猪肺炎支原体抗原定量ELISA检测方法的建立

发布时间：2018-07-13 18:29

【摘要】：为建立猪肺炎支原体抗原的快速检测方法,本研究以抗猪肺炎支原体P46蛋白单克隆抗体为包被抗体,抗P46蛋白多克隆抗体为检测抗体,建立了猪肺炎支原体双抗体夹心ELISA方法。通过优化后的反应条件为:抗猪肺炎支原体P46蛋白单克隆抗体包被浓度为2.5μg/mL,抗P46蛋白多抗及酶标二抗稀释度分别为1∶40 000和1∶10 000。该ELISA的重复性变异系数小于10%,最低检出量为9.754 ng/mL,与猪鼻支原体、猪滑液支原体、絮状支原体等无交叉反应。使用该ELISA方法对人工稀释的培养物中抗原含量进行检测,通过单点法和对样品倍比稀释后进行双平行线拟合两种方法计算,得出两种计算方法结果均与真实值基本相符,但双平行线法检测比单点法检测更加稳定。本研究建立的双抗体夹心ELISA方法为猪肺炎支原体的定量检测提供了有效手段,可用于猪支原体肺炎灭活疫苗生产过程中抗原的检测,为灭活疫苗生产中的质量控制提供了便利。
[Abstract]:In order to establish a rapid method for the detection of mycoplasma pneumoniae antigen, a sandwich Elisa was developed using monoclonal antibody against P46 protein and polyclonal antibody against P46 protein as coating antibody and polyclonal antibody against P46 protein as detection antibody. The optimized reaction conditions were as follows: the concentration of monoclonal antibody against P46 protein of Mycoplasma suis was 2.5 渭 g / mL, the dilution of anti-P46 multi-antibody and enzyme-labeled second antibody were 1:40 and 1:10 000, respectively. The repeatability coefficient of variation of Elisa was less than 10, and the lowest detectable amount was 9.754 ng / mL. There was no cross reaction with mycoplasma suis, mycoplasma synovium and mycoplasma flocculus. The Elisa method was used to detect the antigen content in the artificially diluted culture. The two methods were calculated by single point method and double parallel line fitting after dilution of the double ratio of the sample. The results of the two methods were in good agreement with the real value. But the double parallel line method is more stable than the single point method. The double antibody sandwich Elisa method established in this study provides an effective method for quantitative detection of mycoplasma pneumoniae, can be used for the detection of antigen in the production of inactivated vaccine of mycoplasma pneumoniae, and is convenient for the quality control in the production of inactivated vaccine.
【作者单位】：南京农业大学动物医学院;江苏省农业科学院兽医研究所农业部兽用生物制品工程技术重点实验室国家兽用生物制品工程技术研究中心;江苏省肉类生产与加工质量安全控制协同创新中心;
【基金】：国家自然科学基金面上项目(31370208) 江苏省农业科技自主创新资金项目(CX(14)2040)
【分类号】：S852.62

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