Establishment of a quantitative ELISA capable of determining peptide – MHC class I interaction

Abstract

Many different assays for measuring peptide-MHC interactions have been suggested over the years. Yet, there is no generally accepted standard method available. We have recently generated preoxidized recombinant MHC class I molecules (MHC-I) which can be purified to homogeneity under denaturing conditions (i.e., in the absence of any contaminating peptides). Such denatured MHC-I molecules are functional equivalents of "empty molecules". When diluted into aqueous buffer containing beta-2 microglobulin (beta2m) and the appropriate peptide, they fold rapidly and efficiently in an entirely peptide dependent manner. Here, we exploit the availability of these molecules to generate a quantitative ELISA-based assay capable of measuring the affinity of the interaction between peptide and MHC-I. This assay is simple and sensitive, and one can easily envisage that the necessary reagents, standards and protocols could be made generally available to the scientific community.

HLA-E Allelic Variants

“…Peptide concentrations yielding half-maximum assembly were read directly from the curve and used to compare relative binding to E G and E R . This assay thus gives an indirect estimate of relative affinity as described (28).…”

“…Measurement of Relative Peptide Affinities-Peptide binding to HLA-E G and -E R was compared and quantified using a sandwich enzyme-linked immunosorbent assay method essentially similar to that recently described (28). Briefly, polystyrene (96-well) plates were coated with 100 l of HLA-E-specific monoclonal antibody 3D12 (13) diluted in sodium carbonate buffer (pH 9.6) at a concentration of 20 g/ml.…”

The HLA-A*0201-restricted minor histocompatibility antigen HA-1H peptide can also be presented by another HLA-A2 subtype, A*0206

“…HLA peptide binding assay A quantitative ELISA-based assay capable of measuring the affinity of the interaction between peptide and HLA was carried out as described previously, 20 with some modifications. In brief, purified recombinant HLA molecules in 8 M urea, 10 mM EDTA, 25 mM 2-(N-morpholino)-ethanesulfonicacid and 0.1 mM dithiothreitol were diluted to 4 mg/ml in refolding buffer containing 400 mM arginine, 100 mM Tris pH 8.0, 2 mM EDTA, 5 mM reduced glutathione, 0.5 mM oxidized glutathione, 0.2 mM phenylmethyl sulfonyl fluoride (all from Sigma-Aldrich) and 2 mM purified b2-microglobulin (b2m) on ice.…”

Functional and Structural Characteristics of NY-ESO-1-related HLA A2-restricted Epitopes and the Design of a Novel Immunogenic Analogue

“…This assay does not rely on cell surface stabilization of antibody determinants but rather utilizes an in vitro assembly reaction with quantitation by capture enzyme-linked immunosorbent assay (37,47). As such, this assay is less influenced by cell culture-mediated oxidation and modification of cysteine-containing peptides.…”