WO1993025907A1

WO1993025907A1 - Redox system marked antigen, electrochemical detection immunoassay and assay kit

Info

Publication number: WO1993025907A1
Application number: PCT/FR1993/000561
Authority: WO
Inventors: Chantal Degrand; Ronald Blankespoor; Benoit Limoges; Pierre Brossier
Original assignee: Centre National De La Recherche Scientifique
Priority date: 1992-06-12
Filing date: 1993-06-11
Publication date: 1993-12-23
Also published as: FR2692357A1

Abstract

Antigen marked by a redox system, process for the immunoassay of at least one antigen, and assay kit. The object of the invention is to provide a reliable multiassay homogenous phase process which is speedy, specific and capable of being automated. The object is attained by a process consisting in allowing at least one of the antigens to be assayed (1, 1') to compete with the same antigen (7, 7') marked by a redox system, one of the forms being ionic (3, 3'), against a specific antibody (5, 5') of said antigens (1, 7; 1', 7') present in limited quantity; gathering on an electrode (13), comprising a polyionic film (11) with a polarity opposite to that of the redox system (3, 3'), the marked antigens (7, 7') not bound to the specific antibody (5, 5'), so that said marked antigens (7, 7') become concentrated in said film (11); and measuring the amplified signal supplied by said electrode (13) using electrochemical detection means (15) connected to said electrode.

Description

ANTIGEN MARKED BY A REDOX SYSTEM,

IMMUNOLOGICAL ASSAY PROCESS WITH

ELECTROCHEMICAL DETECTION AND DOSING KIT

DESCRIPTION

The present invention relates to an antigen labeled with a redox system, one of the forms of which is ionic, an immunological assay method with electrochemical detection of at least one antigen and a kit for assaying at least one antigen.

The growing consumption of certain drugs, toxic products or drugs has led to the development of precise, specific and rapid detection methods used in particular in hospitals and analytical laboratories. These methods are intended to allow therapeutic monitoring of drugs prescribed to the patient but also to perform dosages in emergency toxicology. Many drugs such as antidepressants, anti-epileptics, tranquilizers and neuroleptics are administered to patients on a regular basis. However, their therapeutic index (i.e. the range of values between their efficacy threshold and their toxicity threshold) is sometimes extremely low. For example, in the case of antidepressants, there is a risk of intoxication as soon as this drug exceeds a concentration of 2.10 ^{~ 6} M in the serum. Consequently, it is necessary to take regular samples from patients in order to be able to dose and monitor the evolution of the concentration of the drug in biological fluids.

In addition, certain drugs such as amphetamines or cardiotonics may be used for doping purposes. It is important to be able to measure these drugs reliably after a sports competition.

Finally, some patients may voluntarily or accidentally ingest drugs or toxic products. When these patients arrive in a hospital in a state of emergency, it is necessary to carry out very rapid and precise dosages, in order to determine the nature of the product which they have absorbed and to treat them in an appropriate manner.

Numerous, reliable and specific assay methods have been developed. These methods are based on the association of a detector of biological origin, capable of binding in a specific way with the drug which one wishes to dose, of a transducer which generates a signal representative of the binding of the detector. and medication, and an analyzer which transforms the signal obtained into information accessible to the user. Among the assay methods, immunological methods make it possible to assay small amounts of antigen. They are based on the fact that antibodies (Ac) recognize and then bind in a specific way to antigens (Ag), to give an antigen-antibody complex according to the following balanced reaction: Ac + ha ** ~ TAc-Aq free fraction linked fraction

The introduction of radioactive markers in 1959, by the pioneers BERSON and YALOW, was a considerable success and gave birth to radioimmunology (RIA). However, this technique has serious drawbacks. Radioactive tracers are expensive and short-lived. In addition, the use of radioactive material is highly regulated and can only be done by a laboratory approved by CEA.

To avoid these drawbacks, attempts have also been made to develop photosensitive markers whose presence is detected by molecular absorption (infrared or fluorescence) or atomic, infrared or fluorescence. However, these techniques require sophisticated equipment of high cost.

Immunoenzymatic assay techniques are known from the prior art under the name of ELISA Test (registered trademark). Some of these techniques are based on the so-called "competitive assay" principle which consists in introducing and putting in competition a defined quantity of labeled antigens (by an enzyme) and an undefined quantity of unlabeled antigens, facing a limited and known quantity of antibodies. In the ELISA tests, the separation of the free and bound fractions is carried out by transfer of the reaction medium into test tubes or wells, on the walls of which are fixed second specific antibodies. After the first immunological reaction, transfer, decantation and washing steps are carried out before adding the enzyme substrate. This is then colored in a more or less long period of time. The intensity of the coloration is then measured by spectrophotometric assays.

However, the enzymatic labeling of anti¬ genes is delicate and of limited stability. In addition, this technique does not have very good specificity, the results obtained are therefore not entirely reliable. In addition, interference can inhibit the activity of the enzyme. Finally, this approach requires a transfer step to separate the free and bound frac¬ tions, followed by several washes, therefore a long and expensive handling time. This ap- close is characteristic of heterogeneous phase methods.

The object of the invention is to get rid of these conditions and, therefore, relates in particular to a dosing process in homogeneous phase, simpler and faster.

