Immunological and DNA hybridization assays which employ specific antibody and DNA probes are proving to be very valuable in the identification of pathogens and in the diagnosis of genetic diseases. The objective of this project is to improve the sensitivity, safety, and convenience of diagnostic tests by developing a nonradioactive, luciferase based probe technique. To evaluate the usefulness of light emission as a signal in immunological and DNA hybridization assays, the luciferase protein from a marine crustacean will be conjugated to antibody and oligonucleotide DNA. Use of luciferase would avoid the hazards associated with radioisotopes, and light production is convenient to measure. The probe signal can be greatly amplified in comparison to radioisotope decay since light is the product of an enzymatic reaction. In Phase I, methods to covalently link luciferase to protein and DNA will be evaluated, and several prototypic assays incorporating luciferase conjugates will be designed. Should a luciferase probe prove to generate a signal which is superior in terms of simplicity and sensitivity of detection, work in Phase II would proceed to perfecting practical assays and to cloning, expressing, and purifying the luciferase in a recombinant organism for the purpose of providing an abundant supply of this protein.National Institute of General Medical Sciences (NIGMS)
