抗HBsAg表位单克隆抗体的制备及HBV血清型夹心ELISA的建立  

Preparation of anti-HBsAg epitope monoclonal antibodies and establishment of a sandwich ELISA for HBV serotype detection

作　　者：张晓磊[1,2] 聂玉哲[1,3] 蓝兴国[1] 李玉花[1] 

机构地区：[1]东北林业大学生命科学学院,黑龙江哈尔滨150040 [2]大庆麦伯康生物技术有限公司,黑龙江大庆163316 [3]东北林业大学大庆生物技术研究院,黑龙江大庆163316

出　　处：《细胞与分子免疫学杂志》2014年第2期171-175,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：黑龙江省发展高新技术产业专项资金(FW11C201);大庆高新区科技专项资金(DQGX10ZS005)

摘　　要：目的制备抗乙型肝炎病毒(HBV)表面抗原(HBsAg)表位单克隆抗体(mAb),建立HBV血清型夹心ELISA检测方法。方法将HBsAg亚型蛋白分别免疫小鼠,通过细胞融合、高通量筛选ELISA(HTS-ELISA)筛选后得到杂交瘤细胞株并在无血清培养基中扩大培养。采用蛋白A对抗体进行纯化并测定抗体亲和力、亚型、特异性,最后建立双抗体夹心ELISA。结果HBsAg的亚型蛋白ad、adr及ayw分别免疫小鼠后,其血清效价达到1∶105。细胞融合及HTS-ELISA筛选后获得4株杂交瘤细胞株。经过纯化后得到的mAb分别命名为#2-4,#18-3,#7-7,#56-5,亲和力都在1×109L/mol以上。间接ELISA结果显示,#2-4,#18-3,#7-7,#56-5分别特异性地与HBsAg的"d,y,r,w"表位结合。用抗HBsAg"a"表位的mAb#3-11分别与这4株抗体组合,进行夹心ELISA检测。结果显示,不同的ELISA组合能特异性地检测HBsAg的"d,y,r,w"表位。结论成功制备了亲和力高、特异性好的抗HBsAg 4个表位的mAb,建立了检测HBV血清型的双抗体夹心ELISA。Objective To prepare the monoclonal antibodies against HBsAg epitopes and establish a sandwich ELISA system for HBV serotype detection. Methods The BALB/c mice were immunized with HBsAg subtype proteins. The hybridoma cells were cultured in serum-free medium after cell fusion and high throughput screening ELISA （HTS-ELISA）. Monoclonal antibodies were purified with protein A. The specificity, affinity, isotype, and epitope of the mAbs were characterized respectively and the sandwich ELISA system was established. Results The titers of mouse anti-sera reached 1：105. After the cell fusion and HTS-ELISA, the four hybridoma cell lines were obtained. The mAbs were purified and named #2-4, #18-23, #7-7, #56-71, respectively. The mAbs had a high affinity （over 109 L/mol）. Indirect ELISA showed that #2-4, #18-23, #7-7 and #56-71 could recognize HBsAg ＂d, y, r, w＂ epitopes, respectively. The sandwich ELISA was established through using #3-11 （Anti-HBsAg ＂a＂ epitope） as the coating antibody while the HRP labeled mAb as the secondary antibody. The optimized sandwich ELISA was proved to have a good specificity by testing different antibody-antigen reactions. Conclusion The mAbs against HBsAg epitopes we prepared had a good affinity and specificity. The sandwich ELISA for HBV serotype detection we established successfully provided a basis for HBV serotype detection and disease diagnosis.

关 键 词：乙型肝炎表面抗原 单克隆抗体 表位 夹心ELISA 

分 类 号：R392-33[医药卫生—免疫学]