EP0715718A1 - Method for identifying an immunological substance using a multimembrane system - Google Patents

Abstract

An identification method wherein (i) an aqueous mixture of a liquid sample thought to contain the immunological substance to be determined [i.e. X] and a conjugate [anti(X)] thereof coupled to a marker [E*], said mixture being such that the reaction X+anti(X)-E* → X-anti(X)-E* produced a relatively large amount of an X-anti(X)-E* complex, is distributed over (ii) a selective phase consisting of a porous membrane including an immobilised immunological reagent selected from the group consisting of (a) immunological materials that react specifically with conjugate [anti(X)] and the conjugate coupled to a marker [anti(X)-E*] but do not react with the product of said reaction [X-anti(X)-E*], or (b) immunological materials that react specifically with said immunological substance and the product of said reaction [X-anti(X)-E*], but do not react with said conjugate [anti(X)] and said conjugate coupled to a marker [anti(X)-E*].

Description

METHOD FOR IDENTIFYING AN IMMUNOLOGICAL SUBSTANCE USING A MULTI-MEMBER SYSTEM

FIELD OF THE INVENTION The present invention relates to a new method for identifying an immunological substance chosen from the group consisting of antigens and antibodies, said method using a multimembrane system for visually separating and detecting the product of an immunological reaction: antigen + antibody -> antigen-antibody It also relates, as new industrial products, to said multimembrane system, on the one hand, and to the kits or assay kits containing said multimembrane system, on the other hand. PRIOR ART

Technical solutions are known for the identification of an immunological substance chosen from the group consisting of antigens and antibodies, which employ a system of porous lamellar layers.

Patent issued US-A-4,446,232 (Lance A. LIOTTA) recommends a multimembrane device for the identification of an antigen, which comprises: - a first porous zone which contains an antigen and an antibody coupled to a labeling enzyme , said antigens and antibodies being capable of reacting immunologically with one another, said first zone comprising at least two layers: an upper layer containing the antibody coupled to an enzyme, and a lower layer containing the immobilized antigen specific for said antibody, and - a second porous zone containing the substrate of the labeling enzyme and its developer. According to US-A-4 446 232, the antibody coupled to an enzyme, which is an immunological substance comprising two active sites, one of these sites being used for coupling with the labeling enzyme, the other for the bond with the antigen likely to be present in the liquid sample to be analyzed, being such that it (i) crosses the first zone when it reacted with the antigen to be identified if the latter is present in said sample, or (ii) does not cross said first zone due to its fixation on the immobilized antigen when it has not reacted with the antigen to be identified if the latter is not present in said sample. This multimembrane device is also useful, according to US-A-4,446,232, for the identification of an antibody when it is replaced in the first zone. on the one hand the antibody coupled to an enzyme by an antigen coupled to the labeling enzyme, and on the other hand, the antigen not immobilized by an antibody not immobilized.

US-A-4 446 232 further indicates that the two porous zones may each consist of paper, in particular a nitrocellulose paper or a so-called diazobenzyloxylated paper (see column 4 lines 40-47 and example 1).

The main drawback of the multimembrane device according to US-A-4,446,232 lies in the fact that this device has poor sensitivity. First, the liquid sample containing the antigen to be identified passes too quickly through the first zone, in particular through the nitrocellulose membrane where the antibody material coupled to an enzyme is present: consequently the reaction of said antigen to be identified with said antibody coupled to an enzyme either is incomplete or could not be carried out. Secondly, having regard to the operating methods provided in Example 1 of US-A-4,446,232, a non-negligible part of the antibody coupled to the labeling enzyme is immobilized by adsorption on the membrane (s) of the first zone.

Another technical solution is described in the published patent application FR-A-2 666 896 (Jacques TOLEDANO). It implements a multimembrane device for the identification of an antigen (or vice versa of an antibody) capable of being contained in an aqueous liquid sample, said device, in which a plurality of membranes is provided, delaying the passage of said sample. More precisely, according to the indications provided in the description of FR-

A-2 666 896 (see page 3 lines 13-50), this multimembrane device includes:

a filter (A), in particular made of cellulose, polyamide or polycarbonate, intended to receive the liquid sample to be analyzed and capable of containing an antigen to be identified,

a double permeable membrane (B) which is underlying the filter (A) and is formed of a first sheet (B1) comprising a mobile or immobilized reagent (RI) recognizing said antigen to be identified, on the one hand, and a second sheet (B2) which is underlying the first sheet (B1) and comprises a reagent (R2) complementary to (RI), the contact of said antigen to be identified with the reagent (RI) transmitting a signal to the reagent (R2) which in turn activates the mechanisms of the underlying layers, on the other hand,

- a film (Ml) with programmed lysis, which is underlying the sheet (B2), this film (Ml) being at least waterproof vis-à-vis the liquid sample to be analyzed serving here as eluent, then being degraded by said liquid sample so as to allow its passage to the next layer,

- a layer or zone (C), which is underlying the film (Ml) and contains a combination of reagents capable of becoming active in the presence of the signal conveyed by said liquid sample having passed through (Ml), - a film (M2 ) at programmed lysis, which is underlying the layer or zone (C) and similar to the above-mentioned film (Ml), and

- A porous zone (D) comprising the means for revealing the presence or absence of the antigen in the liquid sample to be analyzed.

The technical solution of FR-A-2 666 896 overcomes, thanks to the films Ml and M2, the aforementioned major drawback of the technical solution of US-A-4 446 232 with regard to the completion of the reaction of the antigen to be identified with an antibody coupled to a non-immobilized enzyme, or a sufficient evolution of said reaction. This technical solution which uses a membrane supporting an antibody coupled to a labeling enzyme and not immobilized and an anti-enzyme antibody immobilized on another membrane, has the drawback of giving rise to parasitic reactions with enzymes contained in the sample to be analyzed, either at the level of the immunological reaction of the abovementioned antigen-antibody type, or (above all) at the level of the revelation of the labeling enzyme. On the other hand, when the said membranes M1 and M2 are not used, the technical solution of the multimembrane system of FR-A-2 666 896 is defective in view of the small thickness (less than 0.1 mm) of the membrane (B1 or B2) which comprises the immobilized immunological reagent (RI or R2). When the membranes Ml and M2 are not used. the passage of the liquid through a porous membrane with a thickness of less than 0.1 mm is so rapid that the immunological reaction is poor and that said porous membrane is used at best at 0.01 to 0.1% of its capacity. For example 50 to 100 μg of immobilized DDi retain only 3 to 5 ng of anti-monoclonal antibody conjugate (DDi) coupled to peroxidase.

There is therefore a need to improve the sensitivity and reliability of these technical solutions by more particularly avoiding unwanted phenomena. that constitute (i) immobilization by adsorption of each immunological material which must remain mobile, (ii) the release of each immunological reagent which must remain immobilized, and (iii) parasitic reactions. PURPOSE OF THE INVENTION According to the invention, it is proposed to provide a new technical solution different from the previously known solutions which is more sensitive and more reliable.

