodies (MAbs), but also MAbs cross-reacting between the species. Two MAbs bound normal erythrocyte components. Subcellular localization of the target antigens was studied by immunofluorescence and their molecular identity by immunoblotting after SDS-PAGE. Of the MAbs toP. yoelii, one reacted with a cytoplasmic granule component of 137 k and two others reacted with vacuole-associated antigens of 26 k and 25/70/73 k, respectively. The latter antibodies cross-reacted withP. chabaudi antigens. Of the MAbs toP. chabaudi, all were species specific, one reacting with parasite surface antigens of 79 and 250 k and two with a vacuole-associated antigen of 70 k.     

Development, characterisation and diagnostic application of monoclonal antibodies against Yersinia pestis fibrinolysin and coagulase

A library of monoclonal antibodies (MAbs) which recognised different epitopes of Yersinia pestis fibrinolysin (Fib) was developed. These MAbs were species-specific and demonstrated no cross-reaction in indirect immunofluorescence tests (IIFT) with other gram-negative bacteria possessing plasminogen activator activity. All the MAbs provided equally high levels of immunofluorescence with pPst+ Y. pestis strains cultivated at 37 degrees C and at 28 degrees C. In all cases, the MAbs inhibited both fibrinolytic and coagulase (Coag) activities of Y. pestis in Fib-activity inhibition and coagulase-activity inhibition reactions, and reacted with 35- and 37-kDa proteins of Y. pestis in immunoblotting, demonstrating bifunctional activity possibly similar to the properties of MAbs produced by hybrid hybridomas. On the basis of these and earlier studies, the immunochemical identity of Fib and Coag, two distinct subunits of a bifunctional fusion protein whose specific functional activity depends upon the temperature factor, was established. A new rapid, cheap, strictly specific and safe dot-ELISA based on the use of MAb against Y. pestis Fib (MAb-Fib) for reliable identification of Y. pestis strains was developed. This technique has great advantages over monoclonal diagnostic kits based on the use of MAb against Y. pestis fraction I (FI) because it allows detection of plague bacilli grown at 37 degrees C as well as at 28 degrees C. This dot-ELISA will be valuable as a clinical diagnostic tool and might be applicable to field studies and plague surveillance.     

Antibody interactions with the capsule of Cryptococcus neoformans

Monoclonal antibodies to the encapsulated fungus Cryptococcus neoformans produce different immunofluorescence (IF) patterns after binding to the polysaccharide capsule. To explore the relationship between the IF pattern and the location of antibody binding, two immunoglobulin M (IgM) monoclonal antibodies (MAbs) (12A1 and 13F1) that differ in protective efficacy and IF pattern and one protective IgG1 MAb (2H1) were studied by IF and electron microscopy (EM). Fixing C. neoformans cells in lung tissue for EM resulted in significantly better preservation of the capsule than fixing yeast cells in suspension. The localization of MAbs 12A1 and 13F1 by immunogold EM differed depending on whether the MAb was bound to cells in cut tissue sections embedded in plastic or to cells in solution. In cut tissue sections, MAbs 12A1 and 13F1 bound throughout the capsule, whereas in solution both MAbs bound near the capsule surface. To investigate whether antibody binding to the C. neoformans capsule affected the binding of other primary or secondary reagents, various combinations of MAbs 12A1, 13F1, and 2H1 were studied by direct and indirect IF. The IF pattern and location of binding for MAbs 12A1, 13F1, and 2H1 varied depending on the presence of other capsule-binding MAbs and the method of detection. The results show that (i) binding of MAbs to the C. neoformans polysaccharide capsule can modify the binding of subsequent primary or secondary antibodies; (ii) the IgM MAbs bind primarily to the outer capsule regions despite the occurrence of their epitopes throughout the capsule; and (iii) MAb 2H1 staining of newly formed buds is reduced, suggesting quantitative or qualitative differences in bud capsule.     

Reactivity of monoclonal antibodies to the 41-kilodalton protein of porcine group C rotavirus with homologous and heterologous rotavirus serogroups in immunofluorescence tests.

