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目的提取羊布鲁菌标准强毒株16m膜蛋白,分析其反应原性,并建立血清抗体间接ELISA检测方法。方法利用碳酸盐方法提取羊布鲁菌标准强毒株16m膜蛋白,12%SDS-PAGE分析其相对分子质量范围;用自然感染羊布鲁菌的阳性血清进行Westernblot分析,初步确定膜蛋白组分中能够发生免疫反应的条带;用方阵滴定法确定抗原和抗体的最佳稀释度,建立间接ELISA检测方法,并进行特异性和精密性验证;检测免疫豚鼠的血清抗体效价;对临床上采集的羊血清样品进行检测,并与虎红平板凝集试验结果进行比较。结果分离的羊布鲁菌膜蛋白浓度为9.4μg/μl;SDS-PAGE分析显示,羊布鲁菌膜蛋白的相对分子质量主要集中在17000～80000之间;Westernblot分析显示多条特异性反应条带。膜蛋白抗原的最佳稀释度为1∶1280,抗体最佳稀释度为1∶10。所建立的ELISA方法特异性和精密性良好。ELISA检测豚鼠血清效价为1∶64000。检测临床510份羊血清样品,阳性率为86.3%,高于虎红平板凝集试验检出的阳性率(41.2%)。结论已成功提取了羊布鲁菌膜蛋白,其具有良好的反应原性,并建立了血清抗体间接ELISA检测方法。
Objective To extract the 16 m membrane protein of the standard strain of brucella brucellosis and analyze its reactivity, and to establish an indirect ELISA for detection of serum antibody. Methods The carbonate protein was used to extract the 16M membrane protein of Brucella abortus and the relative molecular mass was analyzed by 12% SDS-PAGE. Western Blot analysis was carried out with the positive serum of naturally infected Brucella brucei, Antibody immunoreactions were detected in the sub-band; the use of square titration to determine the optimal dilution of antigen and antibody, the establishment of indirect ELISA detection methods, specificity and precision verification; detection of guinea pig serum antibody titer; Clinically collected sheep serum samples were tested and compared with tiger red plate agglutination test results. Results The concentration of M. brucei membrane protein isolated was 9.4 μg / μl. SDS-PAGE analysis showed that the relative molecular mass of M. brucei membrane protein was mainly between 17000 and 80 000. Western blot analysis showed that multiple specific reaction strips band. The optimal dilution of membrane protein antigen is 1:1280 and the optimal dilution of antibody is 1:10. The established ELISA method has good specificity and precision. ELISA serum guinea pig titer 1:64000. 510 samples of clinical serum sheep serum samples, the positive rate was 86.3%, higher than the tiger plate agglutination test was positive (41.2%). Conclusion The M. brucei membrane protein has been successfully extracted and has good reactionogenicity. A indirect ELISA method for serum antibody was established.