ASSAY FOR SIMULTANEOUS DETECTION OF MULTIPLE HPV TYPES

THE AIM OF THIS PROJECT IS TO DEVELOP A RAPID, HIGHLY SENSITIVE ASSAY FOR SIMULTANEOUSLY DETECTING THE IN SITU HYBRIDIZATION OF MULTIPLE HUMAN PAPILLOMA VIRUS (HPV) TYPE-SPECIFIC DNA PROBES TO THE HPV DNA IN A SINGLE SPECIMEN. IN PHASE I, A MODEL SYSTEM CONSISTING OF TWO WELL-CHARACTERIZED CERVICAL CARCINOMA CELL LINES WITH KNOWN LEVELS OF HPV TYPE 16 AND 18 DNA WILL BE USED. HYBRIDIZATION OF BIOTIN-AND DNA-LABELED HPV TYPE 16 AND HPV TYPE 18 DNA PROBES WILL BE VISUALIZED BY FLUORESCENCE MICROSCOPY FOLLOWING BINDING OF THE APPROPRIATE FLUORESCENTLY LABELED ANTIBODIES TO THE DNA PROBES. VARIOUSFLUORESCENT LABELS AND ANTIBODY PREPARATIONS WILL BE TESTED. ANTIBODY BLOCKING, BINDING, AND WASHING STEPS WILL BE OPTIMIZED. THE DETECTION SYSTEM, WHICH PROVIDES MAXIMAL SIGNAL INTENSITY AND SPECIFICITY AND MINIMAL BACKGROUND, WILL BE USED IN SUBSEQUENT PHASE II STUDIES IN WHICH THE EFFICACY OF THIS ASSAY FOR IDENTIFYING HIGH-AND LOW-RISK HPV TYPES IN BIOPSY SPECIMENS WILL BE ASSESSED. THESE STUDIES WILL RESULT IN A SIMPLE, CLINICALLY RELEVANT HPV IDENTIFICATION ASSAY THAT IS CAPABLE OF SUPPLYING A CONSIDERABLE AMOUNT OF CRITICAL INFORMATION WITH A MINIMUM OF EFFORT, TIME, AND SAMPLE MATERIAL.