WO1995008110A1 - Enzyme immunoassay method, method for enzyme immunoassaying an antigen using a modified electrode and assay kit - Google Patents

Abstract

Enzyme immunoassay method, method for enzyme immunoassaying an antigen by electrochemical detection, and assay kit used. The object of the invention is to develop assay methods, in which the detection sensitivity of the product formed during the enzymatic reaction is increased. The object is attained using a method consisting in reacting an enzyme (E) with a substrate (S), collecting the ionic or pro-ionic electroactive product obtained at the end of the enzymatic reaction on an electrode (3) with a polyionic compound (1) of opposite charge and measuring the signal produced by said electrode (3).

Description

 ASSAY METHOD OF AN ENZYME AND ASSAY METHOD

IMMUNOENZYMATICS OF AN ANTIGEN USING AN ELECTRODE

 The present invention relates to a method for assaying an enzyme and to an immunoenzymatic method for assaying an antigen, for which the detection is carried out on an electrode modified by a polyonic compound promoting the accumulation of the product of the enzymatic reaction. The invention also relates to a dosing kit, used during the implementation of these methods.

 Enzymes are catalysts involved in many reactions in human and animal metabolism. and as such are present in tissues and organs. More specifically, these are proteins having catalytic activity on specific substrates. For example, alkaline phosphatase is present in various tissues and in particular in the liver or in the bones. This enzyme is involved in the metabolism of carbohydrates. The decrease or disappearance of some of these enzymes generally leads to functional disorders in the body. It is therefore important to carry out assays of these enzymes in order to be able to detect certain pathological anomalies.

In addition, numerous immunoenzymatic assay techniques have been developed in recent years and are used in the medical, veterinary, agro-food or ecological industries. They advantageously replace radioimmunoassay techniques. These techniques make it possible to dose all types of antigens, in particular tcκtques products, drugs and chemical molecules. Thus, ELISA tests are based on the use of an enzyme as a marker. The immunoassay methods allow the determination of antigens even if they are present in small quantities. The assay is based on the fact that the antibodies (Ac) recognize and then bind specifically to the antigens (Ag), to give an antibody-antigen complex, according to the following balanced reaction:

Among the immunoenzymatic assay techniques, a method known as "heterogeneous competitive assay" is known which consists in introducing and putting in. reaction a defined quantity of antigens labeled by an enzyme and an indefinite quantity of unlabeled antigens (antigen to be assayed) to a limited and known quantity of antibodies fixed on the walls of a test tube. After the first immunological reaction, only a fraction of the antigens labeled with an enzyme and of the antigens to be measured reacted with the antibodies, while the rest of the antigens to be determined and of the labeled antigens are in the form of a free fraction inside the test tube. A test tube is then rinsed and washed before adding the specific substrate for the labeling enzyme. The enzyme linked to the antigen and to the antibody then reacts so as to transform the substrate S into a specific product P generally detected by spectroscopic techniques, such as absorbance or fluorimetry measurements. However, absorbance measurements are not always sufficiently sensitive and fluorimetric measurements, although having higher detection limits, are also sensitive to endogenous interference, which decreases the specificity of the assay.

 Also known from the prior art are immunoassay methods in which the enzyme bound to an antigen transforms a non-electro-active (or hardly electro-active) substrate S into an electro-active product P, susceptible to be detected by amperometry. Thus, the article by V.J. RAZUMAS et al. "Kinetic amperometric determination of hydrolase activity", Analytica Chimica Acta, (1980) 117, 387-390, describes one of these detection methods using, on the one hand, alkaline phosphatase as enzyme and the polycatechol phosphate esters and p-aminophenol as substrate and, on the other hand, β-D-glucoside. glucohydrolase as an enzyme and p-aminophenol β -D-glucoside as a substrate. The article by K.R. Wehmeyer et al., "Competitive heterogeneous enzyme imunoassay for digoxin with electrochemical detection", Anal. Chem. (1986), 58, 135-139, describes a heterogeneous competitive enzyme immunoassay for the determination of digoxin (antigen) in human plasma. The enzyme used is alkaline phosphatase and the substrate is phenylphosphate. Patent application EP 0 223 541 describes an electrochemical enzymatic assay method. This method for assaying estradiol uses alkaline phosphatase as an enzyme and the phosphate ester of 4-N-ferrocenoylaminophenol as a substrate. Finally, patent application EP 0 424 586 discloses an immuno-electrochemical assay method for alkaline phosphatase consisting in reacting this enzyme with 1-naphthyl phosphate to form the electroactive 1-naphthol, detected by a carbon electrode.

Unfortunately, in some cases, the substrate and the product obtained have electroactivities neighboring and therefore the assay process does not meet the selectivity criteria imposed.

 Among the immunoenzymatic assay techniques, there is also known a method called "homogeneous competitive assay" which consists in reacting simultaneously in a test tube the antigen to be assayed (Ag), an antigen labeled with a specific enzyme (Ag-E ), the antibody (Ac) and the substrate (S) whose transformation is catalyzed by the abovementioned enzyme. The reaction is as follows:

 Part of the antigens to be assayed and of the antigens labeled with the enzyme react with the antibody to form a complex (Ac / Ag or Ac / Ag-E), while a part of the antigens labeled with the enzyme remain free. . Only this free fraction reacts with the substrate S so as to give a product P.

