洛克沙胂完全抗原的制备及其单克隆抗体的研制

Synthesis of Roxarsone Complete Antigens and Preparation of Corresponding Monoclonal Antibodies

本研究以洛克沙胂(ROX)的结构类似物3-氨基-4-羟基苯胂酸(HAPA)为小分子半抗原,采用碳二亚胺法,分别将其与载体蛋白——牛血清白蛋白(BSA)和鸡卵清白蛋白(OVA)偶联,制备免疫抗原HAPA-BSA和检测抗原HAPA-OVA。经紫外光谱扫描及聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定后,以20μg/只剂量的HAPA-BSA免疫BALB/c小鼠;选择血清效价高、敏感性强的小鼠,取脾脏进行细胞融合,制备杂交瘤细胞株。利用间接ELISA法筛选得到1株可以稳定分泌抗ROX单克隆抗体的细胞株,命名为G12;间接ELISA法测定其细胞上清效价为1:512,间接竞争ELISA测定其半数抑制浓度IC_(50)为3.147 ng/μL。经小鼠体内诱生腹水,辛酸-硫酸铵法纯化获得纯度较高的单克隆抗体,间接ELISA测定其效价为1:102 400,IC_(50)为1.433 ng/μL,且具有良好的特异性。本试验成功合成了ROX人工抗原,并制备了可以分泌高敏感性单克隆抗体的杂交瘤细胞株,为ROX免疫学快速检测方法的建立奠定了基础。

In this research,3-amino-4-hydroxybenzoic acid(HAPA),a structural analogue of Roxarsone(ROX)was adopted as the small molecule hapten.By being conjugated respectively with carrier protein albumin-bovine serum(BSA) and chicken ovalbumin(CVA)through carbodiimide method,the immune antigen HAPA-BSA and detection antigen HAPA-OVA were produced.After UV spectroscopy scan and polyacrylamide gel electrophoresis(SDS-PAGE)verification, HAPA-BSA dosage of 20 μg/per mouse was immunized to BALB/c mice,then the spleens of mice with higher serum titer and sensitivity were taken for cell fusion to prepare hybridoma cell strain.After screening by indirect ELISA method,one cell strain named G12 stably secreting anti-ROX monoclonal antibody was obtained,and the titer in cell supernatant was 1:512, the inhibitory concentration(IC50)was measured as 3.147 ng/μL.Furthermore,the ascites produced in abdominal cavity of mice were purified by caprylic acid-ammonium sulfate method,and higher purity of Abs were obtained, the titer of which was 1:102 400,and IC50 was 1.433 ng/μL and great specificity was showed.In conclusion,ROX artificial antigens were successively synthesized and the hybridoma cell line that could secret high sensitive monoclonal antibody was prepared,which would built a foundation to establish ROX rapid immunological detection method.