Preparation of a Broadly Specific Monoclonal Antibody-Based Indirect Competitive ELISA for the Detection of Benzodiazepines in Edible Animal Tissues and Feed

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• 作者：Juan Wang ; Yulian Wang ; Yuanhu Pan ; Liang Feng ; Dongmei Chen…
• 关键词：Benzodiazepines ; Monoclonal antibody ; Indirect competitive enzyme ; linked immunosorbent assay ; Edible animal tissue ; Feed
• 刊名：Food Analytical Methods
• 出版年：2016
• 出版时间：December 2016
• 年：2016
• 卷：9
• 期：12
• 页码：3407-3419
• 全文大小：741 KB
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Chemistry
  Food Science
  Chemistry
  Microbiology
  Analytical Chemistry
  
• 出版者：Springer New York
• ISSN：1936-976X
• 卷排序：9

文摘

Incorrect use of benzodiazepines could result in serious health problems. To monitor the illegal use of benzodiazepine compounds in animals, a group-specific monoclonal antibody (mAb) was prepared in this study. The obtained 3D7 mAb, which is an IgG1 isotype mAb, displayed an IC₅₀ value of 8.9 ng mL−1 for diazepam and exhibited cross-reaction for diazepam (100 %), nitrazepam (49 %), nordiazepam (140 %), temazepam (32 %), oxazepam (17 %), estazolam (7.5 %), and alprazolam (2.4 %). Based on this mAb, for the first time in this study, an optimized indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) protocol that did not require complicated sample preparation and clean-up was developed. The detection limits of this ic-ELISA for benzodiazepines ranged from 1.2 to 3.3 μg kg−1 in muscle matrix and from 25.2 to 55.4 μg kg−1 in feed matrix. The recoveries ranged from 70.9 to 111.3 % with coefficients of variation below 15.0 %. Good correlations (r > 0.9494) between the results of the ic-ELISA and high-performance liquid chromatography were also observed. This simple method reduced the time required for sample preparation, ensured greater throughput, and met the requirements for benzodiazepine residue analyses. In conclusion, the proposed method is a sensitive and rapid multi-residue technique that offers a cost-effective alternative to current published procedures without any concession in the ability to detect benzodiazepine sedative misuse.