FR2521591A1 - Comtitive binding immuno-enzymatic assay method - with enzymatic amplification cycle to produce malate - Google Patents

Abstract

In a competitive binding assay comprising reaction of substrate (A) and NAD(+) (or deriv.) labelled substrate (A') with a specific binder (esp. antibody) the new feature is that an amplification reaction is carried out using the reactions: A' + G-6-P = A'H + H(+) + 6-phosphogluconate (I) A'H + H(+) + oxaloacetate = A' + malate (II) (G-6-P is glucose-6-phosphate). The first reaction is in presence of G-6-P-dehydrogenase and the second in presence of malate dehydrogenase (MDH), and finally (I) and/or (II) are measured. Measurement of (I) is e.g. by reaction with NADP(+) in presence of (I)-dehydrogenase to give ribulose-5-phosphate (R-5-P) and NADPH and of (II) e.g. by enzymatic reaction with NADP(+) to give pyruvate and NADPH. The reduced coenzyme is measured spectrophotometrically or fluorometrically. The method can be used to assay pharmaceuticals, hormones, proteins, xenobiotic substances, etc. Detection limits are typically 50-100 moles for spectrophotometry or 2.5-10 moles for fluorimetry.

Description

 The present invention relates to competitive assays with labeling by a coenzyme and amplifying enzymatic cycle, as well as an amplifying enzymatic system itself.

 The invention relates more particularly to a method, as well as a system and an assembly for assays in biological fluids by competition with labeling with NAD or one of its derivatives and enzymatic amplifying G-6-PDH / MDH cycle.

 The principle of the assay method according to the invention is based on the competition between a substrate to be assayed and a substrate marked with a coenzyme, that is to say a substrate to which a coenzyme is attached, with respect to a substance. having an affinity for the substrate, such as for example a specific antibody directed against the substrate and, in this case, the assay is an immunochemical assay.

The development of enzymatic amplifier systems and their use for assays are known, in particular from Lowry OH et al., J. Biol. Chem., (1961), 236 (10), pp. 2746-2755 and Kato T. et al., Analytical Biochem., (1973), 53, pp. 86-97. It has also been pointed out that it is advantageous to have the maximum of irreversible reactions so as to increase the amplifying power of the cycle (see
JC Nicolas, J. Chaintreuil, B. Térouanne, B. Descamps and
A. Crastes de Paulet, Ann. Biol. Clin., (1978), 36, pp. 205208).

We have now designed and developed a system for competitive assays with "labeling" and amplifying enzymatic cycle, which is based on a new amplifying enzymatic cycle and involves the following reactions
- competition between the molecule to be assayed (which can be called "substrate") and this molecule labeled with coenzyme (substrate-CEZ +), CEZ + representing NAD + or one of its derivatives vis-à-vis a substance having an affinity for the molecule to be dosed, such as, for example, an anti
specific body directed against the molecule to be assayed and designated by M.

substrate-CEZ + + substrate + M - substrate + (substrate-CEZ +) M
- Determination of unbound CEZ + substrate.

substrat-CEZH + H+ + oxaloacétate

substrat-CEZ+ + malate
Lorsque ce système cyclique a amplifié suffisamment (c'est-à-dire produit suffisamment de malate et de 6phosphogluconate), il reste à arrêter les réactions enzymatiques par un système approprié et à mesurer le malate et/ou le 6-phosphogluconate formés par l'un quelconque des cinq systèmes suivants ::
malate + ou

pyruvate + CO2 + NADPH + malate + NAD+

oxaloacétate + NADH + a+
oxaloacétate + hydrazine - > oxaloacétate hydrazone ou malate + NAD+

oxaloacétate + NADH + oxaloacétate + glutamate

aspartate + α;-cétoglutarate ou
6-phosphogluconate +

ribuloso5-phosphate + CO2
+ NADPH + ou
6-phosphogluconate +

ribulose-5-phosphate + CO2
+ NADPH + ribulos.-5-phosphate

xylulose-5-phosphate xylulose-5-phosphate + ribose -5-phosphate

sédoheptulose- 7-phosphate + glyciraldShyde-3-phosphate glyceraldihyde-3-phosphate +

