Support Used in Bioluminescent Dosing of Enzymes, Substrates or Enzymatic Inhibitors

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 tried a copolymer of polylysine and polyphenylalanine, and obtained improved binding of luciferase. This increase in the adsorption yield is explained by the dual hydrophilic and hydrophobic character of this copolymer, which will hence bind well to the wall of hydrophobic polystyrene through its hydrophobic portion while maintaining the hydrophilic side towards the solution, on which wall the luciferase will become adsorbed. The adsorption yield is hence increased by using a copolymer of hydrophilic polylysine and another, more hydrophobic amino acid. Other polymers of charged hydrophilic amino acids, such as polyhistidine, a copolymer of polyhistidine, polyarginine or a copolymer of polyarginine could also replace the polylysine.

The advantages of this invention are to keep sensitivity and specificity of the luciferase system described above, but to avoid the problems of retention of light and diffusion of substrate caused by the use of the porous support. In the case proposed here, the enzyme is immediately in contact with the reaction solution. Compared with the assay system in a continuous-flow cell developed by Kricka et al. (above), our invention has the advantage of being simpler, of being immediately applicable to the photometers currently on the market and of not having to take into account the requirements imposed by continuous-flow assays: constant flow, suitably modified measuring cell, difficulty of large-scale production.

Compared with the proposal of Mookambeswaren and Sunanda, our invention makes it possible to use immediately the support used for the assay (tube or strip) and to adsorb the enzyme directly on this support via a hydrophilic molecule. We avoid, on the one hand, the covalent modification of the enzyme, which is especially sensitive to chemical coupling agents, and the support used is directly that which is used for the detection apparatus, i.e. a tube for a photometer or a strip for the flat detector, and this enables a ready-to-use assay system to be produced and greatly facilitates the industrial application.

The present invention consists in immobilizing, on the tube or strip which will be introduced into the photometer or placed in front of any other apparatus for measuring photons, a luciferase with, if necessary, one of the NAD(P)H:FMN oxidoreductases and optionally one or more other dehydrogenases or kinases. The tube coated in this manner can be used for assaying the substrate of the dehydrogenases or kinases, the enzymes themselves or an inhibitor of these latter.

For this purpose, it is necessary to add in the requisite proportions, depending on the objective sought, the substrate of a dehydrogenase (or kinase), and NAD (or NADP or ADP), FMN (or nothing) and the substrate of luciferase, usually decanal, these molecules usually being added in a buffer in the presence of molecules which stabilize the enzymes. As a general rule, the test molecule is introduced in a limiting amount with respect to the other constituents of the system. The light emitted will be measured and usually integrated during a given period, in order to increase the accuracy of the assay.

There is a large number of NAD- or NADP-dependent dehydrogenases which can be used consecutively with the NAD(P)H:FMN oxidoreductase and luciferase; by way of example, we mention a few of these below:

lactose dehydrogenase

D-Glucose dehydrogenase

.beta.-D-Galactose dehydrogenase

Glucose-6-phosphate dehydrogenase

Mannitol dehydrogenase

Mannitol-1-P dehydrogenase

Sorbitol dehydrogenase

Polyol dehydrogenase

Pentitol dehydrogenase

D-Xylitol dehydrogenase

2,3-cis-Polyol dehydrogenase

L-Threonic acid dehydrogenase

Lipoyl dehydrogenase

Lactate dehydrogenase

Glyoxylate dehydrogenase

Formaldehyde dehydrogenase

Formate dehydrogenase

Aldehyde dehydrogenase

Alcohol dehydrogenase

Acetaldehyde dehydrogenase

Fucose dehydrogenase

3-.alpha.-Hydroxysteroid dehydrogenase

.beta.-Hydroxysteroid dehydrogenase

7-.alpha.-Hydroxysteroid dehydrogenase

3-.alpha.-20.beta.-Hydroxysteroid dehydrogenase

We give below an example of assay of lactate dehydrogenase and of testosterone using .beta.-hydroxysteroid dehydrogenase. We have shown that the assay of this enzyme could be used for measuring the concentration of an inhibitor of this enzyme, such as diethylstilboestrol (DES). The relationship between the amount of DES and the percentage inhibition of .beta.-hydroxysteroid dehydrogenase is not linear, but the assay of the inhibitor can nevertheless be accomplished by reference to a calibration curve produced with a standard DES preparation.

There is also a large number of kinases which can be used consecutively with the ATP-dependent luciferase. By way of example, we shall mention a few of these below:

Acetate kinase

Pyruvate kinase

Phosphoglycerate kinase

These kinases can be used co-immobilized with the luciferase, or free in solution or alternatively bound to another protein whose concentration it is desired to measure. For example, the kinase can be measured while it is bound to an antibody. This also applies to the dehydrogenases mentioned above.

This list, while not exhaustive, shows the variety of substrates and enzymes which can be assayed by application of the method. The tubes prepared in the manner described can be lyophilized and stored for long periods while retaining a substantial part of their activity.

In this example, we describe in detail the insolubilization of luciferase and of NADH:FMN oxidoreductase. This insolubilization was optimized in respect of various parameters: type of support, use of polylysine, concentration of poly-L-lysine, pH of binding of the enzymes, stabilization of the enzymes. In effect, the adsorption of these two enzymes serves as a basis for extending the discovery in conjunction with other dehydrogenases.

