CH631208A5 - Process for stabilising a labile coenzyme and enzyme - Google Patents

Description

The present invention relates, as a whole, to an improved method for stabilizing labile enzymes or co-enzymes, in a single medium formed by water and an organic solvent.

In the current state of the industrial technique, the reactive ability of enzymes or co-enzymes is stabilized by trapping them in a solid substrate, by freeze-drying, dry mixing of the type used to transform dried powders into tablets, mainly in the field of pharmaceutical diagnostics and related applications, and immobilization by imprisonment of the chemical structure of the enzyme in a solid substrate. Contrary to appearances, these access routes are neither practical nor advantageous, and they are also expensive. The manufacturer is obliged to eliminate the water and supply a partial product, thus abandoning part of the cycle of obtaining quality in the dilution and in the use of the final product. Laboratories are forced to pay the significant costs of packaging, loss of reagents, freeze-drying and dry formulation, and the value of the product is still limited by the packaging methods and by the dimensions.

In addition, good uniformity of the product is difficult to achieve. This is illustrated by the fact that for most of the commercial lyophilized control sera (reference serum), the acceptable variations from one bottle to another, as regards the enzymatic constituents, are fixed at more or less ten for -cent of the average value.

The present invention makes it possible to find a liquid composition containing co-enzymes and / or enzymes which are stabilized in a single container.

It also makes it possible to offer a composition of enzymes and co-enzymes / labiles of the type defined in a medium formed by water and an organic solvent and in which the stabilization of the enzyme and co-enzyme n does not affect enzyme reactivity after a significant period.

Finally, the present invention makes it possible to offer a method for stabilizing labile enzymes and / or co-enzymes in the presence of other labile co-enzymes or other labile enzymes, this composition having a long shelf life.

Labile enzymes and co-enzymes, treated in accordance with the invention, acquire lasting stability without their enzymatic or co-enzymatic reactivity or their photometric absorption being affected. Obtaining enzymatic and co-enzymatic reagents in a stable liquid form promotes the colorimetric application of current methods based on the NAD / NADH couple, as well as other methods, mainly because of the ease of carrying out the separation. ingredients. Stable liquid reagents are particularly advantageous when the consumption of NADH or another co-enzyme is the basis of the measurement and the colored reagent must be separated from NADH and from the reaction medium. In the ultraviolet mode, the liquid medium offers better homogeneity of the reagents and facilitates packaging, just as it offers flexibility of use which contrasts with lyophilized preparations or in dry medium.

The liquid medium which is designed to allow the stabilization of enzymes and co-enzymes as defined below is remarkably formulated so that one or more co-enzymes can be stabilized in this medium. Furthermore, one or more enzymes can be stabilized in the liquid medium. In addition, co-enzymes can be stabilized in the same liquid medium in a single container.

In a preferred embodiment, the stabilization of the enzymes and / or co-enzymes is carried out by dissolving a polymer such as gelatin in distilled water. The gelatin is preferably dissolved in proportions of 0.1% in s

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weight / weight. Then, the gelatin solubilized in water is heated to about 30 ° for its total dissolution. In some cases, an azide can be used which serves not only as a bacteriostatic agent, but also as a stabilizing agent. Then, this solution is allowed to cool essentially to room temperature or to about 20 ° C.

In one case, the co-enzyme, namely nicotinamide-ade-nine-dinucleotide (NAD) is added to the solution together with a buffering agent such as tris- (hydroxymé-thyl) -amino-methane, in order to adjust the pH. The pH is then adjusted approximately between approximately 6.0 and approximately 8.5, its value preferably being equal to 7.5.

After the addition of the co-enzyme, a polyol such as glycerol is advantageously added in an amount of about 30% on a volume basis.

After the addition of the polyol, the pH can again be adjusted to about 7.5.

According to the present invention, more than one co-enzyme can be stabilized in the solution. In this case, the other of these coenzymes can be added before or after the addition of NAD. For example, in one embodiment of the present invention, ATP (ATP) can be added as another co-enzyme.

After the addition of the co-enzymes and when the pH of the liquid has been adjusted, an enzyme such as hexokinase (HK) can also be added. For example, hexokinase should be added in suspension, for example in the form of a suspension in glycerol, or in the form of a suspension in ammonium sulfate. Another enzyme can also be added, such as glucose-6-phosphate dehydrogenase.

