LU83955A1 - COMPOSITION AND METHOD FOR ASSAYS WITH NAD + ENZYMATIC AMPLIFIER CYCLE - Google Patents

Description

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The present invention relates to enzymatic amplifier cycles; it also relates to carrying out immunochemical assays, in particular in biological fluids.

5 The development of enzymatic amplifier systems and their use for assays are known, in particular from Lowry C.H. et al., J. Biol. Chem., (1961), 2 ^ 6 (10), pp. 2746-2755, and Lowry O.H. et al., Analytical Biochem., (1973), 52 ”PP. 86-97 · It has also been reported that there is an interest in having the maximum of irreversible reactions so as to increase the amplifying power of the cycle (see jc Nicolas, J. Chaintreuril, B. Térouanne, b. Descamps, A Paul de Crastes, Ann. Biol, clin., (I978), 36, pp. 205-208.

15 Several enzymatic amplifying cycles allowing assays of small amounts of NAÜ + or NADP + have thus been described. The NAD + amplifying enzyme cycles are based on the following scheme:

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20 S1 + NAD + --—> P1 + NADH + H + E (1) S2 + NADH + H + --— »» P2 + NAD + where E ^ and E2 are the two enzymes respectively used, and S1 and S2 are corresponding substrates.

* The products p ^ and P2 are accumulated and can then

be dosed using a specific reaction of p ^ or P, such as: E

Ρχ + NADH + H + --2-> p3 + NAD (2) 3 ° or.

P2 + NAD + --—> P4 + NADH + H + (3) where E ^ and E ^ are the enzymes used, which lead to the products P ^ or respectively with formation of NAD + or NADH measured by fluorimetry.

for the proper functioning of this type of amplification, several elements must be taken into consideration: 2 - the stages of the cycle (1) must be, as much as possible, irreversible; as these are steps involving dehydrogenases, it is advantageous to make them work in the most favorable direction; 5 - it seems advisable, to make these reactions irreversible, to trap the products p ^ and p ^. However, it should not be forgotten that at least one of the two products p ^ or P2 must still be capable of being dosed; - the revelation step (2) or (3) must also have '10 the maximum irreversibility; - the final measurement of NACH can be done directly by fluorescence, on the other hand, for NAD +, it should be done; find in concentrated alkaline medium. It is therefore advantageous to complete a dosage of NACH (3) so as to limit the blank 15 and the interference.

To date, no known cycle for NAD + uses completely irreversible enzymatic reactions. This is the case of the known LDH-GlDH and ADH-MDH cycles, recalled below, 20 1. LDH-GlDH cycle.

This cycle has been described by Lowry O.H. et al. in J. Biol. Chem. (L961), op. cited.

It is based on the following principle: ~ gj. Lactate + NAD + - £ 22— ^ Pyruvate + NACH + H + -ketoglutarate + NACH + NH ^ * -> Glutamate + NAD +, the revelation reaction can be:

Pyruvate + NADH + H + -> Lactate + NAD +.

30 This cycle makes it possible to dose 1.10- ^ mole of NAD +, ie a starting solution of 10-¾ (on 10 yul of sample): but it has many disadvantages: - no step is irreversible; the LDH involved in the cycle works in the unfavorable direction. It is then necessary to use a very high concentration of lactate which gives very high whites (contamination of the lactate by pyruvate); - the final reading is carried out in 4 M NaOH medium. The 3 blanks are high and the interference is difficult to control; - the amplification of the cycle varies with the concentration of NAD + and a fluorescence / concentration 5 curve is obtained which is not linear.