According to a first aspect of the invention, it is proposed to provide a method for identifying an immunological substance belonging to the set of antigens and antibodies and capable of being contained in a liquid sample to be analyzed.

According to a second aspect of the invention, it is proposed to provide a multimembrane device, as a new industrial product, for the implementation of said method. According to another aspect of the invention, it is proposed to provide a kit, kit or assay kit containing said multimembrane device with if necessary (i) one or more complementary reagents, and / or (ii) a dilution medium.

OBJECT OF THE INVENTION The new technical solution which is recommended according to the invention comprises the distribution (i) of an aqueous mixture of a liquid sample capable of containing an immunological substance to be identified [i.e. X] with a conjugate [anti (X)] of said immunological substance coupled to an enzyme [E *], said mixture being such that the reaction X + anti (X) -E * -> X-anti (X) -E * could have been triggered and lead to the production of a relatively large quantity of X-anti (X) -E complex *, on (ii) a selective phase constituted by a porous membrane comprising an immobilized immunological reagent which is chosen from group consisting of (a) immunological materials which react specifically with the conjugate [anti (X)] and the conjugate coupled to an enzyme [anti (X) -E *], but which do not react with the above-mentioned reaction product [X-anti (X) -E *], or

(b) immunological materials which react specifically with said immunological substance and the product of the aforementioned reaction [X- anti (X) -E], but not reacting with said conjugate [anti (X)] and said conjugate coupled to an enzyme [anti (X) -E *]. The term conjugate, as used here, conforms to the definition given below at the beginning of the chapter entitled "Detailed description of the invention". According to the invention, a method of identifying an immunological substance (X) belonging to all of the antigens and antibodies and therefore capable of being present in a liquid sample to be analyzed is therefore recommended, said method, which highlights performs the reaction of said immunological substance with one of its conjugates [anti (X)] and the use of a multimembrane system, being characterized in that it comprises the steps consisting in

(1 °) bringing into contact the liquid sample capable of containing said immunological substance (X) to be identified with one of its conjugates coupled to an enzyme of formula anti (X) -E * where anti (X) represents a conjugate bifunctional and E a labeling enzyme, so as to obtain the reaction product of formula

X-anti (X) -E * when said immunological substance is present; and, (2 °) passing the reaction medium thus obtained over a multimembrane device comprising:

a first zone comprising a porous membrane provided with an immobilized immunological reagent which is chosen from the group consisting of

(a) immunological materials which react specifically with the conjugate (anti (X)) and the conjugate coupled to an enzyme (anti (X) -E *), but which do not react with the product of the above-mentioned reaction (X-anti (X) -E *), or

(b) immunological materials which react specifically with said immunological substance and the aforementioned reaction product (X-anti (X) -E *), but do not react with said conjugate (anti (X)) and said conjugate coupled to an enzyme (anti (X) -E *), the ratio “weight quantity of said immobilized immunological reagent / surface of the porous membrane provided with said immunological reagent” being between 10 and 150 μg / aτfi, and the active sites of said membrane not used to immobilize said immunological reagent being blocked; and,

a second zone comprising at least one porous membrane, which is underlying the membrane containing said immobilized immunological reagent and which contains a specific substrate for the labeling enzyme (E *) for revealing the presence of said enzyme.

A variant of this method is also recommended according to which a mixture of the sample to be analyzed is prepared in step (1 °) capable of containing the immunological substance X to be identified with a conjugate directed against anti (X) and coupled to the labeling enzyme, this conjugate being a complex corresponding to the formula anti [anti (anti)) - E * and such that

(a) in the absence of X, it reacts with anti (X), and

(b) in the presence of X, it can react with anti (X) only after the complete fixation of X by anti (X), then reacts in step (2 °) said mixture with the anti compound (X ) immobilized at the level of a porous membrane, before proceeding to reveal the anti [anti (anti (X)] - E * complex conjugate not retained by the immobilized anti (X) reagent due to the presence of X (generally in excess ). In other words, according to this variant, a method is provided which comprises the steps consisting in:

(1 °) bringing the liquid sample capable of containing said immunological substance (X) to be identified into contact with a complex conjugate of formula anti [anti (X)] - E * where E * and anti (X) are defined as indicated above, said complex conjugate reacting with the conjugate [anti (X)] directed against said immunological substance (X) in the absence of said substance (X) but not reacting with said conjugate in the presence of said substance (X) free; and,

(2 °) passing said mixture of the sample to be analyzed with said complex anti (X) over a multimembrane device comprising - a first zone comprising a porous membrane comprising the conjugate [anti (X)] as an immunological reagent, the weight quantity of said immobilized immunological reagent / surface area of the porous membrane provided with said immunological reagent being between 10 and 150 μg / cm *****, and the active sites of said membrane not used for immobilizing said immunological reagent being blocked; and, - A second zone comprising at least one porous membrane, which is subjacent to the membrane containing said immobilized immunological reagent and which contains a specific substrate for the labeling enzyme (E) for revealing the presence of said enzyme. According to the invention, a multimembrane system is also recommended which comprises the first and second areas mentioned above.

Finally according to the invention, it is also recommended a kit, kit or assay kit for the identification of an immunological substance in accordance with the above process, said kit, kit or assay kit being characterized in that it includes said multimembrane system and, where appropriate, additional reagents and / or a dilution medium. ABBREVIATIONS

For convenience, the following abbreviations have been used in the present invention. A AEECC denotes 3-amino-9-ethylcarbazole; bCG denotes the gonadotropin of the bovine chorion; BCIP denotes 5-bromo-4-chloro-3-indolyl phosphate; BSA denotes bovine serum albumin (from English: "bovine serum albumin"); C CMMVV denotes a cytomegalovirus; D denotes a particular monomer fragment belonging to the set of FnDPs;

DDi denotes a particular dimeric D fragment belonging to the set of FnDPs;

EIA designates an enzyme immunoassay (from the English: "enzymeimmunoassay");

F (ab) denotes a fragment of an antibody of the Ig type comprising the branches a and b and obtained by cleavage of said antibody by papain; F (ab ') 2 denotes a fragment of an antibody of the Ig type comprising the branches a and b and obtained by cleavage of the said antibody by pepsin; Fc denotes a fragment of an antibody of the Ig type essentially constituted by the branch c and obtained by chemical or enzymatic cleavage; the Fc fragments are homologous but structurally different according to the mode of cleavage [see for this purpose the indications provided in the publication FR-A-2 645 647 as regards the Fc separated from F (ab) and F (ab ') - * ^* *];

FDP designates degradation products of the FgDP or FnDP type; FgDP designates the fibrinogen degradation products; FnDP designates fibrin degradation products; GMV denotes a rubella virus (from the English: "German measles virus");

