A EUROPIUM FLUOROIMMUNOASSAY FOR MEASURING BINDING OF ANTIGEN TO CLASS-II MHC GLYCOPROTEINS

A dissociation-enhanced lanthanide fluoroimmunoassay employing europiu m-streptavidin and time-resolved fluorimetry was developed to measure binding of biotin-labeled peptides to class II MHC proteins. Binding o f biotin-peptides as measured by this assay was saturable and inhibite d in the presence of unlabeled peptide. Background fluorescence was mi nimal and there was a direct relationship between signal and biotin-pe ptide/class II complex concentration from 1.3 pmol to less than 1 fmol total class II. The sensitivity of the assay and the ability to selec tively capture specific class Il proteins from detergent lysates of ce lls with solid phase mAb made it possible to measure formation peptide /class II complexes in live APC cultured with biotin-labeled insulin. This assay is expected to be useful for routine measurement of peptide /class II binding and biochemical analysis of Ag processing events.