鳙鱼变应原小清蛋白单克隆抗体制备及鉴定  

Preparing and immunological characterization of monoclonal antibodies against parvalbumin—a major allergen from bighead carps

作　　者：张强[1] 赵郭存[1] 邹菊[1] 刘志刚[1] 

机构地区：[1]深圳大学过敏反应与免疫学研究所,广东深圳518060

出　　处：《中国公共卫生》2012年第4期548-550,共3页Chinese Journal of Public Health

基　　金：国家自然科学基金(30871752);深圳市深港创新圈项目(CXQ2008026);深圳大学创新科研团队基金(200904)

摘　　要：目的制备、纯化及鉴定抗鳙鱼小清蛋白单克隆抗体。方法以重组鱼主要过敏原小清蛋白(parvalbu-min)为抗原免疫BALB/c小鼠,融合免疫鼠脾细胞和小鼠骨髓瘤NS-1,半固体培养基法结合有限稀释法筛选稳定分泌抗体的杂交瘤细胞株,杂交瘤细胞株诱生小鼠腹水,采用蛋白A亲和层析法获得纯化抗体,间接ELISA方法和w estern blot鉴定抗体效价和特异性以及与其他过敏源的交叉反应性;采用Ig类与亚类鉴定试剂盒鉴定该单克隆抗体的Ig亚型。结果共获得抗parvalbumin细胞株7株,1G1,1B5,1A5,2B9,1H4,1B7,1G3。1G1、1B7、1A5、1H4和1G3效价均高于1∶400 000,P/N>2.1;1B5效价高于1∶200 000,2B9效价较低;Western blot检测表明所有抗体均能识别parvalbumin;抗体亚类为IgG1型,1G1,1A5,2B9,1H4,1B7特异性结合parvalbumin,与其他种类过敏原无交叉反应,1G3和1B5与虾和大豆过敏原有交叉反应性,P/N>2.1。结论获得高效价抗体5株,发现parvalbumin与虾、大豆过敏原存在交叉反应性,为建立parvalbumin检测体系提供依据。Objective To prepare,purify and identify monoclonal antibodies against parvalbumin from bighead carps.Methods Using hybridoma and limit dilution technique,the fusion cell strain secreting antibodies stably was screened.The cells were used to induce ascites in mice and monoclonal antibodies were purified using affinity chromatography on immobilized protein A.The subclass of Ig,speciality,titers,and cross-reactvity of the antibdies were detected with indirect enzyme-linked immunosorbent assay（ELISA） and western blot.Results Seven cell lines secreting antibodies against bighead carp parvalbumin were prepared.The titers of 5 of 7 monoclonal antibodies（1G1,2B9,1H4,1B7,and 1G3） were higher than 1∶ 400 000 and those of 1B5 and 1A5 were higher than 1∶ 200 000（P/N2.1）.Results of western blot indicated that all antibodies could bind to parvalbumin specifically.ELISA showed that the subclass of the Ig was IgG1.1G1,1A5,2B9,1H4,and 1B7 showed specifec binding to parvalbumin,whereas,1G3 and 1A5 had cross-reactivity with allergen from soybean and shrimp（P/N2.1）.Conclusion This research successfully prepared 5 antibodies with high titers for a detection system of parvalbumin and found the allergen composition reacting to the 2 of the 5 monoclonal antibodies from shrimp and soybean.

关 键 词：变应原 小清蛋白 单克隆抗体 

分 类 号：Q786[生物学—分子生物学]