In the field of immunology, there are also known from patent applications EP-0 142 301 and EP-0-167 248, electrochemical assay methods using redox centers such as ferrocene, and a working electrode solid in glassy carbon or metal. In the applications cited, the redox centers may possibly act as mediators of electron transfer in enzymatic reactions, which allows an amplification of the electrical signal and consequently an increase in the sensitivity of the electrochemical method.

Also known from patent application EP 241 309 is a method for assaying theophylline with electrochemical detection using a ferrocene marker. The detection by coulometry is amplified using an enzymatic catalysis and an electrode modified by a polyvinyl ferrocene film whose role is to serve as a relay between the electrode and the marked drug (the latter does not penetrate in the movie).

All of these techniques, in addition to their specific drawbacks, do not allow more than two drugs present in a biological sample to be measured simultaneously. However, it would be desirable when a patient arrives in a state of emergency, to be able to carry out a single multi-dosage of several drugs commonly used and with which the risks of intoxication are frequent.

The invention also aims to remedy the aforementioned drawbacks. To this end, the invention relates to an antigen labeled with a redox system, one of the forms of which is ionic, intended for use in an immunoassay method with electrochemical detection of this antigen, or in an immunoassay kit for this antigen.

According to the characteristics of the invention, this labeled antigen is chosen from the nortriptyline labeled with hexafluorophosphate of cobalticinium carboxylic acid, with pyrrolidinyloxy or with ethyl alkyl viologene dibromide; amphetamine labeled with cobalticinium carboxylic acid tetrafluoroborate or pyrrolidinyloxy; desipramine labeled with cobalticinium carboxylic acid hexafluorophosphate, pyrrolidinyloxy or ethyl alkyl viologene dibromide; or biotin labeled with tetraphenylborate of the cobalticinium carboxylic acid.

These specific antigens were labeled _m is the point when performing processes or immunological assay kits and give particularly interesting results, as will be explained later.

The invention also relates to an immunological assay method with electrochemical detection of at least one antigen comprising at least one functional group. This process includes the steps of

- prepare a solution to be analyzed comprising an aqueous solution buffered at physiological pH,

(preferably pH 7.4) and a biological sample containing at least one of the antigens to be assayed,

- put in competition at least one of said antigens to be assayed with the same antigen marked by a redox system of which one of the forms is ionic (and preferably cationic or procationic), face to an antibody specific for said antigens and present in a limited quantity.

According to the characteristics of the invention, this process then consists in: - collecting on an electrode comprising a polyionic compound of polarity opposite to that of the redox system, (that is to say preferably, polyanionic), the labeled antigens, not linked to the specific antibody, so that these labeled antigens concentrate in said compound during a period of accumulation,

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

A "pro-ionic" redox system is a system one of the forms of which becomes ionic by anodic oxidation ("procationic" redox system) or by cathodic reduction ("pro-anionic" redox system). The electrode is coated with a poly-ionic film. According to a variant embodiment, this polyionic film is polyanionic and comprises a perfluoropolymer carrying anionic sites at physiological pH. This film thus has both hydrophobic and ionic character.

This process allows selective measurements to be made very quickly and with good sensitivity due, on the one hand, to the use of specific antibodies and, on the other hand, to the characteristics of the modified electrode. This is capable of concentrating the labeled antigens even if they are only present in very small quantities inside the analytical solution, which has the effect of amplifying the signal before it is not transmitted to the electrochemical detection means. In addition, this method is very simple to use and of low cost because the apparatus used is extremely simplified. These two characteristics ticks justify the use of this process not only in medical circles, but also in veterinary or agricultural environments, for dosing pesticides for example. In addition, this process is easily self-employable due to the very small number of manipulations to be carried out.

The antigen assayed by this method is a molecule comprising at least one functional group, preferably a primary or secondary amino function or a carboxylic function. This opens up a wide range of possibilities among the substances, pesticides or drugs which can be dosed by this process.

Advantageously, the electrochemical detection means are chosen from amperometry, coulometry, voltammetry, and preferably, square wave voltammetry.

These detection means make it possible to carry out the measurements quickly and reliably, including in a cloudy environment, as is the case when the biological sample of the analyte is a serum for example. This has an advantage over the spectroscopic methods frequently used in immunoassays.

The redox system can be any system corresponding to a rapid electronic exchange at physiological pH (close to 7.4).

Preferably, the redox labeling system is chosen from ferrocene, cobal¬ ticinium, pyrrolidinyloxy or viologene derivatives. These different redox marker systems are cationic or procationic. The cationic systems are already cationic at the start, this is the case for the cobalticinium and viologene derivatives, while the procationic systems are neutral at the start and become cationic when, after having penetrated in the anionic film, they are anodically oxidized by polarization of the electrode; this is the case for ferrocene and pyrrolidinyloxy derivatives. In addition, these marking systems have very different standard redox potentials leading to well individualized electrical signals. Consequently, a multidosage can be envisaged, in which each of the antigens which it is desired to assay is labeled with a redox marker acting at a different potential. The invention also relates to an immunoassay kit for at least one antigen comprising at least one functional group. According to the characteristics of the invention, this kit includes:

at least one support or container having at least one type of antibody-antigen complex labeled with a redox system, one of the forms of which is ionic, the antigen corresponding to that which it is desired to assay, and

- An electrode comprising a polyionic compound of opposite polarity to that of said redox system.

