Direct Covalent Attachment of Small Peptide Antigens to Enzyme-Linked Immunosorbent Assay Plates Using Radiation and Carbodiimide Activation
| Autor: | P. Dagenais, B. Desprez, J. Albert, E. Escher |
|---|---|
| Rok vydání: | 1994 |
| Předmět: |
chemistry.chemical_classification
Calcitonin Gene-Related Peptide Biophysics Enzyme-Linked Immunosorbent Assay Peptide Cell Biology Biochemistry Peptide Fragments Epitope Carbodiimides chemistry.chemical_compound Enzyme chemistry Antigen Gamma Rays Covalent bond Polystyrenes Carboxylate Polystyrene Antigens Molecular Biology Carbodiimide |
| Zdroj: | Analytical Biochemistry. 222:149-155 |
| ISSN: | 0003-2697 |
| DOI: | 10.1006/abio.1994.1466 |
| Popis: | Direct adsorption of small peptide antigens to unaltered, commercially available polystyrene surfaces may be too weak to permit suitable assay by ELISA. We therefore developed a simple method for the covalent attachment of small, potentially single epitope antigens to polystyrene surfaces. Chemical activation of polystyrene plates with carbodiimide considerably improves the total and covalent attachment of radioactive octapeptides. The covalent attachment was demonstrated by washing with hot detergent. A 3.5 Mrad gamma-irradiation of plates also increases total binding, particularly in combination with chemical activation. The covalent attachment presumably occurs through formation and chemical activation of carboxylate functions on the polystyrene surface which form amide bonds with peptides. ELISA test was performed with CGRP and successive smaller CGRP fragments. Covalent attachment of C-terminal peptide fragments as detection antigens allows optimal recognition and sensitivity even for hexapeptides, while decapeptide antigens were already poorly recognized using a conventional antigen plating technique. Repetitive detergent washes and/or prolonged storage of plates with covalently bound antigens did not reduce their ELISA sensitivity. The method with storage and reutilization capacities that we present here will be useful for the development of preplated antibody screening test. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
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