2. ADH-MDH cycle -

This cycle, described by Lowry O.H. et al. in Ann. Biochem. (1973), op. quoted, is as follows:

^ ADH

10 NAD + + Ethanol -> Acetaldehyde + NADH + H (4)

MDH

Oxaloacetate + NACH + H '- "· Malate + MAD4" (5) - Three revelation reactions are possible:

Malate + NADP4 "_» Pyruvate + CO + NAÜPH + H + (6) • * 5 malic d

Malate + NAD + —_> Oxaloacetate + NACH + H + ΤΓΟ f (7)

Oxaloacetate + Glutamate —►Aspartate + o6-Ketoglutaratej 20 Malate + NAD + ——s * Oxaloacetate + NADH + K + (8)

Oxaloacetate + Hydrazine -> Oxaloacetate nydrazone,

Reaction (4) has a certain character of irreversibility due to the formation of volatile 2 ^ acetaldehyde.

But reaction (5) is only relatively favorable. The final assay is a NAü (P) K assay, for which the whites are weak and the interferences are insignificant.

^ We have now found an original enzymatic cycle, based on a completely irreversible reaction. This enzymatic amplifier cycle to: NAD + according to the invention is as follows;

Glucose-6-Phosphate + MAD4 "6-phosphoglucoaate (9) 35 + NADH + H +

EgOg + NADH + H + NADK ~ pQP- .y 2 H20 + NAD + (10)

During the functioning of the cycle, there is accumulation of 6-phosphogluconate, which can be determined according to 4 the classic reaction: β-phosphogluconate + NADP + Ribulose-5-phosp:; ate | (li) + C02 + NAOPH + H +, The first object of the invention is a composition for carrying out an NAD + amplifying enzymatic cycle, characterized in that it comprises, combined in appropriate proportions, the compounds carrying out reactions (9) and (10) ci -above.

The reaction (10) is completely irreversible. However, for the smooth running of the cycle, it is advisable to thermodynamically favor the reaction (9) which is reversible.

This is obtained by choosing a pK zone for carrying out the cycle where the reverse reaction 15 (6-phosphogluononate -> glucose-6-phosphate) has a very unfavorable equilibrium constant, all the more so since found in the presence of a large excess of glucose-6-phosphate compared to the 6-phosphogluconate formed during education.

20 / ”6-phosphoglueonate_7 / ~ NAÜH_7 / ~ K * 7

7 ”NA0 ^ 7 ^” slucose-6-phosphate J

The revelation reaction is also irreversible, which "makes it very sensitive." The final measurement consists of a measurement of the fluorescence of the formed NAOPH - '25 i see the work entitled "Methods of Enzymatic Analysis”, HV Bergmeyer, (1976), pp. 2059-2072, Verlag Chemie GmbH (2nd ed . english} 7 ·

It has been established that the use of this cycle according to the invention makes it possible to dose 25 femtomoles of 30 ITAD + or of NACH or of a derivative of one of these two substances. This cycle has, moreover, a linearity which extends over a zone of concentrations ranging from 25 to 1000 x IO “15 moles of NAO +.

The invention therefore also relates to the application of the aforementioned amplifying cycle to immunochemical assays of substances present in biological fluids, such as, for example, substances known by the abbreviations (the meaning of which will be recalled below) • t 5

Ty T ^, E] / S2 * Ey Testo and others.

The assays using such an amplifying enzyme cycle system i ITAD + have the advantages of not requiring. · 5 - nor radioisotopes to mark the substance to be assayed; the "marker" is then a stable substance; - neither "free-bound" separation.

In practice, the amplifier cycle described is carried out by incubating the various reagents Ί0 and enzymes used and in the appropriate proportions. Given the thermodynamic characteristics of the two enzymes used, a pH zone close to the optimum pH of G-6-PDH is desirable, but not essential.

After a time that a person skilled in the art sets himself (time which is that at the end of which the accumulation of β-phosphogluconate is at least equal to the minimum detectable and which can be easily determined after a few routine tests, if necessary ), the enzymatic reactions are blocked by any suitable means and the reagents allowing the development or the determination of the 6-phospno-glueonate formed are added. When this reaction is complete, the fluorescence of the solutions is measured against a blank.

