A EUROPIUM FLUOROIMMUNOASSAY FOR MEASURING PEPTIDE BINDING TO MHC CLASS-I MOLECULES

A fluoroimmunoassay employing europium-streptavidin and time-resolved fluorimetry was used to measure binding of biotin-labeled peptides to H-2D(k) molecules. A fluorescein-labeled octameric peptide from the mi ddle T (MT) protein of mouse polyoma virus was identified that specifi cally binds to purified D(k)using a previously-established assay based on size exclusion chromatography. A europium immunoassay was adapted to measure binding of a biotin-derivative of this peptide to purified D-k. The assay was found to be sensitive and highly specific. Binding was optimal at pH 5.0 and 24-27 degrees C, and it was not enhanced in the presence of additional beta 2-microglobulin (beta 2m). Excellent r esults were also obtained in experiments with fixed cells that express D-k. This assay is expected to be useful for high volume, routine ana lysis of peptide binding to MHC class I molecules. (C) 1998 Elsevier S cience B.V. All rights reserved.