Three monoclonal antibodies (MAbs) to porcine group C rotavirus immunoprecipitated the major inner capsid protein (41 kDa) but failed to precipitate group A rotavirus proteins. In immunofluorescence tests of rotavirus-infected cell cultures or pig intestines, the MAbs recognized porcine and bovine group C rotaviruses but not group A or B rotaviruses. These MAbs may recognize the group C rotavirus counterpart to VP6 of group A rotaviruses and may be useful as diagnostic reagents.     

Spectrum of monoclonal antibodies to coxsackievirus B-3 includes type- and group-specific antibodies.

Fifteen monoclonal antibodies (MAbs) made to coxsackievirus B-3 were tested against a panel of enteroviruses by indirect immunofluorescence. The MAbs included seven which reacted with coxsackievirus B-3 only, five which reacted with more than one enterovirus included in the panel, one which reacted with broad enteroviral specificity and did not react with any heterologous virus (group specific), and two which reacted with all enteroviruses tested and at least one heterologous virus. The group-specific MAb identified 44 of 45 clinical isolates as enteroviruses, while the two broadly reactive MAbs reacted with all 45 of the clinical isolates. These MAbs are potentially important diagnostic reagents for grouping and typing enteroviruses by indirect immunofluorescence.     

Direct detection of Neisseria gonorrhoeae with monoclonal antibodies characterized by serotyping reagents.

A panel of monoclonal antibodies (MAbs) directed to outer membrane protein I were generated with the ultimate aim of detecting Neisseria gonorrhoeae in patient samples by a direct immunofluorescence (IF) test. In an initial evaluation of the sensitivity of these reagents, a cocktail of six IF MAbs recognized 491 (91%) of 540 gonococci isolates from several centers in Sydney, Australia. IF MAbs designated 185 and 228 recognized serovars of WI serogroup and IF MAbs 208, 210, and 312 recognized serovars of WII/III serogroup. IF MAb 198 recognized serovars within both serogroups. Three additional IF MAbs, designated 322, 323, and 330, were then generated by using strains which failed to react with the original MAb cocktail and which belonged to particular serovars. The new cocktail of nine IF MAbs recognized 96% of the gonococcal isolates, which incidentally contained representatives of serovars shown to have a worldwide distribution in previous studies. Although subtle differences were apparent in the reaction patterns found with coagglutination (serotyping) and IF, there nonetheless seems to be merit in the approach of continually evaluating the sensitivity of diagnostic reagents such as MAbs. This is especially true with an organism such as N. gonorrhoeae, which has the capacity to regularly alter the antigenic structure of its outer membrane proteins.     

Characterization of Monoclonal Antibodies Against Human Sperm Antigens by Immunoassays Including Sperm Function Assays and Epitope Evaluation

ABSTRACT: Fifteen monoclonal antibodies (MAbs) raised against human sperm cells were evaluated for reactions against human sperm by indirect immunofluorescence, immunocytochemistry, agglutination, complement-dependent immobilization, cervical mucus penetration, and hamster egg penetration assays. The MAbs were analyzed for specificity by immunofluorescent reactions with peripheral blood lymphocytes and sperm and classified into three main groups based on regional staining, ie, acrosome, plasma membrane, or tail. One MAb (218) bound to the sperm neck. Three MAbs, (80, 85, and HS-126) were found to react with lymphocytes. Three of five acrosome-reactive MAbs (11, 63, 106), two of five tail-staining MAbs (97, HS-30), and the neck reactor (218) showed significant to highly significant inhibition of sperm penetration of eggs but without significant effects on sperm agglutination, immobilization, or the mucus penetration assay. The three nonspecific MAbs gave strong plasma membrane reactions in the agglutination and immobilization assays and also caused highly significant inhibition of sperm penetration of both cervical mucus and zona-free ova. Preliminary analysis of the complementary antigens suggested that epitopes reacting with MAbs 33 (acrosome) and 85 (plasma membrane) were carbohydrate chains on glycoproteins. Three of five MAbs recognizing tail antigen, the neck-staining MAb, and the nonspecific MAb (HS-126) appeared to be reactive against glycolipid moieties. Seven of the 12 specific MAbs also reacted in indirect immunofluorescence with mouse and rabbit sperm in patterns similar to those observed with human sperm.     

