Development of Competitive Direct ELISA for Gossypol Analysis

• Published in: Journal of agricultural and food chemistry Vol. 53; no. 14; pp. 5513 - 5517
• Main Authors: Wang, Jing, Wang, Xi, Chen, Feng, Wan, Peter J, He, Guoqing, Li, Zhigang
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 13.07.2005
• Subjects: Antibodies, Monoclonal - isolation & purification; Binding, Competitive; Biological and medical sciences; competitive direct enzyme-linked immunosorbent assay; cottonseed meal; Cottonseed Oil - chemistry; enzyme-linked immunosorbent assay; Enzyme-Linked Immunosorbent Assay - methods; Food industries; Fundamental and applied biological sciences. Psychology; gossypol; Gossypol - analysis; Gossypol - immunology; Horseradish Peroxidase; monoclonal antibodies; Terpene; Purification; Development; Monoclonal antibody; Enzyme immunoassay; Detection; Gossypol; ELISA assay; ELISA; immunoassay
• ISSN: 0021-8561; 1520-5118
• DOI: 10.1021/jf050203c

Anti-gossypol monoclonal antibody was purified from cell culturing supernatant by ammonium sulfate precipitation and Protein A AffinityPak. 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 competitive direct enzyme-linked immunosorbent assay (cdELISA) 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 μg/mL, whereas the detection limit for gossypol was 0.005 μg/mL. We also observed a good correlation (R 2 = 0.96, P < 0.05) between the cdELISA method and the AOCS official method for “free” gossypol (extractable gossypol and gossypol derivatives by 70% acetone) analysis of cottonseed meals. This indicates that the newly developed cdELISA could be a valuable and feasible alternative for determination of “free” gossypol, especially when the available sample is limited with relatively low gossypol concentration. Keywords: Gossypol; monoclonal antibody; purification; immunoassay; ELISA