Measuring interactions of MHC class I molecules using surface plasmon resonance

• Published in: Journal of immunological methods Vol. 183; no. 1; pp. 77 - 94
• Main Authors: Khilko, Sergei N., Jelonek, Marie T., Corr, Maripat, Boyd, Lisa F., Bothwell, Alfred L.M., Margulies, David H.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 14.06.1995
• Subjects: AIDS/HIV; Animals; Antibodies, Monoclonal - immunology; Antigen-Presenting Cells - immunology; Biosensing Techniques; CD8-Positive T-Lymphocytes - immunology; Epitope Mapping; Histocompatibility Antigens Class I - immunology; Humans; Kinetics; Major histocompatibility complex; Mice; Monoclonal antibody; Receptors, Antigen, T-Cell - immunology; Spectrum Analysis - instrumentation; Spectrum Analysis - methods; Surface plasmon resonance; k dis, kinetic dissociation rate constant; k ass, kinetic association rate constant; k obs, concentration dependent observed kinetic association rate constant; MHC-I, major histocompatibility complex encoded class I molecule; mAb, monoclonal antibody; HBS, Hepes buffered saline; Monoclonal antibody; SMCC, sulfosuccinimidyl-4-( N-maleimidomethyl)cyclohexane-1-carboxylate; MHC, major histocompatibility complex; Major histocompatibility complex; RU, resonance units; APC, antigen presenting cell; K d, equilibrium dissociation constant; Surface plasmon resonance; β 2m, β 2-microglobulin; SPR, surface plasmon resonance; TCR, T cell receptor
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/0022-1759(95)00033-7

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 k ass (∼ 5000–60000 M −1 s −1) and very small k dis (∼ 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 ( k ass ∼ 10 4–10 6 M −1 s −1 and k dis ∼ 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 k dis ∼ 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.