Product P is detected by the same techniques as those cited for the so-called "heterogeneous competitive assay" method and therefore has the same drawbacks. In addition, if this homogeneous assay technique makes it possible to avoid the transfer, decantation and washing steps, the enzymatic activity of the enzyme present in the complex (Ac / Ag-E) must however be significantly lower. to that of the free antigen labeled with an enzyme (Ag-E), or even negligible, so that the transformation of the substrate S into product P is preferably carried out by the free Ag-E. These disadvantages therefore limit the choice of enzymes which can be used in these techniques.

 There are also known ae heterosis "heterogeneous sandwich" techniques in which the antigen to be assayed is "sandwiched" between an antibody fixed on the walls of a test tube and an antibody labeled with an enzyme. This enzyme subsequently makes it possible to transform a substrate S into product P, capable of being detected. These assay techniques have the aforementioned drawbacks inherent in the type of detection used.

 Consequently, the invention relates to a method for assaying an enzyme and to an immunoenzymatic assay method with electrochemical detection in which the sensitivity of the detection of product P is increased.

 According to the characteristics of the invention, the method for assaying an enzyme comprises the steps consisting in:

 reacting the enzyme to be assayed with a substrate whose transformation into an ionic or proionic electroactive product is catalyzed by said enzyme,

- collecting said ionic or proionic electroactive product obtained, on an electrode provided with a polyionic compound whose charge is opposite to that of said obtained product, so that this product is concentrated in said polyionic compound during a period of accumulation,

 - measure the signal supplied by said electrode at the end of the accumulation period, using electrochemical detection means connected to this electrode.

 A "pro-ionic" electroactive product is a product which becomes ionic by anodic oxidation

("pro-cationic" compound) or by cathodic reduction ("pro-amonic" compound). The signal measured by the electrochemical detection means increases with the quantity of enzyme present in the sample. The polyionic compound of the electrode is capable, thanks to its great affinity for the product obtained at the end of the enzymatic reaction, of accumulating the latter by ion exchange and thus of enhancing the amplifying effect of the enzyme. The association of a sensitive electrochemical sensor with the properties of a specific enzyme leads to a simple analysis technique to implement, inexpensive and extremely efficient since it makes it possible to detect an enzyme at concentrations up to 10 ^{- 12} to 10 ^-13 mol. ^{1 -1} .

 The immunoenzymatic assay method of an anti-gene according to the invention comprises the steps consisting in:

 reacting an enzyme serving as a marker for the antigen to be assayed or for an antibody specific for this antigen to be assayed with a substrate whose transformation into an ionic or proionic electroactive product is catalyzed by this enzyme,

 - Collect the ionic or pro-ionic electroactive product obtained by the enzymatic reaction, on an electrode provided with a polyionic compound whose charge is opposite to that of said product obtained, so that this product is concentrated in said pol yion compound i that during an accumulation period,

 - measure the signal supplied by said electrode at the end of the accumulation period, using electrochemical detection means connected to this electrode.

This assay method can be applied to all categories of immunoassay and in particular to homogeneous or heterogeneous competitive assays or to assays of the "sandwich" type. The substrate / product pair is chosen judiciously so that the accumulation of the product in the polyionic compound is favored to the detriment of the accumulation of the substrate, or even that the accumulation of the substrate is impossible. By preferentially concentrating the product in the polyionic compound, the electrochemical signal detected at the electrode is amplified, which increases the sensitivity of the technique. This assay method quantifies antigens such as drugs, hormones, proteins, pesticides, toxins, with a detection limit of 10 ^-12 to 10 ^-13 mol.1 ^-1 .

 Finally, the invention also relates to a dosing kit characterized in that it comprises:

 - a substrate capable of being transformed into an ionic or proionic electroactive product, under the action of catalysis of an enzyme,

 - an electrode provided with a polyionic compound, the charge of which is opposite to that of the ionic or proionic electroactive product, and in which said product is capable of accumulating,

 - electrochemical detection means connected to this electrode and making it possible to measure the electrical signal supplied by this electrode.

 This dosing kit allows the implementation of the aforementioned dosing methods:

 The invention will be better understood on reading the following description of an embodiment of the invention given by way of illustrative and nonlimiting example. This description is made with reference to the accompanying drawings, in which:

FIG. 1 is a diagram illustrating the method for assaying an enzyme according to the invention, FIGS. 2 and 3 illustrate two alternative embodiments of the electrode used in the metering methods according to the invention,

 FIGS. 4 and 5 are curves representing results obtained by square wave voltammetry,

FIG. 6 is a curve representing the relationship between the peak intensity and the concentration (C) of 2- (N-ferrocenoyl-amino) 4,6-dimethylphenol,

 FIG. 7 is a graph representing the variation of the peak intensity as a function of the concentration of alkaline phosphatase, under various experimental conditions,

 - Figures 8, 9 and 10 are diagrams illustrating three alternative embodiments of the method. immunoenzymatic assay according to the invention,

 FIG. 11 is a calibration curve for test 2 described below,

 FIGS. 12 and 13 are curves respectively representing the variation of the intensity of the peak as a function of the concentration of anti-phenytoin or of phenytoin, and

 - Figure 14 is a standard phenytoin titration curve usable in the context of a heterogeneous competitive assay.

