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.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
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)
作者简介
张长莹（1986-），女，山东济南人，硕士生，主要从事动物传染病预防控制研究，E-mail：2009107066@njau．edu．cn
通讯作者：李玉峰，副教授，E-mail：yufengli@njau．edu．cn；Tel：025—84395504
