Broad‐Spectrum Recombinant Antibody Profiled by Hapten Prediction and Its Application in Immunoassay for Anabolic‐Androgenic Steroids

Illegal use of anabolic-androgenic steroids (AASs) to feed livestock can lead to food residues and endanger public health. Thus, the development of rapid, sensitive detection methods for AASs is urgently needed for AASs to safeguard food safety. In this study, four novel haptens were designed for six AASs (androstenedione, 1,4-androstadienedione, boldenone, methandienone, testosterone, and methyltestosterone). By integrating computer simulation with animal immunization, we identified an optimal hapten combination and generated a hybridoma cell line capable of broadly recognizing all six AASs. Based on cell sequencing, a recombinant antibody (rAb-4E2) was prepared, with half-maximal inhibitory concentrations of 3.37-22.24 ng/mL for the six AASs. Molecular docking revealed TRP106 and VAL99 as key residues for broad-spectrum recognition. A lateral flow immunochromatographic assay strip using rAb-4E2 was developed for AASs detection in milk, beef, and fish, achieving visual detection limits of 2-10 ng/mL, 5-20 µg/kg, and 10-100 µg/kg, respectively. Validation with LC-MS/MS confirmed its reliability for real samples, offering an effective approach for rapid qualitative hormone screening.