glycérate-3-pho.pbate + NADH + H
Le coenzyme utilisé pour le marquage est le
NAD+ ou un dérivé qui peut être dosé avec une grande sensibilité par un cycle enzymatique amplificateur.L'originalité du système selon l'invention tient à l'utilisation d'un nouveau cycle amplificateur et à la possibilité d'effectuer la mesure finale en spectrophototométrie ou en fluorimétrie.substrate-CEZ + + glucose-6-phosphate

substrate-CEZH + H + + oxaloacetate

substrate-CEZ + + malate
When this cyclic system has amplified enough (that is to say produces enough malate and 6phosphogluconate), it remains to stop the enzymatic reactions by an appropriate system and to measure the malate and / or the 6-phosphogluconate formed by the any of the following five systems:
malate + or

pyruvate + CO2 + NADPH + malate + NAD +

oxaloacetate + NADH + a +
oxaloacetate + hydrazine -> oxaloacetate hydrazone or malate + NAD +

oxaloacetate + NADH + oxaloacetate + glutamate

aspartate + α; - ketoglutarate or
6-phosphogluconate +

ribuloso5-phosphate + CO2
+ NADPH + or
6-phosphogluconate +

ribulose-5-phosphate + CO2
+ NADPH + ribulos.-5-phosphate

xylulose-5-phosphate xylulose-5-phosphate + ribose -5-phosphate

sedoheptulose- 7-phosphate + glyciraldShyde-3-phosphate glyceraldihyde-3-phosphate +

glycerate-3-pho.pbate + NADH + H
The coenzyme used for labeling is
NAD + or a derivative which can be measured with high sensitivity by an amplifying enzymatic cycle. The originality of the system according to the invention is due to the use of a new amplifying cycle and to the possibility of carrying out the final measurement by spectrophototometry. or in fluorimetry.

substrat-CEZH + H + oxaloacétate

substrat-CEZ+ + malate, l'arrêt du cycle et la révélation du malate et/ou du 6-phosphogluocnate formés par un système choisi parmi l'un quelconque des 5 systèmes suivants
malate + NADP+ oti

pyruvate + C02 + NaDPH+ + balata + NAD

oxaloacétato + NADH +
oxaloacétate + hydrazine oxaloacêtate hydrazone ou malate + NAD+

oxaloacêtate + NPWE + a+ oxaloacétate + glutamate

aspartate + a-cétcglutarate ou
6-phosphogluconate +

ribulose-5-phosphate + CO2
+ NADPH + 8+ ou
6-phosphogluconate +

ribulos.-5-phosphate + C02 + NADPH * + ribulose-5-phosphate

xylulose-5-phosphate xylulose-5-phosphate + ribose-5-pnosphate

sEdoheptulose-7-phosphate + glycéraldéhyde-3-phosphate glycéraldéhyde-3-phosphate +

glycérate- 3-phosphate
+ NADH + et la mesure finale du NAD(P) H formé en spectrophotométrie ou en fluorimétrie.The present invention therefore has as its first object a method for carrying out assays by competition, with labeling and amplifying enzymatic cycle, characterized in that it comprises the preparation of a molecule labeled with a coenzyme which may in practice be NAD + or a derivative of NAD +, the competition of the molecule to be assayed and of the molecule labeled with the coenzyme vis-à-vis a substance having an affinity for the substrate, the achievement once the balance of competition has been reached in the cycle enzymatic amplifier: substrate-CEZ + + glucose-6-phosphate

substrate-CEZH + H + oxaloacetate

substrate-CEZ + + malate, stopping the cycle and revealing the malate and / or 6-phosphogluocnate formed by a system chosen from any one of the following 5 systems
malate + NADP + oti

pyruvate + C02 + NaDPH + + balata + NAD

oxaloacétato + NADH +
oxaloacetate + hydrazine oxaloacetate hydrazone or malate + NAD +

oxaloacetate + NPWE + a + oxaloacetate + glutamate

aspartate + a-ketcglutarate or
6-phosphogluconate +

ribulose-5-phosphate + CO2
+ NADPH + 8+ or
6-phosphogluconate +

ribulos.-5-phosphate + C02 + NADPH * + ribulose-5-phosphate

xylulose-5-phosphate xylulose-5-phosphate + ribose-5-pnosphate

sedoheptulose-7-phosphate + glyceraldehyde-3-phosphate glyceraldehyde-3-phosphate +

glycerate-3-phosphate
+ NADH + and the final measurement of NAD (P) H formed in spectrophotometry or fluorimetry.

