抗甲型肝炎病毒卵黄免疫球蛋白的制备及应用  

Preparation and application of immunoglobulin of yolk(IgY) against hepatitis A virus

作　　者：夏青娟[1] 李树林 孟凡东 赵淑杰[1] 徐晓霞[1] 惠琪[1] 刘令九[1] 

机构地区：[1]长春生物制品研究所有限责任公司疫苗二室,吉林长春130062 [2]中国生物技术股份有限公司营销中心预防制品部销售一部,北京100029 [3]长春祈健生物制品有限公司疫苗一室,吉林长春130000

出　　处：《中国生物制品学杂志》2013年第12期1793-1796,共4页Chinese Journal of Biologicals

摘　　要：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HAVIgY,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豫;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（IgG） against hepatitis A virus （HAV） and develop a double antibody sandwich ELISA method for HAV antigen content in HA vaccine. Methods IgY against HAV was prepared by immunizing healthy laying hens with purified HAV, purified by several steps, then determined for protein content by ultraviolet spectrophotometry, for relative molecular mass and purity by reduced SDS-PAGE, and for titer, specificity as well as heat, acid and base stabilities by indirect ELISA. A double antibody sandwich ELISA method was developed using the purified IgY as coating antibody, and HRP-labeled monoclonal antibody against HAV as the secondary antibody, and used for determination of HAV antigen content in HA vaccine during production, and the results were compared with those by commercial ELISA kit. Results The IgY concentration after purification by affinity chromatography was 3. 67 mg/ml. The relative molecular masses heavy and light chains of IgY were 66 000 and 27 000 respectively, while the purity was 96. 78%, and the titer was 1 ： 16 000 at most. The purified IgY showed only positive reaction with HAV, while showed no reactions with poliovirus or enterovirus 71 （EV71）. Treatment at 25 - 70 ℃ for 15 rain or with Tris-HCl buffer at pH 3. 0 - 10. 0 for 2 h showed no significant influence on the titer of IgY. The log of HAV concentration at a range of 15. 62 - 2 000. 00 ng/ml was linearly related to the A450 value, with a R2 value of 0. 935. The minimum detection limit of the developed ELISA method was 7. 81 ng/ml, while the coefficient of relationship of detection result was 0. 952 7 to that by commercial kit. Conclusion The prepared IgY showed high concentration, purity, titer, specificity and stability, by which the developed double antibody sandwich ELISA method might be used for determination of HAV antigen content in HA vaccine during production.

关 键 词：肝炎病毒 甲型 免疫球蛋白Y 双抗体夹心酶联免疫吸附测定 

分 类 号：R373.21[医药卫生—病原生物学] R392-33[医药卫生—基础医学]