食品总铬质量浓度icELISA试剂盒的研制及初步应用  

Establishment and Preliminary Application of an Indirect Competitive ELISA Kit for Detecting Total Chromium Mass Concentration in Food

作　　者：王亚楠[1,2] 王晓斐 张雅凌[1,2] 张海棠 王自良[1,2] 

机构地区：[1]河南科技学院动物科技学院,河南新乡453003 [2]动物病毒病防控与药残分析河南省高校重点学科开放实验室,河南新乡453003 [3]河南科技学院新科学院,河南新乡453003

出　　处：《西北农业学报》2017年第12期1868-1876,共9页Acta Agriculturae Boreali-occidentalis Sinica

基　　金："十二五"国家科技支撑计划(2011BAK10B01-15;2014BAD13B05-01)~~

摘　　要：为研制食品中总铬质量浓度检测间接竞争ELISA(icELISA)试剂盒(Cr-Kit)。利用蛋白质连接技术合成免疫原Cr^(3+)-iEDTA-BSA,紫外扫描(UV)和电感耦合等离子体原子发射光谱(ICP-AES)进行鉴定;用Cr^(3+)-iEDTA-BSA免疫BALB/c小鼠,细胞融合技术筛选抗Cr^(3+)-EDTA mAb细胞株,体内诱生腹水法制备Cr^(3+)-EDTA mAb,并对其特性进行分析;应用Cr^(3+)-EDTA mAb研制Cr-Kit,测定其性能并进行初步应用。结果表明,免疫原制备成功,Cr^(3+)-iEDTA-BSA中Cr^(3+)和BSA的质量浓度分别为140.8mg/L和5.81g/L;筛选出2A3C11、2A3D9、2A11G5共3株杂交瘤细胞株,其中2A11G5最好,经6次传代分泌抗体稳定,亲和常数(Ka)为2.69×109 L/mol,与其他重金属离子无交叉反应;Cr-Kit标准曲线的线性范围为1.0~264μg/L,检测限为1.0μg/L,IC50为8.37μg/L,河水样、大米样、面粉样和猪肉样的平均加标回收率分别为101.7%、96.47%、95.8%和84.3%,Cr-Kit与国标(GB5009.123-2014)石墨炉原子吸收光谱法(GFAAS)的符合率为100%。本研究成功研制了Cr-Kit,可满足食品总铬残留的检测要求。Aiming at development of an indirect competitive ELISA（icELISA）kit（Cr-Kit）for measuring total chromium mass concentration in food sample.The method of isothiocyanate（ITC）was used to conjugate Cr^（3＋）-iEDTA to bovine serum albumin（BSA）and immunogen Cr^（3＋）-iEDTA-BSA was obtained,which was identified by Ultraviolet（UV）and inductively coupled plasma atomic emission spectroscopy（ICP-AES）.BALB/c mice were immunized with Cr^（3＋）-iEDTA-BSA and hybridoma strains that secrete Cr^（3＋）-EDTA mAb were established by cell fusion,Cr^（3＋）-EDTA mAb were induced from in vivo method and their immunological properties were measured.Based on Cr^（3＋）-EDTA mAb,the CrKit for measurement of total chromium mass concentration was developed and verified.The results showed that Cr^（3＋）-iEDTA-BSA was synthesized successfully and the quality of Cr^（3＋）and BSA in Cr^（3＋）-iEDTA-BSA were 140.8 mg/L and 5.81 g/L,respectively.Three hybridoma strains of 2 A3 C11,2 A3 D9,2 A11 G5 were picked out and the best one was 2 A11 G5 which secreted stable antibody after six passages and had affinity constant（Ka）for 2.69×109 L/mol and had little or no cross-reactivity with other compounds.The Cr-Kit standard curve of the linear range was from 1.0 to 264μg/L,limit of detection was 1.0μg/L and IC50 value was 8.37μg/L.The average recovery rates of Cr^（3＋）-EDTA spiked in samples of river water,rice,wheat flour and pork were 101.7%,96.47%,95.8% and84.3%,respectively.The coincidence rate of the Cr-Kit was 100% with graphite furnace atomic absorption spectrometry（GFAAS）,which was national standard determination method（GB5009.123-2014）.In this study,Cr-Kit was successfully developed,it can meet the requirements of detection of total chromium mass residue in food.

关 键 词：CR3+ 免疫原 单克隆抗体 间接竞争ELISA试剂盒 总铬质量浓度 

分 类 号：TS207.51[轻工技术与工程—食品科学]