 Preferably, the coenzyme used is NAD +. As a variant, it is possible to use derivatives of NAD +, such as for example N6- (2-carboxymethyl) NAD or N6 (2-carboxyethyl) NAD +, which are advantageously in practice derivatives carrying an accessible group, in particular a group carboxylic, intended to allow or facilitate the fixation on the molecule to be assayed.

 The present invention is not limited to these two derivatives and other derivatives can be used for labeling, depending on the nature of the reaction groups carried by the molecule to be assayed.

After a time that the skilled person is in the process of settling down (time which is that at the end of which the accumulation of 6-phosphogluconate and / or malate is at least equal to the detectable immune by the means used, and which can be easily digested after a few routine tests, if necessary), the enzymatic reactions are blocked by any appropriate means and the malate and / or 6-phosphogluconate are revealed by one of the abovementioned systems, and preferably by system

 oxaloacetate + hydrazinexaloacetate hydrazone, then to a final measurement of the NAD (P) H formed in spectrophotometry or in fluorimetry.

 Those skilled in the art simply have to choose either fluorimetry or spectrophotometry, depending on the range of sensitivity to explore.

 By operating according to the invention, it is possible in practice to reach a detection limit of the order of 5 to 10 × 10 8 8H in substrate in the biological medium with a final measurement in spectrophotometry, while if the final measurement in fluorimetry is carried out , a detection limit of the order of 2.5 to 10.10 9M can be reached.

The invention also relates to the aforementioned amplification system, characterized in that it comprises, associated in appropriate proportions, the products and the enzymes carrying out the following two reactions substrate-CEZ + - + glucose-6-phosphate.

substrat-CEZ+ + malate
En pratique, on réalise le cycle amplificateur décrit en laissant incuber les différents réactifs et enzymes mis en oeuvre, dans les proportions appropriées.substrate-CEZH + H + + oxaloacetate

substrate-CEZ + + malate
In practice, the amplifier cycle described is carried out by allowing the various reagents and enzymes used to incubate, in the appropriate proportions.

+ (substrat NAD+)M, où M est tel que défini plus haut, le système d'amplification susdit, et des moyens de révélation du malate et/ou du 6-phosphogluconate formés et de mesure finale du NAD(P)H formé en spectrophotométrie ou en fluorimétrie.The invention also relates to a metering system associating the means achieving the balance of competition substrate-NAD + substrate +

+ (NAD + substrate) M, where M is as defined above, the aforementioned amplification system, and means for revealing the malate and / or 6-phosphogluconate formed and for final measurement of the NAD (P) H formed in spectrophotometry or fluorimetry.

 As a variant, this system can involve, in the constitution of the means achieving the balance of as a petition, not NAD + itself, but derivatives of NAD +, such as for example N6- (2-carboxyethyl) NAD +; in practice, as derivatives of NAD +, derivatives carrying an accessible group are used, in particular an accessible carboxylic group.

The invention further relates to an assembly or "kit", for carrying out assays in biological fluids according to the above process, comprising, in suitable containers
- a substance with an affinity for the molecule to be assayed, such as for example a specific antiserum
- this molecule labeled with NAD + or with a derivative of NAD as indicated above
- a buffer of the amplifier cycle and the specific substrates of this cycle as well as the enzymes of this amplifier cycle; - a development buffer with the enzyme (s), the development coenzyme (s) and the development substrate (s3) if applicable
- and possibly one (or more) standard (s).

 The method according to the invention was applied to serum hapten assays and this can be illustrated by the assays of 3 antibiotics: gentamycin, tobramycin and amikacin. These are simple examples of application of the invention; they do not in any way limit this, which can equally well be applied for example to the dosages of other antibiotics or, as a general rule, of all substances capable of forming antibodies, for example drugs, hormones, proteins or xenobiotics. It is also possible to envisage the assay of antibodies endogenous to the biological medium using this system.

