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A high-affinity monoclonal antibody (mAb) has been prepared and separately a gold nanoparticle (AuNP)-based and a near-infrared (NIR) fluorescence-based lateral flow immunoassay (LFA) developed for determination of 5-hydroxyflunixin residue in raw milk. The AuNP and IRDye® 800CW were used to label anti-5-hydroxyflunixin mAb to form the AuNP-mAb and NIR dye-mAb conjugates, respectively. Quantitative determination of 5-hydroxyflunixin was achieved by imaging the optical or fluorescence intensity of the AuNP-mAb and NIR dye-mAb captured o