WO1987007725A1 - Cyclic method for the immunoenzymatic dosing of a ligand - Google Patents

Abstract

In this method, an electrode is used to the surface of which an anti-ligand specific to the ligand to be dosed is directly fixed. At the end of the dosing operation, said electrode is subjected to an electrochemical etching in order to re-use it in a new measuring cycle.

Description

 Cyclic immunoenzymatic assay for a ligand.

The present invention relates to a cyclic immunoenzymatic assay for a ligand, in the heterogeneous phase.

We know that the dosage of substances present in low concentration in industrial biological liquids arouses great interest in scientific circles; due to the very numerous applications 10. of these dosages in various technical fields,

Recent techniques use the enzymatic activity as a means of labeling and amplifying the molecules ensuring the molecular recognition processes which intervene in the immunological analysis methods. It has thus been envisaged to label antigens or anti¬ body with enzymes such as peroxidase or glucose oxidase, the activity of which can be easily determined by spectrophotometry.

In these techniques, a distinction is made between assays in homogeneous phase and assays in heterogeneous phase:

- in homogeneous phase assays, the enzymatic activity is a function of the antibody-antigen reaction and it is not necessary to separate the "bound fraction", that is to say the immunoreactor (antigen-enzyme conjugate 5 or antibody-enzyme), which reacted in the immunological reaction, of the "free fraction", which represents the reactive immu¬ neactant;

- in the assays in heterogeneous phase, the enzymatic activity is not modified by the antigen-0 antibody reaction and a separation of the bound fraction and- of the free fraction turns out to be necessary.

It is at these dosages in heterogeneous phase _. that the present invention is interested and we will begin by recalling the principles of these different dosages, which it is customary to classify in competitive and non-competitive methods.

In so-called competitive methods, the conjugate enzy e-ligand (antigen or antibody) is mixed with a sample of the biological medium to be assayed (or with a sample of a standard medium) containing the ligand li¬ bre, in order to compete with it to combine to antiligands fixed in limited quantities on a solid support. Alternatively (assay by inhibition), the ligand can be immobilized on a support and enter into competition with the ligand to be assayed, when they are simultaneously placed in the presence of a limited quantity of antiligand in solution. In these methods, the enzymatic activity is inversely proportional to the concentration in ligand to be assayed.

Among the non-competitive methods, the so-called "sandwich" method is the best known and it applies to the assay of ligands having at least two antigenic sites. The ligand to be assayed is brought into contact with a solid support on which antibodies are fixed. The ligand combines with the antibodies and, after washing of the support, the latter is brought into contact with a determined excess of the same antibodies labeled with an enzyme. These in turn bind to the ligands, which are thus sandwiched between the antibodies attached to the support and those labeled by the enzyme, hence the name of the method. In this case, the enzymatic activity is directly proportional to the concentration of ligand to be assayed.

The use of a solid phase in an enzyme immunoassay is a relatively recent technique, which has the advantage of allowing separation of the free fraction without resorting to physicochemical methods which complicate the assays.

All these methods in the heterogeneous phase require the final detection of the enzymatic activity, which is generally carried out in the homogeneous phase (by spectrophotometric, fluorimetric or bioluminescence methods) or pseudo-homogeneous (by an enzymatic sensor). A very complex optical method, ellipsometry, makes it possible to detect directly on the solid phase the presence, for example, of the antibody used for the assay. However, this method cannot be envisaged for routine measurements.

It has therefore been proposed to use the solid phase itself as a detector for the presence of the enzyme, which makes the process completely heterogeneous and facilitates its implementation.

In processes of this type, after having made reactive sites appear on the surface of an electrode, the latter is brought into contact with an antiligand specific for the ligand to be assayed to form a conductive solid phase which is then exposed to the ligand to be assayed and to an antiligand -enzyme or ligand-enzyme conjugate, the enzyme of this conjugate being capable of reacting with a substrate to provide an electro-active product with respect to the conductive solid phase (see, for example , ϋP-A-0 1 -2 301 and EP-A-

0150999 ^" ).

