高亲和力大豆凝集素单抗制备及免疫学特性鉴定  

Preparation and Immunological Characteristics Identification of Soybean Agglutinin’s Monoclonal Antibody With High Affinity

作　　者：胡骁飞[1] 李燕虹 邢云瑞[1] 胡思宇 孙亚宁 邢广旭[1] 邓瑞广[1] 张改平[1,4] HU Xiaofei;LI Yanhong;XING Yunrui;HU Siyu;SUN Yaning;XING Guangxu;DENG Ruiguang;ZHANG Gaiping(Key Laboratory of Animal Immunology,Henan Academy of Agricultural Sciences,Zhengzhou,Henan 450002;College of Food and Bioengineering,Henan University of Science and Technology,Luoyang,Henan 471003;College of Life Sciences,Henan Agricultural University,Zhengzhou,Henan 450002;College of Animal Science and Veterinary Medicine,Henan Agricultural University,Zhengzhou,Henan 450002)

机构地区：[1]河南省农业科学院动物免疫学重点实验室,河南郑州450002 [2]河南科技大学食品与生物工程学院,河南洛阳471003 [3]河南农业大学生命科学学院,河南郑州450002 [4]河南农业大学牧医工程学院,河南郑州450002

出　　处：《核农学报》2020年第11期2477-2483,共7页Journal of Nuclear Agricultural Sciences

基　　金：“十二五”国家科技支撑计划课题(2014BAD13B05);中国博士后科学基金(2018M632778)。

摘　　要：为制备高亲和力的大豆凝集素(SBA)单克隆抗体,本试验以50μg SBA蛋白/200μL乳化液/只的剂量免疫BALB/c小鼠,间接ELISA和间接竞争ELISA鉴定多抗血清效价和灵敏性。选择血清效价和灵敏性较好的小鼠进行细胞融合。通过有限稀释亚克隆筛选出能稳定分泌SBA单克隆抗体的阳性杂交瘤细胞株。采用体内诱生腹水法批量制备SBA单克隆抗体并对其免疫学特性进行鉴定。结果表明,SBA免疫的4只小鼠中,1号小鼠效价最高且灵敏性最好,半数抑制浓度(IC50)为571.4μg·L^-1。选取该小鼠进行细胞融合,并最终得到1株杂交瘤细胞,命名为5B4E10。制备的SBA单克隆抗体的效价达到1∶409600以上,IC50为82.04μg·L^-1,亚型为IgG1型,亲和力常数(Ka)为1.83×10^9 L·mol^-1。且该抗体与大豆球蛋白、β-伴大豆球蛋白和其他抗营养因子无交叉反应。本研究结果为建立大豆及其制品中SBA的免疫学检测方法奠定了良好的抗体基础。To prepare monoclonal antibodies against soybean agglutinin(SBA)with high affinity.BALB/c mice were immunized with 50μg of protein of SBA in 200μL emulsion,and the antiserum titer and sensitivity were identified by indirect ELISA and indirect competitive ELISA respectively.The mouse with higher serum titer and sensitivity was selected for cell fusing.The hybridoma cell strain secreted monoclonal antibody(mAb)against SBA was selected by limited dilution subcloning.The SBA mAb was prepared by inducing ascites in vivo and the immunological characteristics of which were identified.The results indicated that the NO.1 mouse has the highest serum titer and sensitivity,the IC50 was 574.1μg·L^-1.The NO.1 mouse was selected for cell fusing,and one hybridoma cell strains 5 B4 E10 were screened.The titer of mAb produced by 5 B4 E10 was above 1∶409600,IC50was 82.04μg·L^-1,the subtype was IgG1,the affinity constant Ka was 1.83×10^9L·mol^-1.Furthermore,there was no cross reaction between the prepared mAb and glycinin,β-conglycinin,other anti-nutrient factors.In conclusion,the result of this study would provide an antibody basis for the immunological detection method of SBA in soybean and its products.

关 键 词：大豆凝集素(SBA) 动物免疫 细胞融合 杂交瘤筛选 单克隆抗体 

分 类 号：TS214.2[轻工技术与工程—粮食、油脂及植物蛋白工程]