食品中镉离子胶体金免疫层析快速检测方法的建立及应用

Establishment and Preliminary Application of Colloidal Gold Immunochromatography for Detecting Heavy Metal Cadmium Ion in Foods

目的:建立更加灵敏、特异的食品镉离子胶体金免疫层析快速检测方法。方法:用1-(4-异硫氰苄基)乙烯基二胺-N,N,N’,N’-四乙酸(1-(4-isothiocyanobenzyl)ethylenediamine-N,N,N’,N’-tetraacetic acid,iEDTA)鳌合镉离子合成Cd^(2+)-iEDTA半抗原,异硫氰酯法制备免疫原Cd^(2+)-iEDTA-牛血清白蛋白(bovine serum albumin,BSA)和包被原Cd^(2+)-iEDTA-鸡卵清蛋白(ovalbumin,OVA),电感耦合等离子体发射光谱法和聚丙烯酰胺凝胶法进行鉴定;用Cd^(2+)-iEDTA-BSA免疫Balb/C小鼠,细胞融合技术筛选Cd^(2+)-EDTA单克隆抗体(mAb)杂交瘤细胞株,体内诱生腹水法制备Cd^(2+)-EDTA mAb;应用Cd^(2+)-EDTA mAb建立Cd^(2+)残留胶体金免疫层析快速检测方法 (Cd^(2+)-Strip),并测定其性能。结果:免疫原偶联成功,Cd^(2+)-iEDTA-BSA中BSA与Cd^(2+)的含量分别为7.1 mg/mL和191.7μg/mL;筛选出1A3C11、2B7D8、2E10G9、4F3E7共4株杂交瘤细胞,经9次传代分泌抗体稳定,亲和力最高的2E10G9株亲和常数(Ka)为7.58×10~8 L/mol,对Cd^(2+)-iEDTA的半数抑制浓度为16.3μg/L,与Hg^(2+)-EDTA的交叉反应(cross-reaction,CR)率为18.6%,与其他重金属离子无CR;Cd^(2+)-Strip的检测时间为10 min,检出限为5μg/L,其检测结果与竞争ELISA试剂盒(Cd^(2+)ELISA-Kit)、电感耦合等离子体发射光谱法符合率为100%。结论:制备出了亲和力高、特异性强的Cd^(2+)mAb,建立了灵敏、特异、快速、简便的食品镉离子胶体金免疫层析快速检测方法。

Objective： To establish a colloidal gold immunochromatographic assay（Cd^2＋-Strip） for detecting Cd^2＋ residues in foods. Methods： Artificial hapten Cd^2＋-iEDTA was synthesized by using isotrhiocyanobenzyl-EDTA（iEDTA） to chelate Cd^2＋. The isothiocyanate method was used to conjugate Cd^2＋-iEDTA to BSA to obtain the artificial immunogen Cd^2＋-iEDTA-BSA. The coating antigen Cd^2＋-iEDTA-OVA was obtained in the same way. Both ICP-AES and SDS-PAGE were used to identify Cd^2＋-iEDTA-BSA. Balb/C mice were immunized with Cd^2＋-iEDTA-BSA and hybridoma lines that secreted anti-Cd^2＋-EDTA monoclonal antibody（mAb） were generated by cell fusion. A Cd^2＋ test strip was established with Cd^2＋-EDTA mAb and its traits were tested. Results： Cd^2＋-iEDTA-BSA was synthesized successfully and its concentration of BSA and Cd^2＋ was 7.1 mg/mL and 191.7 μg/mL respectively. Four hybridoma lines, namely 1A3C11, 2B7D8, 2E10G9, 4F3E7, were screened out and 2E10G9 was found to be the best one. The dissociation constant（Ka） of 2E10G9 was 7.58 × 10^8 L/mol and its sensitivity（IC50） was 16.3 μg/L, and it had little or no cross-reactivity with other metal ions, except for Hg^2＋-EDTA with 18.6%. The qualitative detection of Cd^2＋ with the Cd^2＋ test strip could be achieved in 10 minutes, with a limit of detection（LOD） of 5 μg/L. Its sensitivity was the same as that of competitive ELISA-Kit（Cd^2＋ ELISA-Kit） and inductively coupled plasma atomic emission spectrometry（ICP-AES）, and its coincidence rate was 100% as compared with Cd^2＋ ELISA-Kit and ICP-AES. Conclusion： The high affinity and specificity Cd^2＋-EDTA mAb has successfully been generated and used to establish a Cd^2＋ test strip with high sensitivity, specificity, rapidity and briefness. The test strip can be used for the rapid detection of Cd^2＋ residues in foods.