THIS PROPOSAL IS DIRECTED TOWARD DEVELOPING A RAPID DIRECT METHOD FOR DETECTING MUTATIONS IN BLOOD CELLS PROVIDING A POSSIBLE ASSAY FOR THE SCREENING OF LARGE NUMBERS OF INDIVIDUALS.

THIS PROPOSAL IS DIRECTED TOWARD DEVELOPING A RAPID DIRECT METHOD FOR DETECTING MUTATIONS IN BLOOD CELLS PROVIDING A POSSIBLE ASSAY FOR THE SCREENING OF LARGE NUMBERS OF INDIVIDUALS. THE ASSAY IS BASED ON ENUMERATING CELLS BEARING MUTANT HLA MEMBRANE ANTIGENS AMONG PERIPHERAL BLOOD LYMPHOCYTES. THE MUTATIONS WILL BE DETECTED BY A NOVEL SEROLOGICAL TEST WHICH YIELDS A POSITIVE SIGNAL BY USE OF TWO SELECTED MONOCLONAL ANTIBODIES THAT REACT WITH INDEPENDENT REGIONS OF AN HLA HEAVY CHAIN. THIS POSITIVE SIGNAL WILL BE THE RESULT OF ONE OF TWO PROPOSED CONFIGURATIONS IN WHICH THE TWO ANTIBODIES WILL INTERACT NEGATIVELY WITH EACH OTHER. A MUTANT AT ANY SINGLE LOCUS WILL BE DOMINANTLY EXPRESSED WITH THESE ASSAYS AND THE PRESENCE OF MULTIPLE HLA CLASS I LOCI SHOULD INCREASE THE FREQUENCY OF MUTATION AT LEAST FOUR-FOLD COMPARED TO SINGLE ALLELE SYSTEMS. PRELIMINARY WORK HAS RESULTED IN ACQUISITION OF A SERIES OF HLA CLASS I MONOMORPHIC ANTIBODIES WHICH ARE BEING CLASSIFIED BY ADDITION AND COMPETITION EXPERIMENTS. TO TEST THE FEASIBILITY OF THESE ASSAYS, CELLS WITH MUTATIONS IN HLA WILL BE ISOLATED TO USE IN RECONSTRUCTION EXPERIMENTS.