Stability of monoclonal antibody-defined epitopes.
Epitope instability can limit the applications of monoclonal antibody (mAb) technology in laboratory and clinical research. We exposed a group of representative antigens on human hepatocellular carcinoma (HCC) cells to physiochemical insults to study epitope stability as measured by mAb immunoreactivity. Each epitope was found to have a unique pattern of instability which serves to biophysically characterize the antigen and defines the conditions to which the antigen can be exposed during laboratory and clinical investigations. Individual antigens were found to be unstable within a surprisingly well defined window of solvent polarities while being stable on either side of that window. Several antigens were observed to be unstable when exposed to transient changes in pH. When a critical temperature between 42 degrees C and 65 degrees C was achieved, epitopes which were thermosensitive underwent a sudden loss in immunoreactivity. This critical temperature was found to be pH dependent. The effects of polarity, pH, and temperature on epitope stability are consistent with changes in protein structural conformation. In addition, we found that certain fixatives cause a time and concentration dependent loss of epitope immunoreactivity. This study provides a rapid and easy determination of monoclonal antibody-defined epitope stability; the results of which serve to guide further studies on the antigen and to characterize the antigen on the basis of its unique physiochemical stability.
Wilson
B E
BE
Molecular Hepatology Laboratory, Massachusetts General Hospital Cancer Center, Charlestown 02129.
Sun
S
S
Ozturk
M
M
Wands
J R
JR
D000818:Animals; D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm / Q000276:immunology; D000939:Epitopes; D005260:Female; D006863:Hydrogen-Ion Concentration; D008113:Liver Neoplasms / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D013696:Temperature; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D000939:Epitopes