FIG. 1 illustrates the principle of the method for assaying an enzyme according to the invention. This process consists in reacting an enzyme E with a substrate S so that this enzyme catalyzes the transformation of the substrate S into product P and so that this product penetrates and accumulates inside a polyionic compound 1 included in an electrode. 3 consisting for example of a glassy carbon cylinder. The accumulation of the product P in the polyionic compound 1 makes it possible to amplify the signal observed at the output of the electrode 3. This electrode 3 is connected to detection means electrochemical 5 allowing the interpretation of this signal. These detection means 5 comprise a measuring device 7 connected to an apparatus 9 for drawing graphs providing result curves.

 The detection means 5 (or more precisely the measuring device 7) use the principle of amperometry, coulometry, voltammetry or advantageously square wave voltammetry.

 In the embodiment illustrated in FIG. 1, the polyionic compound is in the form of a film covering the external surface of the working electrode 3. However, it would also be possible to use a series of ultra microelectrodes covered with 'a polyionic film or an electrode formed of a. graphite paste impregnated with the polyionic compound as illustrated in Figures 2 and 3 respectively.

FIG. 2 illustrates an exemplary embodiment of an ultra-microdisc electrode, such as that sold by the company Scotland Sensor. This electrode 11 is produced by lithography and consists of 500 carbon microdisks (diameter = 10 μm, total surface Sº = 3.9.10 ^-2 mm ² ) deposited on a support 12 and contained in the circular part 13. The electrode 11 is placed next to a reference electrode 15 in Ag / AgF surrounding the circular part 13. The film of polyionic compound 1 is deposited over the part 13 using a syringe and then dried in the oven for 10 minutes .

FIG. 3 illustrates an exemplary embodiment of an electrode 17 with graphite paste impregnated with the polyionic compound and having a renewable surface. This electrode 17 is mounted at the end of a nozzle 19 and it is connected by a conductor 21 to a rotating Taccusel electrode 23. The end of the electrode 17 can be cut after use for a new use.

 The polyionic compound 1 can be of polyanionic or polycationic nature. The product P is then respectively cationic (or pro-cationic) or anionic (or pro-anionic).

 The polyionic compound 1 can be of mineral or organic origin. As mineral compounds with cation exchange properties, it is possible to envisage, for example, the use of zeolites and clays

(montmorillonite, laponite). Synthetic hydrotalcite clays, on the other hand, have anion exchange properties. As the organic compound, an example of a polyanionic compound is one. perfluorinated polymer to which are grafted sulfonic acid groups. One of these polymers is known under the brand name Nafion (manufactured by E.I. Du Pont de Nemours), and has the following chemical formula:

in which m is between 5 and 13.5, n is approximately 1000 and k is an integer between 1 and 3.

Preferably, the polymer used has a mass of 1100 g per mole of SO ₃ H site. At pH 7.4, this polymer is in polyanionic form. This polymer is also thermally and chemically very stable, insoluble in water alone and therefore in the analytes. It has good ion transfer properties. The fluorinated groups ensure the hydrophobic character and the SO- ₃ groups the anionic character of the polymer.

However, it would also be possible to use other polyanionic organic polymers such as, for example, polypyrrole N-substituted by an alkanoic chain (ex: (CH ₂ ) _n CO ₂ H) or sulphonic, (ex:

(CH ₂ ) _n SO ₃ H).

It would also be possible to use polycationic organic polymers, such as one of those given in Table I below by way of example.

 The choice of the enzyme E and of the substrate S / product P pair is carried out according to the nature of the polyionic compound 1. It is indeed necessary that the product P easily penetrates into the polyionic compound to the detriment of the substrate S, this the latter being or not electroactive.

 When the polyionic compound 1 is of polycationic nature (cf. table 1 above), a hydrolase such as cholinesterase EC-3-1-1-8 for example is advantageously used as an enzyme.

 This enzyme is involved in the following reaction:

Lorsque le composé polyionique 1 est de nature polyanionique, comme le Nafion (marque déposée), par exemple, on utilise avantageusement comme enzyme une oxydoréductase telle que la péroxydase EC-1-11-1-7, une transférase telle que la γ-glutamyltransférase EC-2-3-2-2 ou une hydrolase telle que la β-D-glucuronidase EC-3-2-1-31 ou l'alcaline phosphatase EC-3-1-3-1.

When the polyionic compound 1 is of polyanionic nature, such as Nafion (registered trademark), for example, an oxidoreductase such as peroxidase EC-1-11-1-7, a transferase such as γ-glutamyltransferase is advantageously used as an enzyme EC-2-3-2-2 or a hydrolase such as β-D-glucuronidase EC-3-2-1-31 or alkaline phosphatase EC-3-1-3-1.

 The transformation reactions between the substrate S and the product P following the catalysis reaction of the enzyme are recalled below for each enzyme.

 The examples are chosen so that the substrate S is electro-inactive and / or of global charge. negative, which disadvantages its accumulation in the polyanionic film, and the cationic or procationic substrate P. In the latter case, it becomes cationic during the accumulation period when it enters the film of compound 1 and oxidizes at the anode to a cationic form. It is in this form that it accumulates in the polyanionic film.

