CAMM-Based Rational Hapten Design for Monoclonal Antibodies with Different Specificities against Avermectins and a Sensitive Chemiluminescence Immunoassay

Abstract

Abstract Computer-aided molecular modeling (CAMM) has become an important tool for guiding rational hapten design. Avermectins are a family of macrocyclic lactones with highly similar structures, making monoclonal antibody (mAb) specificity difficult to control. In this work, we used CAMM to analyze the antigenic determinant of avermectin (AVMs), and showed that the macrocyclic lactone and glycosidic linkage are highly reactive. Based on these findings, four haptens (H1−H4) were rationally designed and used to produce mAbs for AVMs. The broadly reactive mAb A1 showed high sensitivity for abamectin B1a, emamectin, eprinomectin, ivermectin, and doramectin, with IC50 values of 5.6, 3.3, 5.1, 13.3, and 18.9 ng/mL, respectively. In contrast, the class-selective mAb A2 showed preferential recognition of abamectin B1a, emamectin, and eprinomectin, with IC50 values of 3.5, 3.8, and 4.6 ng/mL, respectively. Further CAMM analysis indicated that H-Gly42 may contribute to the broad-spectrum recognition of AVMs, whereas exposure of the macrocyclic lactone ring may influence whether the resulting mAbs exhibit broad-spectrum or class-selective recognition. Using mAb A1, a fully automated chemiluminescence enzyme immunoassay was developed, achieving detection limits that met regulatory requirements. In milk samples, the assay showed recoveries of 84.5-104.0% and good accuracy and stability, supporting its application for the rapid detection of AVM residues in dairy products.