检测多杀性巴氏杆菌毒素抗体的单抗竞争ELISA方法的建立  

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

作　　者：卢顺[1] 向敏[1] 吴斌[1] 赵战勤[1] 汤细彪[1] 但汉并[1] 张建民[1] 何华[1] 陈焕春[1] 

机构地区：[1]华中农业大学动物医学院农业微生物学国家重点实验室,武汉430070

出　　处：《农业生物技术学报》2008年第4期555-561,共7页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金项目(No.30471292)资助。

摘　　要：用纯化的产毒素多杀性巴氏杆菌(toxigenie Pasteurella multocida,T^+Pm)毒素基因片段的原核表达产物免疫小鼠(Mus muslucus)制备单抗,并利用表达蛋白和辣根过氧化物酶(HRP)标记的单抗AH12建立了竞争ELISA方法检测多杀性巴氏杆菌毒素抗体。经实验确定抗原包被浓度223 ng/mL,待检血清最佳稀释度1∶2,酶标单抗AH12工作浓度1∶3 200,血清抑制率大于50%为阳性。稳定性试验表明,批间和批内变异系数均小于10%。应用竞争ELISA和细胞毒性中和试验同时检测50份已知血清和82份未知血清,已知血清检测结果显示两者阳性符合率为90%,阴性符合率为100%;未知血清检测结果显示竞争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%。表明所建立的单抗竞争ELISA方法具有敏感、特异、稳定和简便等特点,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。Monoclonal antibodies against Pasteurella multocida toxin （PMT） were produced using Escherichia coli expressed recombinant PMT proteins as immunogens. On the basis of the recombinant protein and HRP-lablled monoclonal antibody AHI2 against Pasteurella multocida toxin, a competitive ELISA was developed to detect antibodies against Pasteurella multocida toxin. Optimium conditions of the ELISA were as following：the concentration of recombinant protein for coating ELISA plates was 223 ng/mL; the best dilution of serum was 1：2; The working titer for HRP-labelled monoclonal antibody AHI 2 was 1：3 200; More than 50% of inhibition rate was positive judging standard. Stability test showed that both inter-assay and intra-assay coefficient variation were below 10%. Both competitive ELISA and in vitro neutralization assay were used to detect 50 known and 82 unknown sera. The results indicated that the positive coincidence and the negative coincidence of 50 known sera were 90% and 100%, respectively; 40.2% of 82 unknown sera were positive detected by competitive ELISA, while 36.6% of them were positive by in vitro neutralization assay, and the coincidence rate was 91.5%. These results showed that the competitive ELISA possessed the advantages of high sensitivity, specificity, stability and briefness. It will be practical for pig diagnosis, immunity effect evaluation and epidemiological survey.

关 键 词：多杀性巴氏杆菌毒素 单克隆抗体 单抗竞争ELISA 抗体检测 

分 类 号：S188[农业科学—农业基础科学]