Development of Monoclonal Antibody-Based Enzyme-Linked Immunosorbent Assay for Gossypol Analysis in Cottonseed Meals

Abstract

A monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed for the analysis of gossypol in cottonseed meals. First, the checkerboard method was used to determine the optimum amount of coating antigen gossypol-BSA (bovine serum albumin) and primary anti-gossypol monoclonal antibody (Mab) needed in the ic-ELISA. Second, the effects of several physical (incubation time and temperature) and chemical (solvent types and concentrations) conditions on the performance of Mab on ic-ELISA were investigated to get a rapid robust assay with high sensitivity. Under the established optimized condition, the concentration of gossypol giving 50% reduction of the maximum ELISA signal (I50) in the competitive standard curve was 0.20 microg/mL, whereas the detection limit for gossypol was 0.024 microg/mL. This ic-ELISA method for the analysis of gossypol extracted by methanol from a variety of cottonseed meals was further compared with the official method of the American Oil Chemists' Society (AOCS). The amounts of gossypol determined by the ic-ELISA had a good correlation with those obtained by the AOCS method (R2 = 0.90).