The invention is described more concretely in the example below, which in no way limits it and in which the abbreviations used have, as in the preceding description, the following meanings - Ac: antibody - NAD: nicotinamide-adenine- dinucleotide - NADH: id., reduced form - G-6-PDH: glucose-6-phosphate-dehydrogenase - MDH: maláte-dehydrogenase - NADP +: nicotinamide-adenine-dinucleotide-phosphate - NADPH: id., reduced form - ASAT : aspartate-aminotransferase - 6-PGDH: 6-phosphogluconate-dehydrogenase - GAPDH: glyceraldehyde-3-phosphate-dehydrogenase - NAD (P) H: NADH or NADPH - CEZ: NAD or substitute derivative
EXAMPLE
An antibiotic, and more specifically gentamycin, was used as an example. The principle diagram would however be strictly the same for each of the two other aminoglycosides which are amikacin and tobramycin.

1. Synthesis of the labeled hapten
Gentamycin cannot be coupled directly to NAD +. We therefore used for the synthesis of the branded product a suitable derivative of NAD +; in this case a derivative carrying an accessible carboxylic group and more precisely N6- (2-carboxyethyl) NAD. 1 g of gentamycin base was dissolved in 5 ml of H2O and the pH was brought to about 7.0. 100 mg of N6- (2-carboxyethyl) NAD + were then added and the pH was adjusted to 4.7-. 500 mg of 1-ethyl- (3-diaminopropyl) carbodiimide hydrochloride dissolved in 2 ml of H2O was then added. The pH was kept at 4.7 for 3 hours, after which it was brought to 7.0 and the solution was applied 1 a on a Dowex C1 exchange resin (2.6 x 40 cm). We eluted with H2O until the exit of the dead volume. The eluted peak absorb band 254 nm was collected and concentrated under reduced pressure.

This solution was then applied to a column of
Sephadex G15 (2.6 x 60 cm). It was eluted with water and the absorbent fraction was recovered at 254 nm. This fraction was concentrated under 0.5 mm Hg at 30 ° C. and applied to a Bio-Rex 70 column (2.6 × 40 cm), previously equilibrated with 20 mM ammonium acetate. We eluted as follows - NH4OCOCH32O mn: until the dead volume left - NH40COCH320 mM to NH4OCOCH3 50mM: according to a linear gradient
4 hour area.

comme décrit par Bergmeyer dans Methoden der enzymatischen
Analyse, 3ème édition, 1974, Verlag Chemie, Weinheim, RFA.In each eluted fraction absorbing at 254 nm, the NAD derivative (coenzymatic activity) and gentamycin were assayed (by radioimmunoassay) and the first significant absorbent peak was recovered at 254 nm and containing NAD + derivative and gentamycin . The fraction was again reduced in volume and applied to Biogel P4. 3 fractions absorbing at 254 nm were obtained and the second fraction, containing NAD + derivative and gentamycin, was collected and concentrated under reduced pressure to a volume of + 5 ml. This gentamycin solution is derived
NAD + was kept as is at -300C. We measured its NAD + derivative content using the classical reaction

as described by Bergmeyer in Methoden der enzymatischen
Analysis, 3rd edition, 1974, Verlag Chemie, Weinheim, RFA.

2. MDH-G-6-PDH amplifier cycle
To carry out this cycle allowing the amplification of a small amount of hapten-NAD +, MDH was used
pork heart and glucose-6-phosphate dehydrogenase
of Leucohostomenteroides, but we might as well have chosen
or any other suitable source of enzyme.

2.1. Purification of enzymes
The enzymes, being contaminated with NAD + and / or
NADR, must have been purified by filtra chromatography
tion on gel (Sephadex G25) (any other suitable method
may also be suitable). The contaminants would be particularly annoying, because their cycle speed is much
more barn than that of hapten-NAD + and they would give
very important whites.

2.2. Cycle realization
The cycle reagent had the following composition
boasts: 0.2 N Tris buffer, glucose-6-phosphate 12 min, oxalo
12 mM acetate, 12 BM mercaptoethanol (which can be replaced
over there. same concentration of dithiothreitol or glutathione
reduced). This reagent can be stored at -30oC for longer
six months. Just before use, we added to
reagent cooled in an ice bath HDH and G-6-PDH
purified to final concentrations of 40 g / ml and 200 g / ml.

