A solid-phase radioimmunoassay for the determination of bacterial-specific antibodies within different immunoglobulin classes: application to bovine Brucella abortus antibodies.

A solid-phase radioimmunoassay (RIA) has been developed for quantitation of class-specific antibodies against bacteria. Brucella abortus cells were used to sensitize glass tubes after sedimentation, drying and methanol fixation. Bovine antibodies that attached to the bacteria were detected by binding of a rabbit antiserum specific for each class or subclass of bovine immunoglobulins followed by binding of 125I sheep anti-rabbit immunoglobulin reagent. This three-step method was adapted as it was very sensitive (less than 0.3 picogram antibody/ml), gave the best discrimination between sample and background counts and elminated the need for several labeled rabbit anti-bovine immunoglobulin class-specific antisera. Parameters affecting quantitation of the assay were investigated: nature and preparation of the test tube, quantity and fixation of Brucella, temperature and incubation time, incubation volume, concentration of reagents. The RIA and the complement fixation test (CFT) were applied to sera obtained at repeated intervals from seven heifers experimentally infected with Brucella abortus during gestation. Rabbit antisera specific for bovine IgG1 were used in the RIA because of antibodies of this subclass are the predominant type implicated in the CFT. The kinetic of appearance of Brucella specific antibodies were very significantly correlated for the two tests (r = 0.056, P less than 0.01) demonstrating the validity of the proposed RIA. This test was 10,000 times more sensitive than CFT.