Characterization of Brucella ovis lipopolysaccharide and its use for diagnosis of ram epididymitis by enzyme-linked immunosorbent assay.

Rough lipopolysaccharide, extracted by a mixture of phenol, chloroform, and petroleum ether from freeze-dried Brucella ovis cells with a yield of 0.71%, contained relatively small amounts of protein and nucleic acid contaminants as compared with lipopolysaccharides from other Brucellae. The crude lipopolysaccharide was suitable as a diagnostic antigen in an enzyme-linked immunosorbent assay for the sensitive and specific detection of ram epididymitis caused by B. ovis infection. In comparative serological tests, the enzyme-linked immunosorbent assay with B. ovis lipopolysaccharide gave better identification of infections and fewer false-negative results than the enzyme-linked immunosorbent assay with sonicated antigen or the complement fixation test.     

Antibody response to Brucella outer membrane proteins in bovine brucellosis: immunoblot analysis and competitive enzyme-linked immunosorbent assay using monoclonal antibodies.

Sera from Brucella-infected bovines were analyzed by immunoblotting by using sonicated cell extracts of B. melitensis or B. abortus and a competitive enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies against outer membrane proteins (OMPs) with molecular masses of 10, 16.5, 19, 25 to 27, 36 to 38, and 89 kDa. Antibody responses against OMPs were compared with antibody responses against smooth lipopolysaccharide. Immunoblot analysis indicated that the antibody response in infected animals was largely different from one animal to another. The antigens of concern were OMPs with molecular masses of 10, 16.5, 19, 25 to 27, 36 to 38, and 89 kDa and other proteins with molecular masses of between 40 and 80 kDa. According to the specificity of the competitive ELISA, OMPs useful for the detection of infected animals are the OMPs of 10, 16.5, 19, 25 to 27, and 36 to 38 kDa. A competitive ELISA with the anti-89 kDa monoclonal antibody was not specific. Results of the competitive ELISA confirmed the individual variability of the humoral immune response against OMPs. It therefore seems that a combination of several protein antigens is necessary for the development of an immunoassay with a sensitivity comparable to that of the smooth lipopolysaccharide ELISA.     

An indirect competitive enzyme-linked immunosorbent assay for acrylamide detection based on a monoclonal antibody

Acrylamide (AA) is formed spontaneously in heated foodstuffs and is a focus of concern in many people due to safety. In this study, we developed an indirect competitive enzyme-linked immunosorbent assay (icELISA) based on a monoclonal antibody (MAb) to detect a derivative, which was generated from 4-mercaptobenzoic acid (4-MBA). As AA is a very small molecule (71.08?Da) and cannot elicit a homologous monoclonal antibody, we coupled the AA derivative (AA-4-MBA) to a carrier protein such as bovine serum albumin (BSA) and ovalbumin (OVA). The conjugates were used as the immunogen and coating antigen. A rapid and sensitive icELISA against AA-4-MBA was obtained by optimizing the experimental parameters. The MAb which had no specificity for AA or 4-MBA, but had high affinity for AA-4-MBA, had a satisfactory IC₅₀ of 32?ng/ml and a limit of detection of 8.87?ng/ml. The quantitative working range was 8.87–112.92?ng/ml (IC₂₀ to IC₈₀). Cross-reactivity with other analogues was lower than 10%. These results indicated that the developed icELISA was a fast and efficient method for detecting AA in food.     

Serodiagnosis of melioidosis by a competitive enzyme-linked immunosorbent assay using a lipopolysaccharide-specific monoclonal antibody

Burkholderia pseudomallei is the causative agent of melioidosis, a severe and potentially fatal infectious disease in humans known to be endemic in Southeast Asia and northern Australia. The infection is also increasingly recognized in various animal species with a potential to spread to humans. With the potential as a biological warfare agent, specific serodiagnosis of melioidosis for surveillance in large populations at risk, humans or animals, would be highly valuable. In this study, a competitive enzyme-linked immunosorbent assay (ELISA) using a lipopolysaccharide-specific monoclonal antibody was developed. The assay provides high specificity, based on a previously described monoclonal antibody to a specific epitope on the lipopolysaccharide (LPS) of B. pseudomallei. The assay sensitivity of 96.0% and specificity of 100% were achieved at a cutoff value of 50% inhibition in human culture-proven melioidosis cases. An optimal cutoff value of 65% inhibition for sera from a melioidosis endemic area was obtained by ROC analysis and resulted in an assay specificity of 86.2%, while maintaining assay sensitivity of 92.0%. A potential application of the assay in the serodiagnosis of melioidosis in animal species was also evaluated usina dolphin sera with satisfactory results.     