In fact, when an enzyme which produces an electroactive species is immobilized in a situation of molecular proximity on a conductive surface, the electrochemical kinetics and the enzymatic kinetics are interdependent, which makes it possible to quantify the presence of the enzyme by the electric electro-chemical detection current of this electroactive species. This state of molecular proximity can be obtained by any of the immunological methods previously described, the antibody being immobilized directly on the conductive surface.

An important consequence of the use of this assay method, which will be designated by the expression

"enzymatic electrocatalysis" is that the quantity of enzyme detectable in this situation is extremely low, which, in its application to an enzyme-linked assay, in heterogeneous phase, leads to a sensitivity greater than that of the previous detection means.

For successive assays on an industrial scale of identical or different ligands, it would however be advantageous to be able to immediately reuse for a new measurement cycle the electrode which was already used in a first implementation of the method. This would save considerable time between successive dosing cycles compared to methods known in the art. It is this problem which the present invention proposes to solve.

To this end, the subject of the invention is a cyclic immunoenzymatic assay method for a ligand, according to which, after having revealed reactive sites on the surface of an electrode by electro-chemical oxidation, ci in contact with an antiligand specific for the ligand to be assayed to form a conductive solid phase comprising said electrode and, fixed to the surface thereof, a substantially monomolecular layer of said antiligand without the interposition of an intermediate support, this solid phase then being exposed to the ligand to be assayed and to an antiligand-enzyme or ligand-enzyme conjugate, depending on whether the so-called "sandwich" or "competitive" assay methods are applied, the enzyme of said conjugate being capable of reacting with a substrate to provide an electroactive product with respect to said solid phase, this process being characterized in that, at the end of the assay, at least the ligands and the conjugates are separated from the electrode tiligand-enzyme or ligand-enzyme attached to said solid phase, with a view to reusing the latter for a new assay cycle.

The separation of the solid phase from the ligands and the anti-ligand enzyme or ligand-enzyme conjugates may be carried out by simple rinsing of the solid phase with a liquid whose pH and / or polarity conditions are different from those of the medium. dosage.

At the end of the assay, the substantially monomoleic layer of anti-ligand can also be separated from the electrode on which it is fixed, with a view to the reuse of this electrode for a new assay cycle, for example in subjecting this electrode to electrochemical pickling by the action of a direct or alternating current.

For this purpose, it is advantageously possible to pass a direct current through an oxidizing solution, between the dosing electrode and a reference electrode, with a potential difference of between 1.7 volts and 2.2 volts.

An alternating current can also be used with a potential difference of + _ 3 volts between the metering electrode and the pickling electrode.

The dosing electrode could be, for example, carbon, platinum or gold _; and can take any form depending on the constraints of introduction of the sample to be assayed (flat, porous or percolating solution electrode).

The antibodies or antigens can be fixed to the electrode by any means known in the art, for example by covalent bond or by physical adsorption, by forming a single molecular layer. In the case of fixing by covalent bond, the electrode will be subjected to a pretreatment comprising, in a manner known per se, a phase of creation of reactive sites on the surface of the electrode by electro-chemical oxidation of this and a phase of activation of these sites by a coupling agent appropriate to the nature of the electrode and to that of the antico ns or the antigen to be fixed, for example an activation by a carbodii ide in the case of a carbon electrode.

In the case of immobilization of the antibodies or antigens on the electrode by physical adsorption, an electrochemical pretreatment of the surface of the electrode will also be carried out before bringing it into contact with said antibodies or antigens.

In each case, the quantity of antibody or antigen to be brought into contact with the electrode can be optimized in order to obtain a substantially mono¬ molecular layer on the surface of the electrode, so that 'she does not do not "screen" in front of the solid phase during the subsequent electrochemical detection of the product of enzymatic catalysis.

During the assay phase, the ligands and / or the anti-ligand enzyme conjugates may only saturate a fraction of the sites of the associated anti-ligands or ligands, which will limit the duration of implementation of the process.

The detection of the product released by the substrate, during the dosing phase, can advantageously be carried out by measuring a current flowing between the electrode and a reference electrode with controlled potential. The attached schematic drawings illustrate the implementation of the invention. In these drawings:

FIGS. 1 to 4 illustrate the preparation of an electroenzymatic electrode according to the invention which can be used in an assay process by a non-competitive method of the "sandwich" type;

Figures 5 and 6 illustrate the preparation of an electrode usable in a competitive method; Figures .7 and 8 are diagrams, respectively, of an electrochemical cell and a metering system usable in the electroenzymatic metering methods according to the invention;

Figures 9 and 10 show swimming standard curves used respectively in the assays of Examples 1 and 2;

Figure 11 is a diagram of an assay cell used in Example 3

Figure 12 is a calibration curve used in Example 3.