When the enzyme and / or co-enzyme solution stabilized in a liquid medium has been prepared, it is preferably dispensed in opaque glass vials which are sealed. In addition, these bottles are usually kept in a refrigerator. The expected lifespan of stabilized enzymes and co-enzymes is up to four years under these conditions, without significant degradation.

In the field of clinical diagnosis, the commercial application of the present invention is illustrated by the diagnostic reagents used to determine the concentration of a substrate, for example concentrations of glucose in biological fluids, etc. Nevertheless, compositions prepared in accordance with the present invention can be used for the qualitative and quantitative analysis of other biological constituents such as the following constituents, contained in biological liquids:

1. Glutamic-oxalacetic transaminase (SGOT) 15 2. Glutamic-pyruvic transaminase (SGPT)

3. Lactic dehydrogenase (LDH-P)

4. Lactic dehydrogenase (LDH-L)

5. Creatine phosphokinase (CPK)

6. a-hydroxybutyric dehydrogenase (a-HBD)

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7. Glucose (via hexokinase nase-G-6-PDH)

The reagents identified above often react similarly, they contain some common labile ingredients, and some of the chemical reactions involved are common.

The following chemical reaction scheme is given as a model to illustrate the general nature of the reactions involved.

Reaction diagram 1 - general model Enzyme 1

(1) SUBSTRATI) c> PRODUCT (S)

pH

Enzyme 2

(2) PRODUCT / SUBSTRATE + NAD-NADH2 ^> NADH2-NAD + pH product

(3) NADH2 + CHROMOGENE Catalyst CHROMOGENg + NAD

(oxidized) <(reduced)

All the enzymatic reactions listed above follow this general scheme, according to which reaction (2) is usually considered to be the coupling reaction, reactions (2) or (3) are the measurement reactions and reaction (1) can be characterized as the primary reaction.

However, it should be noted that these three reactions are not all required for the measurement; in fact, they can be limited to two or just one. In the case of the ultraviolet measurement of the lactic dehydrogenase (LD) activity, only reaction (2) is involved, as follows:

Reaction scheme 2 - LDH LDH

LACTATE + NAD ^> NADH2 + PYRUVATE

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Conversely, other reactions may be involved, in addition to the three that have been mentioned, as in the case of creatine phosphoquinase (CPK):

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Reaction diagram 3 - CPK CPK

(1) GP + ADP ^> ATP + CREATINE

HK

(2) APT + GLUCOSE ^ Z ^ GLUCOSE-6-PHOS. + ADP

(3) GLUCOSE-6-PHOS. + NAD ^ G ~ 6'PP> NADH2 10

(4) NADH2 + INT (ox.) R? LNT + NAD (red.)

Symbols:

CP

= creatine phosphate

CPK

= creatine phosphokinase

ADP

= ATP 5'-diphosphate

AM

= ATP monophosphate

ATP

= ATP

HK

= hexokinase

NAD

= nicotinamide-adenine-dinucleotide

NADP

= nicotinamide phosphate-adenine-dinu-

cleotidos

NADH2

= nicotinamide-adenine-dinucleotide, form

scaled down

GLDH

= glutamate dehydrogenase

G-6-PDH

= glucose-6-phosphate dehydrogenase

G-6-P

= glucose-6-phosphate

INT

= tetrazolium salt

PEP

= phosphoenol pyruvate

PMS

= phenazine methosulfate

PK

= pyruvate kinase

In this case, reactions (2) and (3) can be considered as coupling reactions, reactions (3) or (4) can be considered as measurement reactions, and reaction (1) can be considered as the main reaction.

If one refers to reaction scheme 1 (general model), it is obvious and from the field of general knowledge that the use of this reaction sequence allows the quantitative analysis of substrates and reaction products or catalytic enzymes.

Quantitative analysis of these constituents in biological fluids and a widely accepted diagnostic tool widely used in the diagnosis and treatment of diseases in humans and animals.

