Authenticating Emergent Adulterant 6-Benzylaminopurine in Bean Sprouts: Virtual Hapten Similarity Enhanced Immunoassay

6-Benzylaminopurine (6-BA), a plant growth regulator with cytokinin-like properties, was recently reported to be illegally used in bean sprouts to increase their commercial appearance. It is still nevertheless challenging to quickly detect this adulteration. In this work, four novel haptens (haptens 1-4) of 6-BA were rationally designed with computer-assisted modeling analysis and then synthesized for use as immunizing haptens to produce antibodies. One of two obtained antibodies showed high sensitivity and specificity toward 6-BA. Based on the most sensitive anti-6-BA antibody, an indirect competitive enzyme-linked immunosorbent assay (icELISA) was performed, which demonstrated a 50% inhibition concentration (IC₅₀) of 1.18 μg/L and a limit of detection of 0.075 μg/L. The average recoveries of this icELISA for 6-BA of spiked samples ranged from 87.2 to 95.0% with a coefficient of variation of less than 8.7%. Furthermore, the blind samples were detected simultaneously by the method and HPLC-MS/MS, and the results showed good agreement with each other. Therefore, the proposed icELISA can facilitate the rapid surveillance screening of adulterated 6-BA in sprout vegetables.