 - Oxidoreductases:

e.g. peroxidase

D representing the donor, that is to say for example the leuco form of malachite green which leads to malachite green (D ⁺ ).

 - Transferases:

 e.g. γ-glutamyltransferase

R ² NH ₂ representing for example an amino acid and R ¹ NH ₂ electroactive derivatives of aniline.

 ex: β-D glucuronidase

 ROH representing, for example, one of the alcohols given in Table 2 below. e.g. alkaline phosphatase (A.P.)

;

The remainder R ensures on the one hand the electroactivity of the alcohol and on the other hand, its cationic or procationic character, so as to favor its accumulation in the film of Nafion. Some examples of phosphate esters and alcohols meeting these criteria are given below in Table 2.

For the pairs of substrate S / product PI, 2 and 6, the ferrocenyl residue ensures both the electro-reactivity and the procationic nature of the compound. The anodic oxidation of ferrocene to ferricinium occurs between 0.2 and 0.4 volts (Ag / Agcl electrode). It is the same for the rest NO. in pair 3. Oxidation of NO- to NO ⁺ occurs around 0.5 volts.

 The amine hydrochloride of pair 4 is protonated up to weakly basic pHs. In this case, it is the aminophenol which reversibly oxidizes. For couple 5, cobalticinium reversibly reduces to cobaltocene around -1 volt.

The spirits of couples 1 and 3 are sold. The phosphate ester of pair 3 is also sold and the other esters can be synthesized from the POCI ₃ reagent in pyridine.

 The alcohol of couple 2 was prepared from aminophenoldimethyl and chloride of ferrocene carboxylic acid. The melting point of this alcohol is. at 221 ° C (ethyl acetate / hexane).

Alcohol analysis:

C ₁₉ H ₁₉ NO ₂ Fe (%) calculated: C = 65, 35; H = 5.48;

N = 4.01; Fe = 16.00,

 and

C ₁₉ H ₁₉ NO ₂ Fe (%) found: C = 65, 60; H = 5.55;

N = 3.94; Fe = 15.76.

The phosphate ester of pair 2 has also been prepared and has a melting point varying between 214 and 215 ° C (acetone / CH ₂ Cl ₂ ).

 Ester analysis:

C ₁₉ H ₂₀ NO ₅ FeP (%) calculated: C = 53.17; H = 4.97;

 N = 3.26; P = 7.22, and

C ₁₉ H ₂₀ NO ₅ FeP (%) found: C = 53.38, H = 4.97;

N = 3.32; P = 7.24.

Demonstration of the selective accumulation properties of the electrodes modified by a Nafion film (registered trademark): The selective accumulation properties of Nafion have been demonstrated from the substrate / product pair 2 in Table 2, using alkaline phosphatase as the enzyme.

Carbon electrode covered with a Nafion film:

These measurements were made by square wave voltammetry. The electrolysis cell comprises a central reservoir in which 0.5 to 1 ml of the solution to be analyzed is deposited, containing the substrate S no. 2 or the product P no. 2 diluted in a phosphate buffer pH 7.4. This cell also comprises a platinum wire counter electrode, an Ag / Agcl reference electrode and the working electrode constituted by a glassy carbon disc (diameter = 3 mm; surface S ° = 7 mm ² ), covered a film made from Nafion powder in suspension in an aliphatic alcohol / water mixture, (available from Aldrich, 27,470-4) with a thickness of 0.4 μm. The recording of the voltammograms on a stationary electrode was preceded by a period of accumulation of the substrate No. 2 or of the product No. 2 in the Nafion film lasting 5 minutes. During the accumulation period, the electrode covered with the Nafion film is rotated at the rate of 600 revolutions / min and is maintained at a potential of 0.6 volts. The voltammograms of FIG. 4 were obtained by scanning potentials between 0.6 volts and 0 volts, which corresponds to the reduction of ferricinium to ferrocene.

Curve A illustrates the results obtained with the substrate S n ° 2 (concentration 2.10-5 mol.1 ^-1 in the pH 7.4 buffer) and curve B the results obtained with the product P n ° 2 (concentration 10 ^{- 5} mol.1 ^-1 in pH 7, 4 buffer). From the results of this figure, it can be seen that the ratio between the value of the peak current and the concentration of substrate or product in the buffer is 80 times higher for the product than for the substrate, which shows the high selectivity. of Nafion in favor of product P n ° 2.

Furthermore, curve C corresponds to the voltammogram obtained for product P n ° 2 (concentration 10 ^-5 mol.1 ^-1 in the buffer pH 7.4), in the absence of the Nafion film. The comparison of the peak currents of curve B corresponding to product P no. 2 and of curve C corresponding to product P no. 2 without the Nafion film, shows that the signal is amplified approximately 14 times on the Nafion electrode. compared to one. classic electrode.

 Ultramicroelectrodes:

Measurements were also carried out under identical conditions but with an electrode of the ultramicroelectrode type covered by a Nafion film such as that illustrated in FIG. 2. The attached FIG. 5 represents the square wave voltammograms obtained for the product P n ° 2 ( concentration 2.10 ^-5 mol.l ^-1 in the buffer at pH 7.4). Curve A represents the results obtained immediately after soaking the electrode in the solution while curve B represents the results obtained after an accumulation of product P n ° 2 in the Nafion film for 10 minutes. The peak current for curve A has a value of 0.065 μA and passes to a value of 0.23 μA for curve B. This reflects the accumulation of the product in the Nafion film.