We used polystyrene tubes specially designed for their high adsorption power (Startube, NUNC, Roskilde, Denmark). The adsorption of the poly-L-lysine was carried out by adding per tube 0.5 ml of poly-L-lysine solution at a concentration of 80 .mu.g/ml in a solution of 10 mmol/l NaCl and 50 mmol/l Na phosphate, pH 8. The tubes are incubated for 1 hour at 20.degree. C. with a rotation of 5 revolutions per minute, and then rinsed 3 times with 50 mmol/l Na phosphate buffer at pH 7.5. The enzyme solution (0.2 ml), containing 0.5 mg/ml of luciferase extracted from Vibrio harveyi (Sigma, St. Louis, Mo. L 1637) and 1 unit of NAD(P)H:FMN oxidoreductase extracted from Photobacterium fischeri (Sigma, cat. 476-480) dissolved in 50 mmol/l Na phosphate buffer at pH 7.5 and containing 2 mmol/l of dithiothreitol, is then added to the tube and incubated for 30 min at 4.degree. C. under rotation of 5 revolutions per minute. The tubes are rinsed twice in 10 mmol/l Na phosphate buffer at pH 7.5 containing 5 mg/ml of bovine serum albumin and 2 mmol/l of dithiothreitol, and stored in this buffer.

For the assay, the reaction volume of 0.5 ml comprises 2.5 .mu.mol/l of FMN, 0.0005% of decanal, 2 mmol/l of dithiothreitol, 5 mg/ml of bovine serum albumin, and NADH in increasing concentrations in 10 mmol/l Na phosphate buffer at pH 7.5. The maximal emission of light was measured. It is found that this light emission is proportional to the concentration of NADH present in the reaction medium for a concentration region ranging from 1 to 20,000.times.10.sup.-12 moles in the test. The number of photons emitted per second at the maximum intensity ranges from 20 to 10,000.

In this example, we shall use the tubes on which the luciferase and NADH:FMN oxidoreductase have been adsorbed as described in Example 1, but they will be used for assaying lactate dehydrogenase in solution. For this purpose, the substrates will be added in excess such that the concentration of the enzyme is the limiting factor. The reaction solution of 0.5 ml contains 2.5 .mu.mol/l of FMN, 0.0005% of decanal, 2 mmol/l of dithiothreitol, 5 mg/ml of bovine serum albumin, 0.3 mmol/l of NAD.sup.+ and 0.01 mol/l of L-lactate, and various concentrations of lactate dehydrogenase. The light emitted is measured and integrated during 60 sec. The assay enables lactate dehydrogenase to be measured in a concentration region ranging from 2.times.10.sup.-15 to 50.times.10.sup.-15 moles in the test.

In this example, the three enzymes luciferase, NADH:FMN oxidoreductase and .beta.-hydroxysteroid dehydrogenase will be immobilized on the tube in order to carry out the assay of testosterone.

The adsorption of polylysine and the enzymes on the tube is carried out as described in Example 1, but the enzyme solution contains 0.075 units/ml of .beta.-hydroxysteroid dehydrogenase. For the assay of testosterone, the tubes contain 0.5 ml of 10 mmol/l Na phosphate buffer at pH 7 and 25 .mu.mol/l of FMN, 0.0001% of decanal, 5 mg/ml of bovine serum albumin, 2 mmol/l of dithiothreitol, 1.2 mmol/l of NAD and various concentrations of testosterone. The light intensity is measured and integrated during 60 sec. The testosterone can be measured in a concentration region ranging from 5.times.10.sup.-11 to 5.times.10.sup.-9 moles in the reaction solution.

The polystyrene tubes having a high adsorption power (Startube, NUNC, Roskilde, Denmark) were incubated overnight at 4.degree. C. with a rotation of 5 revolutions per minute for 30 min in the presence of a solution of poly(lysine-phenylalanine) at a concentration of 80 .mu.g/ml in a solution buffered to pH 8 with 0.05 mol/l phosphate buffer. The tubes are then rinsed twice with this same buffer. The solution (0.2 ml) of luciferase extracted from Photinus pyralis (Sigma St Louis, Mo.) contains 40 .mu.g/ml and immunoglobulins G at a concentration of 0.1 mg/ml dissolved in 0.05 mol/l Tris-acetate buffer, 60 .mu.mol/l dithiothreitol, and is incubated for 30 minutes at 4.degree. C. under rotation of 5 revolutions per minute. The tubes are rinsed twice in the enzyme immobilization buffer.

For the assay, the reaction volume of 0.5 ml comprises 400 .mu.l of 25 mmol/l Tris-acetate buffer, pH 7.75, 75 .mu.mol/l dithiothreitol, 125 .mu.mol/l EDTA, 6.25 mmol/l MgCl.sub.2 and 7.5.times.10.sup.-5 mol/l luciferin.

The reaction is started with 100 .mu.l of solution of increasing concentrations of ATP. The light emission is proportional to the concentration of ATP in a concentration region ranging from 1 to 10,000.times.10.sup.-13 mol/l.

As an example of kinase assay, we used acetate kinase which catalyses the following reaction:

This enzyme can be extracted from thermophilic bacteria (Bacillus stearothermophilus) and is especially stable. The optimal conditions for bioluminescence assay of this kinase are as follows:

400 .mu.l of 25 mmol/l Tris-acetate buffer, pH 7.75, 0.125 .mu.mol/l EDTA, 75 .mu.mol/l dithiothreitol, 6.25 mmol/l MgCl.sub.2, 7.5.times.10.sup.-5 mol/l luciferin, 1 mmol/l acetylphosphate; the reaction is started with 10.sup.-6 mol/l ADP (100 .mu.l), the assay of the ATP produced being measured by the ATP-dependent luciferase immobilized on the polystyrene tube. Under these conditions, amounts as small as 10.sup.-17 and 10.sup.-18 .mu.mol of kinase can be assayed in this manner.