Enzymes are complex protein molecules of large molecular weight, usually of unknown chemical structure. They are usually classified according to their catalytic activity and their extreme specificity towards substrates. Enzymes can also be defined as biological catalysts capable of catalyzing a reaction of a single substrate or a reaction of a similar group of substrates.

Co-enzymes are organic chemicals, of relatively low molecular weight and of well-defined structure, the reactions or interactions of which are necessary for a specific titration or enzymatic reaction. They are catalyzed, which results in a reversible modification of their structure and / or of their atomic composition. Coenzymes are very useful in clinical titration methods. Some have a high absorption, their reactions are stoichiometric with the substrate and, consequently, the creation or the disappearance of the absorbent form can be followed by photometry. Nicotinamide-adenine dinucleotide (NAD) and its reduced form (NADH2) are used in many important clinical titrations, for example titrations of S.G.O.T., S.P.G.T. and LHD described above. The compounds NAD and NADH2 have a molecular weight of around 700 and are very complex organic molecules. NADH2 has a strong absorption at 340 nm, while NAD does not absorb at this wavelength.

The substrates are organic chemical compounds of known structure, whose reactions or interactions are catalyzed by enzymes, with, as a consequence, a modification of the structure, the atomic composition or the stereochemical rotation. In general, substrates are prone to microbiological degradation and thus serve as food for bacteria, fungi and other microorganisms. Otherwise, these compounds remain stable in an aqueous medium at a neutral pH or close to neutral, (that is to say in a pH range from 4 to 10). Representative examples of substrates include glucose, lactate or lactic acid, gluconate, etc.

The following reaction schemes illustrate the search for glucose by the use of the co-enzymes ATP and NAD.

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GLUCOSE + ATP ^ ZZZZÎG-6-P + ADP G-6-PDH

n-ft-P + NTAn c ■> NADH + 6- PHOSPHOGLUCONIC ACID

The enzyme that produces the main reaction and the hexoquinase, and the enzyme that triggers the coupling and measurement reaction is the enzyme G-6-PDH. In the above reaction, glucose is measured by measuring the amount of NADH that is formed in the measurement reaction. The reaction is allowed to take place and the quantity formed of the co-enzyme NADH is essentially measured.

The co-enzyme NAD, although unstable in water and in the dry form when exposed to wet environments, is not nearly as unstable as the reduced form NADH2. Therefore, the latter form must be kept away from moisture, while the co-enzyme NAD can be packaged in a container with an aqueous solution, but stabilized according to the process of the invention. Stability is better at an acidic pH, while at

at an alkaline pH, the co-enzyme NAD tends to decompose. The exact mechanism is no more important than the end products, except that the decomposed NAD co-enzyme can no longer function effectively as a co-enzyme, nor does it have the coefficients of extinction at the necessary wavelength.

One of the remarkable advantages of the present invention lies in the fact that all of the components can be stabilized in a single reagent bottle. Generally, there are two main considerations in formulating a stabilized enzyme or co-enzyme. The first of these considerations is to obtain a very stable enzyme or co-enzyme in a liquid medium and the second is to limit the number of packages as much as possible. In the stabilization of co-enzymes such as NAD, it has been observed that co-

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NAD enzyme is much more stable than NADH. Consequently, it is not necessary to use complex stabilization techniques which are essential for NADH. All reagents can therefore be packaged in a single solution.

In the stabilization of the enzymes and co-enzymes according to the present invention, a polymer such as gelatin is advantageously dissolved in distilled water. The polymer is preferably present in the stabilized solution in proportion such that it remains in homogeneous suspension in the refrigerated state, without precipitation. The polymer is for example present in an amount of about 0.01 to about 0.5% based on the total co-composition, preferably in proportions of 0.05 to about 0.25%. All water-soluble polymers which do not inhibit the enzymatic activity and which are capable of trapping the enzyme in the polymer mass are advantageous to use in this embodiment of the present invention. The polymer can be a synthetic or organic material, for example polyvinylpyrrolidone or a dextran of biological origin, for example gelatin, which consists of collagen denatured.

The polymer can be dissolved in water by heating, usually above 30 ° C. The rate at which the polymer is dissolved increases with temperature.

