Study on Detection of Carmine by Indirect Competitive ELISA Based on Highly Specific Monoclonal Antibody

ZHU Lin; CHEN Zi-jian; LUO Lin; WANG Yu; ZHONG Yu-xin; SHEN Yu-dong; SUN Yuan-ming; XU Zhen-lin

doi:10.19969/j.fxcsxb.21040903

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Study on Detection of Carmine by Indirect Competitive ELISA Based on Highly Specific Monoclonal Antibody

Scientific Papers | 更新时间：2023-01-04

- Study on Detection of Carmine by Indirect Competitive ELISA Based on Highly Specific Monoclonal Antibody
- Journal of Instrumental Analysis Vol. 41, Issue 1, Pages: 128-135(2022)
- 作者机构：
  
  1.广东省食品质量安全重点实验室，华南农业大学食品学院，广东广州 510640
  2.广州市食品检验所，广东广州 510410
- 作者简介：
- 基金信息：
- DOI：10.19969/j.fxcsxb.21040903
  CLC：
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朱琳,陈子键,罗林等.基于高特异性单克隆抗体的间接竞争ELISA检测食品中胭脂红的研究[J].分析测试学报,2022,41(01):128-135.

ZHU Lin,CHEN Zi-jian,LUO Lin,et al.Study on Detection of Carmine by Indirect Competitive ELISA Based on Highly Specific Monoclonal Antibody[J].Journal of Instrumental Analysis,2022,41(01):128-135.
朱琳,陈子键,罗林等.基于高特异性单克隆抗体的间接竞争ELISA检测食品中胭脂红的研究[J].分析测试学报,2022,41(01):128-135. DOI： 10.19969/j.fxcsxb.21040903.

ZHU Lin,CHEN Zi-jian,LUO Lin,et al.Study on Detection of Carmine by Indirect Competitive ELISA Based on Highly Specific Monoclonal Antibody[J].Journal of Instrumental Analysis,2022,41(01):128-135. DOI： 10.19969/j.fxcsxb.21040903.

摘要

该研究设计合成了一种胭脂红半抗原，分别采用重氮化法和戊二醛法将半抗原与载体蛋白偶联制备人工抗原，通过免疫Bal b/c小鼠及杂交瘤技术成功筛选制备了胭脂红高特异性单克隆抗体，与苋菜红、柠檬黄等结构类似物无交叉反应。基于该抗体建立了间接竞争酶联免疫分析方法用于检测食品中胭脂红残留。该方法对胭脂红的半抑制浓度（IC,50,）和检出限（IC,10,）分别为10.1 ng/mL和0.98 ng/mL，线性范围为2 ~ 50 ng/mL。将该方法应用于山楂条和雪碧中胭脂红的快速检测，样品加标回收率分别为93.0% ~ 113%和93.0% ~ 100%，相对标准偏差分别为9.7% ~ 12%和3.2% ~ 3.9%。结果与HPLC－UV方法一致，表明该方法可用于食品中痕量胭脂红的快速筛查。

Abstract

Carmine is one of the most widely used synthetic pigments in foods．However，a certain dose of carmine may result in teratogenicity，sensitization，neurotoxicity，reproductive and developmental toxicity．In order to develop a simple，rapid and accurate analytical method for screening carmine residues in food，a novel hapten against carmine was designed and synthesized in this paper. It was conjugated to lactoferrin by diazotization method to obtain immunogen and bovine serum albumin by glutaraldehyde method to obtain coating antigen．After immunizing the Bal b/c mice by immunogen，the spleen cells were fused with SP 2/0 to obtain the hybridoma screening monoclonal antibodies with high specificity to carmine．Negligible cross-reactivity with carmine analogous such as amaranth，lemon yellow，brilliant blue and sunset yellow was obtained．Based on the obtained monoclonal antibody，a simple，rapid and sensitive indirect competitive enzyme-linked immunosorbent assay（icELISA） was developed and validated. After optimizing the working conditions，the proposed icELISA showed a good sensitivity for carmine detection， with a half inhibition rate（IC,50,） of 10.1 ng/mL，a limit of detection（IC,10,） of 0.98 ng/mL and a linear range of 2－50 ng/mL．The proposed assay was then applied to detection of carmine in sprite and hawthorn strips samples．Recoveries of 93.0%－100% for sprite samples and 93.0%－113% for hawthorn strips samples were obtained with relative standard deviations of 3.2%－3.9% and 9.7%－12%，respectively，which were validated by standard high performance liquid chromatography coupled with ultraviolet-visible spectroscopy（HPLC－UV）．Results showed that the proposed icELISA was an ideal tool for the rapid screening of carmine residues in food samples.

关键词

胭脂红半抗原设计单克隆抗体间接竞争ELISA食品

Keywords

carminehapten designmonoclonal antibodyindirect competitive enzyme-linked immunosorbent assay （icELISA）food

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