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Updated: Jul 6, 2025
Multiplexed Fluorometric ImmunoAssay Testing Methodology and Troubleshooting
Published on: December 12, 2011
Antibody Production, Immunoassay Establishment, and Specificity Study for Flunixin and 5-Hydroxyflunixin
Qing Li1, Jiafei Mi1, Yuchen Bai1
1National Key Laboratory of Veterinary Public Health Safety, College of Veterinary Medicine, Beijing Key Laboratory of Detection Technology for Animal-Derived Food Safety and Beijing Laboratory for Food Quality and Safety, China Agricultural University, Beijing 100193, China.
Abstract:
Flunixin (FLU) is a nonsteroidal drug that is widely used in animals, causing severe drug residues in animal-derived foods and environment. The development of antibody-based rapid immunoassay methods is of great significance for the monitoring of FLU and its metabolite 5-hydroxyflunixin (5-FLU). We prepared monoclonal antibodies (mAbs) with different recognition spectra through FLU-keyhole limpet hemocyanin conjugates as immunogen coupled with antibody screening strategies. mAb5E6 and mAb6D7 recognized FLU with high affinity, and mAb2H5 and mAb4A4 recognized FLU and 5-FLU with broad specificity. Through evaluating the recognition of these mAbs against more than 11 structural analogues and employing computational chemistry, molecular docking, and molecular dynamics methodologies, we preliminarily determined the recognition epitope and recognition mechanism of these mAbs. Finally, an indirect competitive enzyme-linked immunosorbent assay for FLU based on mAb6D7 was developed, which exhibited limits of detection as low as 0.016-0.042 μg kg -1 (L-1) in milk and muscle samples.