Purification of a Brucella canis cell wall antigen by using immunosorbent columns and use of the antigen in enzyme-linked immunosorbent assay for specific diagnosis of canine brucellosis.

A cell wall antigen of Brucella canis was purified by immunosorbent columns. The antigen contained two proteins of 30 and 28 kilodaltons and a polysaccharide exhibiting a 12-kilodalton band upon 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antibody to the purified antigen, which specifically reacted with the polysaccharide, was used as the first coating antibody in an enzyme-linked immunosorbent assay (ELISA) for serological diagnosis of canine brucellosis. Dogs inoculated orally with live B. canis were positive and dogs from B. canis-free colonies were negative in the ELISA. Of 199 dogs from a brucellosis-contaminated area, 116 with negative titers in the tube agglutination test (TAT), using heat-inactivated whole B. canis cells as the antigen, were also negative in the ELISA. Seventy-eight of the dogs with questionable titers in the TAT were divided into two groups: 20 dogs that were positive in the ELISA and 58 that were negative. Of five dogs with positive titers in the TAT, three were positive in the ELISA and the gel immunodiffusion test (GD) with crude B. canis extract as the antigen and were also culture positive for B. canis. One dog was positive in the ELISA and GD but gave a negative culture result. Serum from the remaining dog, which was positive with high titer in the TAT but negative in the ELISA and in culture for B. canis, formed a spur precipitate with a homologous precipitate in the GD. These results indicate that the ELISA is a specific serological test for B. canis infection in dogs.     

Development of a competitive enzyme-linked immunosorbent assay for detecting cytomegalovirus antibody

A competitive enzyme-linked immunosorbent assay (ELISA) for detecting cytomegalovirus (CMV) antibody was developed. The competitive ELISA was five times more sensitive than the complement fixation test (CFT) and twice as sensitive as indirect ELISA. Testing of paired sera from cardiac transplant patients taken before and after transplantation showed good correlation between results of competitive and indirect ELISA and CFT. The competitive ELISA was more successful than CFT or indirect ELISA in detecting passively acquired antibody, but detection of CMV antibody by competitive ELISA immediately after primary CMV infection was unreliable, possibly because of the high affinity of the monoclonal antibody chosen for the horseradish peroxidase conjugate. However, competitive ELISA may well prove to be more suitable than indirect ELISA for detecting CMV antibody in blood donations.     

Specific enzyme-linked immunosorbent assay for detection of bovine antibody to Brucella abortus

Six soluble antigens prepared from Brucella abortus were compared with a salt-extractable protein (CSP) antigen in an enzyme-linked immunosorbent assay for the detection of antibody to B. abortus in cattle sera. Of seven preparations tested, antigens from B. abortus soluble antigen (prepared from an autoclaved cell suspension) and CSP were stable on frozen storage. Enzyme-linked immunosorbent assay with CSP antigen under optimal conditions was from 100- to 700-fold more sensitive than the standard agglutination, card, Rivanol precipitation-plate agglutination, and the complement fixation tests in detecting immunoglobulin G antibody. From a practical point of view, however, using the most stringent criteria for determining an "upper negative" value, the enzyme-linked immunosorbent assay with CSP was at least 12-fold more sensitive than the standard agglutination test and any of the other serological tests. Furthermore, the enzyme-linked immunosorbent assay with CSP was specific for antibody to B. abortus.     

Detection of PMV-1 specific antibodies with a monoclonal antibody blocking enzyme-linked immunosorbent assay

A highly reproducible monoclonal antibody (Mab) blocking ELISA (B-ELISA) has been developed and evaluated for the detection of NDV-specific antibodies. The Mab utilised is specific for a conserved PMV-1 serotype-specific epitope, as demonstrated by the indirect immunoperoxidase test. It reacted with all strains representing different serogroups within the PMV-1 serotype, but not with any strain belonging to other PMV serotypes. Sensitivity and specificity of the B-ELISA were compared with the haemagglutina-tion inhibition test (HI). Blocking and HI antibodies were detected in sera of chickens 8 days post-experimental infection. The B-ELISA proved consistently more sensitive than the HI test. In another survey, 62 sera from experimentally vaccinated chickens were tested; 95.2% proved positive by B-ELISA, 85.5% by indirect ELISA and 74% by HI test. When 504 field sera from vaccinated chickens and turkeys were tested, 98% were positive by B-ELISA, and 69% by HI. The specificity was evaluated by testing 1066 samples from NDV-free flocks, all of which proved negative by both methods. Other advantages of the B-ELISA include easy standardization and quality control, and ability to test sera from any species (including exotic or wild birds as well as mammals). The use of low dilution serum or egg-yolk samples makes the test quick and easy to perform and suitable for large-scale screening.     

