摘要
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 the monoclonal antibody（MAb） against hepatitis A virus（HAV） and preliminarily develop a double antibody sandwich ELISA method for quantitative determination of HAV.Methods The MAb was identified for subtype by antibody subtyping kit and for specificity by Western blot.The MAb in ascites was purified by SPA affinity chromatograpy,then tested for antigen epitope,and determined for neutralizing titer by cell culture method.The concentrations of coating antibody and enzyme-labeled antibody for double antibody sandwich ELISA were optimized,based on which a double antibody sandwich ELISA method for HAV was preliminarily developed and evaluated for linear range.Results Of the MAbs secreted by twelve hybridoma cell strains,one was IgM,three were IgG3,three were IgG2a and five were IgG2b.The MAbs secreted by ten strains showed specific binding to HAV VP2 protein,while those by seven strains recognized the same antigen epitopes.The neutralizing titer of MAb No.7 was the highest in all the MAbs,which reached 1 ∶ 4 096.The linear range of the developed method was 15.625 ～ 250 u / ml,with a R2 value of more than 0.97.Conclusion A MAb cell strain secreting IgG2b with high specificity and neutralizing titer was screened,which might be used for preparation of enzyme-labeled antibody and ELISA kit for determination of antigen in hepatitis A vaccine.
出处
《中国生物制品学杂志》
CAS
CSCD
2011年第9期1081-1083,共3页
Chinese Journal of Biologicals
基金
"重大传染病诊断产品质量评价综合技术平台(2009ZX10004-805)"课题资助
