Novel hapten design, antibody recognition mechanism study, and a highly sensitive immunoassay for diethylstilbestrol in shrimp.

Over the past few years, there has been a lack of progress in the quality of diethylstilbestrol (DES) antibodies used in immunoassay. In this study, a new immunizing hapten was designed for remarkably sensitive and specific antibody generation against diethylstilbestrol. By introducing a benzene ring instead of the traditional linear chain alkane as the hapten spacer, a more specific immune reaction was induced in the process of immunization. The developed polyclonal antibodies were characterized using the indirect competitive enzyme-linked immunosorbent assay (icELISA). Under optimized conditions, the half maximal inhibitory concentration (IC50) of the best polyclonal antibody was 0.14 ng/mL and it displayed low cross-reactions (CRs) with the structural analogs such as hexestrol (HEX) and dienestrol (DI). The molecular modeling and quantum chemical computation revealed that the lowest CR of the DES antibody to DI was mainly due to the huge three-dimensional conformational difference between DES and DI. Finally, a highly sensitive icELISA method based on the polyclonal antibody was developed for the determination of DES in shrimp tissue. The limit of detection (LOD) was as low as 0.2 μg/kg in shrimp and the recoveries in the spiked samples ranged from 83.4 to 90.8% with the coefficient of variation less than 13.8%. These results indicated that the use of an aromatic ring as the immunizing hapten spacer arm could be a potential strategy for the enhancement of anti-DES antibody sensitivity, and the established icELISA was applicable to the trace detection of DES in shrimp. Graphical abstract.