Characterization of ELISA Antibody-Antigen Interaction using Footprinting-Mass Spectrometry and Negative Staining Transmission Electron Microscopy
| Autor: | Galahad Deperalta, Aaron T. Wecksler, Matthew D. Callahan, Margaret S. Lin, Denise C. Krawitz |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Models Molecular Glycan medicine.drug_class Enzyme-Linked Immunosorbent Assay CHO Cells Monoclonal antibody Negative Staining Epitope Mass Spectrometry 03 medical and health sciences Mice Cricetulus Western blot Antigen Microscopy Electron Transmission Structural Biology medicine Animals Protein Footprinting Spectroscopy medicine.diagnostic_test biology Chemistry Antibodies Monoclonal Negative stain Footprinting Molecular Docking Simulation 030104 developmental biology Epitope mapping Biochemistry biology.protein Lysophospholipase Epitope Mapping |
| Zdroj: | Journal of the American Society for Mass Spectrometry. 29(5) |
| ISSN: | 1879-1123 |
| Popis: | We describe epitope mapping data using multiple covalent labeling footprinting-mass spectrometry (MS) techniques coupled with negative stain transmission electron microscopy (TEM) data to analyze the antibody-antigen interactions in a sandwich enzyme-linked immunosorbant assay (ELISA). Our hydroxyl radical footprinting-MS data using fast photochemical oxidation of proteins (FPOP) indicates suppression of labeling across the antigen upon binding either of the monoclonal antibodies (mAbs) utilized in the ELISA. Combining these data with Western blot analysis enabled the identification of the putative epitopes that appeared to span regions containing N-linked glycans. An additional structural mapping technique, carboxyl group footprinting-mass spectrometry using glycine ethyl ester (GEE) labeling, was used to confirm the epitopes. Deglycosylation of the antigen resulted in loss of potency in the ELISA, supporting the FPOP and GEE labeling data by indicating N-linked glycans are necessary for antigen binding. Finally, mapping of the epitopes onto the antigen crystal structure revealed an approximate 90° relative spatial orientation, optimal for a noncompetitive binding ELISA. TEM data shows both linear and diamond antibody-antigen complexes with a similar binding orientation as predicted from the two footprinting-MS techniques. This study is the first of its kind to utilize multiple bottom-up footprinting-MS techniques and TEM visualization to characterize the monoclonal antibody-antigen binding interactions of critical reagents used in a quality control (QC) lot-release ELISA. Graphical Abstract ᅟ. |
| Databáze: | OpenAIRE |
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