Measuring interactions of MHC class I molecules using surface plasmon resonance.
To examine the molecular interactions between major histocompatibility complex (MHC)-encoded molecules and peptides, monoclonal antibodies (mAbs), or T cell receptors, we have developed model systems employing genetically engineered soluble MHC class I molecules (MHC-I), synthetic peptides, purified mAbs, and engineered solubilizable T cell receptors. Direct binding assays based on immobilization of one of the interacting components to the dextran modified gold biosensor surface of a surface plasmon resonance (SPR) detector have been developed for each of these systems. The peptide binding site of the MHC-I molecule can be sterically mapped by evaluation of a set of peptides immobilized through the thiol group of cysteine substitutions at each peptide position. Kinetic binding studies indicate that the MHC-I/peptide interaction is characterized by a low to moderate apparent kass (approximately 5000-60000 M-1 s-1) and very small kdis (approximately 10(-4)-10(-6) s-1) consistent with the biological requirement for a long cell surface residence time to permit engagement with T cell receptors. Several mAb directed against different MHC-I epitopes were examined, and kinetic parameters of their interaction with MHC molecules were determined. These showed characteristic moderate association rate constants and moderate dissociation rate constants (kass approximately 10(4)-10(6) M-1 s-1 and kdis approximately 10(-2)-10(-4) s-1), characteristic of many antibody/protein antigen interactions. The interaction of an anti-idiotypic anti-TCR mAb with its purified cognate TCR was of moderate affinity and revealed kinetic binding similar to that of the anti-MHC mAbs. The previously determined interaction of a purified T cell receptor with its MHC-I/peptide ligand is characterized by kinetic constants more similar to those of the antibody/antigen interaction than of the MHC-I/peptide interaction, but is remarkable for rapid dissociation rates (apparent kdis approximately 10(-2) s-1). Such binding studies of reactions involving the MHC-I molecules offer insight into the mechanisms responsible for the initial specific events required for the stimulation of T cells.
Khilko
S N
SN
Molecular Biology Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-1892, USA.
Jelonek
M T
MT
Corr
M
M
Boyd
L F
LF
Bothwell
A L
AL
Margulies
D H
DH
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000938:Antigen-Presenting Cells / Q000276:immunology; D015374:Biosensing Techniques; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D018604:Epitope Mapping; D015395:Histocompatibility Antigens Class I / Q000276:immunology*; D006801:Humans; D007700:Kinetics; D051379:Mice; D011948:Receptors, Antigen, T-Cell / Q000276:immunology; D013057:Spectrum Analysis / Q000295:instrumentation / Q000379:methods*
D000911:Antibodies, Monoclonal; D015395:Histocompatibility Antigens Class I; D011948:Receptors, Antigen, T-Cell