Validation of a second generation competitive enzyme immunoassay (CELISA) for the diagnosis of brucellosis in various species of domestic animals

A second generation competitive enzyme immunoassay (CELISA) for detection of bovine antibody to Brucella abortus was developed to eliminate reagent variables in the assay. This assay was different from earlier CELISA formats in that it used recombinant protein A and protein G immunoglobulin receptors (PAG), labelled with horseradish peroxidase, thus eliminating the requirement for polyclonal anti-mouse-enzyme conjugate for detection. This allowed standardization of the assay. The CELISA uses a monoclonal antibody specific for a common epitope of the O-polysaccharide (OPS) of smooth lipopolysaccharide (SLPS) derived from B. abortus S1119.3. This antibody did not react with PAG. This monoclonal antibody was used to compete with antibody in the bovine test serum to the smooth lipopolysaccharide (SLPS) antigen. Reaction of bovine antibody was then measured directly with the PAG enzyme conjugate. In this case, development of colour in the reaction indicated a positive reaction. The performance characteristics of the new CELISA, sensitivity, specificity and exclusion of antibody of B. abortus S19 vaccinated animals, were very similar to those of the classical CELISA and to the indirect enzyme immunoassay (IELISA) when using sera deemed positive by isolation of the bacterium, either from individual animals or from some animals on the premises. All sera were tested by the buffered antigen plate agglutination test (BPAT) and the complement fixation test (CFT). Only samples positive on both BPAT and CFT were considered as positive and only samples negative on both tests were used considered negative. Sufficient samples from cattle, swine, sheep and goats to validate the test were included based on OIE guidelines suggesting inclusion of a minimum of 300 positive and 1000 negative samples.     

Enzyme-linked immunosorbent assay for differentiation of the antibody response of cattle naturally infected with Brucella abortus or vaccinated with strain 19

Purified O chain of Brucella abortus was passively attached to polystyrene to differentiate antibody responses of cattle vaccinated with B abortus strain 19 from those of naturally infected cattle. In the indirect assay, using O polysaccharide as antigen, a single serum dilution was used and mouse monoclonal antibody to bovine L chain conjugated with horseradish peroxidase was the detection reagent. Measurable antibody was not found in sera of vaccinated cattle, except for 3 sera from cattle that were persistently infected with strain 19. Sera from 25 cattle infected with pathogenic strains contained antibody on the basis of results of indirect enzyme immunoassay, using smooth lipopolysaccharide or O chain as antigens, or results of competitive enzyme immunoassay, using the O-chain antigen. Results in sera from calves with experimentally induced Yersinia enterocolitica serotype 0:9 infection or inoculated with a low dose of B abortus strain 2308 were comparable with those in sera of cattle that were vaccinated with strain 19. The data correlated with those from competitive enzyme immunoassay, using one serum dilution and horseradish peroxidase-conjugated mouse monoclonal antibody to smooth lipopolysaccharide. On the basis of results of the indirect enzyme immunoassay, all sera (except those samples obtained before inoculation) contained antibody to smooth lipopolysaccharide.     

Bovine monoclonal antibody specific for Brucella abortus lipopolysaccharide

The development of a bovine monoclonal antibody against Brucella abortus smooth lipopolysaccharide (BM-8) by interspecies fusion of bovine peripheral lymphocytes from an immunized cow and a murine plasmacytoma cell line is described. The twice cloned cell line secreted bovine IgG1 subclass antibody. Ascites fluid was prepared in pristane treated nu/nu mice by intraperitoneal injection. The pooled ascites fluid was purified by affinity chromatography and the functions of the antibody assessed in various serological tests. The BM-8 antibody did not agglutinate well at a neutral pH, however, under acid conditions it was efficient at agglutinating B. abortus cells. The antibody did not precipitate B. abortus LPS in double agar gel immunodiffusion but was very active in the direct complement fixation test and the indirect enzyme immunoassay, although it was unable to compete with a murine monoclonal antibody in a competitive enzyme immunoassay.     