At 50 μl of the solution thus prepared, we have
added 50 jil, hapten-NAD +, the concentration of NAD +
was between 3.10 8M and 7.10-7M. We went to the bath
marries at 300C for 1 hour. The enzymatic reactions have
been stopped by heating in a boiling water bath for
3 minutes. After cooling, we were able to reveal the products
formed by one of the 5 reactions mentioned above. In the
range of concentrations used, it was easier
to use revelation

 oxaloacetate + hydrazine-Boxaloacetate hydrazone.

To 100 μl of solution, 0.5 ml of development solution was added comprising: aminomethylpropanol O, 1M, hydrazine 0.2. N, NAD + 2 mM and MDH 20 g / ml (pH = 10.0). It was allowed to react for 30 minutes at room temperature so that the malate had reacted. The absorbance was then read in a spectrophotometer at 340 nm. Absorbance was proportional
the amount of hapten-NAD + introduced into the cycle.

3. Assay (in this case, immunochemical assay) 3.1 Inhibition of the cycle by antiserum
It is reasonable to assume that when
hapten-NAD + is linked to the antibody, the NAD + part of
drift loses much of its coenzymatic activity.

We verified this by incubating a constant amount of hap
tbne-NAD + with increasing amounts of antibodies. A
aliquot was then taken up and introduced into a cycle
amplifier. We were thus able to highlight the inhibition
of the cycle by the antibody.

50 jil of gentamycin-NAD derivative + 2.10 6H
derived from NAD +, we added 0, 10, 20, 30, 40, 50
anti-gentamycin antiserum (Atlantic Antibodies GEN 074
01), diluted 10 times in 50 mM Tris buffer, pH = 8.5. We
completed with 100, 90, 80, 70, 60, 50 p1 of the same buffer
Tris 50 mM, pH 8.5. When equilibrium was reached, we
took a 50 μl aliquot which was treated according to
the amplifier cycle as above. We wore the absorber
final bance at 340 nm depending on the amount of antiserum
1/10 added.

The results obtained are reported in the form
graph in Figure 1 attached, which represents the
inhibition curve for the gentamycin-NAD + derivative considered,
namely: gentamycin coupled to N6 - (2-carboxyethyl) NAD +
(curve in solid line).

This derivative was inhibited by an anti-serum
gentamycin.

3.2. Inhibition of gentamycin inhibition
In order for this system to allow a
quantitative dosage, it was necessary to lift the inhibition
by free hapten; To do this, we incubated a quantity
fixed free hapten and hapten-NAD + with quantities
increasing antibodies and, once the balance of compe
tition reached, the amplifier cycle has been carried out.

We. achieved this with gentamycin-derivative
NAD + cited above. The antiserum incubation medium contained 0.17 µg / ml free gentamycin. To 50 l of gentamy cine-NAD derivative + 2.10-6 M in NAD derivative containing 0.5 g / ml of gentamycin, 0, 10, 20, 30, 40, 50? L of diluted anti-gentamycin antiserum were added 10 times in 50 mM Tris buffer,
PH 85. We made up to 150 μl with the same buffer. After 1 hour, 50 μl was taken, which was amplified using the cycle, as indicated above.

The absorbance was then plotted as a function of the quantity of antiserum (curve in dotted line on the
Figure 1).

 As can be seen, the difference between the two curves is the best, for the gentamycin derivative obtained by coupling with N6- (2-carboxyethyl) NAD +, with an amount of 1/10 antiserum of approximately 30 l.

3.3 Performing the assay
A hapten calibration curve was made in serum. For gentamycin, a range of 1- & 20 mg / l was used. Before the assay, the standards and the unknown samples were diluted 11 times in 50 mM Tris buffer, pH 8.5.

 To 50 111 of gentamycin-NAD + derivative 2.10 6 M in derivative of NAD +, 50 μl of standard or diluted sample were added and 50 μl of antiserum diluted 12 times in 50 mN Tris; pH 8.5 .

