大豆Bowman-Birk胰蛋白酶抑制因子单克隆抗体的制备与鉴定  

Preparation and Identification of Bowman-Birk Inhibitor Monoclonal Antibody

作　　者：王耀[1,2] 李燕虹[1,2] 吴佳蓓 邢云瑞 曹金博 陈秀金 李兆周[1] 邓瑞广[2] 胡骁飞[2] Wang Yao;Li Yanhong;Wu Jiabei;Xing Yunrui;Cao Jinbo;Chen Xiujin;Li Zhaozhou;Deng Ruiguang;Hu Xiaofei(College of Food and Bioengineering,National Demonstration Center for Experimental Food Processing and Safety Education,Henan University of Science&Technology,Luoyang 471023,Henan;Henan Key Laboratory of Animal Immunology,Henan Academy of Agricultural Sciences,Zhengzhou 450002)

机构地区：[1]河南科技大学食品与生物工程学院,食品加工与安全国家级实验教学示范中心,河南洛阳471023 [2]河南省农业科学院河南省动物免疫学重点实验室,郑州450002

出　　处：《中国食品学报》2020年第7期216-221,共6页Journal of Chinese Institute Of Food Science and Technology

基　　金：国家自然科学基金项目(31702218);国家科技支撑计划项目(2014BAD13B05);河南省自然科学基金项目(182300410038);河南省高等学校重点科研项目(18B550001);河南省动物免疫学重点实验室开放课题(PKLAI20170603)。

摘　　要：为制备免疫学特性良好的大豆Bowman-Birk胰蛋白酶抑制因子(BBI)单克隆抗体(mAb),以50μg/只的免疫剂量将BBI免疫BALB/c小鼠,采用间接ELISA和间接竞争ELISA试验鉴定多抗血清效价和敏感性。选择血清效价和敏感性较优的小鼠进行细胞融合,多次筛选得到分泌BBI m Ab的稳定杂交瘤细胞株。采用体内诱生腹水法制备BBI mAb并对其免疫学特性进行鉴定。结果表明:1号小鼠经免疫后效价最高且敏感性最好,半数抑制浓度(IC50)为868.58 ng/mL。经细胞融合筛选得到5株杂交瘤细胞,其中3B7-B10 mAb效价达到1∶4.096×10^5以上,亚型为IgG1型,IC50为83.07 ng/mL,具有亲和力较高且特异性强的优点,表明所制备的mAb免疫学特性良好,能够为建立大豆及其制品中BBI的免疫学检测方法提供良好的抗体基础。To prepare monoclonal antibody(mAb) against Bowman-Birk inhibitor(BBI) with good immunological characteristics,BALB/c mice were used to immunize with 50 μg of BBI,and the antiserum titer and sensitivity were identified via indirect ELISA and indirect competitive ELISA.The mice which antiserum has better titer and sensitivity was selected for cell fusing through cell hybridoma technique,stable hybridoma cell lines that could secrete BBI mAb were obtained via multiple screening,and the BBI mAb was prepared by inducing ascitic fluids in vivo and identified the immunological characteristics.The result indicated that the NO.1 mouse was confirmed to produce the antibody with the highest titer and sensitivity,which half inhibitory concentration(IC50) was 868.58 ng/mL.After cell fusing,five hybridoma cell strains were screened.The 3 B7-B10 mAb titer was above 1 ∶ 4.096×105.Its subtype was IgG1 and IC50 was83.07 ng/mL.It had high affinity and specificity,demonstrated the mAb had good immunological characteristics.This study had provided the favorable antibody basis for the establishment of immunological detection of BBI in soybean and its products.

关 键 词：大豆Bowman-Birk胰蛋白酶抑制因子 单克隆抗体 免疫学特性 

分 类 号：TS201.2[轻工技术与工程—食品科学]