The invention therefore has as a second object a method for carrying out immunochemical assays, in particular in biological fluids, characterized in that it comprises the use of the above composition, capable of carrying out an enzymatic cycle , the NAÜ + to be dosed being contained in the biological fluid, with incubation of the various reagents and enzymes at an intermediate pH between the optimal pHs of the two enzymes used, and for a sufficient time to form β-phosphogluconate in an amount exceeding the minimum detectable, and then blocking the cycle and revealing or dosing the 6-pnosphogluconate formed.

Alternatively, the enhancer enzyme cycle (9-10) can be applied to enzyme immunoassays. To carry out such assays, a constant quantity of the same substance, but covalently linked to NAD +, is added to a biological liquid containing the substance to be assayed; we put the two substances in competition with a constant quantity of antibodies directed against the substance to be assayed. The competition reactions 5 can take place and it then suffices to assay the NAü + which has remained liter or the NAD + attached to the antibody. It is not absolutely necessary to provide an intermediate "free-bound" separation step, since NAD + can be inactivated when it is bound to the antibody by means of the substance to be assayed.

For the implementation of the NAD + amplifying enzymatic cycle according to the invention, the NAüH-peroxydesis of Streptococcus faecalis (EC 1.11.1.1) or any other enzyme catalyzing the reaction between NADH and 15 and giving rise to the formation of NAD + is recommended. usable in the second reaction. Generally, an NAÜ-dependent glucose-6-phosphate dehydrogenase will be chosen in order to avoid interference with any NADP-dependent enzyme present in the medium containing the substance to be assayed (in practice, most often a serum ), such as, for example, glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides (EC 1.1.1Λ9).

On the other hand, the white should be made as low as possible to improve the dosages. This blank 25 actually represents the NAD + present in the enzymes used; the quantity of "endogenous" NAD + must therefore be, as far as possible, made minimal, this is conventionally obtained by treatment of the enzymes with charcoal, which unfortunately results in significant losses of enzymes.

We have now succeeded in considerably improving this purification by means of gel filtration chromatography. The blanks provided by such a method are similar to those provided by the wood charoon method, but the procedure is much simpler and above all the loss of enzyme is almost zero.

The following examples illustrate the invention more concretely, without limiting its scope.

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The abbreviations used in the present text, both in the foregoing and in the examples below, have the following meanings: ADH: alcohol dehydrogenase 5 G1DH · * glutamate dehydrogenase LDH: lactate dehydrogenase MDH: malate dehydrogenase NAD +; nicotinamide-adenine-dinucleotide NACH: idem, reduced form "10 NADH-POD: NADH-peroxidase NADP +: nicotinamide-adenine-dinucleotide-phosphate NADPH: idem, reduced form G-β-ΡΟΗ: glucose-6-phosphate-dehydrogenase 6- PC-DH: 6-phosphoglpconate dehydrogenase 15: triiodothyronine: thyroxine E ^: oestrone E ^: oestradiol E_: oestriol 3 20 F: cortisol

Testo: testosterone EXAMPLE 1

This example relates to the production of a cycle in accordance with the invention. 0.1 M Tris and KAc buffer (where Ac denotes acetate group) 0.14 M at pH 8.5 was prepared containing 1 mM glucose-6-phosphate and 1 mM.

Before carrying out the cycle, 100 μg / ml of NADH-POD (Ec 1.11.1.1) and 100 μg / ml of G-β-ΡΟΗ (Ec 1.1.1.49) were added to this buffer; these enzymes had previously been freed of NAD + or NAOH which they could contain by gel filtration.

To 100 μl of the cycle reagent thus produced, 10 μl of the solution containing the NAD + to be assayed was added. It was incubated for 1 hour at 37 ° C. The reactions were then blocked by heating for about 5 minutes at about 100 ° C. After cooling, 1 ml of the developer was added, the composition of which was as follows; 20 mM Tris buffer and 30 mM NH ^ Ac (pH 7.7) containing 8 lf 0.1 mM EDTA, 0.02 fo bovine serum albumin, NAÜP 20 ^ uM and 0.45 ^ ug / ml of 6 -PGDH. The reaction was allowed to proceed for 30 minutes at room temperature, after which the fluorescence was read against a blank obtained according to the above procedure, but with the addition, starting with 10 µl of solution devoid of NAU + instead of 10 µl of the solution containing the NAD + to be dosed.

