拟除虫菊酯类农药单克隆抗体的制备及鉴定  

Preparation and Characterization of Monoclonal Antibodies against Pyrethroid Pesticides

作　　者：徐敦明[1,2] 卢声宇[2] 周昱[1] 杨方[2] 林立毅[1] 杜凤君[1] 张缙[1] 

机构地区：[1]厦门出入境检验检疫局,福建厦门361026 [2]福建出入境检验检疫局,福建福州350001

出　　处：《福建分析测试》2010年第4期5-9,共5页Fujian Analysis & Testing

基　　金：福建省青年人才项目((2006F3002);国家质检总局科技项目(2007IK146);厦门市科技计划项目(3502Z20072003;3502Z20092008)资助

摘　　要：以间苯氧基苯甲酸(PBA)为半抗原,与牛血清白蛋白(BSA)偶联后作为抗原,免疫Balb/c小鼠,取小鼠脾细胞与骨髓瘤细胞(SP2/0)融合,使用PBA与鸡卵清白蛋白(OVA)偶联物作为包被物,间接ELISA法筛选到4株特异性针对PBA的杂交瘤。其中两株细胞2F7和7B1制备腹水并用ProteinG柱纯化抗体,抗体效价均达到1:625000。单抗7B1用于间接竞争ELISA法,PBA检测下限为0.025μg·mL-1,IC50为0.57μg·mL-1,对溴氰菊酯、甲体氯氰菊酯、甲氰菊酯、氰戊菊酯均有特异性识别,IC50分别为0.79μg·mL-1、0.74μg·mL-1、0.63μg·mL-1、0.8μg·mL-1,对联苯菊酯识别弱,IC50为99.7μg·mL-1。该单抗可用于多种菊酯类农药的检测。Enzyme immunoassay was a desirable method for detecting residues of pyrethroids insecticides. Recently many immunoassays methods for pyrethroids have been reported, but few focused on the multi-residue analysis of pyrethroids. To develop a monoclonal antibodies-based determination method for multi-residues of pyrethroids, a generic hapten, 3-phenoxybenzoic acid （PBA）was used to generate broad specificity monoclonal antibodies against a group of pyrethroids. Mixed anhydride reaction （MAR）and active ester method （AEM）were used to couple the PBA to carrier proteins （BSA and OVA）. Characterization of artificial antigens was done by using spectroscopy. BALb/c mice were immunized with the PBA-BSA. The splenocytes of the immunized mice were fused with Sp2/0 myeloma cells by hydridoma technique. The antibodies against PBA were screened by micro-quantity plate agglutination test method and were cloned by end dilution method. Two sets of the specific and affinitive monoclonal antibodies were obtained, including 2F7 and 7B1 with more than 1：625000 of the dilution of antiserum. The 7B1 showed an IC50 value of 0.57μg·mL-1against PBA with a detection limit of 0.025μg·mL-1.This assay showed the cross-reactivities with the structurally closely related compounds including decamethrim, alpha-cypermethrin, fenpropathrin and Fenvalerate with IC50 values of 0.79μg·mL-1,0.74μg·mL-1, 0.63μg·mL-1 and 0.8μg·mL-1.The monoclonal antibody could determinate multi-pyrethroid pesticides.

关 键 词：拟除虫菊酯 农药残留 单克隆抗体 酶联免疫检测方法 

分 类 号：X592[环境科学与工程—环境工程]