Bovine brucellosis: evaluation of field sera by a competitive and superimposable ELISA utilising a monoclonal antibody against Brucella abortus lipopolysaccharide

With the aid of a horseradish peroxidase (HRP) tagged monoclonal antibody against smooth lipopolysaccharide from Brucella abortus (Bruce 1), a competitive and superimposable ELISA test procedure for bovine brucellosis has been evaluated for its ability to discriminate between Strain 19-vaccinated (S19-Vacc) and Biotype 1-infected (B1-Inf) cattle. In the competitive assay, all sera from S19-Vacc animals competed effectively against HRP-Bruce 1 (low HRP activity), while 10 out of 40 B1-Inf animals competed less effectively with Bruce 1 (high HRP activity). Successful competition by cattle antibodies would result in an increased proportion of cattle Igs binding to the assay antigen. This was confirmed by superimposing an alkaline phosphatase conjugated rabbit anti-cattle Ig after the competitive ELISA had been completed. With the superimposable assay, alkaline phosphatase activity was correspondingly high for S19-Vacc animals, and low for 36 out of 40 B1-Inf animals. The superimposable ELISA had therefore improved the discriminatory capabilities of the assay procedure from 75% to 90%.     

A competitive enzyme immunoassay for the detection of bovine antibodies to Brucella abortus using monoclonal antibodies

A competitive enzyme immunoassay (EIA) for the detection of circulating bovine antibodies to Brucella abortus has been developed using horseradish peroxidase conjugated monoclonal antibodies (MAb) raised against B. abortus cell surface antigens. Antibodies present in the serum of either vaccinated or infected cattle can apparently displace the conjugated MAb from the lipopolysaccharide antigen (LPS) in a quantitatively different manner allowing an assessment of immune status of the animal. The results from a panel of sera from animals with a known status of vaccination or infection indicated that the test was more selective in the detection and discrimination of infected from uninfected or immunized animals, than conventional complement fixation, agglutination or indirect enzyme immunoassay procedures.     

Serological relationship between cattle exposed to Brucella abortus, Yersinia enterocolitica O:9 and Escherichia coli O157:H7

Sera from cattle naturally infected with Brucella abortus (n = 160), vaccinated with B. abortus S19 (n = 88) or immunized with Yersinia enterocolitica O:9 (n = 25) or Escherichia coli O157:H7 (n = 80) were collected. The sera were compared for antibody content to the same bacteria by indirect enzyme immunoassay (IELISA), fluorescence polarization assay (FPA) and competitive enzyme immunoassay (CELISA). Cattle sera (n = 523) collected randomly from across Canada were tested in the same tests. Sera from the B. abortus infected group reacted positively in the brucellosis IELISA (IELISA(Br)), CELISA and FPA (FPA(Br)) and the Y. enterocolitica IELISA (IELISA(Ye)) while the Y. enterocolitica FPA (FPA(Ye)) detected antibody in 93.8% and the E. coli IELISA (IELISA(Ec)) 86.9% and the E. coli FPA (FPA(Ec)) 48.1%. About 70% of the sera from B. abortus S19 vaccinated animals reacted in the three IELISAs, 45% in the CELISA, and 37.7% in the FPA(Ec), 21.6% in the FPA(Br) and 5.7% in the FPA(Ye). Sera from E. coli O:157 exposed cattle reacted mainly in the IELISA(Ec) and FPA(Ec) although surprisingly 87.5% reacted in the IELISA(Ye) and only 3.8% in the IELISA(Br). No reactions were observed with these sera in the FPA(Br) and FPA(Ye) but one serum gave a low positive reaction in the CELISA. All sera from Y. enterocolitica O:9 exposed cattle reacted in the IELISA(Br) and IELISA(Ye) and 80% in the IELISA(Ec). In the CELISA, 44% gave a positive reaction and 64% were positive in the FPA(Br), 28% in the FPA(Ye) and 12% in the FPA(Ec). Of the 523 Canadian sera, about 50% reacted in the E. coli tests with only minor reactions in the Y. enterocolitica O:9 and B. abortus assays. From the data, the cross reaction between E. coli O157:H7, Y. enterocilitica O:9 and B. abortus is dependent on the test used. Thus, extensive cross reaction was observed with the IELISA with much less reactivity in the FPA and the CELISA.     