Detection of equine antibody to Babesia equi merozoite proteins by a monoclonal antibody-based competitive inhibition enzyme-linked immunosorbent assay.

A competitive inhibition enzyme-linked immunosorbent assay (CI ELISA) was developed to detect antibody to Babesia equi. One hundred fifty-four equine serum samples from 19 countries were tested for antibody to B. equi by the complement fixation test and by CI ELISA. The CI ELISA and complement fixation test results agreed in 94% (144) of the serum samples tested. The 10 discrepant serum samples were retested and analyzed for ability to immunoprecipitate in vitro translation products from B. equi merozoite mRNA. Five discrepant results were clearly resolved in favor of the CI ELISA, and the remaining five discrepancies were not definitively resolved.     

Enzyme-linked immunosorbent assay with Brucella native hapten polysaccharide and smooth lipopolysaccharide.

Brucella melitensis native haptens (NH) are polysaccharides identical to the O-side chain of the smooth lipopolysaccharide (S-LPS) (E. Moreno, H. Mayer, and I. Moriyón, Infect. Immun. 55:2850-2853, 1987) which precipitate with sera from infected cattle but not from strain 19-vaccinated cattle. In the present work, NH was extracted by the hot-water method (R. Díaz, J. Toyos, M.D. Salvo, and M.L. Pardo, Ann. Rech. Vet. 12:35-39, 1981) and purified free of S-LPS and protein. Purified NH lacked the ability to coat polystyrene and sheep erythrocytes. In contrast, NH acylated with stearoyl chloride bound to both polystyrene and erythrocytes. By hemagglutination and enzyme-linked immunosorbent assay (ELISA), S-LPS and acylated NH gave similar results with blood sera from brucellosis-free, strain 19-vaccinated, and infected cattle. Moreover, a significant correlation between the results of NH ELISA and S-LPS ELISA was demonstrated with milk sera. However, in a competitive ELISA with milk sera, S-LPS in the liquid phase abrogated the binding of antibodies to acylated NH adsorbed to polystyrene, while NH in the liquid phase did not influence the binding of antibodies to polystyrene-adsorbed S-LPS. It is hypothesized that the different precipitations of NH and S-LPS with sera from infected or strain 19-vaccinated cattle are due to differences in the affinity of the antibodies produced upon vaccination or infection and in the physical state of aggregation of NH and S-LPS in aqueous solutions.     

Serological diagnosis of bovine neosporosis by Neospora caninum monoclonal antibody-based competitive inhibition enzyme-linked immunosorbent assay.

Neospora caninum, a protozoan parasite closely related to Toxoplasma gondii, causes abortion and congenital infection in cattle. To investigate specific methods of antemortem diagnosis, the antibody responses of infected cows were evaluated by immunoblot assay and competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA) by using a monoclonal antibody (MAb), MAb 4A4-2, against N. caninum tachyzoites. MAb 4A4-2 bound diffusely to the exterior surface of N. caninum tachyzoites and recognized a single 65-kDa band in immunoblots. MAb 4A4-2 was unreactive to antigens of two closely related apicomplexan protozoa, Toxoplasma gondii and Sarcocystis cruzi. Binding of MAb 4A4-2 was inhibited by mild periodate treatment of N. caninum antigen, demonstrating the carbohydrate nature of the epitope. Immunoblot analysis of N. caninum tachyzoite antigens with sera from cows with confirmed Neospora-induced abortion revealed at minimum 14 major antigens ranging from 11 to 175 kDa. Although the recognized antigens varied from cow to cow, antigens of 116, 65, and 25 kDa were detected in all cows with abortion confirmed to be caused by N. caninum. The binding of MAb 4A4-2 to N. caninum tachyzoite antigen was consistently inhibited by sera from Neospora-infected cows in a CI-ELISA format and was not inhibited by sera from Neospora antibody-negative cows. Furthermore, sera from cattle experimentally infected with T. gondii, S. cruzi, Sarcocystis hominis, or Sarcocystis hirsuta, which had cross-reactive antibodies recognizing multiple N. caninum antigens by immunoblot assay, did not inhibit binding of MAb 4A4-2 in the CI-ELISA. Thus, MAb 4A4-2 binds a carbohydrate epitope on a single N. caninum tachyzoite surface antigen that is recognized consistently and specifically by Neospora-infected cattle.     