Graphite paste electrode:

Finally, a third series of measurements was carried out with the graphite paste electrode such as that illustrated in FIG. 3, (diameter: 3 mm, surface S °: 7 mm ²⁾ . The graphite paste impregnated with Nafion contains inter alia Nafion (available from Aldrich 27,470-4), and graphite powder (available from Ultra Corps CO14145-10 / UCP 1M). The experimental conditions are the same as above. FIG. 6 illustrates the relationship of linearity existing between the peak intensity and the concentration c of product P n ° 2 in table 2. This electrode therefore makes it possible to obtain reliable and reproducible results.

 From the assay principle illustrated in FIG. 1, it is possible to envisage the indirect assay of an enzyme.

Test 1: Determination of an enzyme

An example of an assay for the alkaline phosphatase enzyme was carried out under the following experimental conditions. A sample was prepared by mixing in a NaHCO ₃ buffer (pH 9.6) the substrate S n ° 2 of table 2 at a concentration of 10 ~ ⁵ mol.1 ^-1 , MgSθ4 at a concentration of 10 ^-2 mol. 1 ^-1 and alkaline phosphatase (Sigma P 4252), at concentrations varying between 0.24 and 24 units per liter. The volume of the sample is 1 ml. The sample is maintained at 30 ° C. After 15 minutes of incubation period, the pH is reduced to 7.5 by adding 4 μl 2'4 M hydrochloric acid and the sample is poured into the electrolysis cell described above. The whole is maintained at a temperature of 25 ° C. After an accumulation period of 5 minutes, during which the electrode covered with Nafion is rotated at the rate of 500 revolutions / min and is maintained at a potential of 0.6 V, the signal of the product P is measured. n ° 2 formed during the enzymatic reaction. The results of cyclic voltammetry are represented by the line A in FIG. 7. A second measurement was carried out by slightly modifying the experimental conditions. We use 100 μl of TRIS buffer (pH 10.2) containing 10 ^-4 mol.1 ^-1 of substrate S n ° 2, 5.10 ^-3 mol.1 of NaCl, 10 ^-2 mol.1 ^-1 of MgSθ4 and l alkaline phosphatase at concentrations varying between 0.24 and 1.8 units per liter. After incubation for 30 minutes, the volume of the sample is reduced to 1.0 ml by adding 900 μl of NaHCO ₃ and 4 μl of 4 M hydrochloric acid. The sample was then transferred to the electrolysis cell. identical to that described in the previous examples. Line B of Figure 7 illustrates the results obtained. A detection limit of 0.024 units per liter is reached, which corresponds to a concentration of alkaline phosphatase Sigma P 4252 of 1.3.10 ^-12 mol.1 ^-1 .

On a principle similar to what has just been described for the determination of the enzyme, that is to say by combining a specific enzyme, a pair (substrate S / product P) and a sensor comprising a polyionic compound capable to accumulate the product P formed during the enzymatic reaction, it is possible to carry out immunoenzymatic assays.

 The general principle of the immunoenzymatic assay of an antigen consists in:

 reacting an enzyme E serving as a marker for the antigen to be assayed or for an antibody specific for this antigen to be assayed, with a substrate S so that the enzyme catalyzes the reaction for transforming the substrate S into an ionic electroactive product P or pro-ionic, and

- collect said product P on an electrode provided with one of the polyionic compounds previously described, so that product P concentrates in the polyionic compound and can then be detected.

 The detection principle, the enzymes, the substrates and the products are identical to what has just been described for the enzymatic assay.

 This immunoenzymatic assay principle applies to homogeneous or heterogeneous competitive assays or to sandwich-type assays.

 FIG. 8 illustrates an example of a homogeneous competitive type immunoassay. In this case, the antigen to be assayed (Ag), the enzyme labeled antigen (Ag-E), the antibody (Ac) and the substrate S are reacted. During the reaction, part of the antigens labeled with an enzyme (Ag-E) and assay antigens (Ag) se. fixed on the antibodies (Ac), while only the fraction remained free of enzyme-labeled antigen (Ag-E) reacts with the substrate S to give the product P which then accumulates in the polyionic compound 1. The rest of the device (electrode 3 and detection means) is identical to what has been described for FIG. 1 and has the same references. All the other variants described above concerning the structure of the electrodes, the enzymes, the substrate / product pair and the polyionic compound can also be used here.

FIG. 9 illustrates an example of a heterogeneous competitive type immunoassay. In this case, the antibody (Ac) present in limited quantity is fixed on the walls of a test tube, for example, and a fixed quantity of enzyme labeled antigen (Ag-E) is reacted. in competition with the antigen to be assayed (Ag). After the washing and rinsing steps, the substrate S is added. Only the Ac / Ag-E complexes will react with the substrate S to transform it into product P in proportion to the quantity of labeled antigen present in the form of Ac / Ag-E complex.

 All the other parameters of the assay (enzyme, substrate / product pair, polyionic compound, electrode and detection means) are identical to what has been previously described in relation to FIG. 1 and the assay of the enzyme.

