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目的制备抗甲型肝炎病毒(hepatitis A virus,HAV)卵黄免疫球蛋白(immunoglobulin of yolk,IgY),并建立双抗体夹心ELISA法,用于测定甲肝疫苗中的HAV抗原含量。方法用纯化的HAV免疫健康产蛋母鸡,制备抗HAV IgY,采用多步骤分离纯化IgY,紫外分光光度法测定纯化IgY的蛋白含量,还原型SDS-PAGE分析IgY的相对分子质量及纯度,间接ELISA法检测IgY的效价、特异性及其热稳定性和酸碱稳定性。以纯化的IgY作为包被抗体,HRP标记的抗HAV单克隆抗体作为二抗,建立双抗体夹心ELISA法,对疫苗生产过程中的HAV抗原含量进行测定,并与市售ELISA试剂盒的检测结果进行比较。结果亲和层析纯化后的抗HAV IgY浓度为3.67 mg/ml;重链和轻链的相对分子质量分别为66 000和27 000,纯度为96.78%;效价最高为1∶16 000;纯化的抗HAV IgY只与HAV呈阳性反应,与脊髓灰质炎病毒和肠道病毒71(enterovirus 71,EV71)均不反应;纯化的抗HAV IgY在25~70℃条件下处理15 min及经pH 3.0~10.0的Tris-HCL缓冲液处理2 h,其抗体效价基本无影响。建立的双抗体夹心ELISA法HAV浓度在15.62~2 000.00 ng/ml范围内,HAV浓度的对数值与A450值之间呈线性相关,R2=0.935,最低检测量为7.81 ng/ml,与市售试剂盒检测结果具有良好的相关性,相关系数为0.952 7。结论制备的抗HAV IgY浓度、纯度、效价均较高,特异性较强,稳定性良好,建立的双抗体夹心ELISA法可用于检测甲肝疫苗生产过程中HAV的抗原含量。
Objective To prepare the immunoglobulin of yolk (IgY) of hepatitis A virus (HAV) and establish a double antibody sandwich ELISA for the determination of HAV antigen in hepatitis A vaccine. Methods Immunization of healthy laying hens with purified HAV to prepare anti-HAV IgY, using multiple steps to separate and purify IgY, UV-spectrophotometric determination of purified protein content of IgY, reducing SDS-PAGE analysis of IgY relative molecular mass and purity, indirect ELISA titer, specificity and its thermal stability and acid-base stability. Using purified IgY as coating antibody and HRP-labeled anti-HAV monoclonal antibody as secondary antibody, a sandwich ELISA method was established to detect the content of HAV antigen in the vaccine production process. The results of ELISA and ELISA Compare. Results The concentration of anti-HAV IgY purified by affinity chromatography was 3.67 mg / ml. The relative molecular weights of heavy chain and light chain were 66 000 and 27 000, respectively, with a purity of 96.78%. The highest titer was 1:16 000. Of anti-HAV IgY only reacted positive with HAV and did not react with poliovirus and enterovirus 71 (EV71). The purified anti-HAV IgY was treated at 25-70 ° C for 15 min and pH 3.0 ~ 10.0 Tris-HCL buffer for 2 h, the antibody titer of almost no effect. The established double antibody sandwich ELISA HAV concentration in the range of 15.62 ~ 2000.00 ng / ml, HAV concentration logarithm and A450 linear correlation between the value of R2 = 0.935, the minimum detection of 7.81 ng / ml, and the commercially available The test results of the kit had a good correlation with a correlation coefficient of 0.952 7. Conclusion The prepared anti-HAV IgY has high concentration, purity and titer, strong specificity and good stability. The established sandwich ELISA method can be used to detect the antigen content of HAV during the production of hepatitis A vaccine.