A monoclonal antibody-based competitive enzyme-linked immunosorbent assay for detecting antibody production against avian reovirus protein sigmaA

An assay protocol based on a monoclonal antibody-based competitive enzyme-linked immunosorbent assay (MAb-based c-ELISA) for detection of antibody against avian reovirus protein sigmaA in chicken is described. After the conditions for MAb-based c-ELISA had been optimized, sera collected from birds that received live and inactivated avian reovirus vaccines in different combinations were tested for antibody response against virus protein sigmaA. The results show a high level of antibody against sigmaA was in both vaccinated specific pathogenic free (SPF) and vaccinated commercially reared birds as long as one of the vaccines administered was in an inactivated form. The high level of antibody production is indicated by a high percentage inhibition (PI) values in the sera of the birds; but no antibody production was found in birds which received live vaccine only, as indicated by the low PI values. In serum samples from SPF birds receiving vaccines that include an inactivated form of the vaccine, there is a good correlation between the PI values and serum neutralizing antibody (SN) titers. Again, this correlation was not observed in birds that received only live vaccine. The PI values of commercially reared birds receiving inactivated vaccine were significantly different from those of the mock-treated birds, but this was not the case when the birds received only live vaccine. Taken together, the results suggest that MAb-based c-ELISA may provide an alternative choice for determining the immune status of a vaccinated chicken flock as long as one of the vaccines used was inactivated, and thus would allow a more precise way to predict the appropriate time for vaccination.     

Comparison of lipopolysaccharide and outer membrane protein-lipopolysaccharide extracts in an enzyme-linked immunosorbent assay for the diagnosis of Brucella ovis infection.

Brucella ovis hot saline extracts and petroleum ether-chloroform-phenol lipopolysaccharide were compared in an enzyme-linked immunosorbent assay for the diagnosis of B. ovis ram epididymitis. Hot saline extracts detected greater numbers of infected rams. Chemical characterization of the antigens showed that, although both contained lipopolysaccharide, hot saline extracts also contained outer membrane proteins. These proteins were active as antigens in Western blot tests with sera of infected rams, and therefore they explained the better diagnostic results obtained with hot saline extracts. However, compared with lipopolysaccharide, hot saline extracts showed a higher degree of cross-reactivity with sera from smooth B. melitensis-infected animals. This observation might be explained by the presence of B. ovis outer membrane proteins in hot saline extracts which lack the specificity necessary for serological identification of the Brucella species present.     

Comparative evaluation of the enzyme-linked immunosorbent assay in the laboratory diagnosis of brucellosis.

An enzyme-linked immunosorbent assay was adapted to measure total and Brucella abortus-specific immunoglobulin M antibodies. The results were compared with those of conventional serological tests for B. abortus antibody on the sera of a number of normal controls, apparently healthy occupationally exposed workers, and patients with suspected acute brucellosis. Relative to other tests, the B. abortus enzyme-linked immunosorbent assay was found to be both highly sensitive and highly specific. The serological results obtained in occupationally exposed workers indicate a higher "normal range" for this group and therefore a possibility of false-positive results and overdiagnosis. It is therefore important to establish a separate "normal range" for occupationally exposed workers. Investigation of patients with acute brucellosis showed that the enzyme-linked immunosorbent assay for immunoglobulin M was the most sensitive serodiagnostic test and was likely to be of value in the serodiagnosis of acute brucellosis in occupationally exposed workers.     

Characterization of a monoclonal antibody against neopterin using an enzyme-linked immunosorbent assay with penicillinase as label

An active ester derivative of neopterin was prepared using 4-(N-maleimidomethyl) cyclohexan 4-carboxilic acid N-hydroxy succinimide ester (MCH-NHS), conjugated to bovine serum albumin (BSA) and injected for antibody production (for both monoclonal and polyclonal antibodies). High titer antibody producing spleen cells were removed and fused with myeloma cells of Sp2/0 origin. Neopterin was conjugated to the enzyme penicillinase by a one-step glutaraldehyde method, which was used as tracer. A novel enzyme immunoassay was developed using this conjugate to screen and characterize the monoclonal antibody (MAb) produced in these experiments. After limiting dilutions, it was found that antibody produced by one clone with a Ka value of 7.6 x 10-7 mol/L was specific for a number of structurally related molecules. This clone was found to be of IgG class and IgG2a subclass. The standard curvewas constructed with a sensitivity of 10 pg/well (100 pg/mL) covering up to 1 ng/mL.     

