RAPID CHEMILUMINESCENT DETECTION OF SINGLE CELLS ON FILM

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In this research proposal we describe the development of a chemiluminescent based system for the quantitation of lymphocyte subpopulations in whole blood. The proposed technique will use an alkaline phosphatase based detection system with a chemiluminescent enzyme substrate to quantitate and identify human peripheral blood lymphocytes on instant film. The chemiluminescent substrate 3-(2'adamantane)-4-(3"-phosphoryloxy) phenyl-1, 2-dioxetane, (AMPPDTM) when dephosphorylated by alkaline phosphatase results in a glow of light which may be imaged on Polaroid instant film. Initially, cells which contain the enzyme will be detected by direct incubation with the colorimetric and chemiluminescent substrates. After optimization of quantitation steps we plan to use cell surface labelling with monoclonal antibodies to identify and quantitate lymphocyte subsets within a sample of whole blood. In Phase II of the project we plan to correlate the data obtained on instant film project we plan to correlate the data obtained on instant film with subtyping experiments performed with fluorescent Mabs on the FACS in order to develop a clinical assay for lymphocyte subtyping. This assay will enable the physician to rapidly assess the state of a patient's immune system without expensive instrumentation or special technical expertise.