It is generally a polyionic film.

The invention as a whole will be better understood on reading the following description of a specific embodiment of the invention, given by way of illustrative and nonlimiting example, this description being made with reference to the accompanying drawings, wherein :

FIG. 1 is a diagram illustrating the various stages of the immunoassay method according to the invention,

- Figure 2 is a sectional view of the cell and the electrode used in the dosing process, - Figure 3 shows a theoretical example of a curve obtained by square wave voltammetry,

- Figure 4 shows two curves obtained by square wave voltammetry, respectively with a bare glassy carbon electrode and with a modified electrode according to the invention,

FIG. 5 is a curve illustrating on a logarithmic scale, the evolution of the intensity as a function of the concentration of an analyte containing as labeled antigen alone, amphetamine labeled with cobalticinium,

FIG. 6 is a graph representing the possible interference of the unlabeled antigens on the percentage of electrical signal provided by the labeled antigen,

FIG. 7 is a graph illustrating the influence of normal serum on the accumulation of the labeled antigen inside the film of the electrode,

- Figure 8 is a curve illustrating the percentage of electrical signal supplied by cobalticinium-labeled desipramine as a function of the specific antibody concentration of desipramine, - Figure 9 is a graph representing the results of square wave voltammetry obtained during a test assay,

FIG. 10 is a calibration curve obtained for the modified desipramine dosed with the cobalticinium-labeled desipramine,

- Figure 11 is a curve representing the simultaneous measurement by square wave voltammetry of two labeled antigens and a tracer (case of a multidose), and - Figure 12 is a diagram illustrating the various elements present in the kit assay according to the invention and the immunological reactions taking place during its use.

As illustrated in FIG. 1, the metering method according to the invention consists in bringing into balance at least one antigen to be assayed 1 with the same antigen labeled with a cationic or procationic redox system 3, facing an antibody 5 specific for said antigen 1 and present in a limited quantity. The antigen 1 marked by the redox system 3 carries the global reference 7.

The affinities of antibody 5 for antigens 1 and 7 are similar. Finally, the quantities of labeled anti-genes 7 and of antibodies 5 are known. During the immunoreaction, the labeled antigens 7 react with the specific antibodies 5 so as to form complexes 9 and respectively the antigens 1 and the antibodies 5 form complexes 10. Taking into account the limited quantity of antibodies 5, not all antigens 1 or 7 can react with antibodies 5, and some of them remain free. Among these, however, only the antigens marked 7 can penetrate inside a polyanionic film 11 which covers the surface of an electrode 13 consisting of a glassy carbon cylinder. On the other hand, the complexes 9 labeled antibody-antigen cannot accumulate in the polyanionic film 11, because of their size and their molecular mass. This allows selection and therefore reliable measurements.

Note that the electrode 13 covered with the polyanionic film 11 could be replaced by a carbon paste electrode in which the polyanionic compound is embedded. This variant is not shown in the figures and in the description, only the first variant using the polyanionic film is described in detail.

The accumulation of the marked antigens 7 inside the polyanionic film 11 makes it possible to amplify the signal observed at the output of the electrode 13. This electrode 13 is in fact connected to electrochemical detection means 15 allowing the inter- preparation of this signal. These detection means 15 include, for example, an amperometric, coulometric or voltammetric measurement device 17 which will be detailed later, the latter being connected to an apparatus for drawing graphs 19 providing result curves.

In the embodiment of the invention which has just been described, the film 11 is anionic and the marking system 3 is cationic or procationic, however it is obvious that the reverse could also be achieved without harming the operation of the invention.

The various stages of this process and the means used to carry it out will now be described in more detail.

As explained above, the assay method according to the invention makes it possible to assay for antigens, and more specifically for drugs for which it is necessary to carry out, ie therapeutic monitoring, that is to say determining at regular intervals the quantity present in a biological sample, i.e. an emergency toxicology assay, that is to say to determine the presence or absence thereof in a sample taken from a patient arriving at the hospital.

These antigens, to be able to be labeled with one of the cationic or procatio¬ nic redox systems 3 which will be described later, must have at least one functional group and preferably, a primary or secondary amino function or a carboxylic function. In addition, they must be made up of molecules small enough to be able to penetrate the film.

Among the drugs meeting these characteristics and which can be dosed with the method according to the invention, there will be mentioned in particular:

- tricyclic antidepressants such as desipramine, imipramine, nortriptyline, ami-triptyline, clomipramine, maprotiline and atypical drugs such as nomifensine, mianserine, amineptine, zimelidine, - antiepileptics such as barbiturate derivatives, especially phenobarbital, mephobarbital, or such as hydantoines, in particular phenytoine, mephenytoine, carbamazepine and valproic acid, - tranquilizers derived from benzodiazepines, in particular diazepam, nitrazepam,

- tricyclic neuroleptics such as chlorpromazine and trifluoperazine,

- neuroleptics such as haloperidol, fluospirilene,

- antitumor drugs such as methotrexate, 5-fluorouracil and azathioprine,

- vitamins such as biotin,

- alkaloids, - amphetamines;

- antibiotics,

- cardiotonics and antiarrhythmics,

- neurotransmitters and their derivatives,

- antihistamines, - immunosuppressants such as cyclosporine,

- theophylline, or

- pesticides.

