A HOMOGENEOUS FLUORESCENCE POLARIZATION ASSAY FOR DETECTION OF ANTIBODY TO BRUCELLA-ABORTUS

A homogeneous fluorescence polarization (FP) assay (FPA) was developed for detection of antibody in bovine sera to Brucella abortus. The ass ay used O-polysaccharide prepared from B. abortus lipopolysaccharide i n the molecular weight range of 20-30 kDa which was conjugated with fl uorescein isothiocyanate and used as a tracer. Fluorescence polarizati on was measured with a FPM-1 fluorescence polarization analyzer. Sampl e (20 mu l) was added to 2.0 ml of diluent buffer at ambient temperatu re. A serum blank reading was taken and tracer (10 mu l) to yield appr ox. 1.5 nM fluorescein equivalents was added. The FP of the tracer was determined after a period of greater than 2 min. A positive reaction was indicated by a significant elevation of the FP reading over the ne gative control. In a blind study, 9480 bovine sera were tested in addi tion to sets of four controls which were included with each lot of 100 samples tested. The controls were a strong positive, a weak positive, a negative and a serum derived from a B. abortus strain 19 vaccinated cow. Test sera included 8669 sera from Canadian cattle which were neg ative by routine serological tests, 561 sera from cows from which B. a bortus had been isolated either from tissues or milk and 250 sera from cattle previously vaccinated with B. abortus strain 19 at various tim es, One lot of O-polysaccharide tracer was used for all tests. The ini tial cut-off for negative samples in the fluorescence polarization ass ay was set at 107.2 mP. This resulted in a sensitivity estimate of 98. 1 +/- 1.1% and the specificity was 99.8 +/- 0.09%. After decoding the samples and retesting false positive and negative reactions, the sensi tivity estimate was 98.5 +/- 1.0% and the specificity was 100%. It bec ame evident that the initial cut-off value was set too high and, using ROC analysis, a cut-off of 90 mP increased the sensitivity to 99.02% while the specificity decreased to 99.96%. Of the 250 sera from vaccin ated cattle, 248 were negative giving a point specificity value of 99. 2%.