HBcAb is a core antibody to the hepatitis B virus (in English:

"hepatitis B core antibody");

HBcAg denotes a hepatitis B virus core antigen (in English: "hepatitis B core antigen");

HBsAb denotes a hepatitis B virus surface antibody (in English: hepatitis B surface antibody ");

HBsAg is a hepatitis B virus surface antigen (in English:

"hepatitis B surface antigen"); hCG denotes the gonadotropin of the human chorion, (in English: "human chorionic gonadotropin");

HCV is a hepatitis C virus; HIV denotes a human immunodeficiency virus (in English: "human immunodeficiency virus");

HSV denotes a herpes virus (in English: "herpes simplex virus");

I denotes an immunoglobulin;

^IV A denotes an influenza A virus (in English: "influenza A virus");

IV B denotes a type B influenza virus (in English: "influenza B virus");

MES denotes 2- (N-morpholino) ethanesulfonic acid; MPB denotes methoxynaphthyl-galactoside; NTB denotes tetrazonium blue (in English: "nitro-blue tetrazonium");

OD denotes optical density; PAI denotes an inhibitor of plasminogen activators; PAI-1 denotes inhibitor 1 of plasminogen activators. complex-forming substance (tPA-PAI-1) and (uPA-PAI-1) with tPA and uPA respectively;

PBS denotes a physiological phosphate buffer solution (in English: "phosphate-buffered saline");

PVP denotes polyvinylpyrrolidone;

RF stands for rheumatoid factor;

RIA means a radioimmunoassay (from English:

"radioimmunoassay"); RSV is a respiratory syncinesia virus (in English:

"respiratory sincitial virus");

RT indicates the ambient temperature (15-25 ° C), preferably RT will be between 18 and 25 ° C;

TMB denotes 3, 3 ',, 5' -tetramethylbenzidine; tPA designates any tissue type plasminogen activator (English: "tissue plasminogen activator") and includes sctPA (tissue type plasminogen activator with single-strand structure, in English: "single-chain tissue plasminogen activator") and tctPA (tissue type plasminogen activator with double strand structure, in English: "two-chain tissue plasminogen activator");

TV denotes a toxoplasmosis virus; uPA denotes any urokinase-type plasminogen activator (from English: "urokinase plasminogen activator") and includes scuPA (urokinase-type plasminogen activator with single-strand structure, in English: "single-chain urokinase plasminogen activator", also called prourokinase) and tcuPA (urokinase-type plasminogen activator with double-stranded structure, in English:: two-chain urokinase plasminogen activator ", also called urokinase);

9C3 monoclonal antibody specific for DDi sold by the company known as DIAGNOSTICA STAGO);

2F7 monoclonal antibody specific for DDi sold by the company called DIAGNOSTICA STAGO); 7D4 monoclonal antibody specific for PAI-1 (marketed by the company called DIAGNOSTICA STAGO); 7F5 monoclonal antibody specific for PAI-1 (marketed by the company called DIAGNOSTICA STAGO). BRIEF DESCRIPTION OF THE DRAWINGS

In the accompanying drawings, in no way limiting, FIGS. 1-5 schematically illustrate multimembrane systems according to the invention, in which the immunological reagent is immobilized essentially on the upper surface and / or in the mass of the porous membrane comprising it;

- Figures la-4a and lb-4b respectively illustrate the reaction mechanisms involved in the multimembrane systems of Figures 1-4 corresponding, when the latter are used in a "negative" test (absence of immunological substance X in the sample to analyze) and respectively in a "positive" test (presence of said immunological substance in the sample to be analyzed);

- Figures 5a and 5b schematically illustrate the mechanisms involved in the multimembrane system of Figure 5, when the concentration of the immunological substance X, present in the sample to be analyzed is lower (Figure 5a: "negative" test) or higher (Figure 5b: "positive test") at a predetermined concentration; and,

- Figures 6, 7 and 8 schematically illustrate in section multi-membrane systems according to the invention, in which the immobilized immunological reagent is disposed essentially in the mass of the porous membrane comprising it.

DETAILED DESCRIPTION OF THE INVENTION

By the expression "immunological substance" is meant here any element of a couple (antigen / antibody) involved in an immunological reaction: antigen + antibody> antigen-antibody Couples (antigen / antibody) include here not only couples

(antigenic substance / antibody) but also the immunological couples (agomste / antagonist) or (conjugated / anticonjugate) such as the couples (biotin / avidin) and (activator / inhibitor) [in particular the couples (tPA / PAI)].

By "antigenic substances" here is meant the antigens themselves, on the one hand, and the substances from which antibodies can be generated, on the other hand. Among these latter substances, mention may in particular be made of haptens, peptides, medicaments comprising at least one peptide fragment. alkaloids and generally any substance with an immunological structure. By "conjugate" of a given substance (X or respectively anti-X) is meant a partner [anti-X or respectively anti (anti-X)] specific for said substance in an immunological reaction. According to this definition, the term "conjugate does not necessarily imply that said pair is coupled to a marker, of the enzyme type for example. The preferred pairs (antigen / antibody) according to the invention are the pairs

(antigenic substance / antibody) and (antibody / anti (antibody)).

The immunological substance to be identified may be an antigenic substance. such as in particular DDi, FgDP, FnDP. Protein C, Protein S. α- antitrypsin, hCG, bCG, PAI (in particular PAI-1), tPA (especially sctPA and tctPA), uPA (especially scuPA and tcuPA), an antigen of bacterial or viral origin such as HBcAg , HBsAg, IV ^ antigen, IVg antigen or RSV antigen, or an antibody present in or directed against an envelope or nucleus poπion of bacteria, mold or virus such as HBcAb, HBsAb, CMV antibody, GMV, HCV , HIV (especially HIV-1, HIV-2 or HIV-3), HSV, MCV, RCV or TV.

In practice, said immunological substance is here an immunological material intervening (i) in the mechanisms of hemostasis, (ii) in the bacteriological or virological field, (iii) in the mechanisms of fertilization / implantation, or also (iv ) the food industry (research in particular for the presence of an antigen or antibody specific for the strains of

Neisseria).

The conjugate of said immunological substance is advantageously here a monoclonal antibody directed against (in English: "raised against") said immunological substance. Said monoclonal antibody as used according to the invention is a highly purified and bifunctional material. The monoclonal antibody conjugate coupled to an enzyme of formula anti (X) -E as used herein must be purified. It is indeed important that this antibody coupled to an enzyme is essentially devoid of free enzyme to avoid parasitic staining of the developer of the labeling enzyme. The sample likely to contain the immunological substance to be identified is an aqueous liquid, in particular a body liquid such as in particular blood (mainly plasma, and if necessary serum), urine, saliva, sweat, milk, or an aqueous liquid, synthetic or semi-synthetic medium containing a product, in particular food, of vegetable or animal origin, in liquid or ground form.