Among these, the chemical formula of:

- nortriptyline

- desipramine:

- amphetamine:

- biotin:

H

° ^NH D „* -CH -CH -CH -CH -CH -C00H

H _{2 2} 2 2

As explained above, for the immunological reaction which has been described in FIG. 1 to proceed correctly, it is necessary that the redox labeling system 3 does not disturb the recognition by the antibody 5, of the labeled antigen 7 .

The labeling systems meeting these requirements and capable of binding with a substance or a drug having at least one functional group and in particular a primary or secondary amino function or a carboxylic function, are chosen from derivatives of the pairs:

- ferrocene / ferricinium (Fc / Fc ⁺ ):

F * ë> - cobalticinium / cobaltocene (Cc ⁺ / Cc):

<S $

<S>

- pyrrolidinyloxy / pyrrolidinyloxonium

(N0- / N0 ⁺ )

0 ^* I

- ethyl alkyl viologene (in the form of dibromide) / radical viologene (EV ²⁺ / EV- ⁺ ):

Br Br

and their derivatives.

Among these derivatives, mention may, for example, be made of salts such as hexafluorophosphate, tetraphenylborate or tetrafluoroborate of carboxyic acid of cobalticinium.

Table 1 below illustrates four examples of drugs which have been labeled with some of the various redox poten¬ tial labeling systems. The marked drugs obtained are numbered from 1 to 10. The table also gives the elementary analysis of these marked drugs.

Table 1: Compounds prepared and their elemental analysis

Marked drug Marking system: N 'Calculated Elementary Analysis (Found) M: Formula

(Table 1 continued)

The antigens marked No. 1 to 3 and 5 to 10 were developed during the carrying out of the assay method and are also particularly suitable for the assay kit described later.

The method of labeling drugs with one of the aforementioned redox systems is carried out as follows.

Medicines with a primary or secondary amino function can be symbolized by the formula Med-NRlϋ, while the redox labeling system M is in the form of acid chloride or hydroxysuccinimide ester. In this case, the labeling process can be carried out according to reaction (1) (which was used to prepare the labeled drugs

1, 5 and 7 of Table 1) or according to the two reactions (1 ') and (2 ") (which were used to prepare the drugs

2, 4, 6 and 8 of table 1):

MCC1 + Med-NRlH -HC1. Med-NR! CM (1)

S ^ Il O

or

where DCC is dicyclohexylcarbodiimide

(2 ')

Medicines with a carboxylic function can be symbolized by the formula R ² C00H. They are previously treated with the hydrazine NH2-NH2 according to reaction (3) and the hydrazine thus obtained reacts with an acid chloride according to the preceding reaction (1):

R ² C0H + H2-NH2> R ² CNHNH2 + H ₂ 0 (3)

O O

This reaction served to mark the medicament 10 in Table 1.

Finally, the labeling with viologene is carried out in two stages (4) and (5) which made it possible to prepare the drugs 3 and 9 of table 1:

Med-NR ¹ !. + Br (CH2) 5CCl -HCl ^ Méd-NR ^ CI ^) 5-Br (4)

O O

FIG. 2 illustrates the structure of the cell in which the accumulation on the modified electrode 13 is carried out, then the amperometric measurement after the antigen-antibody reaction has taken place.

This cell consists for example of a central reservoir 21 with an internal diameter of 13 mm, inside which is deposited 0.5 to 1 ml of solution to be analyzed 23 containing the biological sample to be analyzed (serum, urine , etc.) diluted in a pH 7.4 buffer. This sample is generally present in an amount of a few tens of microliters. The reservoir 21 further comprises two lateral tubes 24 in which are disposed a platinum wire counter electrode 25 and an Ag / AgCl reference electrode 27. The electrode 13 in vitreous carbon covered with a layer of anionic polymer film 11 and on which the reaction of the invention takes place is immersed in the tank 21.

The glassy carbon cylinder 13 for single use is placed in a sleeve 29 of Teflon (registered trademark). This set is fixed to a 31 Tacussel EDI 101T rotating electrode. The contact between the carbon cylinder 13 and the electrical conductors 33 of the electrode 31 is ensured by a spring 35.

Due to the cationic nature of the markers, the polymer film will be polyanionic in nature.

Advantageously, this polymer is a perfluorinated polymer to which are grafted sulfonic acid groups. It is known by the brand name

Nafion (manufactured by E.I. DUPONT 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 SO3H site. At pH 7.4, it is in polyanionic form.

The electrode will be manufactured to present reproducible results from a solution tion of Nafion 117 sold by Aldrich, (reference 27, 470-4).

This polymer is thermally and chemically very stable, insoluble in water alone and therefore in the analytes. It has good ion transport properties. The fluorinated groups ensure the hydrophobic character and the Sθ3 ^~ groups, the ionic character of the polymer.

