摘要
目的建立重组戊型肝炎病毒(Hepatitis E virus,HEV)抗原双抗体夹心ELISA定量检测方法,用于HE疫苗中HEV抗原含量的检测。方法以重组HEV病毒样颗粒作为免疫原,免疫母鸡,制备抗HEV-IgY多克隆抗体,纯化后作为包被抗体,HRP标记的抗HEV单克隆抗体作为检测抗体,建立HEV抗原双抗体夹心ELISA定量检测方法,并对其进行验证。用建立的方法检测3批HE疫苗成品的HEV抗原含量,计算HEV抗原对氢氧化铝的吸附率。结果建立的ELISA方法线性范围为2～128 ng/ml,最低定量限为2 ng/ml;该方法检测冻干甲肝减毒活疫苗、重组人白介素-2(IL-2)、乙型肝炎表面抗原(HBsAg)、人血白蛋白、小牛血清及健康人血清均无交叉反应;该方法检测3个浓度的HEV抗原内部参考品的回收率在95.13%～104.50%之间,试验内及试验间变异系数均<15%;包被抗HEV-IgY的酶标板于37℃放置5 d,其检测HEV抗原内部参考品的A450值及敏感性均未发生显著变化;3批HE疫苗成品中HEV抗原对氢氧化铝的吸附率均大于95%,符合相关质控要求。结论已成功建立了HEV抗原双抗体夹心ELISA定量检测方法,可用于HEV抗原含量的检测。
Objective To develop a double antibody sandwich ELISA method for quantitative detection of recombinant hepatitis E virus （HEV） antigen. Methods Hens were immunized with virus-like particles （VLPs） of recombinant HEV, based on which a double antibody sandwich ELISA method was developed using the prepared polyclonal antibody against HEV-IgY as coating antibody and HRP-labeled monoclonal antibody against HEV as detection antibody, and verified. The HEV antigen contents in three batches of final products of HE vaccine were determined by the developed ELISA method, and the adsorption rate of aluminium hy- droxide with HEV antigen was calculated. Results The linear detection range and minimum detection limit of developed ELISA method were 2 - 128 and 2 ng/ml respectively. The prepared ELISA kit showed no cross reactions with freeze-dried live attenuated hepatitis A vaccine, recombinant human IL-2, HBsAg, human serum albumin, calf serum or healthy human serum. The internal references for HEV antigen, at three concentrations, were detected by the developed ELISA method, and the results showed that the recovery rates were 95. 13% ～ 104. 50%, while both the intraand inter-coefficients were less than 15%. No significant change was observed in the A45o value or sensitivity of internal reference for HEV antigen detected by using the HEV-IgY coated ELISA plate after storage at 37℃ for 5 d. All the adsorption rates of aluminium hydroxide with HEV antigen in three batches of final products of HE vaccine were more than 95%, which met the relevant requirements for quality control. Conclusion A double antibody sandwich ELISA method for quantitative detection of HEV antigen was successfully developed.
出处
《中国生物制品学杂志》
CAS
CSCD
2011年第11期1363-1365,1372,共4页
Chinese Journal of Biologicals