When the polymer has completely dissolved in water, an azide such as sodium azide can be added, preferably in an amount of about 0.1% w / w. However, the amount of azide added can range from about 0.01 to about 0.5%. It has been found that the azide has the surprising effect of promoting the stability of the enzymes. Previously, azide was thought to play the role of a bacteriostatic or bactericidal agent. However, although the complete stabilization mechanism with azide, in combination with the other ingredients, is not fully understood, it has been established that azide provides better stability. In many cases, azide is not necessary and can be removed. Thus, the polymer and the organic solvent in an aqueous medium are often sufficient to stabilize the labile components. In a few cases, the azide must be removed since it may tend to interfere with stabilization, or otherwise materially affect a substrate such as glucose, for example. In addition to the above, other bactericidal agents or other fungicides which do not chemically react with a substrate or which do not inhibit the enzymatic reaction can be used. For example, some of the agents that can be used in addition to sodium azide are benzoic acid, phenol, thymol or pentachlorophenol.

In some cases, it may be desirable to use a metal such as magnesium, which facilitates the initiation of a reaction when the stabilized composition is used. One of the preferred agents for this purpose is magnesium, in the saline form of magnesium chloride. This agent does not have to be incorporated into the stabilized compositions and can be added at the time of use.

This agent which activates the coupling enzymes should be used in an amount of from about 0.01 to about 1% and preferably in an amount of about 0.03%.

At this stage of the process, the solution can be cooled to a temperature of the order of room temperature, for example between about 20 and about 25 ° C in a water bath. When the solution has cooled, a buffering agent such as tris (hydroxymethyl) aminomethane can be added. This buffering agent is added, for example, in an amount of from about 50 to about 200 mmol, but at least sufficient to maintain the pH in a range of 6.0 to about 8.5. Other known buffering agents and other forms of buffering agents can also be used in the process. In some cases, buffer salts of the type defined below may be used. The buffer salt is added in an amount necessary to maintain the pH between 6.0 and 8.5. Generally, the buffer consists of a combination of 0.1 to 1% of an alkali metal hydroxide and 0.5 to 3% of an acidic alkali metal carbonate or phosphate. The total salt content also affects the amount of polymer required.

For a higher salt content, for example above 4% by weight, less polymer is required because of the electrostatic stabilization produced by the salt. However, for a high salt content, the polymer can cloud the solution or precipitate, and its redissolution requires heating the solution.

When the pH of the solution has been adjusted to the desired value, the first of the co-enzymes, for example ATP or NAD, etc., can be added. In this case, the co-enzyme ATP is added on a basis of about 0.3 to about 30 mmol, based on the total composition.

As indicated in the above, it is possible to form solutions of the stabilized enzymes and co-enzymes. Thus, two or more co-enzymes and mild or more enzymes can be stabilized in the same solution. For example, the co-enzyme ATP can be stabilized as described herein. Furthermore, the co-enzyme NAD can also be individually stabilized as described herein. However, when two or more co-enzymes are stabilized, these co-enzymes can generally be added simultaneously or in any order. The co-enzyme NAD is preferably added in a proportion of between about 0.6 and about 60 mmol based on the total composition.

At this stage of the process, the pH should again be adjusted to a value at least in the range of 6.5 to about 8.0 or less and preferably to a value of 7.5.

After adjusting the pH, a suitable organic solvent such as glycerol can be added. It is then added in a proportion included in the range of 25 to 40% by volume / volume, although in the most advantageous case, the proportion added of organic solvent is 30% by volume / volume. However, the amount of organic solvent can range from about 5 to 70% v / v.

The organic solvent should have the following characteristics:

1. pH range from 4 to 10

2. Liquid at room temperature and at refrigerator temperature

3. No reaction with NAD or ATP, etc., except for the formation of electrostatic bonds (ie hydrogen bonds)

4. Miscible with water

5. Normal free solvolysis energy is low (normal resonance is established)

Solvents of interest to be used are generally stable organic solvents such as ethers, ketones, sulfones, sulfoxides and alcohols such as methanol, ethanol, propanol, butanol, acetone, dioxane, dimethyl sulfoxide, dimethyl sulfone and tetrahydrofuran. However, solvents of the liquid polyol type have greater activity for a lower concentration of solvent in the treatment step. Preference is given to liquid polyols containing two to four hydroxyl groups and two to ten carbon atoms, such as glycerol, ethylene glycol, propylene glycol or butane-

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diol. Glycerol, propylene glycol, 1,2-propane diol all have these qualities and are the solvents of choice.

