基于重组P113蛋白的山羊抗绵羊肺炎支原体抗体间接ELISA检测方法的建立及应用

Development of an Indirect ELISA for detection of Mycoplasma ovipneumoniae antibody in goats based on recombinant protein P113

绵羊肺炎支原体(MO)是引起绵羊、山羊等小反刍动物非典型肺炎的病原。为建立基于黏附素P113蛋白的检测MO抗体的间接ELISA方法,本研究采用原核表达并纯化的MO重组P113蛋白(rP113)作为包被抗原,经优化条件后建立了间接ELISA方法(rP113-ELISA),并对该方法的特异性、敏感性和重复性进行评价。特异性试验结果显示该方法对MO之外的常见支原体和其他病原的抗体检测结果均为阴性,特异性较强。敏感性试验结果显示,13200稀释的阳性血清仍能检出,敏感性高。该方法重复性试验结果显示组内和组间变异系数均小于5%,重复性较好。利用rP113-ELISA和间接血凝方法(IHA)对MO疫苗免疫山羊的抗体和80份临床样品进行检测,结果显示检测MO疫苗免疫抗体的消长规律结果与IHA检测结果一致;对80份临床山羊血清样品检测结果与IHA检测结果阳性符合率为87.5%,阴性符合率为82.1%,rP113-ELISA敏感性高于IHA。本研究为MO抗体检测提供了一种特异、灵敏和稳定的方法。

Mycoplasma ovipneumoniae(M.ovipneumoniae)can cause non-typical pneumoniae in sheep,goats and other small ruminants.The objective of this study was to develop an indirect ELISA(iELISA)for detecting antibodies against M.ovipneumoniae based on its adhesin protein P113.The purified recombinant protein P113(rP113)of M.ovipneumoniae was used as coating antigen.The iELISA,named rP113-ELISA,was established by optimizing the reaction conditions to detect the specific antibodies against M.ovipneumoniae.Subsequently,specificity,sensitivity and repeatability of the rP113-ELISA were evaluated.Furthermore,the rP113-ELISA was validated and compared with IHA method by detecting antibodies against M.ovipneumoniae from the serum of vaccinated goats and clinical goat samples.The specificity test results showed that the rP113-ELISA presented good specificity.The method could only exhibit positive results for sera against M.ovipeumoniae,while showing negative results for all sera against other pathogens.The sensitivity test result indicated good sensitivity of the rP113-ELISA,with the detection limit for positive serum dilution 1􀏑3200.The rP113-ELISA also demonstrated good reproducibility with intra-and inter-assay CV of less than 5%.The vaccinated goat sera detection results of both methods showed the same growth and decline pattern.For 80 clinical samples,both methods' positive and negative coincidence rates were 87.5%and 82.1%,respectively.The rP113-ELISA was more sensitive than IHA.The rP113-ELISA developed in this study provided a specific,sensitive and reliable method for detecting and quantifying antibodies against M.ovipeumoniae.