 FIG. 10 illustrates an example of a sandwich type immunoassay. In this case, the antibody (Ac) present in excess is fixed on the walls of a test tube, for example. The antigen to be assayed (Ag) is added and reacts with the antibodies (Ac). Then, antibodies labeled with an enzyme (Ac-E) are added in excess and sandwich the antigens linked to the antibodies. The. substrate S is added after rinsing the test tube.

 All the other parameters of the assay (enzyme, substrate / product pair, polyionic compound, electrode and detection means) are identical to what was previously described in relation to FIG. 1 and the assay of the enzyme.

 These different types of enzyme immunoassay according to the invention can be used to assay any antigen capable of being linked to an enzyme meeting the requirements of the invention or any antigen having a specific antibody capable of being linked to such an enzyme.

Two examples of carrying out competitive assays of homogeneous and heterogeneous type will be given below, applied to the determination of phenytoin. This product is an antiepileptic whose therapeutic index is between 2.10 ^-5 mol.1 ^-1 and 8.10 ^-5 mol.1 ^-1 .

Test 2: Competitive dosage in homogeneous phase

For this assay, phenytoin labeled with alkaline phosphatase (Phen-AP, Interchim Ref. FIT) is used. 80-IP30, commercial solution of 50 units / ml in TRIS buffer).

 - calibration curve

 This is illustrated in FIG. 11. It was carried out according to the experimental conditions of curve B in FIG. 7.

 - verification of the affinity between the alkaline phosphatase-labeled phenytoin and the antiserum

 The affinity between the antigen and the antibody is checked as follows.

 An immunoreaction is carried out at 37 ° C. for 30 minutes between 100 units per liter of labeled antigen

(phenytoin labeled with alkaline phosphatase AP) and antiserum containing anti-phenytoma antibodies in a volume of 250 μl of NaCl (5.10 ^-3 mol.1 ^-1 ) in the presence of normal rabbit serum.

The enzymatic reaction is then carried out at 37 ° C, after addition of 250 μl of TRIS buffer containing NaCl (5.10 ^-3 mol.1 ^-1 ), MgSO ₄ (2.10 ^-2 mol.1 ^-1 ) and 2 10 ^-5 mol .l ^-1 of substrate n ° 2 of table 2 (phosphate ester of 2- (N-ferrocenoylammo) 4,6-dimethylphenol). The reaction lasts 15 minutes.

Finally, 500 μl of NaHCO ₃ (pH 9.6), 7 μl of 4 M HCl are added and a period of accumulation of product No. 2 is carried out for a period of 5 minutes in the measuring electrode.

 The determination is made by square wave voltammetry under the same experimental conditions as that of curve A or B in FIG. 4.

This reaction is repeated by adding increasing amounts of antiserum, the total volume of antiserum and normal rabbit serum being kept constant at 6 μl and the volume of the final solution containing the labeled phenytoin, the substrate and the antiserum being 1 ml. In the presence of increasing additions of anti-phenytoma antiserum, the amount of anti-phenytoma / phenytoma complex labeled with AP increases at the expense of free AP-labeled phenytoin, and the decrease in the electrical signal confirms that the enzymatic transformation of the substrate n ° 2 in product n ° 2 can only be done through the phenytoin marked by free AP.

From the experimental conditions corresponding to point A of FIG. 12, a standard phenytoin titration curve (FIG. 13) was obtained by adding increasing amounts of phenytoin at the level of the immunoreaction step. This calibration curve is directly usable for dosing clinical samples after dilution, and it was used for the determination of an E1 sample, by introducing 1.5 μl of E1, 1.5 μl of normal rabbit serum and 3 μl anti-phenytoma at the immunoreaction stage. After accumulation and voltammetric determination, a signal of 6.3 μA is obtained for the peak current corresponding, according to the calibration curve of FIG. 13, to a phenytoin concentration of 4, 0.10 ^-8 mol.1 ^-1 , i.e. a concentration of phenytoin, in the sample E1, of 2, 7.10 ^-5 mol.1 ^-1 . The theoretical value determined in a hospital laboratory by high performance liquid chromatography is 3.2.10 ^-5 mol.1 ^-1 .

Trial 3: Competitive dosage in heterogeneous phase

To immobilize the antiserum on the walls, one maxisorp test tube (Polylabo ref. 470319), the anti-phenytoma antibody is diluted 100 times in a tarpon phosphate pH 7.4, then 200 μl of this solution are introduced into the test tube. After 15 hours of contact at 4 ° C., the test tube is rinsed with a phosphate / Tween 20 buffer mixture in volume 0.1. [Aldrich 27, 434-8] (400 μlx3 for 10 minutes). The evaluation of the enzymatic activity of the phenytoin marked by alkaline phosphatase, when all the antibodies present on the walls of the tube are complexed by the marked phenytoin, is carried out according to the following protocol:

 1 ° The introduction into the tube of 10 μl of labeled phenytoin (5000 units per liter) and 190 μl of phosphate buffer, is followed by 60 minutes of incubation at 37 ° C, then two rinses for 10 minutes in buffer phosphate / Tween 20 and two rinses with TRIS buffer.

 It was verified that the liquid phase (approximately 200 μl) extracted with the syringe after incubation and. before rinsing, contains excess free labeled phenytoin.