As seen ^on Figure 1 has the electrode 1, after having undergone a treatment suitable to allow immobilisa¬ tion to its antibody 2 surface by covalent bonding or by physical adsorption, is contacted with a solution containing these antibodies. These, which are antibodies specific for the antigen to be assayed, bind to the surface of the electrode by forming a monolayer, which completely or partially covers the treated surface, depending on the treatment conditions and the desired result.

The resulting modified electrode is then brought into contact with a solution of the antigen 3 to be assayed (FIG. 2) which is fixed on the antibodies 2. The conditions for bringing into contact are such that the antibodies are not saturated with antigens and that part of the antibody sites remains vacant. The number of antigen-antibody bonds formed will depend on the concentration of antigens in the test solution.

The time for bringing the solid phase into contact with the antigen solution, or incubation phase, is of the order of a few minutes, whereas, in most iπjiunoenzymatic assay methods, the durations of incubation bation are around 1 hour. In fact, this method is much more sensitive than the usual methods, for the reasons indicated above, and it is therefore not necessary to saturate all the antibody sites, which makes it possible to significantly reduce the contact time. The electrode is then exposed to the antibody-enzyme conjugate 4-, which selectively binds to the anti-genes 3 (FIG. 3) "The conditions for contacting can be such that the conjugates 4- saturate all the antigenic sites , but this is not an obligation, if one proceeds by comparison with an internal standard produced under the same conditions. The incubation time can therefore be considerably reduced compared to prior techniques. At the end of the phase illustrated in FIG. 3, the antibody-enzyme conjugate 4- is in a situation of enzymatic electro-catalysis at the surface of the electrode 1. It suffices, in fact, to introduce in the medium to be assayed one or more substances (or "substrates") capable of reacting with the enzyme of the conjugate 4-, as seen in FIG. 4-, to detect and assay using the electrode 1 the product 6 of the enzymatic reaction. Thus with acetylcholinesterase used with an artificial substrate of the acetylthiocholine type the catalyzed reaction is: acetylthiocholine> thiocholine + acetate The thiocholine (R-SH) produced is electrochemically detected if the potential of the electrode is at least at 0.7 V with respect to a reference electrode of the Ag / Ag Cl type:

2 R-SH ^• > RSS-R + 2H ⁺ + .2e ^" It is possible, from the oxidation current obtained, to calculate the number of enzymatic molecules in an enzymatic electrocatalysis situation and thus measure, with the using an internal calibration, the concentration of the antigen 3 "Figures 5 and 6 illustrate the same process of conditionning of enzymatic electrocatalysis for an antigen-enzyme conjugate 7 used in a competitive method.

In this use, the enzyme-ligand conjugate 7 is put in competition with the free ligand 3 of the biological medium to be assayed to combine with the antibodies 2 immobilized by forming a monolayer on the electrode 1 (FIG. 5) “As before, if the 'one then introduces into the medium to be assayed one or more substances 5 (or substrates) capable of reacting with the enzyme E of the conjugate 7 _{' it} is possible, as can be seen in FIG. 6, to detect and assay with the using electrode 1 the product 6 of the enzymatic reaction.

In accordance with the invention, in order to be able to reuse the electrode for a new dosing cycle, after the detection phase illustrated in FIGS. 4- to 6, the electrode is brought back to its starting state, before fixing the antibody 2.

To this end, an electrochemical pickling is carried out under conditions analogous to those of the pretreatment of the electrode mentioned above.

Note that this pickling of the electrode is not effective only because the layer of antibodies adhering to it is a substantially monomolecular layer.

