本研究以洛克沙胂（ROX）的結構類似物3-氨基-4-羟基苯胂酸（HAPA）爲小分子半抗原，采用碳二亞胺法，分别將(jiāng)其河厭與載體蛋白——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(jīng)紫外光譜掃描及聚丙烯酰胺凝膠電泳（SDS-PAGE）鑒定後(hòu)，以20 μg/隻劑量的HAPA-BSA免疫BALB/c小鼠；選擇血清效價高、敏感性強的小鼠，取脾髒進(jìn)行細著學胞融合，制備雜交瘤細胞株。利用事城間接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，命名爲G12；間接ELISA法測定其細胞上清效價爲1:512，間接競争ELISA 測定其半數抑制濃度 IC50爲3.147 ng/μL。經(jīng)小鼠體内誘生腹水，辛酸-硫酸铵法純化獲得純度較高的單克隆抗體，間接ELISA測定其效價爲1:102 400，IC50爲1.433 ng/μL，且具有良好(hǎo)的特異性。本試驗成(chéng)功合成(chéng)作些了ROX人工抗原，并制備了可以分泌高敏感性單克隆抗們電體的雜交瘤細胞株，爲ROX免疫學(xué)快速檢測方法的建立奠定了基礎。
Synthesis of Roxar暗呢soneComplete Antigens
and Preparation ofCorrespon開可ding Monoclonal Antibodies
In this research，3-amino-4-hydroxybenzo數民ic acid（HAPA），a structural ana做慢logue of Roxarsone（ROX）was adopted as the small molecule hapt廠如en. By beingconju不熱gated respectively with carrie短農r protein albumin-bovine ser市技um（BSA）and chicken ovalbumin（CVA）through carbodiimide method，the immune antigen HAPA-BSA and現雨 detection antigenHAPA-OVA were p道費roduced. After UV sp科靜ectroscopy scan東店 and polyacrylamide gelelectrophoresi媽習s（SDS-PAGE）verification，HAPA-BSA dosage of 20 μg/per mouse was im冷員munized to BALB/c mice，then the spleens of 廠森mice with higher serumtiter an鐘外d sensitivity were ta謝暗ken for cell fusion 門高to prepare hybridoma cellstrai短地n. After screening by i放長ndirect ELISA method，one cell strain named G12 stably secr歌兵eting anti-ROXmonoclonal antibody was 了銀obtained，and the titer in cell sup筆厭ernatant was 1:512，the inhibitory concentr山機ation（IC50）was measured as 3.147制美 ng/μL. Furthermore，the ascites pro有票duced in abdominal cav秒內ity ofmice were purifie報器d by caprylic acid-ammonium sulfate met內中hod，and higher purity of Abs were還資 obtained，the titer of which was 1:102 400，and IC50 was 1.433 ng/μL and great specificity was showe冷物d. Inconclusion，ROX artificial antigens were success為說ivelysynthesized and the hybri拍通doma cell line that could secret h了吧igh sensitivemonoclonal antibod舊睡y was prepared，which would built a foundation to esta黃一blish ROX rapidimmun從些ological detection method.
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