In the selective phase, the immobilized immunological reagent is placed on the upper surface of the porous membrane and / or in the mass of said porous membrane. In a first variant, the immunological reagent immobilized at the level of the selective phase is chosen from immunological materials which react specifically with said anti (X) conjugate and said conjugate coupled to an anti (X) -E enzyme but do not react with the product of the reaction of step (1 °), namely X-anti (X) -E *. In this variant, the immobilized immunological reagent is then

- the immunological substance (X) itself,

a conjugate of said conjugate, namely (i) an antibody [anti (anti (X))] directed against the antibody [anti (X)] of step (1 °), or (ii) a protein or a peptide reacting specifically with respect to said conjugate [anti (X)]. The anti [anti (X)] reagent, which is immobilized at the level of the porous membrane of the first zone, will advantageously be a monoclonal or polyclonal antibody directed against a fragment or an active site of the anti (X) antibody, namely especially :

- an anti [F (ab ') 2] antibody, - an anti-kappa chain antibody, or

- an anti-idiotype antibody.

The use of a protein, a peptide or a peptidominetric compound of the anti-idiotype poπion, reacting specifically with anti (X) and anti (X) -E but not reacting with X-anti (X) -E makes it possible to improve, like the aforementioned anti [anti (anti)) reagents, the sensitivity of the determination of the immunological substance X.

According to this first variant, the negative results (absence of X) result in an absence of coloration, and the positive results (presence of X in the sample to be analyzed) result in the development of coloration. In a second variant, the immobilized immunological reagent is chosen from antibodies directed against the immunological substance to be identified, and advantageously specific to an epitope different from the epitope against which the anti (X) antibody intervening in the reaction was generated. step (1 °). According to this second variant, the negative results (absence of X) result in the development of a coloration, and the positive results (presence of X in the sample to be analyzed) result in an absence of coloration.

When finally the complex conjugate X- [anti (anti (X))] - E intervenes in step (1 °) in admixture with the liquid sample to be analyzed, it will advantageously be obtained by coupling with the labeling enzyme d '' a specific antibody, namely:

- a monoclonal or polyclonal anti [F (ab ') 2_ antibody,

- an anti-kappa chain monoclonal antibody, or

- an anti-idiotype monoclonal or polyclonal antibody, then reaction with substance X from the sample to be analyzed.

In general, the anti (X) material, which is used according to the invention, is a bifunctional or divalent immunological substance, that is to say a substance comprising two active sites: a first active site for reacting with X or anti [anti (X)] and a second active site serving for the binding with the labeling enzyme or for immobilization at the level of the porous membrane of the selective phase.

The membrane of the selective phase has according to the invention a thickness greater than or equal to 0.1 mm and in general less than or equal to 10 mm. A thickness of 0.1 mm constitutes a threshold below which the porous membrane of the selective phase gradually loses its capacities and is therefore unusable.

Preferably, a porous membrane having a thickness between 0.2 and 10 mm and better a thickness between 0.2 and 1 mm will be used for the selective phase.

In addition, the porous membrane of the selective phase has a porosity (evaluated by comparison with the diameter of its pores) of between 0.2 and 10 μm and preferably between 0.3 and 6 μm, and better still a porosity of less than 1 μm. (in particular 0.3-0.8 μm).

This membrane is used for fixing either by covalence or by adsorption of the immunological reagent to be immobilized on its upper face and / or in its mass. Among the membranes useful for immobilization by covalent, mention may in particular be made of porous membranes of the polymer or copolymer type such as VERSAPOR ^R (acrylic membranes sold by the company called GELMAN), LOPRODYNE ^R (polyamide membranes sold by the company called PALL), TRUFFIN ^R , SUPPOR ^R and THERMAPOR ^R (polysulfone membranes sold by the company called GELMAN).

The membranes specially adapted for the field of immunodiagnosis and which are advantageous according to the invention, comprise in particular nitrocellulose membranes, for immobilization by adsorption (in particular the mtrocellulose membranes sold by the company known as SCHLEICHER & SCHUELL), and for immobilization by covalence of the membranes IMMUNODYNE ^R (porous polyamide membranes marketed by the company called PALL), IMMOBILON ^R (porous membranes in hydrophilic polyfluorocarbon marketed by the company called MILLIPORE) and ULTRABIND ^R (porous polysulfone membranes marketed by the company called GELMAN) . These membranes can be suppoπées. The suppoπ (constituted by a polymer material, for example made of polyamide, making the membranes more manipulable and less fragile), is in particular incorporated in the nitrocellulose membranes and the IMMUNODYNE ^R membranes (which allows use on both sides, a of the faces being however more reactive than the other), or linked to one of the faces as is the case in the ULTRABIND ^R membranes (in this case one can only use for immobilization the other face).

The reaction mixture, which is distributed at the level of the porous membrane of the selective phase passes through by (i) capillarity or (ii) by capillarity and gravity, said porous membrane is perpendicularly (ie in the thickness direction, it is that is to say in a direction perpendicular to the plane of the membrane) or, more advantageously, longitudinally (ie in the direction of the length of the membrane). As indicated above, the weight quantity of immunological reagent immobilized at the level of the selective phase, compared to the surface unit (ie "surface density" or "surface concentration"), is between 10 and 150 μg / cm ² . The surface of the membrane of the selective phase is here that of the upper or lower face; it is not that of the edge of said membrane. Advantageously, this surface density will be between 20 and 100 μg / cm ^. From a practical point of view, the surface density is a function of the immobilization method chosen. When a membrane is immersed in a liquid medium containing the immunological material which one wishes to immobilize in such a way that the height of the liquid extends up to approximately 2 mm above the surface of the membrane to be treated, it is found in many cases that covalently the surface density of the immobilized product increases up to a certain value and that it decreases or is constant beyond this value; there is a phenomenon of salting out or saturation even before all the active sites of the membrane are completely saturated. In short, by covalence, an impregnation liquid containing more than 150 μg / ml of immunological reagent cannot be used. When the immobilization of the immunological reagent by adsorption is carried out under the same conditions, it is found that the immobilization is no longer reliable when the impregnation liquid contains more than 400 μg / ml of immunological reagent. For this purpose, see the immobilization tests on the membrane provided below.

In general, immobilization by adsorption provides a surface concentration in general greater than immobilization by covalence, however immobilization by adsorption is more sensitive to the phenomena of salting out than immobilization by covalence. This explains why it is advisable to limit the surface concentration of immunological reagent immobilized by covalence or adsorption.

Finally, it is particularly important that the active sites of the membrane of the selective phase remaining still free after the immobilization of the immunological reagent are essentially all blocked. This blocking or saturation is required to avoid any parasitic immobilization during the implementation of the method of the invention. Among the means which are suitable for blocking said active sites which are still free, there may be mentioned in particular those which are indicated in US-A-4 175 112. The blocking means suitable for this purpose are, in order of preferably increasing, TWEEN ^R 20, polyvinylpyrrolidone, casein, serum bovine albumin and diet milk powder. REGILAIT ^R diet milk powder used at a concentration of 1 to 5% w / v has proven to be the most effective means here.