To carry out this electrochemical pickling, in the case of a glassy carbon electrode, for example, it will suffice to bring the electrode to a potential of approximately 2 volts relative to a reference electrode Ag / Ag Cl in a solution oxidizing, for a few seconds. It may also be that after the assay phase, we simply want to reuse the fixed antibodies. In this case, the antibodies used (for example monoclonal) will have been selected so that rinsing, under physicochemical conditions different from the measurement, ensures the rupture of the antigen-antibody bonds, without denaturing of the antibody immobilized on the solid phase. The reuse of the electrode, in accordance with the invention, applies to all the usual immunoenzymatic assays in heterogeneous phase, whether competitive methods, sandwich or by inhibition, of any molecule allowing a immunological recognition (protein, enzyme, hormone, hapten, etc.), through the use of an appropriate medium and dosage conditions.

It will be noted that, in a known manner if the molecule to be assayed is an enzyme catalyzing a detectable electrochemical reaction, the assay is considerably simplified and only requires an antibody to this enzyme. This will appear in particular from Example 4 which will be described below.

As is known, the enzyme used for the preparation of the conjugates is necessarily an enzyme having as substrate or product of catalysis an electroactive redox couple. This redox couple, which can be physiological or artificial, is chosen so that it has good electrochemical reactivity on the electrode used, in order to obtain optimal sensitivity.

The corresponding enzymes are generally oxidoreductases, but can also be enzymes of another class, meeting the previous condition, for example hydrolases.

The table below brings together some enzymes which can be used for the preparation of the conjugates detectable by enzymatic electrocatalysis.

BOARD

The overall sensitivity of the assay procedures depends on three factors:

- the intrinsic sensitivity of solid phase detection, - the contact time between the sample to be assayed and the solid phase,

- the quality of the immunological material used. For the first factor, the detection sensitivity is linked to the molecular catalytic activity of the enzyme used for the amplification (for example, approximately 10 000 s ^" for a conjugate obtained with acetylcholinesterase from Electricus Electrophorus). case, the enzymatic electrocatalysis is capable of detecting the presence of a

- 1 fi minimum of 10 ^"" mole of conjugate per cm of real surface of the solid phase.

This exceptional sensitivity is explained by the molecular proximity between the enzymatic site and the electrochemical site, which reduces the distance that the product has to travel to be detected to a few nanometers.

It is important to note, in this regard, that the detection sensitivity of the conjugate is entirely independent of the surface of the solid phase. The size and shape of the electrode used can therefore be adapted to the amount of antigen which it is desired to measure.

This point will be illustrated in the examples offered.

As regards the contact time between the solid phase and the sample, this contact time determines a "capture rate" T, which may be less than 1, if all the antigens have not been captured, the rate of capture being defined as the ratio between the number of antigenic molecules captured by the solid phase and their total number in the sample.

T rate is also sensitive to the ratio between the volume of the sample and the surface of the solid phase ^*. One of the characteristics of the invention is that the detection is sufficiently sensitive for it to be possible to accept low capture rates (for example T = 0.03 in Example 1). Finally, the practical sensitivity of the assay method is a function, as with any immunological method, of the quality of the antibodies used as well as the non-specific parasitic adsorption on the solid phase. Rinses in appropriate buffers, known to the skilled person, limit the adsorption not spe _¬ cific. However, it will be noted that the very short incubation time allowed by the proposed procedure considerably reduces these non-specific adsorptions.

The overall sensitivity obtained is ultimately a modular parameter depending on the analytical problem treated. The examples presented below illustrate effective sensitivities of up to 2.10 - ^" 12 mole per liter or 2.10 - 21 mole in the test portion.

The following examples illustrate various forms of implementation of the invention. EXAMPLE 1

This example illustrates the assay of an immunoglobin

G by the sandwich method. on a solid phase with a medium surface (glass carbon υlane electrode ô with a surface of approximately 0 * 1 cm). 1) Preparation of reagents

The anti rabbit 1 gG was ourni ^* by BYOSIS laboratories of Compiègne (Prance). It was obtained from sheep and purified according to known procedures. The glucose oxidase-anti conjugate 1gG is prepared by the glutaraldehyde method of AVRAMEAS et al (Immunochemistry, Pergamon Press 6, 4-3-52 (1969). After purification, the conjugate is enzymatically active at $ 95 and im unologically active 2) Apparatus The measurement could, for example, be carried out in an electrochemical cell 9 with circulation, of the type illustrated in FIG. 7 "

In this cell, the liquid arrives via a canalization 10, circulates in a thin strip delimited by a film 11 of plastic material, for example of PTPE, and is discharged via a pipe 12. The cell also comprises a working electrode 13, for example made of vitreous carbon, a counter electrode 14-, for example of the Ag / Ag Cl type, and a pickling electrode 15, for example in vitreous carbon. The electrodes are sheathed over their entire lateral surface by an insulating material and are in contact with the liquid to be metered only by their end face. Of course, such a cell is only of exemplary value and one could use any other system designed by those skilled in the art.