When the organic solvent chosen is a polyol, it is not necessary to use azide or other acterostatic agents, since the polyol effectively plays the role of a bacteriostatic agent. However, although the chosen solvent and the polymer provide the required stability in an aqueous solution, azide is sometimes preferable, provided that it seems to favor the coupling between the polymer and the enzymes.

When glycerol or another polyol has been added, the pH of the solution thus formed is readjusted. For example, the pH may be slightly basic and can therefore be adjusted by adding normal hydrochloric acid. Likewise, if the pH is slightly acidic, a suitable base can be added so that it reaches the value of 7.5.

The co-enzyme NAD should be excess. As indicated, the glucose assay is performed by measuring the formation of NADH from NAD. The NADH form is unstable in an acid medium and degrades at a pH equal to 6; its degradation is even extremely rapid at a pH equal to 4. The pH of the solution is therefore maintained above a neutral pH (above 7). Although the co-enzyme NAD is actually more stable in an acid medium, it has been found that it does not degrade notably in a slightly basic medium with a pH equal to 7.5. However, the co-enzyme NAD is preferably added in considerable excess, so that there is always a sufficient amount of non-degraded NAD, even after a stay of several years in this liquid medium.

Generally, all the co-enzymes are present in an amount at least sufficient to carry out the desired assay. There is normally no upper limit on the amount of co-enzyme that is present, the maximum amount however being limited by commercial conditions.

After the co-enzymes have been added to the liquid liquid solution, the chosen enzymes can be introduced. As with co-enzymes, enzymes can be added in any order. Again, one or more enzymes can be added to the solution. According to the preferred aspect of the invention, and in accordance with the enzyme system identified above, the two enzymes are HK and G-6-PDH. The HK enzyme is also added, preferably in an amount which is not less than 111 international units per liter (pH 7.6.25 ° C). However, it is best to add at least 1000 international units of HK per liter.

The G-6-PDH enzyme should preferably be formed from L-mesenteroides type bacteria and should be concentrated in a range of about 100 to about 30,000 international units per liter or more. According to an advantageous aspect of the invention, approximately 3000 international units of G-6-PDH of this type are normally used, at a pH of approximately 7.8 at 25 ° C.

The enzymes should be present, each in an amount of at least 100 international units per liter, although in most commercial reagents, the enzyme such as hexoquinase must be present in at least an amount of 1000 international units per liter. In normal commercial packaging, the enzyme is present in an amount of approximately 1000 to 10 000 international units at a pH equal to 7.6 and at a temperature of approximately 25 ° C. However, the maximum amount of enzyme is unlimited, although normally, in almost all applications the amount of enzyme does not exceed 100,000 international units.

Although the entire mechanism for accomplishing stabilization of enzymes and co-enzymes is not fully understood, it is assumed that the chosen solvent stabilizes the enzyme in the liquid medium by protecting the site of a functional group, it that is, the part of the molecule where a reaction can actually take place with a substrate or is otherwise catalyzed.

It is assumed that stabilization takes place by protecting the enzymes and co-enzymes from microbial contamination and, therefore, from degradation.

The co-enzyme NAD differs from the co-enzyme NADH in that NAD does not dissolve in significant quantities in the chosen solvent such as propylene glycol. However, the co-enzyme NAD is more stable in water and does not seem to be stabilized by the polyol. A pure polyol denatures the enzymes, but in the presence of an aqueous solution such as a mixture of water and a solvent, the enzymes are not denatured. Apparently, a popular group must be present in the organic solvent to keep the active sites of the enzymes in a stable state. Obviously, some form of physical or chemical reaction takes place in the concentrated medium formed of water and an organic solvent, provided that the enzymes and co-enzymes retain their catalic activity and do not degrade at the specified concentrations.

In addition to the indications given above, the polymer appears to react in a certain way with the azide to form an electrostatic bond or covalent bond between the enzymes and the polymer. It may also appear that the polymer elongates by encapsulating in a way, and consequently by protecting, the active sites of the enzymes. In this way, denaturation of enzymes or any other form of degradation is inhibited or does not take place.

