Abstract: 【Objective】 To develop a high-throughput indirect ELISA diagnostic method for detecting antibodies against bovine brucella. 【Method】 The indirect ELISA method was developed by using the purified LPS from brucella S2 strains as the coating antigen, and confirming coating concentration of antigen, coating buffer, coating time, blocking buffer, sample diluent buffer, serum dilution, rabbit anti-bovine antibody diluent buffer, rabbit anti-bovine antibody diluting concentration, and the reaction time of substrate by chessboard titration. We detected 176 brucella positive samples and 132 negative samples using the developed method. The detecting result was analyzed by SPSS17.0. At the same time, ROC curve and area under the curve, sensitivity and specificity values, the critical value by Youden index was also constructed. The into-batch and inter-batch repeatability were also evaluated by using the positive and negative control serum. We detected the 1 200 clinical samples to compare the coincidence rate between bovine brucellosis indirect ELISA antigen kits and the commercial imported kits. 【Result】 The optimal conditions for each of the components of the indirect ELISA kit was described below. The coating concentration of antigen is 10 μg·mL^-1, the coating buffer is carbonate buffer solution, the coating time is 16 hour in 2-8℃, the serum degree of dilution is 1﹕50, the blocking buffer is PBS containing 3% gelatin, and the sample dilution buffer is PBS containing 0.5%sucrose, the rabbit anti-bovine antibody diluent buffer is PBST containing 5% horse serum, the rabbit anti-bovine antibody diluting concentration is 1﹕20 000 and the reaction time of substrate is 15 minutes. The critical value is P%=20% (P%=OD₄₅₀ sample/OD₄₅₀ positive control×100%). In this critical value, the sensitivity and specificity values of the developed method is 97.7% and 95.5% respectively. By detecting the 1 200 clinical samples, it is showed that the coincidence rate was 96.25% between our indirect ELISA antigen kit and the commercial imported kit. 【Conclusion】 We established the indirect ELISA method for detecting bovine brucella antibody with good specificity and sensitivity.

Key words: brucella, indirect ELISA, specificity, sensitivity