Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1997-02-28
1998-11-17
Budens, Robert D.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 4, 435 8, 435 792, 435 793, 435 794, 435 795, 436501, 436547, 436548, 5303913, C12Q 168, C12Q 166
Patent
active
058374655
DESCRIPTION:
BRIEF SUMMARY
This is a 35 U.S.C. 371 of PCT/GB95/02038, filed Aug. 30, 1995.
The resent invention relates to a method of labelling chemical materials, particularly biological materials, for the purpose of chemical, and particularly biological assay, and more particularly specific binding assay, such as immunoassay and hybridisation probing techniques and for incorporation of labels into specific amplification products e.g. using PCR in assay formats.
BACKGROUND OF THE INVENTION
The firefly luciferase mediated cleavage of luciferin (Eqn 1) has a high quantum yield and stable light output allowing the enzyme itself to be detected at very low concentrations using relatively simple instruments (see McCapra. Potential applications of bioluminescence and chemiluminescence in Turner et al (Edit.) Biosensors: fundamentals and applications: Oxford University Press, (1988): 617-37). Many methods have been developed using luciferase as an indirect label (see Wannlund and DeLuca, `Bioluminescence immunoassays: Use of luciferase antigen conjugates for determination of methoxylate and DNP` in Deluca and McElroy (Edits). Bioluminescence and Chemiluminescence: Basic chemistry and analytical applications. London: Academic Press, (1981): 693-696; Geiger and Miska, Bioluminescence enhanced enzyme immunoassay: New ultrasensitive detection system for enzyme immunoassay, Clin. Chem. Clin. Biochem. J. (1987) 25. 31-38 and Murakami et al, Development of a bioluminescent detection system using adenylate kinase and firefly luciferase in Szalay et al (Edits.) Bioluminescence and chemiluminescence: Status Report, Chichester: John Wiley and Sons, (1993) 296-300.
The present inventors have noted that assay design could be much simplified whilst maintaining sensitivity by using luciferase as a direct label, but that methods of coupling it to assay binding agents are required that do not result in inactivation of what is well known to be a very labile enzymatic activity. The standard covalent coupling reagents such as glutaraldehyde, SMCC and SMPB rapidly and irreversibly inhibit luciferase activity.
Luciferase, eg that from Photinus pyralis, contains four cysteine residues (see de Wet et al (1987) Molecular and Cellular Biology) 7, 725-737 two of which are near to or part of the active site (see Deluca and McElroy (1978) Methods in Enzymology, 57, 3-15. The present inventors have determined that binding of covalent coupling reagents to these residues may cause the observed inactivation and have provided a method for coupling luciferase to assay agents that protects the enzyme from irreversible inhibition.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a plot of luminescence v. time as described in Example 1, and
FIG. 2 is a plot of luminescence v. amount of ricin as described in Example 1.
DESCRIPTION OF THE INVENTION
Thus in a first aspect of the present invention there is provided a method for conjugating firefly 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, such that the concentrations of magnesium ions and adenosine triphosphate are greater than 0.2 mmol/l and 0.05 mmol/l respectively and (b) performing a covalent coupling reaction between the luciferase and the chemical entity using a 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.sup.2+ ATP), optionally together with D-luciferin. Preferably only one of Mg.sup.2+ ATP or D-luciferin is present. If all three of Mg.sup.2+, ATP and luciferin are present then it is preferable to exclude oxygen from the reaction mixture.
In a second aspect of the present invention there is provided a labelled chemical entity comprising a chemical entity conjugated to active firefly luciferase as provided by th
REFERENCES:
patent: 5583024 (1996-12-01), McElroy et al.
Analytical Biochemistry, vol. 175, No. 1, -1988 New York, NY U.S.A., pp. 14-21, XP 000563627, L.J. Kricka, "Clinical and biochemical applications of luciferase and luciferins".
Wannlund et al., Meth. Enzymol. 92:426-432, 1983.
Harlow et al., Antibodies:A Laboratory Manual, Cold Spring Harbor, 1988, pp. 553-558.
Clin. Chem. 40/3 347-357 (1994) Kricka Selected Strategies for Improving Sensitivity and reliability of Immunoassays.
Clin. Chem. 37/9, 1472-1481 (1991) Kricka "Chemiluminescent and Bioluminescent Techniques".
Bioluminescence and Chemiluminescence Fundamentals and Applied Aspects pp. 171-178 Kricka "The Clinical and Research Potential of Bioluminescence and Chemiluminescence in Medicine".
Series C: Mathematical and Physical Sciences vol. 226 pp. 237-248 Analytical Uses of Immobilized biological Compounds for Detection, Medical and Industrial Uses Coulet et al "Immobilized Biological Compounds in Bio-and Chemiluminescence Assays".
Murphy Melanie Jane
Squirrell David James
Budens Robert D.
The Secretary of State for Defence in Her Britannic Majesty's Go
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