Development Of An Enzyme Linked-Immunosorbent Assay For The Determination Of Phenylurea Herbicides

An enzyme-linked immunosorbent assay (ELISA) was systematically studied. Based upon the produced monoclonal antibody (McAb). an indirect competitive ELISA for a set of phenylurea herbicides (PUHs) was established. A method of computer-aided molecular modeling (CAMM) was established in quantitative structure-activity relationship (QSAR) studies to obtain a deeper insight into the PUHs antibody interactions on how and which molecular properties of the analytes quantitatively affect the antibody recognition.A PUH hapten was coupled to carrier protein (Bovine Serum Albumin, BSA) to obtain immunogen, which was applied to immunize mice. Hybridoma was produced with the fusion of mouse spleen cell and myeloma cell. Optimal McAb was produced by limited cloning and screening. Conditions of coating amount, antibody amount, amount of secondary antibody, and blocking buffer were optimized, and an indirect competitive ELISA was established for twelve PUHs with the half-maximum inhibition concentration (IC50) of1.7to920.7ng/mL, and the detection limits were0.4to76.9ng/mL. Phosphate Buffer Saline was applied to10-fold dilute Haihe River water, which was spiked with PUHs, and the recoveries were77.05-121.71%, indicating the reliability of the established ELISA.CAMM-based QSAR analyses were employed to study the effect of PUHs’structures, substructures, and other properties on antibody recognition. It was revealed that (1) the most important impact factor of the antibody recognition was the PUHs’hydrophobicity, which provided a quadratic correlation to the antibody recognition.(2) The frontier-orbital energy parameterumo was also demonstrated as a related determinant for this reaction.(3) Hapten-carrier-linking groups were less exposed to antibodies during immunization, thus groups of the analytes in the same position were generally considered to be less contributive to antibody recognition during immunoassay. But the results of substructure level analysis showed that these groups played an important role in the antigen-antibody interaction.This is the first time introducing Hansch equation, substructure level2D-QSAR and Hologram QSAR to the study of antigen-antibody interaction. It is demonstrated that CAMM-based QSAR analyses were reliable tools on the study of antibody recognition.