Production and partial characterization of monoclonal antibodies to four Chlamydia psittaci isolates.

Monoclonal antibodies (MAbs) were produced to four Chlamydia psittaci isolates: NJ1 and TT3 from turkeys, VS1 from a parakeet, and B577 from an ovine abortion. MAbs were tested for reactivity with each isolate by the indirect immunofluorescent antibody technique and for neutralization by an inclusion reduction neutralization technique in tissue culture. Two genus-specific and 14 serovar-specific MAbs were produced. Genus-specific MAbs reacted with all avian and mammalian isolates; however, each failed to neutralize its homologous chlamydial isolate. Turkey isolates NJ1 and TT3 were antigenically similar; serovar-specific MAbs produced to each reacted equally with both isolates yet showed little or no reaction with other serovars. Serovar-specific MAbs to the parakeet and abortion isolates were distinct; no cross-reactions were seen with other serovars. None of the serovar-specific MAbs reacted with an ovine arthritis isolate. Of the 14 serovar-specific MAbs, 13 partially neutralized homologous strains with or without the addition of complement. Because of the high specificity, the serovar-specific MAbs used with the immunofluorescence technique provided a rapid and precise method to identify three serovars of C. psittaci.     

Development of monoclonal antibodies to highly pathogenic avian influenza H5N1 virus and their application to diagnostics, prophylaxis, and therapy

A panel of 17 monoclonal antibodies (MAbs) against highly pathogenic avian influenza virus (HPAIV) A/Duck/Novosibirsk/56/05 A/H5N1 (subclade 2.2) isolated in Russian Federation was developed. Immunoblot analysis showed that 12 MAbs were specific for the hemagglutinin (HA) and 5 MAbs for nucleoprotein (NP). All anti-HA MAbs were reactive in ELISA and immunofluorescence (IF) test and 10 of them were reactive in hemagglutination-inhibition (HI) and neutralization tests. Quantitative competitive ELISA revealed that anti-HA MAbs recognized at least 4 non-overlapping antigenic determinants and anti-NP MAbs recognized at least 3 non-overlapping antigenic determinants. Four sandwich ELISA procedures were developed using the obtained MAbs. These procedures are useful for 1) identification of avian, human, and swine influenza A viruses, 2) differentiation of avian influenza virus (AIV) from human and swine influenza viruses, 3) differentiation of AIV H5 from other AIV subtypes, and 4) differentiation between 2.2 and 2.3.2 subclades of H5N1 influenza viruses. Prophylactic and therapeutic efficacy of anti-HA MAbs with high neutralization activity was tested in BALB/c mice. A complete protection was achieved by single injection of MAbs (20 mg/kg) 24 hrs before challenge with 10 LD50 of HPAIV H5N1. Therapeutic efficacy was 90% that was similar to those of Rimantadine and Tamiflu.     

Identification of porcine alveolar macrophage glycoproteins involved in infection of porcine respiratory and reproductive syndrome virus

Summary.  The aim of this study was to identify the receptor(s) for PRRSV on porcine alveolar macrophages (PAMs) by producing monoclonal antibodies (MAbs) against these cells. Hybridoma supernatants were selected for their ability to block PRRSV infection. Four MAbs, 1-8D2, 9.4C7, 9.9F2, and 3-3H2 inhibited infection and recognised cell surface, PAM-specific antigens as shown by immunofluorescence and immunoperoxidase monolayer assay. These MAbs were then used to identify cellular proteins involved in PRRSV infection by radioimmunoprecipitation assays (RIPAs). MAbs 1-8D2 and 9.9F2 each recognised a 150 kDa-polypeptide doublet, while MAbs 9.4C7 and 3-3H2 both recognised a 220 kDa-polypeptide. Glycosidase treatment demonstrated all these polypeptides to be N-glycosylated. Thus, multiple glycoproteins appear to be involved in infection of PAMs by PRRSV. Received April 17, 2002; accepted July 25, 2002     

Modulation of expression of mouse macrophage surface antigens by monoclonal antibodies.

