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洛克沙胂完全抗原的制备及其单克隆抗体的研制

点击数:4212  时间:2019-03-04 15:01:45  来源: 中国动物检疫      作者: 刘红亮等

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20 μg/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 IC503.147 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 400IC501.433 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。


Synthesis of RoxarsoneComplete Antigens

and Preparation ofCorresponding Monoclonal Antibodies

In this research3-amino-4-hydroxybenzoic acidHAPA),a structural analogue of RoxarsoneROXwas adopted as the small molecule hapten. By beingconjugated respectively with carrier protein albumin-bovine serumBSAand chicken ovalbuminCVAthrough carbodiimide methodthe immune antigen HAPA-BSA and detection antigenHAPA-OVA were produced. After UV spectroscopy scan and polyacrylamide gelelectrophoresisSDS-PAGEverificationHAPA-BSA dosage of 20 μg/per mouse was immunized to BALB/c micethen the spleens of mice with higher serumtiter and sensitivity were taken for cell fusion to prepare hybridoma cellstrain. After screening by indirect ELISA methodone cell strain named G12 stably secreting anti-ROXmonoclonal antibody was obtainedand the titer in cell supernatant was 1:512the inhibitory concentrationIC50was measured as 3.147 ng/μL. Furthermorethe ascites produced in abdominal cavity ofmice were purified by caprylic acid-ammonium sulfate methodand higher purity of Abs were obtainedthe titer of which was 1:102 400and IC50 was 1.433 ng/μL and great specificity was showed. InconclusionROX artificial antigens were successivelysynthesized and the hybridoma cell line that could secret high sensitivemonoclonal antibody was preparedwhich would built a foundation to establish ROX rapidimmunological detection method.

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