2种不同无色孔雀石绿单克隆抗体的制备  

Preparation of two different monoclonal antibodies against leuco malachite green

作　　者：陈力[1] 胡鲲[1] 姜有声[1] 黄宣运[2] 安健[3] 杨先乐[1] 

机构地区：[1]上海海洋大学,国家水生动物病原库,上海201306 [2]中国水产科学研究院东海水产研究所,上海200090 [3]连云港市海洋与水产科学研究所,江苏连云港222044

出　　处：《西北农林科技大学学报（自然科学版）》2011年第7期28-34,共7页Journal of Northwest A&F University(Natural Science Edition)

基　　金：现代农业产业技术体系建设专项资金项目(NYCYTX-49-17);公益性行业(农业)科研专项经费项目(nyhyzx07-042);公益性行业(农业)科研专项经费项目(200803013)

摘　　要：【目的】制备无色孔雀石绿（LMG）单克隆抗体,并分析其生物学特性,为建立LMG残留的免疫学检测方法奠定基础。【方法】通过改进的碳二亚胺法制备无色孔雀石绿-牛血清白蛋白偶联物（LMG-BSA）与副品红-牛血清白蛋白偶联物（PA-BSA）,用紫外扫描、质谱法方法鉴定偶联物。利用制备的LMG-BSA与PA-BSA作为免疫原,免疫4~5周龄Balb/c小鼠,用杂交瘤技术制备LMG单克隆抗体,并对其效价、亲和力、灵敏度和特异性等进行分析。【结果】LMG-BSA与PA-BSA的偶联比率分别为7∶1和37∶1;经间接酶联免疫吸附（ELISA）法筛选,由LMG-BSA和PA-BSA抗原各获得1株杂交瘤细胞,分别命名为L-1、P-1。这2株细胞株分泌的单克隆抗体纯化后,经测定效价分别为1.25×105和6.25×106,亲和力常数分别为4.92×109和1.44×108L/mol,半抑制质量浓度（IC50）分别达8.03和10.5μg/L。L-1分泌的单克隆抗体仅与孔雀石绿存在交叉反应,交叉反应率为100%;P-1分泌的单克隆抗体与孔雀石绿及副品红均存在交叉反应,交叉反应率分别为80%与100%。【结论】成功获得了2株LMG单克隆抗体,其均可用于水产品中LMG残留的快速检测。【Objective】 The research was conducted to generate monoclonal antibodies of leuco malachite green（LMG） and analyze its biological traits to establish a determination method for LMG residues in food tissues.【Methods】 The conjugation of leuco malachite green-bovine serum albumin（LMG-BSA） and paraleucaniline-bovine serum albumin（PA-BSA） were prepared via a modified carbodiimide method.Features of the conjugation were identified by ultraviolet spectrometry and mass spectroscopy.In order to generate monoclonal antibody（Mab） against hapten-leuco malachite green（LMG）,LMG-BSA and PA-BSA were used as immunogen to immunize for four to five-week-old Balb/c mice.The Mabs were obtained by hybridoma technology and their titer,affinit,sensibility and specificity were identified.【Results】 LMG-BSA and PA-BSA coupling ratio was 7∶1 and 37∶1;The titers of purified Mabs secreted by L-1 and P-1were 1.25×105,6.25×106,respectively.Characterization studies of these two Mabs showed that they had high affinity for LMG,with the affinity of 4.92×109,1.44×108 L/mol,and the IC50 was 8.03 and 10.5 μg/L.Mabs secreted by L-1 exhibited high cross-reactivity（100%） with malachite green;Mabs secreted by P-1 exhibited high cross-reactivity（80% and100%） with malachite green and paraleucaniline.【Conclusion】 These two monoclonal antibody were useful for determination of LMG residues in aqua-production.

关 键 词：无色孔雀石绿 副品红 半抗原 抗体 免疫 

分 类 号：S852.4[农业科学—基础兽医学]