Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer reviewed journal
Publication Acceptance Date: 10/1/2004
Publication Date: 12/29/2004
Citation: Wang, X., Chen, F., Wan, P.J., Huang, G. 2004. DEVELOPMENT OF MONOCLONAL ANTIBODY-BASED ENZYME-LINKED IMMUNOSORBENT ASSAY FOR GOSSYPOL ANALYSIS IN COTTONSEED MEALS. Journal of Agricultural and Food Chemistry. 52(26):7793-7797. Interpretive Summary: Gossypol is a natural pigment present in cottonseed and other cotton plant tissues. The naturally existing pigment was known to possess biological activities, such as, antitumor, antiviral, insecticidal, antioxidative, antinutritional effect in different levels. Over consumption of gossypol containing feed by livestock could cause problems such as malnutrition, pulmonary edema, cardiac irregularity, etc. Precise determination of the gossypol content in cottonseed products is important. Current analysis of gossypol uses either spectrophotometric approach as defined by the American Oil Chemists' Society (AOCS) official method (Ba 7-58, 1987; Ba 8-78, 1987) or HPLC method. These methods require up to grams quantity of sample and have limited detection limit of gossypol at 0.5 ' 10 ppm level. Immunochemical assay using a monoclonal antibody offers an alternative approach to determine gossypol. It has shown the benefits over the traditional methods. This Mab could measure gossypol directly without complicated chemistry. It requires milligram quantity of sample and is able to detect gossypol less than 20 ppb which is 25 ' 500 times more sensitive than the HPLC and AOCS methods.
Technical Abstract: A monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed for the analysis of gossypol in cottonseed meal. 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 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 0.25 microgram/mL, whereas the detection limit for gossypol was 0.02 microgram/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 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).