According to a variant (variant A) of implementation of the method of the invention, one or more drops of the mixture (which consists of the liquid sample and the conjugate of the immunological substance [X] coupled to a enzyme, of formula [anti (X) -E *], which contains the product of the immunological reaction [X-anti (X) -E *] when the immunological substance X is present in said sample, and which was prepared in a suitable reactor, in particular a flask, a cuvette or a tube) at the level of the membrane of the selective phase comprising the immobilized immunological reagent.

According to another variant (variant B), said mixture is produced by pouring one or more drops of the liquid sample, capable of containing the immunological substance to be identified, onto another porous membrane situated above the membrane of the selective phase. In this case, this other membrane has its essentially saturated active sites and comprises a deposit on its upper face, either entirely or by pad, of the conjugate coupled to an enzyme of formula anti (X) - E which is not immobilized. To slow the passage of the liquid sample over said conjugate coupled to an enzyme, it is recommended, where appropriate, to have above (preferably) and / or below (if necessary) the porous membrane supporting said deposit [ie anti (X) -E * not immobilized] one or more porous membranes of which all the active sites are substantially blocked and which are devoid of any immunological material.

In variant B, it is essential that the conjugate coupled to an enzyme [i.e. the anti (X) -E *] product is deposited without being immobilized on its suppoπ, the porous membrane.

In addition, it is advantageously recommended to coat said conjugate coupled to an enzyme with a water-permeable polymer or with a glass fiber membrane, in order to protect it (after crosslinking of the polymer when it is used) during storage and handling by the user. In general, in the absence of protection, it should not touch with the finger said conjugate coupled to a non-immobilized enzyme to avoid distorting the dosage by removal of said conjugate.

We also proceed according to the methods of variant B above when we replace in step (1 °) the non-immobilized immunological reagent of formula [anti (X)] - E * by the non-immobilized reagent of formula anti [anti (X)] - E * above.

Furthermore, according to variant B, one can also provide at the top of the multimembrane device an additional anticoφs as illustrated below in the description of FIGS. 5, 5a and 5b, from a side, and from FIG. 8, d 'other side. The labeling enzyme, in particular peroxidase, an alkaline phosphatase, β-galactosidase or urease. is revealed by means of its specific substrate. For example, when the labeling enzyme is peroxidase, the revealing complex will comprise a substrate, namely in particular the D-glucose / glucose oxidase mixture which supplies hydrogen peroxide, according to the mechanism: glucose oxidase D-glucose + 2H2O - O2> gluconic acid + H2O2 on a membrane underlying that of the selective phase, and the colored indicator, TMB, on a layer underlying that containing the substrate. Alternatively, instead of the system with two revealing membranes, a system comprising three membranes may be used: a first membrane for D-glucose, a second membrane for glucose oxidase and a third membrane for TMB. The TMB can also be replaced by the AEC. When the labeling enzyme is an alkaline phosphatase, the developer may be a BCIP / NBT mixture.

When the labeling enzyme is β-galactosidase, the colored developer may be MPB.

It is wise to protect the porous membrane comprising the colored indicator suπout from light when it is the TMB. For this purpose, the multimembrane device according to the invention will be housed in an opaque envelope, or else the reading window which must reveal the variation in color will be closed during storage by an opaque non-stick tape which must be peeled before use.

The choice of the labeling enzyme generally depends on the coφoreal liquid medium capable of containing substance X to be identified. Thus, if said coφorel liquid may contain peroxidase and / or catalase (as may be the case in the field of identification of substances involved in the mechanisms of hemostasis), it is important not to use as a labeling enzyme peroxidase. Indeed the peroxidase or catalase present in the sample would then react with the substrate of the peroxidase at the level of the second zone. In addition, since the glucose oxidase used for revealing peroxidase is also sensitive to catalase, it is important that said glucose oxidase is substantially pure.

Furthermore, like certain coφoral liquids, such as blood, can also contain an alkaline phosphatase, the preferred labeling enzyme according to the invention will be a β-galactosidase, that is to say an enzyme which is not found in blood, plasma or serum.

If the liquid sample likely to contain substance X to be determined contains an identical enzyme or which may interfere with the marker E *, it is possible to treat said sample with one of the inhibitors of said enzyme before implementing the method of l 'invention.

Furthermore, the labeling enzyme can be replaced by an equivalent labeling means. Thus E * can represent, according to the invention, colloidal gold, colloidal silver (in particular colloidal gold doped with silver) or particles of colored latex with a particle size of less than 0.1 μm (for example colored latex particles having a particle size of 25-60 nm). in this case the second zone may either be deleted or be made up of a porous membrane devoid of an enzyme substrate and which is similar in nature or analogous to the membrane of the selective phase of the first zone. The porous membranes which can be used for supposing the non-immobilized immunological reagent will have a thickness and a porosity similar to those of the porous membrane of the selective phase. From a practical point of view, it is preferred that the porosity of these membranes be greater than 1 μm and advantageously between 3 and 6 μm. The additional porous slowing membranes will have a porosity similar to that of the selective immunological phase.

For convenience, in the multimembrane systems of Figures 1-5, la-5a and lb-5b, the labeling enzyme is peroxidase. a section, and the revelation of this enzyme is carried out by means of a set of two porous membranes, one comprising the generator of H2O2 (peroxidase substrate), namely the D-glucose / glucose oxidase mixture, the other underlying component comprising the developer, namely TMB, on the other side; (we can of course use a set of three membranes here as indicated above). Of course, as already indicated above, the labeling enzyme can be different from the peroxidase and the development can be obtained with one or more membranes for the substrate / colored developer assembly. As indicated above, when the labeling enzyme is alkaline phosphatase the colored developer will be the BCIP / NBT mixture, and when the labeling enzyme is β-galactosidase the colored developer will be MPB, β-galactosidase being the preferred labeling enzyme according to the invention for the reasons given above. Still for convenience, in said FIGS. 1-5, la-5a and lb-5b, the immunological substance to be identified is presented as being an antigenic substance.

Reference is made to FIGS. 1, 1a and 1b. The multimembrane device 1 according to the invention comprises a first porous membrane 10 whose upper surface 11 and at least one poπion of the mass comprise covalently bonded (preferably) or by adsoφtion an immobilized antigen 5 identical to the antigenic substance (X) to identify and likely to be contained in a sample 4a or 4b of coφoreal fluid to be tested, of a section, and a set of two porous membranes 2 and 3, the porous membrane 2 comprising (in its mass) the substrate D- glucose / glucose oxidase, the porous membrane 3 comprising (in its mass) the colored developer TMB, on the other side.

