检测多杀性巴氏杆菌毒素抗体的单抗竞争ELISA方法的建立

本研究以纯化的多杀性巴氏杆菌毒素基因片段的原核表达产物作为抗原免疫小鼠制备单抗，并利用表达蛋白和多杀性巴氏杆菌毒素单抗酶结合物建立了竞争ELISA方法检测多杀性巴氏杆菌毒素抗体。经过研究确定抗原包被浓度为223ng／mL，待检血清最佳稀释度为1：2，酶标单抗工作浓度为1：3200，血清抑制率大于50%为阳性。应用单抗竞争ELISA和细胞毒性中和试验同时对82份血清进行猪多杀性巴氏杆菌毒素抗体检测，竞争ELISA的检出率为40.2％，细胞毒性中和试验检出率为36.6％，两者符合率达91.5％。试验结果表明，该ELISA方法特异性强，敏感性高，稳定性和重复性好，操作简便。本方法的建立在实验室诊断的标准化、猪群萎缩性鼻炎疫苗免疫效果的评价及流行病学调查方面具有应用价值。

Establishment of a McAb-based Competitive ELISA for the Detection of Antibodies against Pasteurella multocida Toxin

Abstract：Monoclonal antibodies against Pasteurella multocida toxin(PMT) were produced by using E.coli expressed recombinant PMT proteins as immunogens.on the basis of recombinant protein expressed by E.coli and HRP-labled monoclonal antibody against Pasteurella multocida toxin,a competitive ELISA was developed to detect antibodies against.Pasteurella multocida toxin.Optimium conditions of the ELISA Were developed as following：the concentration of recombinant for coating ELISA plates is 223 ng /mL;the best dilution of serum to be tested was 1:2; the working titer for HRP-labelled monoclonal antibody was 1:3200;>50% of inhibition rate was the positive judging standard.A total of 82 serum samples were detected in parallel by both the competitive ELISA and in vitro neutralization assay.40.2％ of them were detected as positive by competitive ELISA,while 36.6％ of them were positive by in vitro neutralization assay.The coincidence rate of the two assays is 91.5%.These results showed that the cELISA has the advantage of higher sensitivity，specificity，reproducibility and stability.It would be very useful in diagnosis，surveillance of PMT antibodies and epidemiological survey.