As indicated above, it is possible to stabilize at least two or more of two co-enzymes or at least two or more of two enzymes in the same solution.

Furthermore, and more importantly, it is possible to stabilize both enzymes and co-enzymes in the same solution. It is assumed that the aqueous solution of the organic solvent is the main factor in stabilizing the co-enzyme, although the polymer does not appear to exert a stabilizing effect. In the stabilization of the enzyme, the organic solvent and the polymer seem to be the main factors from which the stabilization results. In addition, and in many cases, the azide helps to improve stabilization. In both cases, it can be seen that the enzymes and co-enzymes are still both in the same single solution.

Some of the other reactions which have been carried out with the stabilized enzyme and co-enzyme compositions are indicated below. The reaction involving the phosphorylation of creatine is as follows:

CK

CREATINE + ATP ^> CP + ADP ^ ph 8-9

The other reactions all explain themselves and refer to the list of symbols given above. For a NADP reaction, the scheme is as follows:

G-6-PDH

G-6-P + NADP t *> NADPH + Acid

6- phosphogluconic for an ADP reaction, the scheme is as follows:

CK

CP + ADP /> CREATTNE + ATP

1 ph 6-7

For the following scheme, the starting creatine + ATP reaction would be used to produce the ADP co-enzyme:

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PK

ADP + PF.P ^ ^ ATP + pyruvate LDH

Pyruvate + NADH ^ ZZZZ ^ Lactate + NAD

The following reactions illustrate the use of the enzymes urease and GLDH in stabilized liquid compositions.

UREASE UREE c *> 2NH4 ++ CO2

pH 6-8

GLDH

NH4 ++ a-ketoglutarate + NAD ^ ZZZZZZZZÉ Glutamate +

NADH

The invention is further illustrated by the following examples.

Example 1

About 0.7 g of a gelatin polymer is added to about 700 ml of water. This solution is then heated above 30 ° C to dissolve the gelatin polymer.

After the addition of the polymer, the solution is immersed in a water bath so as to reduce its temperature to about 22 ° C.

The pH is then adjusted between 6.5 and 8.0. When the temperature has been reduced and the pH has been adjusted, about 2.0 g of ATP is added to the solution and then 4.0 g of NAD. 3 g of magnesium chloride are added at the same time as the co-enzyme NAD. 300 ml of glycerol are then added.

After the addition of glycerol, the pH is adjusted to about 7.5 by addition of IN hydrochloric acid. After complete dissolution, the solution is poured into a plastic or glass container, which is then closed. The containers are sealed and stored in the refrigerator. It has been found that a stabilized coenzyme composition prepared in this way has storage stability of up to four years without significant degradation.

Example 2

The sample produced in accordance with Example 1 is added with the enzyme exoquinase and then placed in the sealed glass container. The same shelf life is obtained, without significant degradation.

Example 3

The same sample from Example 2 is also added with the enzyme G-6-PDH and then sealed in the glass container; the same longevity is obtained, without appreciable degradation.

Example 4

About 1.0 g of a dextran polymer is added to about 700 ml of water. This solution is then heated above 30 ° C to dissolve the polymer. The solution is kept in a water bath to reduce the temperature to about 22 ° C.

When the temperature has been reduced, about 2.2 g of NADP is added to the solution and then 4.0 g of ATP. The pH of the solution is adjusted to the range of 6.5 to 8.0. Then 325 ml of glycerol are added.

After the addition of glycerol, the pH is adjusted to about 7.5 by addition of IN hydrochloric acid. When total dissolution has been obtained, the solution is introduced into a plastic or glass container which is then closed. The containers are sealed and stored in the refrigerator. It has been found that a stabilized coenzyme composition is about as effective as the composition of Example 1, although the azide was not added.

The following examples are presented schematically to indicate the reactants and the amounts added in the various important stages of production of the stabilized compositions of the present invention.