The cell 9 is incorporated in a fully automated system of the type, that of FIG. 8, which allows an internal calibration ensuring the precision of the assay. The electrochemical cell 9 is supplied by the line 10 from a fluid circuit 16, itself supplied with reagents by the line 17 _" with possible taking of sample by the line 18. The electrodes 13, 14 and 15 are connected to a power supply system 19, which simultaneously serves as an electronic control and acquisition interface, coupled to a micro computer 20. 3) Metering

In this case, the principle of the "sandwich" method described with reference to FIGS. 1 to 4 is used. The introduction and circulation of the reagents is ensured by the fluid circuit 16 in the following manner:

L'électrode 15 est utilisée comme cathode. Cette phase de décapage de l'électrode régénère la surface de la phase solide et facilite l'adsorption de l'anticorps₀ The working electrode 13 is oxidized for 5 seconds in a nitrochromic mixture (10% by weight of HNO5 + 2% by weight of + water) by passing a current of 5 πiA.

The electrode 15 is used as the cathode. This stripping phase of the electrode regenerates the surface of the solid phase and facilitates the adsorption of the antibody ₀

After rinsing with buffer (0.01 M phosphate, pΞ 7, - »0.15 M NaCl), an antibody solution (300 g / l in the same buffer) is brought into contact for 5 minutes with the solid phase. Rinse for 2 minutes with blocking buffer (0.01 M phosphate, ^' pH 7,''^' gelatin 0.25 + Tween 20.0.1 (polyoxyethylenesorbitan onolaurate sold under this name and used as surfactant) + 0.15 M NaCl) makes it possible to remove the excess antibody. 0.2 cπr of the solution containing the anti¬ gene to be dosed is introduced with a flow rate of 0.1 cπr per minute. This phase is followed by 2 minutes of rinsing with a blocking buffer.

The conjugate (100 μg / cm- ⁵ in the blocking buffer) is introduced for 1 minute, then rinsed with a solution of benzoquinone (10 M in 0.1 M acetate buffer, pH 5.6 + 0.15 M NaCl) .

The current of the electrode, brought to 350 mV relative to the electrode 14-, is then sampled for 1 minute by the data acquisition system 19 and 20. The introduction of 0.2 M glucose into the same solution causes a current jump, due to the detection of the enzymatically formed hydroquinone. This current jump, practically instantaneous, is directly proportional to the quantity of conjugate in the electrocatalysis - enzymatic position. 11 is connected by a calibration curve (FIG. 9) the concentration of 1 gG in the sample.

The calculated capture rate is here of the order of 0.03 and the sensitivity of the order of 2.1 O ^"1 mole / 1, in antigen, ie 4-, 10 ^" ^ mole of antigen in the taking of trial. The measurement cycle can then start again by pickling the solid phase. The whole cycle here lasts 20 minutes including the adsorption of the antibody on the solid phase. To immediately reuse the electrode 13 in a new measurement cycle, it suffices to subject it to the same pickling operation as that which initiated the previous cycle.

EXAMPLE 2 This example relates to the determination of a steroid, 176 oestradiol, by a so-called "competitive" method, on a solid phase of medium surface (flat carbon electrode 0.1 cm in surface) "

1) Reagents

The rabbit anti-estradiol antibody was obtained from a specialized laboratory. The estradiol-acetylcholinesterase conjugate was prepared according to the method of

ERLANGER et al (Methods in immunology and immunochemistry, Académie Press, vol 1,144-, (1967).

2) Apparatus

The same apparatus is used as in Example 1.

