Surface engineering: optimization of antigen presentation in self-assembled monolayers.
The formation of self-assembled monolayers (SAMs) on gold surfaces containing an antigenic peptide (NANP)6 and HS(CH2)11OH, and the specific binding of a monoclonal antibody to these layers were investigated by surface plasmon resonance (SPR). Peptides were synthesized by solid-state phase synthesis and were linked either to cysteine or to an alkyl-thiol to allow covalent attachment to gold. The content of the peptide in the SAMs was systematically varied, and the binding properties of the monoclonal antibody were compared with those measured by microcalorimetry in solution. At a critical peptide concentration in the SAM an optimal antibody binding and complete surface coverage was attained. At lower peptide concentrations, the amount of adsorbed antibody decreased; at higher peptide concentrations, the binding constant decreased. These effects can be explained if the accessibility of the antigenic epitopes depends on the peptide density. Addition of free antigen induced the desorption of bound antibodies and allowed accurate measurements of the dissociation rate constant. Binding constants obtained from steady-state measurements and from measurements of the kinetic rate constants were compared.
Duschl
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Institut de Chimie Physique IV, Ecole Polytechnique Fédérale de Lausanne, Switzerland. duschl@icphp1.epfl.ch
Sévin-Landais
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Vogel
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D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D017951:Antigen Presentation; D001386:Azides / Q000737:chemistry / Q000276:immunology; D001665:Binding Sites; D001698:Biomedical Engineering; D055592:Biophysical Phenomena; D001703:Biophysics; D002151:Calorimetry; D006046:Gold; D066298:In Vitro Techniques; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000276:immunology; D013499:Surface Properties
D000911:Antibodies, Monoclonal; D001386:Azides; D008024:Ligands; D011732:Pyridoxal Phosphate; D006046:Gold; C044823:N-4-azido-2-nitrophenylpyridoxyl-5-phosphate