In FIG. 1, the antigens marked 7 can bind, via an ion exchange, either to the surface of the film 11, or in depth, after the immunological reaction has taken place. The modified electrode 13 is immersed in the solution 23 (see FIG. 2) and this for a determined period, called "accumulation time", so that the antigens marked 7 have time to concentrate in the film 11 and that the electrode 13 can thus provide an ampli¬ fied signal, by electrochemical detection means 15. During this accumulation period, the electrode 13 is maintained in open circuit when the marker is cationic (cobalticinium and viologen, for example) or at a constant anodic potential for procationic markers (ferrocene and pyrrolidinyloxy, for example). In addition, advantageously, the modified electrode is driven in a rotational movement so as to facilitate the path of the labeled antigens 7 towards the film 11. Then, the rotation is interrupted to carry out the electrochemical measurement.

It should however be noted that once the measurement has been made, the polymer is polluted by the labeled antigen and that it can no longer be used for a new measurement. It is therefore necessary to replace the carbon cylinder 13 covered with the film 11, at each measurement.

As described above, the electrochemical detection means 15 comprise a measuring device 17 which can be a device for measuring by amperometry, by coulometry or voltammetry, and advantageously, by square wave voltammetry. Square wave voltammetry uses the. counter electrode 25, the reference electrode 27 and the working electrode 13 modified by a Nafion film, illustrated in FIG. 2.

As illustrated in FIG. 3, square wave voltammetry consists in carrying out a voltage sweep between the reference electrode 27 and the modified electrode 13. We obtain peaks of intensity A whose maximum value H is measured.

In order to show the effectiveness of the Nafion film, a comparative test was carried out between an electrode covered with a Nafion film and an electrode made of bare vitreous carbon (see FIG. 4).

Square wave voltammetry measurements were carried out with a known concentration (0.9.10 ^~ M) of amphetamine labeled with cobalticinium, alone, in solution. In Figure 4 attached, curve C1 illustrates the results obtained with an electrode made of bare vitreous carbon and curve C2 shows the results obtained by an electrode covered with a Nafion film, after accumulation for 5 minutes. The value of the peak current of the curve C1 is 1.5 uA while the value of the peak of the curve C2 is 63.05 uA.

It can therefore be seen that for an identical concentration of products present in a sample, the electrode covered with the Nafion film makes it possible to amplify the signal approximately 40 times, in this case. The sensitivity of the results is therefore greatly improved by the presence of the Nafion film, which also makes it possible to detect extremely small quantities of products present in the initial sample. To carry out the method according to the invention, one of the preliminary steps consists in carrying out a calibration curve for the labeled drug, alone in solution and for measuring the different peak currents obtained as a function of known increasing concentrations of labeled drug. FIG. 5 is a calibration curve obtained for amphetamine labeled with cobalticinium, alone, in a pH 7.4 buffer, for an accumulation time of 5 minutes and an electrode rotation speed of 600 revolutions / minutes. This curve shows that the linearity domain is important (logarithmic scale).

Various tests have been carried out in order to prove the effectiveness of the method according to the invention. Labeled antigen / unlabeled antigen interaction test

Under the immunoassay conditions, that is to say at pH 7.4, the layer 11 of Nafion polymer is able to accumulate, not only the antigens 7 labeled by a cationic or procationic redox system 3, but possibly also the protonated form of antigens 1 when these correspond to the formula Med-NR - ^ - H. Tests performed on amphetamine have shown that the signal produced by amphetamine labeled 7 is not modified by the increasing intake of amphetamine 1 alone. On the other hand, it has been observed, in the case of nortriptyline and desipramine, that this interference resulted in a reduction in the electrical signal observed at the output of the electrode 13. In view of these results, it is desirable to chemically transforming the initially cationic antigen 1 into an anionic form which cannot react with the anionic film 11. This transformation is carried out by adding succinic anhydride to this Med-NR ^ -H antigen chemical reaction (6). The modified acid forms of nortriptyline and desipramine are thus obtained:

Med-NRlH + O- (6)

It will be noted that, conversely, if an anionic antigen 1 were used, it would be treated so as to have it in a cationic form which does not react with a cationic film.

This change in medication removes the interference and therefore restores the original signal.

FIG. 6 represents the influence exerted by increasing concentrations of simple desipramine (curve C1) and of desipramine modified to present itself in an anionic form (curve C2), on the percentage of electrical signal which the desipramine labeled with cobalticinium provides. The latter is present at a concentration of 2.10 ^" ^ M. It is found that in the presence of simple desipramine (curve Cl), the latter occupies anionic sites of the film 11, in an abusive manner and that the desipramine labeled with cobalticinium does not can react completely with the film 11 sites. Consequently, the percentage of electrical signal due to redox systems decreases, on the contrary, in the presence of the modified drug in anionic form (curve C2), the percentage of signal is not influenced. In addition, this chemical transformation of the drug (antigen) has a second advantage: the antibody 5 (see FIG. 1) has a better affinity for the antigen 1 when the latter is in anionic form. sensitivity dosing.