As a variant, the multimembrane device 1 can also comprise a porous membrane 12 which comprises on its upper surface 13 a conjugate 14. The conjugate 14 is a monoclonal anticoφs 15 (directed against the antigenic substance X, referenced 5, to be identified) coupled to the labeling enzyme 6, for example peroxidase. The conjugate 14, which corresponds to the formula anti (X) -E *, is simply deposited on the upper surface 13 of the membrane 12 without being immobilized both by covalence and by adsorption. In practice, the active sites which are still free from the porous membrane 10 and all the active sites which are free from the porous membrane 12, when the latter is present, are completely and previously saturated or blocked by an appropriate means, preferably by means of REGILAIT diet milk powder. in order to substantially avoid any parasitic immobilization. The deposition of the conjugate 14 of anti (X) -E * formula on the surface 13 is carried out by zones (one or more pads, in practice a single pad is sufficient) or over the entire said surface 13. When the deposition question is produced by studs, the stud (s) are advantageously identified by a caπouche, in particular a circumference, drawn and materialized on said surface 13. When a porous membrane 12 is used as suppoπ of the anti (X) -E * conjugate, if necessary, it is recommended to have at least one additional porous membrane (not shown) above or below said membrane 12. The presence of one or more additional porous membranes identical or similar to the porous membrane 12 (in particular a porous glass fiber membrane), with blocked or saturated active sites, but lacking the conjugate 14 seπ to slow the passage, through said membrane 12, of the sample 4a or 4b to be analyzed, and to ensure sufficient production for the following stages of the X-anti (X) -E * complex produced according to the reaction:

X + anti (X) -E *> X-anti (X) -E * The figure illustrates diagrammatically the reaction mechanisms which intervene when the sample to be analyzed does not contain the antigenic substance X referenced 5; and, FIG. 1b schematically illustrates the reaction mechanisms which take place when the sample to be analyzed contains said antigenic substance X. A mixture (generally 1 drop) of ((generally 1 drop)) is distributed according to arrow 7 on the upper surface 11 of the porous membrane 10. i) the sample to be analyzed 4a not containing the antigenic substance X or 4b containing the said antigenic substance X to be identified with (ii) the conjugate 14 of formula anti (X) -E *, the reaction medium containing either the said conjugate 14 ( absence of the antigenic substance (X)) according to FIG. 1a, ie said conjugate 14, the antigenic substance X and the reaction product of formula X-anti (X) -E (presence of the antigenic substance (X) in the sample to be analyzed) according to Figure lb.

As a variant, it is possible to distribute, on at least one poπion (or pad) of the upper surface 13 of the porous membrane 12 coated with the non-immobilized conjugate 14, the sample 4a or 4b, the resulting reaction medium passing through said membrane 12 and then flowing according to arrow 7.

At the selective phase constituted by the membrane 10 which comprises the immobilized antigenic substance X referenced 5, the mechanisms involved are as follows. According to FIG. 1a, the conjugate 14, which could not react with the absent antigenic substance X and which therefore has a free reactive function, binds to the antigenic substance X, referenced 5, immobilized and does not cross the membrane 10. Said conjugate 14 thus captured cannot be detected by the substrate / developer assembly of the porous membranes 2 and 3. The assay according to FIG. 1a constitutes a non-colored negative test.

According to FIG. 1b, the X-anti (X) -E complex formed and contained in the reaction medium is entrained without being retained by the antigenic substance X, referenced 5, in excess and immobilized, as soon as the two reactive functions of the anticoφs anti (X), referenced 15 are occupied in said complex X-anti (X) -E by the labeling enzyme and substance X. The immobilized substance X fixes the conjugate 14 which has not reacted with X. The X-anti (X) -E * complex which does not react with immobilized substance X crosses the porous membrane 10 and its enzymatic activity is demonstrated by the action of labeling enzyme on the substrate assembly revealing the porous membranes 2 and 3. The assay according to FIG. 1b constitutes a positive colored test.

We refer to Figures 2, 2a and 2b. The multimembrane device 20 according to the invention compoπe. like the device 1 of FIG. 1, porous membranes 10, 2 and 3. The porous membrane 10 comprises a monoclonal anticoφs conjugate 15 of anti formula (X) directed against the antigenic substance X referenced 5. This monoclonal anticoφs is immobilized by covalence (preferably) or by adsoφtion on the upper surface 11 and at least one poπion of the mass of the membrane 10.

As a variant, the multimembrane device 20 can also comprise a porous membrane 12 disposed above the membrane 10. On the upper surface 13 of said membrane 12 or on a poπion of said surface 13, is deposited non-immobilized a conjugate 24 conjugate 15. Conjugate 24, which is of the anti [anti (X)] - E * type, is constituted here by a monoclonal or polyclonal anti [F (ab ') 2] referenced 25 directed against the F (ab ') 2 of the anti-monoclonal anticoφs anti (X) 15 and coupled to the labeling enzyme 6. If necessary, one or more additional porous slowing membranes are arranged above and / or below the porous membrane 12; each additional slowing membrane is identical or different from the membrane 12 but is devoid of the anti-anti [F (ab ') 2].

As indicated above, the deposition of the conjugate 24 can be carried out on the entire upper surface 13 of the membrane 12 or on a poπion of said surface 13.

The active sites still free of the membrane 10 after immobilization of anti (X) and the active sites of the other membranes (ie the membrane 12 and the additional porous slowing membranes) when these are present, are saturated, preferably by means REGILAIT ^R , prior to mounting or assembling the multimembrane device.

The conjugate 25, namely the anticoφs of formula anti [F (ab ') 2] ^a ^ c * ¹⁰ *** ⁵ * according to its affinity with respect to anti (X) compared to that of X. In the absence of X in the sample, anti [F (ab ') 2l is fixed on anti (X). In the presence of X in the sample, anti [F (ab ') 2] can only be fixed on anti (X) after the fixation of substance X available on anti (X). In other words, anti [F (ab ') 2] has an affinity for anti (X) lower than that of X. Thus, according to the multimembrane device illustrated in FIGS. 2, 2a and 2b, when the reaction medium contains the anti [F (ab ') 2] -E * conjugate without containing the antigenic substance X, the anti-monoclonal anticoφs anti (X) fixes said anti [F (ab') 2] -E * conjugate present , and when the reaction medium simultaneously contains the antigen X and the anti [F (ab ') 2] -E * conjugate, said anti-monoclonal anticoφs anti (X) preferentially fixes the antigen X. It then suffices to have an excess of X so as not to fix the anti [F (ab ') 2] -E * conjugate on the immobilized anticoφs, referenced 15, anti (X). Is distributed according to arrow 7 on the upper surface 11 of the porous membrane 10 a mixture (in general 1 drop) of (i) the sample to be analyzed referenced 4a not containing the antigenic substance X or referenced 4b containing said antigenic substance X to be identified with (ii) conjugate 24 of anti formula [F (ab ') 2] -E *, the resulting reaction medium containing either said conjugate 24 (sample 4a devoid of X) or the mixture of said conjugate 24 and said substance antigenic X (sample 4b containing X).

