Second generation competitive enzyme immunoassay for detection of bovine antibody to Brucella abortus

Evaluation of Indirect Enzyme-Linked Immunosorbent Assays and IgG Avidity Assays Using a Protein A-Peroxidase Conjugate for Serological Distinction between Brucella abortus S19-Vaccinated and -Infected Cows

“…In addition, it is possible that the residual percentages of the positivity rates found with animals from GIV may be due to cross-reactivity, due mainly to the fact that the antigen preparation used in the present work was constituted by B. abortus S-LPS. It was already demonstrated in the literature that a number of bacteria induce antibody responses that cause false-positive reactions in tests for brucellosis, mostly when using B. abortus S-LPS (28)(29)(30)32). As the serum samples of group IV came from animals with epidemiological characteristics well defined, i.e., unvaccinated animals from a brucellosis-free region, the possibility of infection or residual vaccination interference in the results obtained in the present work is low.…”

“…in serum samples from various species of domestic animals, including cattle, sheep, goats, and pigs (30). Although the PAG conjugate is available from several commercial sources and despite its added advantage over the anti-immunoglobulin conjugate, in addition to the fact that S-LPS can also be standardized, iELISAs using this universal reagent were able to exclude partially antibodies in vaccinated cattle, since vaccinate specificity was around 50% (29). The use of monoclonal antibodies that do not react with PAG has been standardized in a second generation of the cELISA for detection of bovine antibody to B. abortus, thus eliminating reagent variables as the requirement for polyclonal anti-mouse enzyme conjugate for detection.…”

“…Immunoglobulin-binding proteins, such as protein A, protein G, or recombinant protein A/G labeled with horseradish peroxidase (HRPO), have been used as valuable tools in the ELISA for detection of anti-Brucella antibodies in various animal species (29,30). While protein G reacts with both bovine IgG subclasses (IgG1 and IgG2), protein A reacts more specifically with the IgG2 subclass, whose levels increase with the intensity of antigen exposure in Brucella infection (22,32).…”

“…The optical density (OD) was read at 405 nm using a plate reader (Titertek Multiskan Plus; Flow Laboratories, McLean, VA). Antibody titers were expressed as previously described (29,30) as the reactivity index (RI) relative to the mean OD of positive-control sera, according to the following formula: RI ϭ (mean OD sample/mean OD positive control) ϫ 100.…”

Development and Comparative Evaluation of a Competitive ELISA with Rose Bengal Test and a Commercial Indirect ELISA for Serological Diagnosis of Brucellosis

“…The sensitivity and specificity estimate on comparison with I-ELISA were 92.3-97.5% and 90.3-94.0% respectively. Standardization and harmonization of serological tests used for the presumptive diagnosis of infectious diseases has always been difficult due to variability in reagents and subjective assessment [10]. However, development of such assays with monoclonal antibodies is time consuming and expensive.…”