Labeled antigen / normal rabbit serum interaction test

In the absence of antibodies 5, the non-specific interactions (protein bonds) of the serum with the labeled drugs must be evaluated, since the electrical signal corresponds to the n ¹ molecules which do not interact. FIG. 7 illustrates the influence of normal rabbit serum on the accumulation in the electrode of the labeled antigen, here the nortriptyline marked respectively with cobalticinium (curve C1), pyrrolidinyloxy (curve C2) and ferrocene (curve C3). When drugs are labeled with cobalticinium, the addition of normal rabbit serum has little effect on the electrical signal. This is illustrated by the curve C1 where the concentration in labeled drug is 5.10 ~ ⁷ M. This results from the ionic character, therefore hydrophyle of the marker. On the other hand, when this drug at a concentration of 10 ~ ^ M is labeled with pyrrolidinyloxy (curve C2) or ferrocene (curve C3), the drop in signal is greater due to the hydrophobic nature of the labeled drugs. Antibody / labeled antigen interaction test

In order to verify that the immunological reaction is carried out correctly, it is necessary to show that the specific antibodies 5 are not only effective vis-à-vis the antigens 1 alone but also vis-à-vis the marked antigens 7 and that the presence of the redox labeling system 3 does not modify the interaction with the antibody. FIG. 8 il¬ illustrates the assay of the antiserum, that is to say of the serum containing the antibody. (It should be noted that before the immunoassay itself, it is necessary to determine the quantity of antibodies which will have to be added later during the assays to be in antibody defect).

Increasing amounts of antibody 5 were added to a solution containing a fixed quantity of antigens labeled 7 (here desipramine labeled with cobalticinium, concentration 2.7 × ¹⁰ -7 M) and in which electrode 13 was immersed. addition of antibodies causes the electrical signal obtained at electrode 13 to drop due to the depletion of the free labeled desipramine solution, the only one capable of penetrating inside the polyanionic film 11. In other words, the complexes antibodies / antigens marked 9 cannot enter this film 11 and are therefore no longer counted, which explains the decrease in the percentage of electrical signal. As a purely illustrative example, the SCATCHARD method made it possible to determine the antibody affinity constant for desipramine labeled with cobalticinium, and this constant is equal to 5.10 ⁷ M " ¹ . process according to the invention

As in any immunoassay, before undertaking determinations of unknown concentrations on biological samples, it is necessary to establish a calibration curve for a range of given concentrations. For this, five solutions have been prepared, these are:

- solution A: labeled antigen, here desipramine labeled with cobalticinium, (concentration 10 ^{~ 5} M), dissolved in ethanol,

solution B: antigen alone, modified, that is to say the desipramine modified in its anionic form and dissolved in a buffer pH 7.4, (that is to say solution E) concentration 10 ^~ ^ M , - solution C: normal diluted rabbit serum 1/5 in the pH 7.4 buffer (i.e. solution E),

solution D: antibody, here anti-rabbit serum diluted 1/5 in the pH 7.4 buffer, (that is to say solution E),

- solution E: buffer pH 7.4 (2.7 g of Na HP0 ₄ , 12 H2θ + 0.338 g NaH2Pθ4, 2 H2θ + 1.64 g NaCl + water up to 500 ml).

Then, 3 test tubes were prepared in which the following mixtures were carried out: control tube (1): solution A + solution C + solution E, zero tube (2): solution A + solution D + solution E, tube standard (3): solution A + solution

B + solution D + solution E.

The solutions were homogenized, then incubated for example at 37 ° C for 60 minutes, so as to reach the equilibrium of the antibody-antigen reaction.

They were then transferred successively to the electrolysis cell 21, itself immersed in a bath thermostatically controlled at 25 ° C. The measurements are carried out by immersing the electrode 13 coated with the Nafion film 11 in the cell and by imposing on it a rotation of 600 revolutions per minute for 5 minutes (period of accumulation of the antigens marked free). The square wave voltammetry measurement is then carried out. Obtaining a calibration curve involves three measurements for each point. Tube 1: Ag labeled + serum - peak current Ii, Tube 2: Ag labeled + Ac - ^ peak current 12, Tube 3: Ag labeled + Ag alone + Ac— ^ peak current I3. The results are illustrated in the figure

9 attached. In the first case, all of the labeled antigens can bind to the electrode and concentrate in the polymer film, which explains the high value of the peak current Iι "In the second case, part of the labeled antigens reacts with the antibodies and only the excess labeled antigens are concentrated on the electrode. This explains the low value of the peak current 12. Finally, in the third case, the competition reaction between the labeled antigens and the unlabeled antigens, against the antibodies takes place. A smaller fraction of the free labeled antigens than in the second measurement, concentrates on the electrode. The peak current 13 is therefore an intermediate value between Ii and I2.

The percentage of signal is defined by (I00.l2) / Iι and the signal B / B ₀ (%) is equal to 100 (I ₁ -I) / (I ₁ -I ₂ ⁾ .

From this type of results, a calibration curve can be drawn, such as the one illustrated in FIG. 10. This curve can be used for the analysis of a sample containing an unknown quantity of modified (anionic) desipramine. .

Furthermore, the method according to the invention makes it possible to envisage the realization of multidoses. Indeed, as illustrated in FIG. 1, it is possible to assay ^' in parallel not only an antigen 1 but also a different antigen l' and to react the latter in competition with an antigen 1 'marked by a different redox system 3' facing 5 'antibodies specific for the antigen 1'. In fact, the derivatives of ferrocene, cobalticinium, pyrrolidinyloxy and viologene exhibit peaks of intensity at potentials sufficiently different from each other so that a succession of peaks can be detected on a results curve.

