Novel methods to rapidly and sensitively analyze antigenic peptide binding to MHC class II molecules.

One of the important steps for antigen presentation by MHC class II molecules involves binding of a peptide fragment of the antigen to the class II molecule followed by recognition of the resulting complex by T cells. The most commonly used methods for studying binding of peptide to MHC II are: equilibrium dialysis, gel filtration chromatography, HPLC and polyacrylamide gel electrophoresis. Each of these methods has some limitations and is time consuming. In addition, each requires a considerable amount of native MHC class II, which is always difficult to obtain. In this report, we describe three different sensitive methods using radiolabeled peptide to study peptide binding to murine MHC class II molecules. These are: nitrocellulose filter binding, thin-layer chromatography (TLC) using plate-supported silica gel or PEI cellulose, and paper electrophoresis using Sepraphor cellulose polyacetate paper. All three methods are rapid, highly sensitive and require only ng quantities of affinity pure MHC class II molecules and peptides. These methods can be used to calculate the peptide occupancy of MHC class II molecules.