3) Dosage

The principle of the antigen competition method vis-à-vis ^" the antigen labeled for the solid phase is used, described with reference to FIGS. And 6. The introduction and circulation of the reagents is ensured automatically by the fluid circuit 16 according to the following chronology:

The stripping and adsorption phases of the anti-estradiol body are identical to the first two phases of Example 1. " ^•

During the competition phase, an antigen-enzyme conjugate solution of appropriate concentration is introduced with the solution to be assayed. The flow rate of this sample / conjugate mixture is 0.1 cπ / min. and this phase lasts 3 minutes.

After rinsing for one minute with phosphate buffer (0.01 M pH 7.5 + 0.15 M NaCl), the acetylthiα-choline solution (5.10 "M in the same buffer) is introduced.

The current jump measured by electrode 13, previously brought to + 700 mV relative to electrode 14-, corresponds to the oxidation of the thiocholine produced by the conjugate in the position of enzymatic electrocatalysis.

In this case, the measured current is inversely proportional to the concentration of antigen in the sample to be assayed. FIG. 10 represents a calibration curve.

For a new measurement, just proceed to a new stripping of the electrode 13.

EXAMPLE 3 This example relates to the assay of an immunoglobin G by the "sandwich" method, using a solid phase of large surface (carbon felt electrode of real surface 50 cm).

1) Reagents

The antibodies have the same origin as those of Example 1. The conjugate is obtained here by coupling the acetylcholinesterase from Electricus electrophorus to the antibody by the carbodii ide method described by Goodfried TL et al., Science 1964-, 14-4-, 13 -4- - 4-6.

2) Apparatus

The apparatus used is shown diagrammatically in Figure 11. The solid phase is a carbon felt cyclinder 22 (for example, that bearing the reference RYC 2000 sold by the company Le Carbone Lorraine) 7 mm long by 5 πim in diameter, corresponding to a

2 real carbon fiber surface of the order of 50 cm. The electrical contact with this working electrode is provided by the annular electrode 23 made of platinum. The solution, introduced by 24-, percolates through the electrode 22 and is evacuated by the outlet 25 "

The reference electrode 26 and the stripping electrode 27 are identical to those of FIG. 7. This electrochemical micro-reactor 21 is incorporated in place of the electrochemical cell in the automated system described in FIG. 8.

This micro reactor is particularly effective for very dilute solutions which require a high capture rate. It also makes it possible to further reduce the contact times between solutions and solid phase due to the large surface / volume ratio obtained.

3) Dosing In principle, the dosing process is identical to that of Example 1, with the following differences: - the adsorption time of the antibody is reduced to 2 minutes;

est su vie d une phase e r nçage au tampon phosphate 0,01, M,pH 7,4-).- the sample to be dosed (volume 10 cnr ⁵ ) is introduced with a flow rate of 5 cπr / minute; - the potential of electrode 22 during the measurement phase is + 700 m? relative to the electrode 26; the current jump is obtained by introduction of the enzyme substrate, here acetylthiocholine 51C ^" M in a 0.01 M phosphate buffer, pH 7.5 / NaCl 0.15 MB The phase stripping procedure solid is different because of its large surface area. In this case, the power supply 18 imposes an alternating current on the frequency of the sector of approximately ^ 3V ^* in amplitude between the working electrode 22 and the electrode 27 "This phase, carried out in the presence of the nitro- mixture _" ^lasts 5 seconds.

is known to have a phase of erosion with phosphate buffer 0.01, M, pH 7.4-).

The entire measurement cycle lasts 15 minutes and an example of a calibration curve is given in FIG. 12. The sensitivity is of the order of 2.10 −2 mol / 1 with a capture rate of between 0.5 and 0 , 9. It will be noted that, if less good sensitivity is desired, it is sufficient to reduce the volume of the sample. EXAMPLE 4-

This example concerns the detection and localization of a protein in vivo using a microelectrode (carbon fiber with a diameter of 10 αm).

In this example, the solid phase is used to detect the presence of free acetylcholinesterase located at the periphery of a nerve cell in the spinal cord of the rat. The anti-acetylcholinesterase antibody is immobilized on the carbon fiber, which is introduced under microscopic control into the tissues ^' , according to the usual methods in electrophysiology. 1) Reagents ^' The anti-acetylcholinesterase monoclonal antibody suitable for this measure is not marketed and was supplied by a specialized university research laboratory.

