Binding to native proteins by antipeptide monoclonal antibodies.
mAb raised against synthetic peptides derived from cholera toxin, myohemerythrin, and sickle hemoglobin were analyzed by both solid-phase and solution-phase methods. Antipeptide mAb against cholera toxin (mAb TE32 and TE33), against myohemerythrin (mAb B13I2, B13C2, and B13F2), and against sickle hemoglobin (mAb HuS-1 and HuS-2), had been previously described and used for vaccine development, structural characterization, or identification of a specific antigenic determinant, and each was apparently capable of binding both peptide and native Ag. In this study, all were found to bind whole protein when tested against immobilized Ag in a standard solid-phase assay (ELISA), yet none of the antibodies recognized the Ag in its true native form, failing to bind when tested in several solution-phase assay systems, including size exclusion HPLC. This discrepancy may be the result of modifications of the epitope created by interaction and possible denaturation of the protein on the solid-phase matrix. As a consequence, binding of these antibodies to peptides, either immobilized or in solution, or to immobilized protein, cannot be used to infer that the peptide has assumed a conformation that corresponds to that of the cognate sequence in the native protein. A re-evaluation of binding data that relates antipeptide mAb to native structural characteristics may be necessary.
Spangler
B D
BD
Biological and Medical Research Division, Argonne National Laboratory, IL 60439-4833.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions / Q000502:physiology*; D001667:Binding, Competitive; D002772:Cholera Toxin / Q000276:immunology; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004797:Enzyme-Linked Immunosorbent Assay; D006422:Hemerythrin / Q000031:analogs & derivatives / Q000276:immunology; D006451:Hemoglobin, Sickle / Q000276:immunology; D006801:Humans; D010455:Peptides / Q000276:immunology*; D011485:Protein Binding
D000911:Antibodies, Monoclonal; D006422:Hemerythrin; D006451:Hemoglobin, Sickle; D010455:Peptides; C008571:myohemerythrin; D002772:Cholera Toxin