ARS | Publication request: Development of competitive direct ELISA for gossypol analysis

Submitted to: Journal of Agriculture and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 1, 2005
Publication Date: June 8, 2005
Citation: Wang, J., Wang, X., Chen, F., Wan, P.J., He, G., Li, Z. 2005. Development of competitive direct ELISA for gossypol analysis. Journal of Agriculture and Food Chemistry. 53(14):5513-5517.

Interpretive Summary: Gossypol, a naturally occurring polyphenolic compound in cotton tissues and seed, is a powerful antioxidant with many beneficial functions as well as an antinutrient for livestock. Traditionally, its determination relies on spectrometric or high performance liquid chromatography method. Both methods are reproducible. But their detection limit often requires large sample sizes. This can be alleviated by using immuno assay. This report presents a direct competitive enzyme-linked immunosorbent assay (dc-ELISA) for gossypol analysis. I50 value, the concentration of gossypol causing 50% inhibition of the maximum ELISA signal in the competitive standard curve, was 0.067 'g/mL, whereas the detection limit for gossypol was 0.005 'g/mL (~5 ppb). We also observed a high correlation (R2=0.96, P<0.05) between the direct competitive ELISA method and the AOCS official method for Free Gossypol (extractable gossypol by 70% acetone) analysis of cottonseed meals. This indicates that the newly developed dc-ELISA can be a valuable alternative for determination of Free Gossypol, especially, when the available sample is limited with relatively low gossypol concentration and the number of sample is huge.

Technical Abstract: Anti-gossypol monoclonal antibody was purified from cell culturing supernatant by ammonium sulfate precipitation and Protein A AffinityPackTM. The antigen (i.e., gossypol) was labeled with horseradish peroxidase through Schiff-base formation. Both the purified antibody and the enzyme-labeled gossypol were employed to develop a direct competitive enzyme-linked immunosorbent assay (dc-ELISA) for gossypol analysis. I50 value, the concentration of gossypol casing 50% inhibition of the maximum ELISA signal in the competitive standard curve, was 0.067 'g/mL, whereas the detection limit for gossypol was 0.005 'g/mL (~5ppb). We also observed a high correlation (R2=0.96, P<0.05) between the direct competitive ELISA method and the AOCS official method for Free Gossypol (extractable gossypol by 70% acetone) analysis of cottonseed meals. This indicates that the newly developed dc-ELISA can be a valuable and feasible alternative for determination of Free Gossypol, especially, when the available sample is limited with relatively low gossypol concentration and the number of sample is huge.