2) Apparatus A manual method is used here.

The microelectrode used (for example that bearing the reference MPC1 marketed by SOLEA-TACUSSΞL) is sectioned so that the useful length of the fiber is approximately 50 μm. Taking into account the diameter (10 µm), this corresponds to an external surface of the fiber of the order of 1, 6.10 ^" ^ cm ² _β

During the contact phase with the sample, the electrode is mounted on a micromanipulation stand. During the enzymatic activity measurement phase, the electrode is placed as the working electrode in a conventional electrochemical assembly with three electrodes. The current is measured using a pico-amperometer, because of the low currents output.

3) Dosage The micro-electrode is pretreated by passing

10 pA for 5 seconds in the nitrochromic mixture of Example 1. After rinsing with phosphate buffer (0.01 M, pH 7.4), the anti-acetylcholinesterase antibody is adsorbed by simple soaking of the fiber in a solution 10 pg / cm in the same buffer.

After rinsing with the blocking buffer of Example 1, the electrode is fixed on a micromanipulator and the tip of the carbon fiber is inserted into the preparation for 2 minutes. Localization is carried out under a _β microscope

The detection of the acetylcholinesterase thus "taken up by the antibodies fixed on the electrode can then be carried out, in deferred fashion, in the electrochemical cell. The currents measured are of the order of 10 to 100 pico A, depending on the quantity of enzymes to be detected and the contact times in vivo. The sensitivity, expressed here in quantity of enzymes and not in concentration, is of the order of 2.10—21 mole of acetylcholinesterase. The method is semi quantitative, provided that contact times with witnesses are standardized.

It will be noted that the method can be used in vitro according to the same principles, in the case of very small sample volumes (for example of volume less than 1 μl). This proves the very high sensitivity of the detection method.

Claims

1.- Cyclic immunoenzymatic assay for a ligand, according to which, after having revealed reactive sites on the surface of an electrode by electrochemical oxidation, the latter is brought into contact with an antiligand specific for the ligand to be assayed for forming a solid conductive phase comprising said electrode and, fixed to the surface thereof, a substantially monomolecular layer of said antiligand without the interposition of an intermediate support, this solid phase then being exposed to the ligand to be assayed and to an anti-conjugate -ligand- enzy e or ligand-enzyme depending on whether the meth des so-called "sandwich" or "competitive" meth des is applied, the enzyme of said conjugate being able to react with a substrate to provide an electroactive product vis- with respect to said solid phase, this process being characterized in that, at the end of the assay, at least the ligands and the antiligand-enzy and conjugated ligand-enzyme conjugates are separated from the electrode on said solid phase, with a view to the reuse of this latter- for a new dosing cycle. 2.- Method according to claim 1, characterized in that the separation of the solid phase from the ligands and the antiligand-enzyme or ligand-enzyme conjugates is carried out by rinsing said solid phase with a liquid whose conditions of pΞ and / or polarity are different from those of the assay medium. 3.- Method according to claim 1 _? characterized in that, at the end of dosing, said layer of said anti¬ ligand is also separated from the electrode on which it is fixed, with a view to the reuse of said electrode for a new dosing cycle. 4-o- A method according to claim 3, characterized in that the separation of said anti-ligand layer is carried out by subjecting said electrode to an electrochemical pickling by the action of a direct or alternating current. 5.- Method according to claim 4-, characterized in that, for the pickling of the electrode, a direct current is passed through an oxidizing solution, between the metering electrode and a reference electrode with a potential difference between +1.7 volts and 2.2 volts. 6.- Method according to claim 4-, characterized in that, for the pickling of the electrode, an alternating current is used, with a potential difference of + 3 volts between the metering electrode and the pickling electrode . 7.- Method according to one of claims 1 to 6, characterized in that, during the assay phase, the ligands and / or the antiligand-enzyme or ligand-enzyme conjugates only saturate a fraction of the sites of the εntiligands or associated ligands. 8.- Method according to one of claims 1 to 7, characterized in that the detection of the product released by said substrate during the dosing phase is carried out by measuring a current flowing between said electrode and a reference electrode at- controlled potential.