It was thus possible to assay 25 fM (femtomoles) of NAD + or of NAD + derivative. It should be noted that the amplification of the cycle thus produced is linear in a large concentration range, which ranges from about 25 to 1000 femtomoles.

EXAMPLE 2

This example relates to the prior purification of the enzymes used. The enzymes used in the amplifier cycle according to 1Tinvention must contain as little NAD + or NASH as possible; the commercially available preparations of NADH-POD and of G-6-PDH were then purified then used in accordance with the operating procedure described in Example 1.

Gel enzymes were involved, in practice on Sephadex G 25 gel, in 0.1 M Tris buffer, pH 8.5.

The losses in enzymatic activity are zero and the enzymes thus purified can be stored at + 4 ° C. and then used directly in a cycle such as that described in Example 1. The blanks obtained by this method are of the same order of magnitude as those obtained by the conventional method of purification with charcoal.

EXAMPLE 3.

This example relates to the calculation of the amplification of the cycle, with reference to the signals given in fluorescence by known quantities of 6-phosphogluconate revealed according to Example 1.

It was thus possible to assay 0.9 nM / ml of 6-phosphogluconate. this allows to calculate the amplification of the cycle, which is then 6,000 per hour according to example l.

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When performing a cycle for assaying NAD + in biological media, it is not necessary to carry out a revelation curve for 6-phos-phogluconate. It suffices to carry out some MAD 5 standards which will be used to draw a calibration curve. The choice of the value and the number of these standards depends on the precision to be obtained and the area of concentration explored. It is also advisable to include a blank, as described above, in each series.

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Claims (7)

1. Composition for carrying out an NAD + amplifying enzymatic cycle using an irreversible reaction, with a view to an assay, characterized in that it comprises, combined in appropriate proportions, the compounds making it possible to carry out the reactions : Glucose-6-phosphate + NAD + 6-phosphogluconate + NADH + H + H202 + NADH + H + NA.?S.TJL9P ..> 2 H20 + NAD +, 10 where G-6-PDH and NADH-POD respectively denote glucose- 6-phosphat'e-dehydrogenase and a NADH-peroxidase, and accumulates 6-phosphogluconate which is the dosable substance. 2. Method for carrying out immunochemical assays, in particular in biological fluids, characterized in that it comprises the use of the composition capable of carrying out an enzymatic cycle according to claim 1, the NAD + being that contained in the medium to be assayed, incubation of the various reagents and enzymes at a pH intermediate between the optimum pHs of the two enzymes used, and for a time sufficient to form 6-phosphogluconate in an amount exceeding the minimum detectable , and then blocking the cycle and revealing or dosing the 6-phosphogluconate formed. 3. Method according to claim 2, characterized in that it uses a NADH-peroxidase from Streptococcus faeca- 2. lis (EC 1.11.1.1) and a glucose-6-phosphate dehydrogenase NAD-dependent and not interfering with any NADP-dependent enzyme present in the medium containing the substance to be assayed. 4. Method according to claim 3 ", characterized in that use is made of glucose-6-phosphate-dehydrogenase from Leuconostoc mesentroids (EC 1.1.1.49). 5. Method according to any one of claims 2 to 4, characterized in that the enzymes are purified beforehand by subjecting them to gel filtration chromatography, 6. A method of immunoenzymatic assay, characterized in that it comprises the addition, to a biological liquid contained i) ‘. '-' It denants the substance to be dosed, of a constant quantity of the same substance, but covalently linked to NAD +, the competition of the two substances with a constant quantity of antibodies directed against the substance to be dosed, and mi-4 implements the immunochemical assay method according to any one of claims 2 to 5. 7. Composition according to claim 1, characterized in that the revelation of 6-phosphogluconate implements a conversion to nbulose-5-phosphate by means of NADP and in the presence of 10-phosphogluconate dehydroginase. t r