The conformation-sensitive immunoassay: a membrane based ELISA system for identifying antibodies sensitive to alterations of protein conformation.
A Conformation-Sensitive Immunoassay (CSI) has been developed for identification of antibodies that are sensitive to alterations in protein conformation. The method involves covalently coupling proteins to an activated hydrophilic membrane support. The membrane bound proteins are then treated under conditions known to alter protein conformation, immobilized in a non-native state via additional covalent interactions with the support, and subsequently probed using conventional ELISA techniques. This method has been validated using several well characterized conformation-sensitive antibodies to horse cytochrome c and sperm whale myoglobin. A panel of monoclonal antibodies directed against bovine somatotropin (bSt) has been partially characterized using this validated CSI procedure. Each of these antibodies to bSt has been shown to detect conformational alterations of bSt structure. Data are also presented that demonstrates that the accuracy of CSI analysis is superior to that of Western blotting for characterizing conformation-sensitive antibodies.
Pfund
W P
WP
Control Biotechnology Development, Upjohn Company, Kalamazoo, MI 49001.
Bourdage
J S
JS
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D015153:Blotting, Western; D002417:Cattle; D003574:Cytochrome c Group / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D013006:Growth Hormone / Q000276:immunology; D006736:Horses; D008567:Membranes, Artificial; D009211:Myoglobin / Q000276:immunology; D011487:Protein Conformation; D011489:Protein Denaturation; D011506:Proteins / Q000276:immunology* / Q000648:ultrastructure; D014907:Whales
D000911:Antibodies, Monoclonal; D003574:Cytochrome c Group; D008567:Membranes, Artificial; D009211:Myoglobin; D011506:Proteins; D013006:Growth Hormone