Rapid immunofiltration assay based on colloidal gold–protein G conjugate as an alternative screening test for bovine and ovine brucellosis

A non-enzymatic rapid immunofiltration assay (NERIFA) was developed as an alternative field test for rapid detection of anti-Brucella antibody in bovine and ovine sera. The assay was based on Brucella abortus lipopolysaccharide as diagnostic antigen and colloidal gold particle–protein G conjugate as detection reagent. Its diagnostic performance was evaluated using undiluted well-defined positive and negative serum samples in comparison with Rose Bengal test (RBT), complement fixation test (CFT) and a commercial and an in-house indirect enzyme-linked immunosorbent assay (ELISA). A perfect test agreement was found between NERIFA and ELISAs by kappa statistics. In addition, McNemar’s analysis of the results showed that the RBT for bovine sera and the CFT for ovine sera were found significantly less performant than indirect ELISAs and NERIFA. The results of the present study indicated that the NERIFA could be considered as a simple, rapid, and accurate field test for screening of ovine and bovine brucellosis. Therefore, this test constitutes a high potential to be used as an alternative model particularly in brucellosis prevalent tropical and subtropical geographical areas.     

The serological response of cattle immunized with Yersinia enterocolitica O:9 or O:16 to Yersinia and Brucella abortus antigens in enzyme immunoassays

Sera from calves immunized with Yersinia enterocolitica serotypes O:9 or O:16 were tested by indirect enzyme linked immunosorbent assay (ELISA) using lipopolysaccharide (LPS) preparations from Brucella abortus or Y. enterocolitica O:9 or O:16 for their antibody content of the IgG1 or IgG2 subclasses. High IgG1 responses were present with the three antigens in both groups although some individual variations between animals were noted. The IgG2 responses were modest and in some cases not above background 'noise'. Thus IgG2 antibody was not measurable in sera from serotype O:9 injected calves when using serotype O:16 LPS or in serotype O:16 injected calves when using B. abortus or serotype O:9 LPSs. A competitive ELISA using B. abortus O-polysaccharide and a monoclonal antibody to B. abortus LPS (initially designed to differentiate the antibody responses of cattle naturally infected with B. abortus from those vaccinated with strain 19) was used on sera from both groups of calves. Using this test, no antibody was detected in the group immunized with serotype O:16 and except for one animal in the serotype O:9 immunized group, only low levels of antibody were transiently in evidence. One animal in this group responded with quite high levels of competing antibody which, however, declined towards the end of the test period. The competitive ELISA may prove a useful serological tool for differentiating vaccinal and field infection titers to B. abortus and also to eliminate cross-reactions observed with Y. enterocolitica serotypes.     

Use of hydrophobic cloths as antibody adsorbents for enzyme immunoassay: detection of Brucella antigens

A cloth-ELISA (C-ELISA) antigen capture assay for the detection of Brucella melitensis and B. abortus was developed. Segments (6-mm squares) of hydrophobic polyester cloth coated with diluted serum from a B. abortus-infected cow were incubated with saline suspensions of heat-killed Brucella cells, or with cultures of bovine or sheep blood or bovine tissue homogenates that had been inoculated with B. abortus or B. melitensis added to trypticase soy broth (TSB) and incubated for 2-3 days. The captured antigen was detected by a bovine anti-Brucella antibody-horseradish peroxidase conjugate system. The enrichment culture technique detected as few as three Brucella colony-forming units (c.f.u.) in 0.5 ml of bovine blood and was positive in cultures in which the Brucella concentration had reached 3 X 10(6) c.f.u. ml-1 (after 2 or 3 days incubation). The combined enrichment-cloth-ELISA method gave complete correlation with cultural isolation and results were available 3 days before colonies appeared in conventional culture. Hydrophobic cloths have potential use in diagnostic procedures since they provide simple, rapid and economical assays.     

Enzyme immunoassay for the diagnosis of brucellosis: chimeric Protein A-Protein G as a common enzyme labeled detection reagent for sera for different animal species

A recombinant protein combining the immunoglobulin binding sites of Proteins A and G, conjugated with horseradish peroxidase was used as a universal detection reagent for the assessment of antibodies against Brucella spp. The reagent was applied in an indirect enzyme immunoassay for detection of antibodies to smooth lipopolysaccharide antigen in sera from Brucella spp. exposed and non-exposed cattle, sheep, goats and pigs and to antibodies to rough lipopolysaccharide in sheep, dogs and cattle. The results were similar to those obtained when murine monoclonal antibody-enzyme conjugates were used. An added advantage was that a universal cut-off for all tests using the proteins A and G detection reagent could be established, simplifying diagnostic interpretation of the data.     

Cleavage of bovine immunoglobulin G1 in whey by an extracellular material from Brucella abortus.

