Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving luciferase
Patent
1987-02-20
1990-05-22
Warden, Robert J.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving luciferase
435 4, 435 7, 435 15, 435 26, 435174, 435175, 435180, 435181, 435805, 436807, 422 52, C12N 1108, C12Q 166, G01N 3353
Patent
active
049277529
DESCRIPTION:
BRIEF SUMMARY
The invention relates to an assay system consisting of a wall on which is adsorbed at least one luciferase which, by acting consecutively with one or more adsorbed or unadsorbed enzymes, enables a series of substrates, enzymes or enzyme inhibitors to be assayed by bioluminesence. This invention enables a series of biological molecules to be assayed, and is hence applicable in biochemical analyses and in medical and veterinary diagnosis.
Some luciferases are enzymes extracted from bacteria and which catalyse the reduction of flavin mononucleotide (hereinafter designated FMN) with the concomitant production of photons. These photons can be detected and measured quantitatively in photometers or by any light-sensitive detector. These luciferases can be combined with an NADH:FMN oxidoreductase or with an NADPH:FMN oxidoreductase (NADH=nicotinamide adenine dinucleotide in its reduced form; NADPH=nicotinamide adenine dinucleotide phosphate in its reduced form). Thus, it is possible, by means of these two enzymes working simultaneously, to assay NADH or NADPH in concentration regions ranging from 1 to 1000.times.10.sup.-12 moles in the test (Stanley, P. E. Methods in Enzymology, vol. 57, pages 215-222, 1978; Lavi et al., J. Clin. Chem. Clin. Biochem., vol. 19, pages 749 and following pages, 1981).
Another luciferase is isolated from fireflies, and enables photons to be produced from the cleavage of adenosine triphosphate (ATP) to adenosine monophosphate (AMP) and pyrophosphate.
Two developments of this method have been proposed: on the one hand, the insolubilization of these two enzymes on agarose beads by coupling with cyanogen bromide, followed by the combination of these first two enzymes with a third NADH- or NADPH-dependent dehydrogenase, which enables the substrate of this latter enzyme to be measured. The main achievements are those of De Luca and his group, relating to the assay of testosterone, L-malate, D-glucose, 6-P-gluconate, L-lactate, L-alanine and L-glutamate, with a minimal assay region ranging from 1.5 to 10.times.10.sup.-12 moles (U.S. Pat. No. 4,234,681 of M. A. De Luca-Mc Elroy; Weenhausen, G. and De Luca, M., Anal. Biochem., vol. 127, 380, 1982; Ford, J. and De Luca, M., Anal. Biochem., vol. 110, 43, 1981; Jablonski, E. and De Luca, M., Methods in Enzymology, vol. 57, 202, 1978). The 12 biliary .alpha.-hydroxy acids can also be measured (Scholemerich et al., Anal. Biochem., vol. 133, 244, 1983). The method can also be used to measure these dehydrogenases (Haggerty, C. et al., Anal. Biochem., vol. 88, 162, 1978). While this system is sensitive and specific, it nevertheless has the drawbacks of introducing a solid support (agarose or the like) into the reaction system, and this causes some retention of the emitted light and is very sensitive to agitation at the time of measuring, since the use of the porous support (agarose) requires the diffusion of the substrates into the meshes of the gel. One of the solutions proposed for these problems is to pass the solution continuously through a cell containing the gel to which these enzymes are bound (Kricka et al., Anal. Biochem., vol. 129, 392, 1983). This solution, while it deals with the disadvantages mentioned above, involves making the assay technique more complex through the use of a special apparatus.
On the other hand, recently, V. Mookambeswaren and Sunanda (European Patent 0,169,767) have proposed covalent binding of luciferase to a gel made from albumins linked to each other with glutaraldehyde.
The objective of the present invention is to remedy the disadvantages of the various systems described above, in the following manner: the invention, as characterized in the claims, consists in adsorbing the enzymes on the walls of the tube which will be introduced into the photometer, or on a strip of shape and size suitable to the measuring apparatus which can be a simple photographic film. This adsorption will be accomplished via a layer of polylysine or of a copolymer of polylysine with another charged hydrophilic molecule. We have successfully
REFERENCES:
patent: 4234681 (1980-11-01), De Luca-McElroy
Spiegel Carol A.
Warden Robert J.
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