title: Spectroscopic evidence that monoclonal antibodies recognize the dominant conformation of medium-sized synthetic peptides.
abstract: Spectroscopic methods have amply documented that small- and medium-sized peptides tend to assume unordered conformations in water. The conformational tendencies, however, manifest in halogenated alcohols, and the preferred secondary structures are apparent from the circular dichroism (CD) spectra. Here we report the results of immobilizing peptide and protein antigens from various mixtures of trifluoroethanol and water during enzyme-linked immunosorbent assays. The increased recognition by the appropriate monoclonal antibodies (mAbs) is correlated with the increase of the alpha helical, beta turn, or beta pleated sheet content of the peptides presented in the different solvent mixtures. Remarkably, the antibody binding can be detected at considerably lower antigen levels if the antigen is immobilized from trifluoroethanol. The antigens we used corresponded to fragments of normal human neurofilaments and tau protein found in the paired helical filaments of Alzheimer's disease, and the nucleoprotein of rabies virus. The conformation of myoglobin is as stable in water as in trifluoroethanol, and therefore acted as a negative control. Indeed, the recognition of myoglobin did not increase upon increasing the trifluoroethanol concentration in the solvent used to apply the antigen to the plate. The possibility of imperfect binding to the plastic carrier or nonspecific binding to irrelevant antibodies is excluded by using control experiments. We offer the first direct evidence that the mAbs recognize the secondary structure of epitopes, and that it is possible to correlate the binding conformation of the epitopes with CD measurements made in trifluoroethanol-water mixtures.
authors.affiliation: Wistar Institute of Anatomy and Biology, Philadelphia, PA 19104.
authors.forename: E
authors.initials: E
authors.lastname: Lang
authors.forename: G I
authors.initials: GI
authors.lastname: Szendrei
authors.forename: V M
authors.initials: VM
authors.lastname: Lee
authors.forename: L
authors.initials: L
authors.lastname: Otvos
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000937:Antigen-Antibody Reactions / Q000276:immunology; D002213:Capsid / Q000138:chemical synthesis / Q000276:immunology; D002942:Circular Dichroism; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D006801:Humans; D009211:Myoglobin / Q000276:immunology; D016900:Neurofilament Proteins / Q000138:chemical synthesis / Q000276:immunology; D010446:Peptide Fragments; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D014270:Trifluoroethanol; D014758:Viral Core Proteins / Q000138:chemical synthesis / Q000276:immunology; D016875:tau Proteins / Q000138:chemical synthesis / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D009211:Myoglobin; D016900:Neurofilament Proteins; D010446:Peptide Fragments; D010455:Peptides; D014758:Viral Core Proteins; D016875:tau Proteins; D014270:Trifluoroethanol