抗柯萨奇病毒A组2型单克隆抗体制备及ELISA抗原检测方法的建立

目的  制备抗柯萨奇病毒A组2型( coxsackievirus A2,CV-A2)单克隆抗体（单抗），建立CV-A2抗原检测方法。方法  用纯化后的CV-A2全病毒颗粒免疫小鼠，筛选获得抗CV-A2单抗。建立CV-A2抗原检测方法，确定线性范围，对其准确度、精密度、稳定性、专属性进行验证。用 ELISA检测病毒颗粒纯化过程中样品的抗原含量。结果  制备了高效价的抗CV-A2单抗并建立 ELISA抗原检测方法，检测范围为5.00~320.00 ng/ml。高、中、低3个浓度样品准确度验证回收率在89.58%~104.78%之间。重复性验证变异系数分别为2.10%、2.47%、6.18%。中间精密度验证变异系数分别为2.89%、2.69%、1.94%。耐用性验证回收率在84.26%~114.21%之间。包被微孔板于37℃放置3d,样品回收率在90.31%~103.11%之间。专属性验证结果显示该方法只识别CV-A2抗原，与其他抗原均无交叉反应。结论  建立并验证了CV-A2 ELISA抗原检测方法，可应用于病毒纯化过程中样品的抗原检测，还可应用于含CV-A2的手足口病多价疫苗的CV-A2抗原含量检测。

Generation of anti-coxsackievirus A2 monoclonal antibodies and establishment of quantitative antigen ELISA

Objective To generate monoclonal antibodies (mAbs) against coxsackievirus A2(CV-A2)and to establish a quantitative antigen ELISA. Methods Mice were immunized by purifiedCV-A2 virions to obtain hybridomas secreting anti-CV-A2 mAbs. Linear range of established CV-A2ELISA was determined, and its accuracy, precision, stability and specificity were validated. Samples in all purification steps of CV-A2 particles were detected. Results High binding-efficiency mAbs againstCV-A2 were generated. A quantitative ELISA for antigen detection was established. The detection range was 5. 00-320. 00 ng/ml. The recovery rates for accuracy verification of samples with high, medium andlow concentrations were between 89. 58% and 104. 78%. Their respective coefficients of variation (CVs)of repeatability verification were 2. 10%, 2. 47% and 6. 18%, and CVs of intermediate precision verification were 2. 89%, 2. 69% and 1. 94%. The recovery rates of durability were 84. 26%-114.21%. Coated microtiter plate was placed at 37 ℃ for 3 d and samples recovery rates were90.31%-103.11%. Specificity verification showed that the method only recognized CV-A2 antigen, and did not cross react with other antigens. Conclusion A CV-A2 quantitative ELISA is established, which can be applied to antigen detection in samples at different purification stages of CV-A2, and in multivalent hand-foot-mouth disease vaccines containing CV-A2.