2 ° After introduction into the tube of 10 μl of substrate No. 2 (10 ^-3 mol.1 ^-1 ) and 190 μl of TRIS buffer containing NaCl (5.10 ^-3 mol.1 ^-1 ) and MgSO ₄ (10 ^{- 2} mol.1 ^-1 ), the enzymatic transformation of substrate No. 2 into product No. 2 is carried out for 15 minutes at 37 ° C.

3 ° The electrochemical detection of substrate no. 2 is carried out after transfer of the 200 μl of solution into the electrolysis cell then addition of 100 μl of NaCl (5.10 ^"3 mol.1 ^-1 ), 300 ml of NaHCO ₃ and 6 μl of 4 M HCl, and finally an accumulation on an electrode coated with a Nafion film, for 5 minutes at 0.6 V. The peak current of the square wave voltammogram obtained is 14.3 μA.

The standard phenytoin titration curve in Figure 14 was obtained by adding 6 μl of phenytoin diluted in phosphate buffer at phase 1 of the protocol (composition of the sample: 10 μl of alkaline-labeled phenytoin phosphatase (5000 units per liter), 184 μl of phosphate buffer and 6 μl of phenytoin). This curve can be used directly in zone I to assay clinical samples after dilution to 1/1000 (addition of 1 μl of sample in phase 1 of the protocol).

Application to the assay of 2 hospital samples E ₂ and E ₃ :

 Note that E2 and E3 also contain du-phenobarbital and 7 (or 8) drugs.

Finally, the invention relates to a dosing kit capable of being used for the implementation of the two methods previously described. This dosing kit includes:

 - a substrate capable of being transformed into an ionic or proionic electroactive product, under the action of catalysis of an enzyme,

 - an electrode provided with a polyionic compound whose charge is opposite to that of said product and in which said product is capable of accumulating,

 - electrochemical detection means connected to this electrode and making it possible to measure the electrical signal supplied by this electrode.

The enzymes, the substrate / product pairs, the measurement electrode, the polyionic compound and the detection means are identical to those which have been described previously. When the enzyme is free, the assay kit allows this enzyme to be assayed. When the enzyme is linked to a given antigen or to an antibody specific for a given antigen, the assay kit makes it possible to assay this antigen.

Claims

1. Method for assaying an enzyme (E), characterized in that it comprises the steps consisting in:  reacting the enzyme (E) to be assayed with a substrate (S) whose transformation into an ionic or proionic electroactive product (P) is catalyzed by said enzyme (E),  - collecting said ionic or proionic electroactive product (P) obtained, on an electrode (3, 11, 17) provided with a polyionic compound (1) of mineral or organic origin, the charge of which is opposite to that of said product ( P) obtained, so that this product (P) concentrates in said polyionic compound (1) during a period of accumulation,  - measure the signal supplied by said electrode (3) at the end of the accumulation period, using electrochemical detection means connected to this electrode.  2. Method for immunoenzymatic assay of antigen, characterized in that it comprises the steps consisting in:  - react with an enzyme (E) serving as a marker for the antigen to be assayed or for an antibody specific for this antigen to be assayed, with a substrate (S), the transformation of which into an ionic or proionic electroactive product (P) , is catalyzed by this enzyme (E), - collecting the ionic or pro-ionic electroactive product (P), obtained by the enzymatic reaction, on an electrode (3, 11, 17) provided with a polyionic compound (1) of mineral or organic origin whose charge is opposite to that of said product (P) obtained, so that this product (P) is concentrated in said polyionic compound (1) during a period of accumulation,  - measure the signal supplied by said electrode (3) at the end of the accumulation period, using electrochemical detection means connected to this electrode.  3. Assay method according to claim 1 or 2, characterized in that the product (P) is cationic or pro-cationic and in that the polyionic compound (1) is of polyanionic nature and is chosen from zeolites, montmorillonite , laponite, polypyrroles N-substituted by an alkanoic or sulphonic chain, or a perfluorinated polymer of formula:

