本研究以洛克沙胂（ROX）的結(jié)構(gòu)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(lián)，制備免疫抗原HAPA-BSA和檢測(cè)抗原HAPA-OVA。經(jīng)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(jià)高、敏感性強(qiáng)的小鼠，取脾臟進(jìn)行細(xì)胞融合，制備雜交瘤細(xì)胞株。利用間接ELISA法篩選得到1株可以穩(wěn)定分泌抗ROX單克隆抗體的細(xì)胞株，命名為G12；間接ELISA法測(cè)定其細(xì)胞上清效價(jià)為1:512，間接競(jìng)爭(zhēng)ELISA 測(cè)定其半數(shù)抑制濃度 IC50為3.147 ng/μL。經(jīng)小鼠體內(nèi)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(cè)定其效價(jià)為1:102 400，IC50為1.433 ng/μL，且具有良好的特異性。本試驗(yàn)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(xì)胞株，為ROX免疫學(xué)快速檢測(cè)方法的建立奠定了基礎(chǔ)。
Synthesis of RoxarsoneComplete Antigens
and Preparation ofCorresponding Monoclonal Antibodies
In this research，3-amino-4-hydroxybenzoic acid（HAPA），a structural analogue of Roxarsone（ROX）was adopted as the small molecule hapten. By beingconjugated respectively with carrier protein albumin-bovine serum（BSA）and chicken ovalbumin（CVA）through carbodiimide method，the immune antigen HAPA-BSA and detection antigenHAPA-OVA were produced. After UV spectroscopy scan and polyacrylamide gelelectrophoresis（SDS-PAGE）verification，HAPA-BSA dosage of 20 μg/per mouse was immunized to BALB/c mice，then the spleens of mice with higher serumtiter and sensitivity were taken for cell fusion to prepare hybridoma cellstrain. After screening by indirect ELISA method，one cell strain named G12 stably secreting anti-ROXmonoclonal 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 ofmice 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. Inconclusion，ROX artificial antigens were successivelysynthesized and the hybridoma cell line that could secret high sensitivemonoclonal antibody was prepared，which would built a foundation to establish ROX rapidimmunological detection method.
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