Preparation of high-specificity monoclonal antibody based on rational hapten design and development of immunoassay for accurate detection of dexamethasone in milk and animal tissues

Dexamethasone, a long-acting glucocorticoid, exhibits considerable structural similarity to other glucocorticoids. To avoid false-positive results in immunoassays due to cross-reactivity of antibodies with structural analogues, the preparation of highly specific antibodies is crucial. In this study, a novel hapten DEX-GA was designed and synthesized, and the monoclonal antibody (mAb) 3D1 was successfully prepared based on the hapten. The mAb 3D1 has the 50 % inhibitory concentration of 0.38 ng/mL for dexamethasone, with cross-reactivity below 7.45 % against twelve glucocorticoids including betamethasone, prednisone, triamcinolone and beclomethasone. Based on this antibody, indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and colloidal gold immunochromatography assay (GICA) were developed for accurate detection of dexamethasone in milk and animal tissues. The limits of detection of ic-ELISA in milk and animal tissues ranged from 0.132 to 0.215 μg/kg, which was a 1-3-fold increase in sensitivity compared with the previously reported ic-ELISA method. The GICA method established in this study had a visual limit of detection of 0.3 μg/L in milk. Notably, this constitutes the first application of GICA in animal tissues, demonstrating a visual detection limit of 0.2 μg/kg. The accuracy and reliability of both ic-ELISA and GICA were validated using liquid chromatography-tandem mass spectrometry.