Broad-specificity antibody profiled by hapten prediction and its application in immunoassay for fipronil and major metabolites

Pollution by the pesticide fipronil (FPN) and three potentially stronger toxicological metabolites (FPNs) threatens ecological balance and human health. Thus, broad-specificity antibodies and immunoassays are required for FPNs determination. Here, we clarified the common skeleton of FPNs by molecular overlay and predicted the effectiveness of haptens by computational chemistry before chemical synthesis; finally, confirmation was obtained by an immunization experiment. One novel monoclonal antibody (mAb), 19F8, with broad specificity to FPNs was obtained based on the predicted hapten H3 that lacks the heterogeneous −SOCF3 group of FPNs and is stretched by a 6-carbon-long spacer arm at the −NH2 position. The molecular recognition study showed that the broad specificity of mAb 19F8 to FPNs originated from hydrogen/halogen bonds and pi-pi stacked/pi-alkyl interactions, demonstrating the reliability of the hapten prediction strategy. Finally, a rapid, reliable, and sensitive immunoassay was established based on mAb 19F8 with IC50 values of 1.85 − 4.89 ng mL−1 and limits of detection of 1.13 − 3.69 μg kg−1 for total FPNs in river water and egg samples. This work not only established a rapid screening method for total FPNs in environmental and food samples but also proposed an effective hapten prediction strategy to improve the efficiency of antibody preparation.