食品科学

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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

王亚楠1,王晓斐2,牛琳琳1,雷 壮1,张海棠1,王自良1,*   

  1. 1.河南科技学院动物科技学院,河南 新乡 453003;2.河南科技学院新科学院,河南 新乡 453003
  • 出版日期:2016-09-25 发布日期:2016-09-26
  • 通讯作者: 王自良
  • 基金资助:

    “十二五”国家科技支撑计划项目(2011BAK10B01;2014BAD13B05)

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

WANG Yanan1, WANG Xiaofei2, NIU Linlin1, LEI Zhuang1, ZHANG Haitang1, WANG Ziliang1,*   

  1. 1. College of Animal Science and Veterinary Medicine, Henan Institute of Science and Technology, Xinxiang 453003, China;
    2. Xinke College, Henan Institute of Science and Technology, Xinxiang 453003, China
  • Online:2016-09-25 Published:2016-09-26
  • Contact: WANG Ziliang

摘要:

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Cd2+-iEDTA半抗原,异硫氰酯法制备免疫原Cd2+-iEDTA-牛血清白蛋白(bovine serum albumin,BSA)和包被原Cd2+-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Cd2+-iEDTA-BSA免疫Balb/C小鼠,细胞融合技术筛选Cd2+-EDTA单克隆抗体(mAb)杂交瘤细胞株,体内诱生腹水法制备Cd2+-EDTA mAb;应用Cd2+-EDTA mAb建立Cd2+残留胶体金免疫层析快速检测方法(Cd2+-Strip),并测定其性能。结果:免疫原偶联成功,Cd2+-iEDTA-BSA中BSA与Cd2+的含量分别为7.1 mg/mL和191.7 μg/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7.58×108 L/mol,对Cd2+-iEDTA的半数抑制浓度为16.3 μg/L,与Hg2+-EDTA的交叉反应(cross-reaction,CR)率为18.6%,与其他重金属离子无CR;Cd2+-Strip的检测时间为10 min,检出限为5 μg/L,其检测结果与竞争ELISA试剂盒(Cd2+ 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Cd2+ 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。

关键词: 镉离子, 免疫原, 单克隆抗体, 胶体金免疫层析, 快速检测

Abstract:

Objective: To establish a colloidal gold immunochromatographic assay (Cd2+-Strip) for detecting Cd2+ residues
in foods. Methods: Artificial hapten Cd2+-iEDTA was synthesized by using isotrhiocyanobenzyl-EDTA (iEDTA) to chelate
Cd2+. The isothiocyanate method was used to conjugate Cd2+-iEDTA to BSA to obtain the artificial immunogen Cd2+-
iEDTA-BSA. The coating antigen Cd2+-iEDTA-OVA was obtained in the same way. Both ICP-AES and SDS-PAGE were
used to identify Cd2+-iEDTA-BSA. Balb/C mice were immunized with Cd2+-iEDTA-BSA and hybridoma lines that secreted
anti-Cd2+-EDTA monoclonal antibody (mAb) were generated by cell fusion. A Cd2+ test strip was established with Cd2+-
EDTA mAb and its traits were tested. Results: Cd2+-iEDTA-BSA was synthesized successfully and its concentration of
BSA and Cd2+ 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 × 108 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 Hg2+-EDTA with 18.6%. The qualitative detection of Cd2+ with the Cd2+ 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 (Cd2+ ELISA-Kit) and
inductively coupled plasma atomic emission spectrometry (ICP-AES), and its coincidence rate was 100% as compared
with Cd2+ ELISA-Kit and ICP-AES. Conclusion: The high affinity and specificity Cd2+-EDTA mAb has successfully been
generated and used to establish a Cd2+ test strip with high sensitivity, specificity, rapidity and briefness. The test strip can be
used for the rapid detection of Cd2+ residues in foods.

Key words: cadmium ion, immunogen, monoclonal antibody, colloidal gold immunochromatographic assay, rapid detection

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