Fluorescence polarization assay for the diagnosis of anti-Brucella abortus antibodies in cattle serum: adaptation for its use in microplates.

GUTIÉRREZ, SILVINA ELENA; LUTZELSCHWAB, CLAUDIA; RODRIGUEZ, EDGARDO; DIAZ, ALEJANDRA; ESTEIN, SILVIA

Ciudad Autónoma de Buenos Aires

Congreso; Brucellosis 2011, International Research Conference; 2011

Asociación Argentina de Microbiología

Bovine brucellosis has been present in Argentina for many years. The national Programme of Control and eradication of bovine brucellosis involves the vaccination of calves between 3 and 8 month of age with B. abortus S19 and identification and culling of infected cattle. BPA is used as screening test, while SAT and 2 ME tests are used as confirmatory, and results of Complement Fixation test are considered conclusive of infection. These tests have widely contributed to the control of the disease, but have some disadvantages like subjectivity, and the delay of 48 h in having the result. To overcome this problems primary binding assays like ELISAs and Fluorescence polarization assay (FPA) have been developed and recently incorporated into the Official Control Programme(1). The FPA assay has been proposed as an obligatory test in the international commerce of cattle. The aim of the present work was to evaluate a commercially available kit for FPA assay in a 96-well microplate test using different dilutions of serum and antigen, and a panel of positive and negative sera from different regions of Argentina. Serum samples were obtained from 124 cows and 20 bulls belonging to free and infected herds. A commercially available antigen (the O-polysaccharide from B. abortus 1119-3 conjugated with FITC) was obtained from a local laboratory. The test was carried out in black 96-well microplates following the recommendations of the manufacturers. Two dilutions of reagents were assayed: a 1:100 dilution of both serum and antigen, and a combination of a 1:10 dilution of serum with 1:20 dilution of antigen (200 µl final volume). The FP was measured with a Beckman DTX 880 multimode reader. Samples were considered positive when the mP obtained was over 105, negative if it was below 94 mP, and indeterminate if they were between 94 and 105 mP. One hundred fifteen of the 144 samples scored negative in the two variants of the microplate test, 18 scored positive in both tests and 4 samples scored positive when tested at a 1:100 dilution, but negative if tested at a 1:10 dilution. These differences were not statistically significant (Mc Nemar´s test, p=0,1336). In conclusion, the microplate FPA assay conducted at a 1:100 dilution of both serum and antigen (conditions recommended for the single tube analyzer) was as efficient as the test performed with 1:10 and 1:20 dilutions of serum and antigen respectively. (1) Nielsen K. y col. (1998) Veterinary Immunology and Immunopathology  66: 321-329