Recombinant nucleocapsid protein-based IgG enzyme-linked immunosorbent assay for the serological diagnosis of SARS

The recombinant nucleocapsid protein (rNP) of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) was expressed in a baculovirus system. The purified SARS-CoV rNP was used as an antigen for detection of SARS-CoV antibodies in IgG enzyme-linked immunosorbent assay (ELISA). The ELISA was evaluated in comparison with neutralizing antibody assay and the authentic SARS-CoV antigen-based IgG ELISA. Two-hundred and seventy-six serum samples were collected from health care workers in a hospital in which a nosocomial SARS outbreak took place and used for evaluation. The SARS-CoV rNP-based IgG ELISA has 92% of sensitivity and specificity compared with the neutralizing antibody assay and 94% sensitivity and specificity compared with the authentic SARS-CoV antigen-based IgG ELISA. The results suggest that the newly developed SARS-CoV rNP-based IgG ELISA is a valuable tool for the diagnosis and seroepidemiological study of SARS. The SARS-CoV rNP-based IgG ELISA has an advantage over the conventional IgG ELISA in that the antigen can be prepared by laboratory workers without the risk of infection.     

Development of a blocking enzyme-linked immunosorbent assay for the serological diagnosis of chicken anaemia virus

The development and evaluation of an enzyme-linked immunosorbent assay (ELISA) for the detection of serum antibody to chicken anaemia virus (CAV) are described. This test depends on the abilities of CAV-specific antibodies present in convalescent chicken serum to block the reaction between virus antigen, adsorbed to the ELISA plate. and a CAV-specific mouse monoclonal antibody (MAb), 2A9, that has been conjugated to horseradish peroxidase. The 2A9 MAb has been shown to react with 10 geographically different field isolates of CAV, a finding which indicates that the test will find worldwide application. In comparative experiments involving 525 serum samples from specific pathogen free and commercial breeder flocks, there was 98.5% agreement between the results obtained with the blocking ELISA and those obtained with an indirect ELISA developed previously in this laboratory. The blocking ELISA was found to have advantages in terms of speed and cost compared with the indirect ELISA format.     

A monoclonal antibody capture enzyme-linked immunosorbent assay for detecting antibodies to infectious bursal disease virus.

A monoclonal antibody capture enzyme-linked immunosorbent assay (mAb-ELISA) for antibodies to infectious bursal disease virus (IBDV) in chicken sera was developed and compared with conventional ELISA. When sera from farm chickens were tested by the two ELISAs and serum neutralization (SN), the correlation rate between SN and mAb-ELISA was 100% (49/49), and that between SN and conventional ELISA was 81.6% (40/49). In mAb-ELISA, all of the sera that were antibody-negative by SN had low absorbance values (below 0.05), and the absorbance values correlated closely with the SN titers. In the conventional ELISA, however, the sera antibody-negative by SN had various absorbance values ranging from 0.06 to 0.32. mAb-ELISA had much lower non-specific reactions than the conventional ELISA against sera from IBD-negative chickens.     

Monoclonal antibody enzyme-linked immunosorbent assay for specific identification and typing of subgroup F adenoviruses.

Monoclonal antibody specific for subgroup F enteric adenoviruses (EAds) was prepared by fusing P3-NS1/Ag4-1 mouse myeloma cells with lymphocytes from BALB/c mice immunized with G1105, an adenovirus type 41 (Ad41) strain. Monoclone 3F11/2H9, which specifically recognized Ad41, was successfully used as detector antibody in an enzyme-linked immunosorbent assay (ELISA). Additionally, previously prepared monoclones 5D8/2C2 and 2H6/1E11, recognizing Ad40 plus Ad41 and Ad40 alone, respectively, were used to study stool and/or tissue culture specimens from 106 patients with adenovirus-positive gastroenteritis. By ELISA, 91 had EAds (22 were Ad40 and 69 were Ad41) and 15 had non-EAds. ELISA results were in concordance with restriction endonuclease results for 38 of 39 specimens, with dot blot data for 19 of 20 specimens, and with neutralization test results for 74 of 78 specimens. ELISA was at least 10-fold more sensitive than direct electron microscopy was for the detection of EAds in stool specimens.