Structural analysis of bovine somatotropin using monoclonal antibodies and the conformation-sensitive immunoassay.
Bovine somatotropin was studied with respect to thermal stability, quantitative thermal denaturation kinetics, and refolding potential following thermal denaturation using a panel of 6 monoclonal antibodies and the Conformation-Sensitive Immunoassay (CSI). The antibody panel consisted of 4 conformation-dependent and 2 sequence-specific antibodies. Each of the antibodies revealed unique thermal stability profiles for their respective epitopes suggesting that they each recognize different antigenic determinants. Comparing the thermal stability profiles generated with these antibodies allowed the stability of bovine somatotropin to be "dissected" based on individual structural features. The degree to which bovine somatotropin is stabilized by disulfide bonds was examined using CSI-based quantitative thermal denaturation kinetics profiles generated under reducing and nonreducing conditions. All of the conformational epitopes unfolded faster under reducing conditions indicating that the two disulfide bonds within the somatotropin molecule impart some degree of global stabilization. The ability of bovine somatotropin to refold after reducing or nonreducing thermal denaturation was also examined using the antibody panel and the CSI. The results show that, although significant refolding was evident for some epitopes, bovine somatotropin cannot refold to the native state following thermal denaturation under either reducing or nonreducing conditions.
Pfund
W P
WP
Analytical Research & Specification Development, Pharmacia & Upjohn, Inc., Kalamazoo, Michigan 49001, USA.
Bourdage
J S
JS
Farley
K A
KA
D000818:Animals; D000911:Antibodies, Monoclonal; D000937:Antigen-Antibody Reactions; D002417:Cattle; D004220:Disulfides; D000939:Epitopes; D013006:Growth Hormone / Q000737:chemistry* / Q000276:immunology*; D007118:Immunoassay / Q000379:methods; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013816:Thermodynamics
D000911:Antibodies, Monoclonal; D004220:Disulfides; D000939:Epitopes; D013006:Growth Hormone