For the drugs 1 to 10 of table 1, the values of the potentials corresponding to the intensity peaks of the different redox systems are given in table 2 below. Table 2

Marked drug Peak Potential marking system (V) Sensitivity ip / C (Almol ^-1 )

nortriptyline cobalticinium - 1.13 51 ACO

31 accumulation at ^' + 0.7V

2 pyrrolidinyloxy nortriptyline + 0.34 13 accumulation at + 0.8V nortriptyline viologene - 0.78

ferrocene amphetamine + 0.20 cobalticinium amphetamine - 1.13 pyrrolidinyloxy amphetamine + 0.34 cobalticinium desipramine - 1.13 pyrrolidinyloxy desipramine + 0.34

desipramine viologene - 0.78

10 biotin cobalticinium - 1.07 ACO

Ag / AgCl; Cl ^" (0.05M)

*: no sensitivity measurement carried out

ACO: Accumulation in Open Circuit

Thus, by using at least two different labeling systems, it is possible to measure simultaneously in a single measurement, at least two drugs present in a single sample originating from a patient. It is well understood that in emergency toxicology, this type of screening tests can rapidly detect the presence and concentrations of several drugs in common use.

FIG. 11 precisely illustrates a triple assay carried out on a sample containing simultaneously nortriptyline labeled with cobalticinium, amphetamine labeled with ferrocene and dimethyl viologene.

The measurements were carried out at a temperature of 25 ° C., with an electrode coated with Nafion, rotating at 600 rpm, after 5 minutes of accumulation at 0.6 V. During the measurement, one swept the potential between + 0.6 V and - 1.4 V and three peaks numbered PI, P2 and P3 were observed respectively. The PI peak located near 0.19 V corresponds to the drug labeled with ferrocene, the P2 peak located at -0.77 V corresponds to the reduction of dimethyl viologene and the P3 peak located at approximately -1.11 V corresponds to a drug marked with cobalticinium. By plotting the intensity values obtained on a preset calibration curve, it would be possible to deduce the concentrations of the various drugs. Here the concentration of nortriptyline marked with Cc ⁺ was 5.0.10 ~ ⁷ mol.l- !, that of amphetamine marked with Fe of 3.45.10 ^{~ 7} mol.l ^" ! And that of methyl viologene of 5, 2.10 ^" ° mol.l ^" !.

The invention also relates to a dosing kit using the principle of the method which has just been described. This kit is shown diagrammatically in FIG. 12. It includes a container 40 which contains at least one type of antibody 5'-5'-antigen complex labeled with the ionic redox system 3'3 '. This complex may or may not be attached to the container. The antigen corresponds to that which one wishes to assay. In this figure 12, the container 40 takes the form of a test tube and makes it possible to carry out a sequential analysis. However, it is obvious that this container could also be in the form of a multi-well assay plate. Furthermore, it is also possible to carry out a continuous flow analysis and in this case, the Ac / Ag complex labeled 9, 9 ′ is fixed on beads of various natures which play the role of support 40. These different types of containers or supports are given by way of illustrative example. They are for single use only.

The assay kit according to the invention also comprises an electrode 13 coated with a poly¬ ionic film 11, of polarity opposite to that of the system 3, 3 'and advantageously polyanionic. Advantageously, this film 11 is the polyanionic film of Nafion (registered trademark) previously described and the redox labeling system 3, 3 ′ is of cationic or procationic nature, such as those which have been previously described. One could also use a kit comprising Ac / Ag complexes marked by an anionic or proanionic redox system and an electrode coated with a polycationic film.

Finally, the kit can possibly include test tubes 42, 44 containing standard samples of known quantities of antigen to be assayed 1 and 1 respectively. These tubes allow the calibration curve to be produced according to the principle previously described. The user can also prepare his standard samples himself. When using this dosing kit, the biological sample 23, containing the antigens 1, 1 ', possibly treated to be presented in anionic form, is poured into the tube 40. The competition reaction takes place and certain antigens 1, 1' will dislodge the antigens labeled 7, 7 'to attach in their place on the antibodies 5, 5'. The rest of the reaction is identical to what has been described above for the process, in particular as regards the electrochemical detection means and the analysis of the results on the basis of the calibration curve.

Claims

1. Antigen marked by a redox system, one of the forms of which is ionic, intended for use in an immunoassay method with electrochemical detection of this antigen or in an immunoassay kit for this antigen, characterized in that l the labeled antigen is chosen from the nortriptyline labeled with cobalticinium carboxylic acid hexafluorophosphate, pyrrolidinyloxy or ethyl alkyl viologene dibromide; amphetamine labeled with cobalticinium carboxylic acid tetrafluoroborate or pyrrolidinyloxy; cobalticinium carboxylic acid hexafluorophosphate labeled desyramine, pyrrolidinyloxy or ethyl alkyl viologene dibromide; or tetraphenylborate-labeled biotin of cobalticinium carboxylic acid.

