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    • 75. 发明授权
    • LUCIFERASE LABELLING METHOD
    • 荧光素酶标记方法
    • EP0778945B1
    • 2001-10-04
    • EP95929977.7
    • 1995-08-30
    • The Secretary of State for Defence
    • SQUIRRELL, David, JamesMURPHY, Melenie, Jane
    • G01N33/535C12Q1/68G01N33/58G01N33/543G01N21/84
    • C12Q1/6825G01N33/535
    • A method is provided for conjugating luciferase to a chemical entity, particularly to a specific binding agent such as an antibody, antigen or a nucleic acid, and more particularly an antibody, comprising (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 wherein 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 the 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 (Mg ATP), optionally together with D-luciferin. In a second aspect of the invention there is provided a labelled chemical entity comprising a chemical entity conjugated to active luciferase as provided by the method of the present invention. 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 hybridisation probe or a chain extension primer, e.g. a PCR primer. Most advantageously the entity is an antibody as previous attempts to couple antibodies to luciferase have resulted in inactivity. Test kits are further provided.