Culture extracts of in vitro grown Brucella abortus were demonstrated to cleave a part of the Fc portion of bovine immunoglobulin G1 in whey but not in serum or as a purified protein from serum. Supernates from Strains 19 and 2308 of B. abortus were both capable of this hydrolysis whereas living cells were not. The cleavage process was independent of antibody activity to B. abortus, appeared to require factor(s) found only in some whey samples and was ineffective with the other bovine immunoglobulins.     

Immunomodulation in cattle immunized with Brucella abortus strain 19

Regulation of the bovine immune response to immunization with Brucella abortus Strain 19 (S19) was investigated through application of a modification of an assay to measure suppressor T lymphocyte activities in humans and through development and characterization of antigen-stimulated T lymphocyte lines in vitro. A total of nine of steers were alloted into two groups: control (n = 4) and S19-immunized (n = 5). Peripheral blood mononuclear cells (PBMC) from each animal were cultured in vitro with mitogens (concanavalin A (Con A) and pokeweed mitogen (PWM], B. abortus antigens (B. abortus soluble antigen (BASA) and whole heat-killed B. abortus cells (HKC)) and media alone periodically from days 4 through 49 of the experiment. Supernates from these cultures were assayed for immunomodulatory activity(s) by addition to indicator cultures stimulated with suboptimal concentrations of Con A. Supernates from PBMC of S19-immunized steers generated with B. abortus antigens significantly (P less than 0.05) suppressed indicator cell responses as compared to those from control steers on days 35 and 49 post-immunization. This suppressive activity from PBMC of immunized cattle with respect to that of control cattle could also be induced through mitogenic stimulation with Con A or PWM. On day 49 of the study, suppressive activity was spontaneously released from the PBMC of immunized cattle. T lymphocyte lines were initiated from two S19-immunized steers at 2 and 9 weeks post-immunization. These T cell lines were characterized with respect to proliferative responses to B. abortus antigens through in vitro assay and surface marker expression through indirect immunofluorescence with a limited panel of monoclonal antibodies. Results from the present study indicated that S19 immunization induces a subpopulation(s) of cells in the PBMC of cattle capable of regulating the in vitro response to B. abortus. This regulatory activity is detectable by in vitro assay as early as 7 weeks post-immunization. Furthermore, the regulatory cell(s) appear to involve BoCD8+ T, lymphocytes which are specific for B. abortus antigens.     

Monoclonal antibodies to Brucella surface antigens associated with the smooth lipopolysaccharide complex

Hybridomas producing antibodies to determinants associated with the lipopolysaccharide (LPS) of Brucella abortus and B melitensis were obtained by polyethylene glycol fusion of the SP2/0 myeloma cell line with B lymphocytes harvested from a Sprague-Dawley-derived rat previously immunized with whole B abortus strain 1119 organisms. Two clones, BRU38 and BRU28 , were selected for their ability to react with whole B abortus organisms and purified smooth-LPS ( f5p ). The BRU38 monoclonal antibodies were absorbed with live, rough strain 45/20 and smooth strains of B abortus and B melitensis organisms, whereas only smooth strains absorbed the antibody activity from BRU28 . Complete inhibition of the monoclonal's activity could be achieved with crude smooth-LPS, a purified f5p fraction, and a water-soluble acid degraded polysaccharide. Absorption of BRU38 and BRU28 with rough Brucella LPS, polysaccharide-B antigen, keto- deoxyoctanoic acid, or with several sugars and fatty acids known to be components of the Brucella LPS complex had no effect on the monoclonals. The data indicate that antigenic determinants are associated with the smooth LPS complex, probably with the O-side chain, and are expressed patchwise and in different quantities on several strains of B abortus and B melitensis. The B abortus rough strain 45/20 contains surface determinants which lead to the agglutination of smooth strain 1119 organisms. The potential use of monoclonals in competitive enzyme-linked immunosorbent assays for diagnostic purposes is discussed.     