2. A method of immunological assay with electrochemical detection, of at least one antigen comprising at least one functional group, comprising the steps consisting in:

- prepare a solution to be analyzed comprising an aqueous solution buffered at physiological pH and a biological sample (23) containing at least one of the antigens to be assayed (1, l '),

- put at least one of said antigens to be dosed in competition (1, l ') with the same antigen (7, 7') labeled with a redox system one of the forms of which is ionic (3, 3 '), to an antibody (5, 5 ') specific for said antigens (1, 7; 1', 7 ') and present in a limited quantity, this method being characterized in that it also consists in:

- collect on an electrode (13) comprising a polyionic compound (11) of opposite polarity that of the redox system (3, 3 '), the labeled antigens (7, 7') not linked to the specific antibody (5, 5 '), so that these labeled antigens (7, 7') penetrate the inside said polyionic compound (11) and concentrate therein during a period of accumulation,

- measure the signal supplied by said electro¬ (13) at the end of the accumulation period, using electrochemical detection means (15), connected to this electrode.

3. Immunoassay method according to claim 2, characterized in that the electrode (13) is coated with a polyionic film (11).

4. Immunological assay method according to claim 3, characterized in that the redox system (3, 3 ') is cationic or procationic and in that the film (11) covering the electrode (13) is polyanionic.

5. Immunological assay method according to claim 4, characterized in that the poly¬ anionic film (11) covering the electrode (13) is a perfluoric poly¬ mother, carrying anionic sites, at physiological pH.

6. Immunoassay method according to claim 5, characterized in that the poly¬ anionic film (11) covering the electrode (13) is a perfluoropolymer of formula;

- "

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

7. Immunological assay method according to claim 2, characterized in that the antigen (1, l; 7, 7 ') is a molecule comprising at least, a primary or secondary amino function or a carboxylic function.

8. Immunological assay method according to claim 2 or 7, characterized in that the antigens (1, l ') to be assayed are chosen from:

- tricyclic antidepressants such as desipramine, imipramine, nortriptyline, ami-triptyline, clomipramine, maprotiline and atypical drugs such as nomifensine, ianserine, amineptine, zimelidine,

- antiepileptics such as derivatives of barbiturates, in particular phenobarbital, mephobarbital, or such as hydantoines, in particular phenytoine, mephenytoine, carbamazepine and valproic acid,

- tranquilizers derived from benzodiazepines, in particular diazepam, nitrazepam,

- tricyclic neuroleptics such as chlorpromazine and trifluoperazine, - neuroleptics such as haloperidol, fluospirilene,

- anti-tumor drugs such as methotrexate, 5-fluorouracil and azathioprine,

- vitamins such as biotin, - alkaloids,

- amphetamines,

- antibiotics,

- cardiotonics and antiarrhythmics,

- neurotransmitters and their derivatives, - antihistamines. - immunosuppressants such as cyclospons.

- theophylline, or

- pesticides.

9. Immunological assay method according to claim 4, characterized in that the redox labeling system (3, 3 ') is chosen from ferrocene, cobalticinium, pyrrolidinyloxy or viologene derivatives.

10. Immunological assay method according to claim 3, characterized in that before being put in competition, the antigen to be assayed (1, l ') is treated so that it is in an ionic form of the same polarity as that of the film (11) covering the electrode (13).

11. Immunological assay method according to claims 4 and 10, characterized in that before being put in competition, the antigen to be assayed (1, l ') is treated with succinic anhydride so as to take a form anionic.

12. Immunological assay method according to claim 2, characterized in that the electrochemical detection means (15) are chosen from amperometry, coulometry, voltammetry or square wave voltammetry.

13. Immunological assay method according to claims 4 and 6, characterized in that the detection means are square wave voltammetry.

14. Immunological assay method according to claim 4, characterized in that the redox system (3, 3 ') is procationic and in that the electrode (13) is maintained at a positive potential during the accumulation period.

15. Immunological assay method according to claim 3, characterized in that the system redox (3, 3 ') is cationic and in that the electrode (13) is kept in open circuit during the accumulation period.

16. Immunoassay method according to any one of the preceding claims, charac¬ terized in that the electrode (13) coated with the poly¬ ionic film (11) is for single use.

17. Immunological assay kit for at least one antigen (1, l ') comprising at least one functional group, characterized in that it comprises:

- At least one support or container (40) having at least one type of complex (9, 9 ') anti¬ body (5) -antigen (7, 7') marked by a redox system one of the forms of which is ionic (3, 3 '), the antigen (7, 7') corresponding to that (1 „l ') which one wishes to assay and

- An electrode (13) comprising a polyionic compound (11) of opposite polarity to that of said redox system (3, 3 ').

18. Dosing kit according to claim

17, characterized in that the electrode (13) is coated with a polyionic film (11).

19. Dosing kit according to claim

18, characterized in that the redox marking system is cationic or procationic and in that the film

(11) covering the electrode (13) is polyanionic.

20. Assay kit according to claim

19, characterized in that the polyanionic film (11) covering the electrode (13) is a perfluoropolymer of formula:

- [(CF ₂ -CF ₂ -

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

21. Dosing kit according to claim 17, 18, 19 or 20, characterized in that it comprises:

- A series of sample tubes (42, 44) containing known quantities of at least one of the anti-genes (1, l ') to be assayed, in order to allow the production of a sampling curve.

22. Dosing kit according to claim

17, characterized in that the redox labeling system (3, 3 ′) is chosen from ferrocene, cobalticinium, pyrrolidinyloxy or viologene derivatives.

23. Assay kit according to claim 17, characterized in that the antigen (1, l; 7, 7 ') is a molecule comprising at least one primary or secondary amine function or a carboxylic function.