 After 1 hour, 50 r1 were removed and added to 50 μl of cycle reagent. After 1 h of incubation, the cycle was stopped by heating, 3 minutes in a boiling water bath. After cooling, 0.5 ml of developer reagent was added. When the revelation was finished, the absorbance was measured at 340 nm.

 The results can be read in FIG. 2 of the attached drawing plates, where the difference As340 nm -A 340 nm representing the absorbance of the standard minus the absorbance of zero is plotted on the ordinate, as a function of the concentration in gentamycin. The unknown samples are then measured by interpolation on the curve obtained.

Claims (9)

 1. Method for carrying out assays by competition, with labeling and amplifying enzymatic cycle, characterized in that it comprises the preparation of a molecule marked with a coenzyme chosen from NAD + or a derivative of NAD + (noted CEZ +), the competition between the molecule to be assayed (substrate) and the molecule labeled with the coenzyme (substrate-CEZ +) vis-à-vis a substance having an affinity for the substrate, the production, once the equilibrium of competition has been reached, of the enzymatic cycle of substrate-CEZ + amplifier + glucose-6-phosphate

 substrate EZE + H + + oxaloacetate

 substrate-CEZ + + malate, stopping the cycle and revealing the malate and / or 6-phosphogluconate formed by a system chosen from any one of the following 5 systems  malate + or

 pyruvate + CO2 + ANADPH + malate + NAD +

 oxaloaceate + NADE +  oxaloacetate + hydrazine -> oxaloacetate hydrazone or malate +

 oxaloactate + NADH + oxaloacetate + glutamate or

 aspartate + α; - ketoglutarate  6-phosphogluconate + NADP + or

 ribulose-5-phosphate + CO2  + NaDPH + H + 6-phosphogluconate + NADP +

 ribulose-5-phosphate + C02  + NADPH + B + ribulose-5-phosphate

 xYlUlose-5-phosphate xylulose-5-phosphate + ribose-5-phosphate

 Sedoheptulose- 7-phosphate + glyceraldehyde-3-phosphate glyciraldshyde-3-phosphate +

 glycdrate-3-phosphate + NADH + and the final measurement of NADPH (NADH) formed in spectrophotometry or fluorimetry.  2. Method according to claim 1, characterized in that the molecule to be measured the substrate is contained in a biological fluid and the substance having an affinity for the substrate is a specific antibody directed against it.  3. Method according to one of claims 1 or 2, characterized in that the coenzyme is a derivative of NAD + carrying an accessible carboxylic group, and in particular 16- (2-carboxymethyl) NAD + or N6 - (2-carboxyethyl) NAD +.  4. Amplification system, characterized in that it comprises 1 associated in appropriate proportions, the products and the enzymes carrying out the following two reactions: substrate-CEZ + + glucose-6-phosphate

 substrate EZE + H + + oxaloacetate

 + malate  5, Assay system, characterized in that it combines the means achieving the competitive balance substrate-CEZ + + substrate + N substrate-N + (substrate-CEZ +) N, where N represents a substance having an affinity for the substrate while CEZ + represents NAD + itself or a derivative thereof, with the amplification system according to claim 4 and with means for revealing the malate and / or the 6-phosphogluconate formed and for final measurement of the NADP (MATH) trained in spectrophotometry or fluorimetry.  6. Dosing system according to claim 5, characterized in that the substance having an affinity for  is the substrate, the molecule to be measured, the specific antibody directed against it.  7. Assay system according to one of claims 5- or 6, characterized in that the NAD + derivative is -choisi parsi'les NAD + derivatives carrying an accessible carboxylic group, and in particular N6 - (2-carboxymethyl) NAD + or N6 - (2-carboxyethyl) NAD +.  8. Together for carrying out dosages in  biological fluids by the method according to any one of claims 1 to 3, characterized in that it comprises, in suitable containers  - a substance with an affinity for the molecule to be assayed  - this molecule marked with NAD or with a derivative of NAD +;  - a buffer of the amplifier cycle and the specific substrates of this cycle, as well as the enzymes of this amplifier cycle  - a development buffer with the enzyme (s), the development coenzyme (s) and the development substance (s) if necessary;  - and possibly one (or more) standard (s).  9. An assembly according to claim 8, characterized in that the substance having an affinity for the substrate to be assayed is an antiserum.