Competitive enzyme-linked immunosorbent assay for heartwater using monoclonal antibodies to a Cowdria ruminantium-specific 32-kilodalton protein

Hybridomas producing monoclonal antibodies (mAb) to Cowdria ruminantium were raised. Four mAbs of the IgG isotype reacted in western blots with a 32-kilodalton Cowdria protein (Cr32), which had previously been shown to be conserved and immunodominant. A fifth mAb of the IgM isotype recognized a 40-kDa Cowdria protein. The latter mAb was negative in an indirect fluorescent antibody test (IFA), whereas the other four were positive. mAb No. 4F10B4 showed the strongest signal in western blots using three different stocks of Cowdria. Immuno-gold labeling of Cowdria organisms in vitro using 4F10B4 showed that Cr32 has surface-exposed antigenic determinants. Using mAb 4F10B4, a competitive ELISA was developed which detected specific Cowdria antibodies in goat, sheep and cattle sera. Antibodies in animal sera competed with binding of mAb 4F10B4 to a crude sonicated Cowdria antigen obtained from infected endothelial cell cultures. The competition ELISA (CELISA) detected antibodies in 55 out of 70 (79%) goats experimentally infected with one of eight different Cowdria stocks. Fourteen out of the 15 sera which were shown negative in the CELISA were also negative in the IFA. Nevertheless, all 15 sera recognized some epitopes of the immunodominant Cowdria-specific 32 kDa protein as judged from their reaction with this protein in western blots. Overall, there was 89% agreement between CELISA and IFA considering all 70 goat sera. Moreover, antibodies were detected in nine out of nine sheep infected with one of three different stocks of Cowdria and in sera from calves experimentally infected by two different strains of heartwater. There were no cross-reactions with Ehrlichia phagocytophila antibodies in goat sera, nor with Anaplasma marginale antibodies in bovine sera. Lack of cross-reactivity and detection of antibodies to eight geographically widely distributed stocks of Cowdria, makes the competition ELISA a promising test for use in heartwater endemic areas.     

Bovine respiratory syncytial virus-specific monoclonal antibodies

Five hybridomas were produced which secreted monoclonal antibodies to bovine respiratory syncytial virus (BRSV). Two antibodies (8G12, 15C7) neutralized the virus and inhibited syncytia formation in vitro. These monoclonal antibodies also stained, by indirect fluorescent assay, an external envelope protein of living virus-infected cells, and recognized the 48k subunit of the viral fusion protein by Western blot analysis of bovine respiratory syncytial virus-infected cell lysates. Three other monoclonals (6A12, 14D3, 14E3) stained, by indirect fluorescent assay, acetone-fixed virus-infected cells but not living cells. Three hybridomas (6A12, 8G12, 15C7) secreted monoclonal antibody of isotype IgG1, k; two hybridomas secreted monoclonal antibody of isotype IgG2a, k. This apparently is the first report of monoclonal antibodies specific for BRSV glycoproteins.     

The long-term culture of bovine monocyte-derived macrophages and their use in the study of intracellular proliferation of Brucella abortus.

Although the immune response to Brucella abortus is multifaceted, the key event in contending with this pathogen appears to be the interaction of the organism with cells of the mononuclear phagocyte system. A cell culture system was developed which allowed the long-term maintenance of blood monocyte-derived macrophages in Teflon culture vessels in a relatively unstimulated state. The assay system was optimized for timing of bacteria-macrophage interaction and numbers of bacteria and macrophages used in each assay. Interaction of B. abortus strain 2308 with bovine mononuclear phagocytes from animals phenotypically resistant and susceptible to infection with B. abortus was investigated. This cell culture and assay system should provide a useful model for the investigation of intracellular parasitism in cattle.     

Immunopotentiation of cattle vaccinated with a soluble Brucella abortus antigen with low LPS content: an analysis of cellular and humoral immune responses.

The adjuvant effects of dimethyl dioctadecyl ammonium bromide (DDA) alone or in combination with trehalose dimycolate (TDM) or muramyl dipeptide (MDP) on bovine humoral and cellular responses to a soluble protein extract of gamma irradiated Brucella abortus strain 19 (SPEBA) were investigated. Thirty-five beef steers were randomly allotted to nine groups. Three of these groups received SPEBA (2 mg protein per dose) subcutaneously in combination with adjuvants, one group received the reduced dose of B. abortus strain 19 (S19), and one group received SPEBA alone. Controls included groups receiving adjuvant preparations only or no vaccine. Immune responses to the various immunizations were assessed sequentially for 56 days using various in vitro and in vivo assays. The humoral response to B. abortus was measured using standard serologic tests, an enzyme-linked immunosorbent assay, and a quantitative fluorometric immunoassay. The cell-mediated immune (CMI) response was measured by antigen-specific lymphoproliferation (LP), interleukin 2 (IL 2) production, and soluble suppressor factor release. Skin testing at day 35 for delayed-type hypersensitivity (DTH) to SPEBA was also performed. Minimal humoral responses were induced with SPEBA alone. The highest and most sustained serum antibody responses to B. abortus antigens were elicited by the S19 vaccine. A combination of SPEBA with DDA + TDM induced higher antibody levels than SPEBA with DDA or SPEBA with DDA + MDP. Responses to DTH among the groups receiving SPEBA were most notable in the SPEBA with DDA + TDM groups. Increased IL 2 production was greatest in the S19 and SPEBA with DDA + TDM vaccinates. The results indicated that a combination of DDA + TDM best potentiated immune responses to a soluble B. abortus antigen preparation and may be useful as adjuvants for future vaccines.     