Example 5 ADP, AMP and NAD stabilized about 700 ml of water 0.5 g of gelatin hot dissolve 0.7 g of sodium azide allow to cool to room temperature 50 g of creatine phosphate 4 g of ADP 20 g of AMP 15 g of NAD

dissolve and adjust the pH between 7 and 9 300 ml of glycerol shake and readjust the pH

pour into a bottle and seal tightly

Example 6

ADP, AMP, NAD, HK and G-6-PDH stabilized About 300 to about 15,000 international units of G-6-PDH per liter are added and about 100 to about 10,000 international units of HK per liter are added to the solution. 'example 5, before packing.

Example 7

1.5 g of NAD

dissolve in 5ml of water add 5 ml of glycerol adjust the pH to a value below 5

Example 8 Stabilization of NAD and HK 1.5 g of NAD

Dissolve in 10 ml of buffer (ph 7) of piperacid

0.1 M zine- [bis] -ethane-sulfonic

adjust the pH to 6-7

add 10 ml of glycerol readjust the pH

add and dissolve 10 mg of HK of activity equal to 150 international units per milligram.

Example 9

Stabilization of creatine, ATP and PEP 1000 ml of water add 12.1 g of tris (hydroxymethyl) aminomethane add 1.0 g of gelatin dissolve by heating above 30 ° C

allow to cool to room temperature add 2.0 g ATP

add 2 g of PEP

add 10.0 g of dissolved creatine and adjust the pH to 9.

Example 10

To the stabilized solution of Example 9 100 is added to

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10,000 international units of LDH per liter and 100,000 to 100 international units of DK per liter, before packaging.

The compositions of Examples 5 to 10 all have the same longevity, without any appreciable degradation. In addition, each of the foregoing examples was based on samples actually prepared and tested in accordance with the present invention.

B

Claims (10)

631208 2 1. Method for stabilizing a composition containing a labile co-enzyme, a labile enzyme or a mixture of the two used in assays for biological diagnosis, this enzyme and this co-enzyme cooperating to act on the reactivity of a constituent biological agent used in an assay for biological diagnosis and being normally unstable in an aqueous medium, method characterized in that it consists: (a) mixing water with an inert organic solvent miscible with water to form an aqueous solution of said organic solvent, said solvent being liquid at least at room temperature; (b) adding an at least sufficient amount per liter of a labile co-enzyme, a labile enzyme or their mixture to said solution to carry out an assay, by dissolving in said solution and participating in a assay reaction, (c) adjusting the pH in a range of 6.0 to 8.5 and (d) placing the composition in sealed packaging. 2. Method according to claim 1, characterized in that (a) the solvent is present in a proportion of 5 to 70% by volume per volume of solution, (b) adding at least 100 international units per liter of the labile enzyme or the labile co-enzyme. 3. Method according to claim 1 or 2, characterized in that said enzyme is chosen from glucose-6-phosphate-de-hydrogenase, hexokinase, glutamate dehydrogenase, creatine-phosphokinase, pyruvate kinase and a Alkaline phosphatase and said co-enzyme is chosen from nicotina-mide-adenine-dinucleotide, adenosine triphosphate, adenosine 5'-diphosphate, nicotinamide-adenide-dinucleotide phosphate and adenosine monophosphate. 4. Method according to claim 1, characterized in that it also consists in adding a second enzyme or co-enzyme to said solution, which is also stabilized there, after adjustment of the pH. 5. The method of claim 1, further characterized in that the solvent has a pH of 4 and 10, is liquid at room temperature and at refrigerator temperature, does not react with enzymes or co-enzymes other than by formation of electrostatic bonds (that is to say hydrogen bonds), is miscible with water and has a low energy free of solvolysis (normal resonance). 6. Method according to one of claims 1 to 5, characterized in that it consists in dissolving in said composition a water-soluble polymer which does not substantially inhibit the enzymatic activity. 7. Method according to claim 6, characterized in that the polymer and gelatin present in the solution in a proportion of between 0.01 and 0.5%. 8. Method according to one of claims 1 to 7, further characterized in that the solvent is added so as to be present in an amount 25 to 50 and preferably 25 to 40% by volume. 9. Method according to one of claims 1 to 8, characterized in that the solvent is a polyol containing two to 10 carbon atoms and 2 to 4 hydroxyl groups. 10. Method according to one of claims 1 to 9, characterized in that a bacteriostatic agent is added, for example an azide, which also plays the role of an agent stabilizing the enzyme.