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洛克沙胂完全抗原的制備及其單克隆抗體的研制

點擊數:7450  時間:2019-03-04 15:01:45  來源: 中國動物檢疫      作者: 劉紅亮等

本研究以洛克沙胂(ROX)的結構類似物3-氨基-4-羥基苯胂酸(HAPA)為小分子半抗原,采用碳二亞胺法,分別將其與載體蛋白——牛血清白蛋白(BSA)和雞卵清白蛋白(OVA)偶聯,制備免疫抗原HAPA-BSA和檢測抗原HAPA-OVA。經紫外光譜掃描及聚丙烯酰胺凝膠電泳(SDS-PAGE)鑒定后,以20 μg/只劑量的HAPA-BSA免疫BALB/c小鼠;選擇血清效價高、敏感性強的小鼠,取脾臟進行細胞融合,制備雜交瘤細胞株。利用間接ELISA法篩選得到1株可以穩定分泌抗ROX單克隆抗體的細胞株,命名為G12;間接ELISA法測定其細胞上清效價為1:512,間接競爭ELISA 測定其半數抑制濃度 IC503.147 ng/μL。經小鼠體內誘生腹水,辛酸-硫酸銨法純化獲得純度較高的單克隆抗體,間接ELISA測定其效價為1:102 400IC501.433 ng/μL,且具有良好的特異性。本試驗成功合成了ROX人工抗原,并制備了可以分泌高敏感性單克隆抗體的雜交瘤細胞株,為ROX免疫學快速檢測方法的建立奠定了基礎。


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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