猪嗜血支原体酶标单抗阻断ELISA检测方法的建立  

Development of Blocking ELISA for Mycoplasma suis with Peroxidase-Labelled Monoclonal Antibody Against MSG1 Protein

作　　者：张长莹[1] 李玉峰[1] 姜平[1] 

机构地区：[1]南京农业大学动物疫病诊断与免疫重点开放实验室,南京210095

出　　处：《畜牧兽医学报》2011年第11期1591-1597,共7页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：公益性行业(农业)科研专项项目(200903036-10)

摘　　要：为确定猪嗜血支原体(M.suis)在临床的感染情况和进行流行病学调查提供检测方法,本研究以纯化的猪嗜血支原体重组MSG1蛋白和辣根过氧化酶标记的MSG1蛋白单抗建立了检测猪嗜血支原体的阻断ELISA方法。经筛选确定,抗原最佳包被浓度为0.5μg.mL-1,待检血清最佳稀释度为1∶1,作用时间为37℃1.5h,酶标单抗最佳稀释度为1∶10 000,作用时间为37℃1h。通过对100份M.suis阴性血清阻断ELISA检测结果进行统计学分析,确定本方法的判定标准为当阻断率PI≥23.71%时判为阳性,PI≤36.35%时判为阴性,23.71%<PI<36.36%时判为可疑。敏感性、特异性和重复性试验证明该检测方法敏感性高、特异性强、可重复性好,可用于M.suis流行病学调查和疾病诊断。To provide a serological method for detecting antibodies against Mycoplasma suis（M.suis）,a blocking ELISA was developed based on a monoclonal antibody（MAb） to M.suis-MSG1 protein.The conditions for each step were optimized.The optimal concentration of the coating antigen was 0.5 μg·mL-1;sera samples diluted 1 fold and incubated 1.5 h at 37 ℃;MAb-HRP dilution is 10 000 fold and incubated 1 h at 37 ℃.The blocking results of one hundred M.suis negative sera samples were statistically analyzed,and the cutoff of blocking ELISA was determined that the samples presenting a percentage inhibition of ≥ 23.71% were considered positive;samples with a calculated percentage inhibition of ≤ 36.35% were rated negative and those presenting a blocking effect between 23.71% and 36.35% were considered inconclusive.The Blocking ELISA proved to be specific,sensitive and it showed high reproducibility and low variability.This method will be useful in clinical detection and epidemiological study on M.suis.

关 键 词：猪嗜血支原体 MSG1蛋白 酶标单抗 阻断ELISA 

分 类 号：S852.62[农业科学—基础兽医学]