As a variant, it is possible to distribute, on at least one poπion (or pad) of the upper surface 13 of the porous membrane 12 coated with the non-immobilized conjugate 24, the sample 4a or 4b, the resulting reaction medium passing through said membrane 12 and then flowing according to arrow 7.

At the selective phase that constitutes the membrane 10 which compoπe on its upper surface and in at least one poπion of its mass the immobilized anticoφs anti (X) referenced 15, the mechanisms involved are as follows. According to FIG. 2a, the anti [F (ab ') 2] poπion of the anti [F (ab') 2J-E * conjugate is recognized by the immobilized antiicoφs anti (X) and the labeling enzyme 6 of the resulting complex cannot be revealed at the porous membranes 2 and 3. The assay according to FIG. 2a constitutes a non-colored negative test.

According to FIG. 2b, the antigen X comes to saturate the immobilized anti-monoclonal anticoφs (X) by preventing the attachment of the anti conjugate [F (ab ') 2] -E *. Consequently, anti [F (ab ') 2] -E * crosses the porous membrane 10 and its enzymatic activity is demonstrated by its action on the substrate / developer assembly of the porous membranes 2 and 3. The assay according to the figure 2b constitutes a positive colored test. The multimembrane device 30 illustrated in FIGS. 3, 3a and 3b constitutes a variant of the device 20 in FIGS. 2, 2a and 2b, according to which the anti-monoclonal anti-antiφs (X) and anti-monoclonal or polyclonal antiφφs [F (ab ' ) 2] have been interveπis. More specifically, the selective phase of the device 30 comprises an anticoφs anti [F (ab ') 2] immobilized on the upper surface and in at least one poπion of the mass of the porous membrane 10.

Is distributed according to arrow 7 on the upper surface 11 of the porous membrane 10 a mixture (generally 1 drop) of (i) the sample to be analyzed 4a not containing the antigenic substance X or the sample 4b containing said antigenic substance X to be identified with (ii) the conjugate 34 of formula anti (X) - E, which is an anti-monoclonal anticoφs anti (X), referenced 15, directed against the antigenic substance X, referenced 5, and coupled to the labeling enzyme 6. The resulting reaction medium contains in particular said conjugate 34 according to FIG. 3a in the absence of X, or the X-anti (X) -E * complex according to FIG. 3b in the presence of X.

As a variant, as indicated above for the device 20 of FIG. 2, it is possible to distribute, over at least one poπion (or pad) of the upper surface 13 of the porous membrane 12 coated with the non-immobilized conjugate 34. sample 4a or 4b, the resulting reaction medium passing through said membrane 12 and then flowing along arrow 7.

Of course, as indicated above, one or more additional porous membranes can be provided for slowing down above and / or below the porous membrane 12; the deposition of the conjugate 34 can be carried out on the entire upper surface 13 of the membrane 12 or on a poπion of said surface 13; and the active sites still free of the membrane 10 after immobilization of anti (X) and the active sites of the other membranes (ie the membrane 12 and the additional porous slowing membranes) when these are present, are saturated, preferably at REGILAIT, prior to mounting or assembling the multimembrane device. At the selective phase constituted by the membrane 10 which comprises on its upper surface and in at least one poπion of its mass the anti-monoclonal or polyclonal immobilized anti [F (ab ') 2], referenced 25, the mechanisms involved are the following.

According to FIG. 3a, due to the absence of antigenic substance X in sample 4a, the conjugate 34, which is an anti-monoclonal anticoφs (X), referenced 15, coupled to the labeling enzyme 6, is retained by the monoclonal or polyclonal antico l'ants immobilized anti [F (ab ') • ->] referenced 25. Said conjugate 34 thus immobilized cannot be revealed by the assembly substrate / developer of porous membranes 2 and 3. The assay according to FIG. 3 a constitutes a non-colored negative test.

According to FIG. 3b, due to the presence of substance X in sample 4b, the X-anti (X) -E * complex is not recognized by the immobilized anti-monoclonal or polyclonal anticoφs [F (ab ' ) 2]. This complex therefore crosses the porous membrane 10 and its enzymatic activity is demonstrated in the membranes 2 and 3. The assay according to FIG. 3b constitutes a positive colored test. In devices 20 and 30, the anti-monoclonal or polyclonal anti [F (ab ') - ^* - *] can in particular be a rabbit or goat anticoφs directed against the F (ab') 2 fragment of an anticoφs of mouse.

Of course, the anticoφs anti [F (ab ') 2] can be replaced here by an anticoφs anti [anti (X)] equivalent having regard to the relative affinity by rappoπ to X vis-à-vis anti (X ). Among the anti [anti (anti (X)] anticoφs which are suitable for this purpose, there may be mentioned in particular the monoclonal anticoφs (in particular of rat) anti¬ kappa chain (in particular of mouse), of a pan, and the monoclonal or polyclonal anticoφs anti -idiotype of a monoclonal anticoφs (in particular of mouse) produced in another animal (in particular goat or rabbit), of other side.

Reference is made to FIGS. 4, 4a and 4b. The multimembrane device 40 is designed to implement an EIA sandwich method by means of two monoclonal antibodies reacting on sites distinct from the antigenic substance X. The device 40 of FIG. 4 comprises, like that of FIG. 2, a selective phase consisting of a porous membrane 10 comprising on its upper surface 11 and in at least one poπion of its mass an immobilized monoclonal anticoφs 15 of formula anti (X) ** -, a pan, and a revelation zone constituted here of porous membranes 2 and 3 for the substrate / developer assembly, on the other side. Said anti (X) *** monoclonal anticoφs, referenced 15, is directed against a first active site or epitope of the antigenic substance X referenced 5.

The mixture of the sample to be analyzed (sample 4a devoid of substance X, or sample 4b containing said substance X) with a conjugate 44, which is a monoclonal anticoφs of formula anti (X) 2, referenced 45, directed against a second site active or epitope of the antigenic substance X and coupled to the enzyme marking 6. is distributed according to arrow 7 on the upper surface 11 of the selective phase. This mixture contains, when the substance X to be identified is present in the sample to be analyzed, the product of the immunological reaction: X + anti (X) ² -E * -> X-anti (X) ² -E * As a variant , it is possible to distribute, on at least one poπion (or pad) of the upper surface 13 of the porous membrane 12 coated with the non-immobilized conjugate 44, the sample 4a or 4b, the resulting reaction medium passing through said membrane 12 and s' then flowing according to arrow 7.

If necessary, one or more additional porous membranes for slowing down can be arranged, as indicated above, above and / or below the porous membrane 12.

