Monoclonal Elisa for 2,4-Dichlorophenoxyacetic Acid: Characterization of Antibodies and Assay Optimization

Twelve monoclonal antibodies (MAbs) against the widely used herbicide 2,4-D were produced by hybridomas from two fusions of murine myeloma cells and spleen cells isolated from BALB/c mice immunized with hapten conjugated via the carboxyl group to thyroglobulin. To evaluate the sensitivity and selectivity of MAbs, competitive indirect ELISA was used. MAb E2/G2 exhibited the highest sensitivity toward 2,4-D (IC50 = 0.8 ng/mL) and a favorable selectivity toward 18 structurally related substances. Besides the expected highcross-reactivity with methyl ester 2,4-D (104.8%),cross-reactivity with MCPA (13.8%) and with 2,4,5-T (9.5%) was found. Cross-reactivity with other structural analogs did not exceed 2.7%. Optimization studies showed that in competitive ELISAs for 2,4-D coating conjugates with hapten densities of 2.3 and 3.3 mol of 2,4-D/mol of BSA were more sensitive than conjugates with hapten densities of 15.9 and 26.5 mol of 2,4-D/mol of BSA. The best dose-response curves presented in this study were almost linear in the concentration range 0.2-10 ng/mL