 in which m is between 5 and 13.5, n is approximately 1000 and k is an integer between 1 and 3.  4. Assay method according to claim 1 or 2, characterized in that the product (P) is anicmque or pro-anionic and in that the polyionic compound (1) is polycationic in nature and is chosen from synthetic clays of the type hydrotalcite, polytyramine, poly-4-vinylpyridine, poly-4-vinyl-1-methylpyridinium, polypyrrole or polypyrrole N-substituted by an alkylammonium chain. 5. Assay method according to claim 1 or 2, characterized in that the enzyme is chosen from oxidoreductases, transferases or hydrolases. 6. Assay method according to claim 1 or 2, characterized in that the polyionic compound (1) is of polycationic nature, in that the enzyme (E) is cholinesterase and in that the electropositive substrate (S) is an ester of choline chloride and the electronegative product (P) is an electroactive carboxylic acid.  7. Assay method according to claim 1 or 2, characterized in that the polyionic compound (1) is of polyanionic nature and in that the enzyme (E) is chosen from peroxidase, γ-glutamyltransferase, β- D-glucaronidase or alkaline phosphatase. 8. Assay method according to claim 3, characterized in that the enzyme is alkaline phosphatase, in that the substrate (S) is chosen from phosphate esters of the ROPO ₃ ^2- type and in that the product ( P) is chosen from ROH alcohols, R being a cationic or procationic group.  9. Metering method according to claim 8, characterized in that the substrate (S) / product pairs (P) are chosen from the pairs: ferrocenylmethanol phosphate ester / ferrocenylmethanol, 2- (N-ferrocenoylamino) -4,6-dimethylphenol / 2- (N-ferrocenoylamino) -4,6-dimethylphenol ester phosphate ester 4-hydroxy-TEMPO / 4-hydroxy-TEMPO phosphate, 2- (N, N-dimethylaminomethyl) 4-aminophenol hydrochloride phosphate ester / 2- (N, N-dimethylaminomethyl) 4-aminophenol hydrochloride, phosphate ester 4-N-cobaltocenoylaminophenol hexafluorophosphate / 4- (N-cobaltocenoylaminophenol hexafluorophosphate or 4-ferrocenylphenol / 4-ferrocenylphenol phosphate ester. 10. Assay method according to claim 2, characterized in that the enzyme (E) serves as a marker for the antigen to be assayed and in that the assay is of homogeneous or heterogeneous competitive type.  11. Assay method according to claim 2, characterized in that the enzyme (E) serves as a marker for the antibody and in that the assay is of the "sandwich" type.  12. The assay method according to claim 1 or 2, characterized in that the electrochemical detection means (5) are chosen from amperometry, coulometry, voltammetry or square wave voltammetry.  13. The assay method according to claim 1 or 2, characterized in that the electrode is in the form of an electrode (3) made of glassy carbon covered. of a polyionic compound film (1).  14. The assay method according to claim 1 or 2, characterized in that the electrode (11) is in the form of carbon microdisks (13) deposited on a support (12) and covered with a film of the polyionic compound ( 1).  15. The assay method according to claim 1 or 2, characterized in that the electrode (17) is in the form of a graphite paste impregnated with the polyionic compound (1) and electrically connected to a rotating electrode (23).  16. Dosing kit characterized in that it comprises:  - a substrate (S) capable of being transformed into an ionic or proionic electroactive product (P), under the action of catalysis of an enzyme (E), - An electrode (3, 11, 17) provided with a polyionic compound (1) whose charge is opposite to that of the ionic or pro-ionic electroactive product (P), and in which said product (P) is susceptible to accumulate, - electrochemical αetection means (5) connected to this electrode and making it possible to measure the electric signal supplied by this electroαe.  17. Assay kit according to claim 16, characterized in that the product (P) is cationic or pro-cationic and in that the polyionic compound is of polyanionic nature and is chosen from zeolites, montmoπllonite, laponite, N-substituted polypyrroles with an alkanoic or sulphonic chain or a perfluorinated polymer of formula:

 in which m is between 5 and 13.5, n is approximately 1000 and k is an integer between 1 and 3.  18. Assay kit according to claim 16, characterized in that the product (P) is anionic or pro-anionic and in that the polyionic compound (1) is of polycationic nature and is chosen from synthetic clays of the hydrotalcite type, polytyram, poly-4-vmylpyrιdιne, poly-4-vιnyl-1-methylpyndιnιum, polypyrrole or polypyrrole N-substituted with an alkylammonium chain.  19. Assay kit according to claim 16, characterized in that the polyionic compound (1) is of polycationic nature, in that the enzyme (E) is cholinesterase and in that the electropositive substrate (S) is an ester choline chloride and the electronegative product (P) is an electroactive carboxylic acid. 20. Assay kit according to claim 16, characterized in that the polyionic compound (1) is polyanionic nature and in that the enzyme (E) is chosen from peroxidase, γ-glutamyltransferase, β-D-glucaronidase or alkaline phosphatase.  21. Assay kit according to claim 18, characterized in that the enzyme (E) is alkaline phosphatase and the substrate (S) / product (P) pairs are chosen from the pairs: phosphate ester of 2- (N -ferrocenoylamino) -4,6-dimethylphenol / 2- (N-ferrocenoylamino) -4,6-dimethyl-phenol, 4-hydroxy-TEMPO / 4-hydroxy-TEMPO phosphate ester, 2- (N hydrochloride phosphate ester , N-dimethylaminomethyl) 4-aminophenol / 2- (N, N-dimethylaminomethyl) 4-aminophenol hydrochloride, hexafluorophosphate phosphate ester. 4-N-cobaltocenoylaminophenol / 4- hexafluorophosphate (N-cobaltocenoylaminophenol or the phosphate ester of 4-ferrocenylphenol / 4-ferrocenylphenol.  22. Assay kit according to claim 16, characterized in that the electrochemical detection means (5) are chosen from amperometry, coulometry, voltammetry or square wave voltammetry.  23. Assay kit according to claim 16, characterized in that the electrode is in the form of an electrode (3) of vitreous carbon covered with a film of polyionic compound (1).  24. Assay kit according to claim 16, characterized in that the electrode (11) is in the form of carbon microdisks (13) deposited on a support (12) and covered with a film of the polyionic compound (1) . 25. Assay kit according to claim 16, characterized in that the electrode (17) is in the form of a graphite paste impregnated with the compound polyionic (1) and electrically connected to a rotating electrode (23).