Protection of mice against Brucella abortus infection by inoculation with monoclonal antibodies recognizing Brucella O-antigen

Monoclonal antibodies recognizing the O-polysaccharide portion of Brucella abortus strain 2308 provided BALB/c mice with passive protection against challenge exposure with the homologous strain. Numbers of colony-forming organisms in the spleen were reduced by IgM and IgG monoclonal antibodies. Active immunization of mice, using B abortus 2308S lipopolysaccharide, resulted in production of IgM antibody at 14 days. Clearance of organisms in the actively immunized mice after challenge exposure at 14 days was nearly identical to that in passively immunized mice. Mice either passively or actively immunized were effectively protected from 0 to 28 days. Bacterial colonization of the spleen was observed to increase in both groups of mice at 56 days and indicated that humoral responses were effective in eliminating the organism in the early stages of infection, but other immune mechanisms were necessary for protection of mice in the later stage of infection with virulent strains of B abortus.     

Evaluation of three serum i-ELISAs using monoclonal antibodies and protein G as peroxidase conjugate for the diagnosis of bovine brucellosis

Three i-ELISAs using LPS, the immunodominant component of Brucella abortus, were developed with three different conjugates: monoclonal antibodies 1C8 (anti-bovine IgG(1)) and 3H3 (mainly specific for bovine IgG(2) but also reacting with IgG(1)) and protein G (reacts with both bovine IgG subclasses). Using a cut-off value of 2.5U/ml, the i-ELISA with 3H3 as conjugate had a specificity (95% CI: 98.32-99.63%) that was significantly higher than the same assay with 1C8 (95% CI: 96.08-98.26%) or PG (95% CI: 95.83-98.09%). In areas where false positive serological reactions (FPSR) were common, the specificity of the i-ELISAs decreased significantly. The specificity of the i-ELISAs increased with the age of the animals tested, irrespective of the conjugate. The specificity of the i-ELISAs and traditional tests was also examined using sera from animals infected per os with bacteria bearing LPS similar to the Brucella LPS. It appeared that Yersinia enterocolitica O:9, Xanthomonas maltophilia and Salmonella urbana infections induced FPSR both in the i-ELISAs and in the traditional tests, but the 3H3 assay was significantly less prone to produce false positive reactions than the 1C8 and PG assays. The i-ELISAs were more sensitive, allowed earlier detection, and were more persistent than the traditional serological tests both in experimentally and naturally Brucella-infected animals. Weekly i-ELISA monitoring of experimentally infected pregnant heifers (previously vaccinated or not) allowed a prediction of abortion. Furthermore, the 1C8 assay showed significantly higher titres irrespective of day post-infection and vaccination status. The accuracy of the assay could be improved by making use of additional information (e.g. animal age or conjugate) and by selecting appropriate cut-off points on the basis of the prevailing epidemiological situation. The i-ELISAs appear an appropriate choice in order to maintain an official brucellosis-free status because of their sensitivity, early detection and long persistence and, for the same reasons, seem especially valuable for the detection of latent carriers (i.e. animals classified negative by classical serological tests) among imported animals.     

Enzyme immunoassay for detection of Mycoplasma bovis antigens in bull semen and preputial washings

A capture enzyme immunoassay was developed for the detection of Mycoplasma bovis antigens in bull semen or preputial washings. IgG prepared from rabbits immunised with M bovis was passively adsorped to 96-well polystyrene plates. This antibody captured M bovis antigens which were then detected by using an IgG preparation from an immunised cow and murine monoclonal antibody to the bovine L-chain conjugated with horseradish peroxidase. The sensitivity of the assay was approximately 200 colour changing units (ccu)/ml and the specificity was excellent in that other species of mycoplasma, ureaplasma or acholeplasma did not react. A blind study of bull semen experimentally contaminated with M bovis detected all specimens with more than 200 ccu/ml.