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

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+-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+-iEDTA-BSA免疫BALB/c小鼠，细胞融合技术筛选抗Cr+-EDTA mAb细胞株，体内诱生腹水法制备Cr+-EDTA mAb，并对其特性进行分析；应用Cr+-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+-iEDTA-BSA中Cr+和BSA的质量浓度分别为140.8 mg/L和5.81 g/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，与其他重金属离子交叉反应；Cr-Kit 标准曲线的线性范围为1.0～264 μg/L，检测限为1.0 μg/L，IC50为8.37 μg/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+-iEDTA to bovine serum albumin(BSA) and immunogen Cr+-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+-iEDTA-BSA and hybridoma strains that secrete Cr+-EDTA mAb were established by cell fusion,Cr+-EDTA mAb were induced from in vivo method and their immunological properties were measured. Based on Cr+-EDTA mAb,the Cr-Kit for measurement of total chromium mass concentration was developed and verified. The results showed that Cr+-iEDTA-BSA was synthesized successfully and the quality of Cr+and BSA in Cr+-iEDTA-BSA were 140.8 mg/L and 5.81 g/L,respectively. Three hybridoma strains of 2A3C11,2A3D9,2A11G5 were picked out and the best one was 2A11G5 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 264 μg/L,limit of detection was 1.0 μg/L and IC50 value was 8.37 μg/L. The average recovery rates of Cr+-EDTA spiked in samples of river water ，rice,wheat flour and pork were 101.7%,96.47%,95.8% and 84.3%,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.