Process for conjugating luciferase to a chemical entity chemical entity labeled specific binding test test kit and process for conjugating luferase to a chemical entity

摘要

PCT No. PCT/GB95/02038 Sec. 371 Date Feb. 28, 1997 Sec. 102(e) Date Feb. 28, 1997 PCT Filed Aug. 30, 1995 PCT Pub. No. WO96/07100 PCT Pub. Date Mar. 7, 1996Luciferase is conjugated to a chemical entity, particularly to a specific binding agent such as an antibody, antigen or a nucleic acid, and more particularly an antibody, by (a) mixing the luciferase with one or more of D-luciferin, magnesium ions and adenosine triphosphate and (b) performing a covalent coupling reaction between the luciferase and the binding reagent using a covalent coupling reagent where the amount of D-luciferin, magnesium ions and/or adenosine triphosphate is sufficient to protect the luciferase activity against inhibition by the covalent coupling reagent. Preferably, step (a) is carried out by mixing the luciferase with its substrates in solution and preferably both magnesium and adenosine triphosphate are present as magnesium adenosine triphosphate (Mg2+ ATP), optionally together with D-luciferin. Also disclosed is a labeled chemical entity comprising a chemical entity conjugated to active luciferase as formed by the method. Preferably, the chemical entity is a specific binding agent suitable for use in a specific binding assay, preferably being an antibody, antigen or nucleic acid. When the binding agent is a nucleic acid, it is preferably an oligonucleotide, but may be a polynucleotide or a nucleoside, and may be used as a hybridization probe or a chain extension primer, e.g., a PCR primer. The entity is an antibody as previous attempts to couple antibodies to luciferase have resulted in inactivity. Test kits are further provided.