Mouse macrophages from peritoneal cavity were exposed to monoclonal antibodies (MAbs) directed against cell surface antigens and the effect on antigen expression was investigated. The two Mabs used, 3A33 and 3A35, were produced by cell fusion between a mouse plasmacytoma and rat lymphocytes immunized against mouse macrophages. The binding of the MAbs to cell surface was measured by immunofluorescence and flow cytometry or by a radioimmunological technique. When injected i.p. the MAbs diminished the expression of the corresponding antigens but did not alter it when added to cultures of adherent macrophages. Antigenic modulation, however, could be produced in vitro either by inhibiting macrophage adherence during incubation with MAbs or by using a second antibody layer. MAb 3A33 (IgG2a) was more effective than 3A35 (IgM) in provoking modulation. The appearance of re-synthesized antigens on cell surface was not affected by macrophage adherence. The modulated antigens were found to internalize into cytoplasmic vacuoles.     

Production and characterization of monoclonal antibodies to Mobiluncus species.

Members of the genus Mobiluncus are anaerobic motile curved rods which are associated with bacterial vaginosis (BV). Murine monoclonal antibodies (MAbs) to the ATCC type strains of M. curtisii subsp. curtisii, M. curtisii subsp. holmesii, and M. mulieris were produced and characterized by enzyme-linked immunosorbent assay and indirect immunofluorescence assay. Four MAbs were subspecies specific and reacted with M. curtisii subsp. curtisii but not with M. curtisii subsp. holmesii; four were specific for M. mulieris. The remaining antibodies demonstrated some cross-reactivity: three were species specific and reacted with both subspecies of M. curtisii, and one defined an epitope shared by M. curtisii subsp. holmesii and M. mulieris but not by M. curtisii subsp. curtisii. None of the MAbs reacted with a panel of other bacteria commonly present in the vaginas of normal women or women with BV. Examination of the molecular specificities of the antibodies by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting revealed four antibodies which were specific for an 82,000-dalton molecule of M. curtisii subsp. curtisii and five antibodies which bound a major band of M. mulieris at 93,000 daltons. Selected MAbs reacted in the indirect immunofluorescence assay with 24 of 25 Mobiluncus spp. clinical isolates from local women with BV and could be used for direct detection of Mobiluncus spp. in vaginal fluid from a patient with BV.     

Development of monoclonal antibodies against Ureaplasma urealyticum serotypes and their use for serotyping clinical isolates

Monoclonal antibodies (MAbs) against Ureaplasma urealyticum serotype 2, 5, 7, 8, 10, 11, 12, and 13 reference strains were developed. The reactivities of these MAbs with the 14 serotype reference strains was verified by colony immunofluorescence assay and Western blot assay. MAbs against serotypes 2, 7, 10, 11, and 12 were serotype specific, whereas MAbs against serotypes 5, 8, and 13 showed cross-reactivity. All MAbs against serotype 5 were cross-reactive with serotype 2, and one showed, in addition, cross-reactivity to serotypes 9 and 10. Mutual cross-reactivities were observed between MAbs against serotypes 8 and 13. The usefulness of the MAbs for the serotyping of U. urealyticum strains was evaluated by serotyping 21 selected clinical isolates. A complete set of MAbs (the newly developed MAbs and the previously described MAbs against serotypes 1, 3, 4, 6, 9, and 14) as well as a complete set of polyclonal antibodies (PAbs), PAbs 1 to 14, were used. MAbs were able to identify 18 of 21 isolates including 2 isolates with mixed serotypes. Polyreactivity, which occurred with 19 of the 21 isolates with PAbs, was not observed by the use of MAbs. MAbs seem to be a more valuable tool than PAbs for serotyping and could help in investigating a possible link between the expression or variability of the serotype-specific antigens and pathogenicity.