Development of competitive direct ELISA for gossypol analysis.
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 microg/mL, whereas the detection limit for gossypol was 0.005 microg/mL. We also observed a good correlation (R2= 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.
Department of Food Science and Human Nutrition, Clemson University, Clemson, South Carolina 29634, USA.
Jing
J
Wang
Xi
X
Wang
Feng
F
Chen
Peter J
PJ
Wan
Guoqing
G
He
Zhigang
Z
Li
D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D001667:Binding, Competitive; D003369:Cottonseed Oil / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006072:Gossypol / Q000032:analysis* / Q000276:immunology; D006735:Horseradish Peroxidase
D000911:Antibodies, Monoclonal; D003369:Cottonseed Oil; D006735:Horseradish Peroxidase; D006072:Gossypol