Specific binding reactions monitored with ligand-cofactor conjugates and bacterial luciferase

✍ Scribed by Hartmut R. Schroeder; Robert J. Carrico; Robert C. Boguslaski; James E. Christner

• Publisher: Elsevier Science
• Year: 1976
• Tongue: English
• Weight: 572 KB
• Volume: 72
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(76)90531-5

✦ Synopsis

A unique rapid method for assaying specific binding reactions with ligandcofactor conjugates and a bioluminescent reaction is described. Biotin and 2,4dinitrofluorobenzene were coupled covalently to the free amino residue of nicotinamide 6-(2-aminoethylamino) purine dinucleotide (AENAD) to produce the enzymatically active conjugates biotinyl-AENAD and DNP-AENAD. After reduction with alcohol dehydrogenase and ethanol these two conjugates were measured quantitatively by means of light produced in a bioluminescent reaction employing luciferase from Photobacterium jsheri.

Light production by biotinyl-AENADH

and DNP-AENADH was inhibited by the specific binding proteins avidin and antibody to DNP. respectively. The counter ligands, biotin and DNP-6-aminocaproate, reversed the inhibition by the respective binding proteins in competitive binding reactions. Thus, specific binding reactions can be assayed rapidly without separation of free and bound labeled ligands.