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目的筛选甲型肝炎病毒(Hepatitis A virus,HAV)单克隆抗体(MAb),并初步建立HAV双抗体夹心ELISA定量检测方法。方法用抗体亚型鉴定试剂盒鉴定MAb亚型;通过Western blot法鉴定MAb的特异性;SPA亲和层析法纯化MAb腹水,检测MAb的抗原识别表位;细胞培养法检测MAb的中和效价。确定双抗体夹心法包被物与酶标MAb的浓度,初步建立HAV双抗体夹心ELISA定量检测方法,并考察方法的线性范围。结果 12株HAV单抗中,1株为IgM型,3株为IgG3型,3株为IgG2a型,5株为IgG2b型;10株MAb可特异性结合HAV的VP2蛋白;7株MAb识别相同的抗原表位;7号MAb中和效价最高,可达1∶4 096。建立的HAV双抗体夹心ELISA定量检测方法的线性范围为15.625～250 u/ml,R2﹥0.97。结论 筛选出1株特异性好、中和效价较高的IgG2b型MAb,可用于制备酶标抗体和ELISA检测试剂盒,应用于甲肝疫苗中抗原的检测。
Objective To screen monoclonal antibody (MAb) of Hepatitis A virus (HAV) and establish a quantitative ELISA assay for HAV double antibody sandwich ELISA. Methods The subtype of MAb was identified by using the antibody subtype identification kit. The specificity of MAb was identified by Western blot. The ascites of MAb was purified by SPA affinity chromatography and the epitope of MAb was detected. The neutralization effect of MAb was detected by cell culture price. To determine the concentration of double-antibody sandwich coating and enzyme-labeled MAb, HAV double antibody sandwich ELISA quantitative detection method was initially established and the linear range of the method was investigated. Results Among the 12 HAV McAbs, one was IgM type, three were IgG3 type, three were IgG2a type and five were IgG2b type. Ten MAbs could specifically bind VP2 protein of HAV. Seven MAbs recognized the same Antigen epitope; No. 7 MAb and the highest titer, up to 1: 0960. The established HAV double antibody sandwich ELISA quantitative detection of linear range of 15.625 ~ 250 u / ml, R2> 0.97. Conclusion A strain of IgG2b MAb with good specificity and high neutralization titer was screened for the preparation of enzyme-linked immunosorbent assay (ELISA) and ELISA kit for the detection of antigens in hepatitis A vaccine.