RAPID CHEMILUMINESCENT DETECTION, QUANTITATION, AND IDENTIFICATION OF SINGLE CELLS ON INSTANT FILM

THIS RESEARCH WILL DEVELOP A CHEMILUMINESCENT-BASED SYSTEM FOR THE QUANTITATION OF LYMPHOCYTE SUBPOPULATIONS IN WHOLE BLOOD. THE TECHNIQUE WILL USE AN ALKALINE PHOSPHATASE-BASEDDETECTION SYSTEM WITH A CHEMILUMINESCENT ENZYME SUBSTRATE TO QUANTITATE AND IDENTIFY HUMAN PERIPHERAL BLOOD LYMPHOCYTES ON INSTANT FILM. WHEN DEPHOSPHORYLATED BY ALKALINE PHOSPHATASE, THE CHEMILUMINESCENT SUBSTRATE 3-(2'-ADAMANTANE)-4-(3"-PHOSPHORYLOXY)PHENYL-1,2-DIOXETANE (AMPPD) RESULTS IN A GLOW OF LIGHT THAT MAY BE IMAGED ON POLAROID INSTANT FILM. INITIALLY, CELLS THAT CONTAIN THE ENZYME WILL BE DETECTED BY DIRECT INCUBATION WITH THE COLORIMETRIC AND CHEMILUMINESCENT SUBSTRATES. AFTER OPTIMIZATION OF THE QUANTITATION STEPS, CELL SURFACE LABELING WILL BE USED WITH MONOCLONAL ANTIBODIES TO IDENTIFYAND QUANTITATE LYMPHOCYTE SUBSETS WITHIN A SAMPLE OF WHOLE BLOOD. IN PHASE II OF THE PROJECT, TROPIX, INC., WILL CORRELATE THEDATA OBTAINED ON INSTANT FILM WITH SUBTYPING EXPERIMENTS PERFORMED WITH FLUORESCENT MONOCLONAL ANTIBODIES 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.