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

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

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3 d,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。

Objective To generate monoclonal antibodies(mAbs)against coxsackievirus A2(CV-A2)and to establish a quantitative antigen ELISA.Methods Mice were immunized by purified CV-A2 virions to obtain hybridomas secreting anti-CV-A2 mAbs.Linear range of established CV-A2 ELISA 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 against CV-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 and low 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 were 90.31%-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.