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 assay used O-polysaccharide prepared from B. abortus lipopolysaccharide in the molecular weight range of 20-30 kDa which was conjugated with fluorescein isothiocyanate and used as a tracer. Fluorescence polarization was measured with a FPM-1 fluorescence polarization analyzer. Sample (20 microliters) was added to 2.0 ml of diluent buffer at ambient temperature. A serum blank reading was taken and tracer (10 microliters) to yield approx. 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 negative control. In a blind study, 9480 bovine sera were tested in addition 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 negative by routine serological tests, 561 sera from cows from which B. abortus had been isolated either from tissues or milk and 250 sera from cattle previously vaccinated with B. abortus strain 19 at various times. One lot of O-polysaccharide tracer was used for all tests. The initial cut-off for negative samples in the fluorescence polarization assay 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 sensitivity estimate was 98.5 +/- 1.0% and the specificity was 100%. It became 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 vaccinated cattle, 248 were negative giving a point specificity value of 99.2%.

Comparison of serological tests for the detection of ovine and caprine antibody to Brucella melitensis.

The indirect enzyme-linked immunosorbent assay (IELISA), the competitive enzyme-linked immunosorbent assay (CELISA) and the fluorescence polarisation assay (FPA) were evaluated with sera from sheep experimentally infected with Brucella melitensis and negative Canadian sheep. The sensitivity and specificity of the assays were as follows: IELISA: 91.7% and 97.6%, CELISA: 75.0% and 99.8% and FPA: 91.7% and 89.5%. Sera from the same experimental population were divided according to serological reaction in the rose bengal agglutination test (RBT) and the complement fixation test (CFT). Reactivity relative to the RBT positive and CFT positive sera were as follows: IELISA: 99.7%, CELISA: 93.2% and FPA: 99.1%. Since sera from goats with proven B. melitensis infection were not available, 699 sera from goats judged positive in the buffered antigen plate agglutination test (BPAT) and CFT and 982 BPAT/CFT negative Canadian goats were used. The sensitivity and specificity of the assays relative to the BPAT and CFT positive sera were: IELISA: 99.4% and 98.0%, CELISA: 95.4% and 97.1% and FPA: 92.7% and 99.8%.

Validation of the fluorescence polarization assay and comparison to other serological assays for the detection of serum antibodies to Brucella abortus in bison.

A number of serological tests were compared for the detection of antibodies to Brucella abortus in bison (Bison bison). The performance of the fluorescence polarization assay (FPA) in both the preliminary evaluation and a subsequent blind validation indicated that this test was the most suitable for serological diagnosis of brucellosis in bison. The sensitivity and specificity in the preliminary evaluation were 92.1% and 99.4%, respectively. The sensitivity and specificity in a subsequent blind study were 96.3% and 97.6%, respectively. In a double blind study conducted on bison vaccinated with B. abortus strain 19, the data suggests that the FPA can differentiate bison infected with B. abortus from bison vaccinated with B. abortus strain 19. Both the indirect immunoassay (IELISA) and the competitive immunoassay (CELISA) performed nearly as well as the FPA. The buffered antigen plate agglutination test (BPAT) and the complement fixation test (CFT) did not perform as well as the FPA, CELISA or the IELISA in both studies. The FPA is a homogeneous assay eliminating the washing steps and reducing incubation to minutes rather than hours saving on time, equipment, materials, reagents and cost. These attributes, together, with its excellent sensitivity and specificity make the FPA an attractive test for the detection of serum antibodies to Brucella abortus in bison.

Evaluation of the fluorescence polarization assay and comparison to other serological assays for detection of brucellosis in cervids.

The complement fixation test (CFT), competitive enzyme immunoassay (CELISA), indirect enzyme immunoassay (IELISA) and fluorescence polarization assay (FPA) were evaluated for the detection of antibodies to Brucella abortus and Brucella suis biotype 4 in caribou (Rangifer tarandus caribou), elk (Cervus elapus), red deer (Cervus elapus), and reindeer (Rangifer tarandus tarandus). When combining the data the FPA and the CELISA were determined to be the most suitable tests for serodiagnosis of Cervidae. The overall actual sensitivity of the CFT and the IELISA was 100%. The overall actual sensitivity for the CELISA and FPA was 99%. The overall relative specificity of the CFT (including treatment of anti-complementary data as positive or negative for analysis), the CELISA, the IELISA and the FPA were 65%, 93%, 99%, 99%, and 99%, respectively. The specificities of the buffered plate agglutination test (BPAT), the CFT, the CELISA, the FPA and the IELISA for 55 elk vaccinated with B. abortus strain 19 and tested 4 mo post vaccination were 14%, 31%, 51%, 84%, and 2%, respectively. The FPA is the diagnostic test of choice because it has sensitivity and specificity values comparable to the CELISA; it has the capability to distinguish vaccinal antibody and antibody resulting from exposure to cross-reacting organisms such as Yersinia enterocolitica 0:9 from antibody to Brucella spp. in most cases; it is technically simple to do; it is adaptable to field use and it is relatively inexpensive.