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.
Wang
Jing
J
Department of Food Science and Human Nutrition, Clemson University, Clemson, South Carolina 29634, USA.
Wang
Xi
X
Chen
Feng
F
Wan
Peter J
PJ
He
Guoqing
G
Li
Zhigang
Z
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