摘要
通过双功能螯合剂iEDTA螯合Cd2+并偶联卵白蛋白(OVA)和牛血清白蛋白(BSA),合成免疫抗原Cd-iEDTAOVA和筛选抗原Cd-iEDTA-BSA;以Cd-iEDTA-OVA注射BALB/c小鼠,制备Cd2+单克隆抗体,以Cd-iEDTA-BSA通过酶联免疫吸附法(ELISA)进行筛选,并分析抗体性质。结果表明:以Cd2+和OVA含量分别为174.6230μg/L、1.7892mg/mL的Cd-iEDTA-OVA抗原免疫小鼠,Cd2+和BSA含量分别为48.1881μg/L、1.8065mg/mL的Cd-iEDTA-BSA抗原筛选,可获得7F4和7E8两株特异性较好的杂交瘤细胞;7E8E5、7E8G9、7F4B8、7F4D6和7F4H8共5株高纯度单抗,均属IgG1型,其中7E8E5效价最高,达1:512000,亲和常数也最高,达4.55×108L/mol。建立了Cd2+间接竞争ELISA法,IC50为1150ng/mL,检测限为260ng/mL(R2=0.9916);与Hg2+有较强交叉,与Zn2+、Cu2+、Ca2+交叉较弱,与Mg2+、Ni2+、Al3+、Pb2+、Ba2+几乎无交叉。
Bifunctional chelator isotrhiocyanobenzyl-EDTA （iEDTA） was coupled with Cd2＋ and conjugate with ovalbumin （OVA） and bovine serum albumin （BSA） to obtain Cd-iEDTA-OVA as an immune-antigen and Cd-iEDTA- BSA as a screening antigen, respectively. Cd-iEDTA-OVA was injected to BALB/c mice to produce mAb against Cd2＋, and Cd-iEDTA-BSA was utilized to screen the monoclonal antibodies （mAb） by enzyme-linked immunosorbent assay （ELISA）. Results showed that specific hybridoma cell lines 7F4 and 7E8 were obtained after immunizing mice with Cd-iEDTA-OVA containing 174.6230 μg/L Coa＋ and 1.7892 mg/mL OVA, and screening antibody with Cd-iEDTA-BSA containing 48.1881 μg/L Cd2＋ and 1.8065 mg/mL BSA. Five high purity mAbs were obtained, such as 7E8E5, 7E8G9, 7F4B8, 7F4D6 and 7F4H8, whose subtypes belong to IgG1. Among them, the titer of 7E8E5 was the highest, which was 1：512000; the affinity constant （Ka） of 7E8E5 was 4.55 × 10^8 L/mol. As a result an indirect competitive ELISA method for Cd2＋ was established, and the IC50 and detection limit were 1150 ng/mL and 260 ng/mL （R2 = 0.9916）, respectively. However, it showed strong cross reactivity with Hg2＋, weak cross reactivity with Zn2＋, Cu2＋ and Ca2＋, and almost no cross reactivity with Mg2＋, Ni2＋, A12＋, Pb2＋ or Ba2＋.
出处
《食品科学》
EI
CAS
CSCD
北大核心
2013年第21期248-253,共6页
Food Science
基金
湖南省科技计划重大专项(2011FJ1002-4)
国家自然科学青年基金项目(31301404)
“十二五”国家科技支撑计划项目(2012BAD34B08)
长沙市科技计划项目(K1207176-21)
关键词
镉
抗原
杂交瘤细胞
单克隆抗体
酶联免疫吸附法
cadium
antigen： hybridoma cell
monoclonal antibody （mAb）
enzyme linked immunosorbent assay （ELISA）