As indicated above, the deposition of the conjugate 44 can be carried out on the entire upper surface 13 of the membrane 12 or on a poπion of said surface 13. In addition, the active sites still free of the membrane 10 after immobilization of anti ( X) *** and the active sites of the other membranes (ie the membrane 12 and the additional porous slowing membranes) when these are present, are saturated, preferably by means of REGILAIT, prior to the assembly or assembly of the multimembrane device .

At the level of the selective phase, the mechanisms involved are as follows.

According to FIG. 4a, the conjugate 44, which could not react with substance X, due to the absence of X in the sample 4b, is not recognized by the immobilized monoclonal anticoφs 15; it therefore crosses the porous membrane 10 of the selective phase and its enzymatic activity is revealed by the revealing substrate assembly of the porous membranes 2 and 3. The assay according to FIG. 4a constitutes a colored negative test.

According to FIG. 4b, due to the presence of substance X in the sample 4b, the complex formed X-anti (X) ² -E * is recognized and fixed by the immobilized anticoφs anti (X) - * - according to the El A sandwich mechanism: μanti (X) l + X-anti (X) ² -E * -> | -anti (X) lX-anti (X) ² -E * where the symbol | - represents immobilization by covalence or adsorption at the porous membrane 10.

Due to the capture of the X-anti (X) ² -E * complex, the enzymatic activity of the labeling enzyme 6 thereof cannot be revealed by all substrate revealing the porous membranes 2 and 3. The assay according to FIG. 4b constitutes a positive non-colored test.

FIGS. 5, 5a and 5b illustrate a particular technique for identifying an antigenic substance X, referenced 5, which implements the methods described above with regard to the device 1 of FIG. 1.

In short, the operating methods envisaged with the multimembrane device 50 of FIG. 5 include, for the determination of a greater than normal amount of an antigenic substance X naturally present in a sample of coφorel fluid (this case occurs in particular in the field of identification of DDi in plasma),

(a) bringing the sample to be analyzed into contact 54a (which contains substance X at a naturally normal concentration) or 54b (which contains said substance X at a concentration above normal) with a conjugate 55 which is anticoφs monoclonal of formula anti (X) ¹ directed against the antigenic substance X referenced 5, to form a complex of formula X-anti (X) ¹ ;

(b) bringing the resulting reaction medium into contact with a second conjugate of formula anti (X) ² -E * and referenced 14, which is a monoclonal antibody 15 of formula anti (X) ² -E * coupled to the enzyme marking 6, the monoclonal anticoφs 15 which is identical or different from the monoclonal anticoφs 55 fixing the excess of X (by comparison with the normal concentration); and, as indicated in the description of FIGS. 1, la and lb above,

(c) the distribution, according to arrow 7, of the reaction medium resulting from the selective phase constituted by the porous membrane 10, the upper surface 11 and at least a portion of the mass of which contain substance X immobilized by covalence or adsorption; then,

(d) the revelation of the labeling enzyme 6 of the X-anti (X) ² -E * complex, neither recognized nor retained by the membrane 10 of said selective phase, by means of the substrate / developer assembly of the porous membranes 2 and 3.

Steps (a) and (b) can be carried out in a flask, microcuvette or tube. They can also be carried out directly in the multimembrane device, in this case, there is provided above the membrane 10 a set of two layers, namely (i) a second selective phase consisting of a porous membrane 510 comprising the anticoφs anti (X) ² , referenced 55, immobilized on its upper surface 511 and in at least a portion of its mass, and (ii) a porous membrane 12 comprising the conjugate 14, which is a monoclonal anti (X) ² anticoφs, referenced 15, coupled to the labeling enzyme 6 and corresponding to the formula anti (X) ² -E *. said conjugate 14 being deposited on the upper surface 13 of said membrane 12 and not immobilized on said surface 13.

Steps (c) and (d), which include (i) the passage of the reaction medium having passed through the membrane 12 or having been prepared in a reactor (flask. Microcuvette or tube) external to the multimembrane device. through the selective phase of the membrane 10, then (ii) the revelation of the labeling enzyme 6, are carried out as indicated above (see description of Figures 1, la and lb).

In short, the mechanisms involved are as follows. According to FIG. 5a, the antigen X existing in the sample 54a is picked up by the immobilized monoclonal antibody 55 of formula anti (X) ** -, and the conjugate 14 of formula anti (X) ² -E * is retained during its passage on the selective phase by the antigen X immobilized on the upper surface 11 and in at least one poπion of the mass of the porous membrane 10. As a result, there is no revelation of the enzymatic activity of the labeling enzyme 6 at the porous membranes 2 and 3. The assay according to FIG. 5a constitutes a non-colored negative test.

According to FIG. 5b, a part of the total amount of antigen X is retained by the immobilized monoclonal anticoφs 55, the rest combines with the conjugate 14 which diffuses through the porous membrane 10 without being immobilized. The enzymatic activity is then demonstrated by its action on the substrate / developer assembly of the porous membranes 2 and 3. The assay according to FIG. 5b constitutes a positive colored test.

From a practical point of view, for the identification of the factors intervening in the mechanisms of hemostasis, any of the devices 1, 20, 30, 40 and 50 of FIGS. 1, 2, 3, 4 can be used. and 5; for the identification of antigenic materials (or anticoφs) of bacterial or viral origin, it is recommended rather to use the multimembrane devices 20, 30 and 40 of Figures 2, 3 and 4 to avoid any risk of contamination of the personnel in contact with said multimembrane devices, by an antigen (or anticoφs) X, which would be immobilized at the level of the membrane 10.

Without being within the scope of the invention, the selective phases of the multimembrane devices of FIGS. 1, 1a and 1b to 5, 5a and 5b can comprise the immobilized immunological reagent either in their masses or on their upper faces. FIGS. 6 to 8 relate to multimembrane devices which each comprise a selective phase traversed longitudinally by the mixture constituted by the sample to be tested and the anti (X) or anti [anti (anti)) conjugate coupled to a non-enzyme immobilized. Reference is now made to FIG. 6 which illustrates a multimembrane device 60 according to the invention. This device 60 includes:

a well 66 formed in a covering sheet 67 and into which one or more drops (1 to 5 μl) of the sample to be analyzed are introduced, capable of containing the immunological substance X to be identified and previously mixed with a conjugate coupled to an enzyme, for example anti (X) -E * or anti [anti (X)] - E *, as a variant said conjugate coupled to an enzyme can be deposited not immobilized on a porous membrane suppoπ housed in said well 66;

- a porous membrane (called affinity) 61 intervening as a selective phase, which contains in its mass an immobilized immunological reagent, which is in capillary relation by one of its ends 63 with the well 66 and which is supplied with liquid (test sample / conjugate mixture coupled to a non-immobilized enzyme) through said well 66 when it has been loaded with said liquid;

one or more porous membranes 62 for revealing the labeling enzyme, this or these porous revealing membranes being observable by means of an opening 68 formed on the opposite side 69 of the cover sheet 67 and longitudinally offset by compared to well 66, said porous membrane (s) 62 being in capillary relation via the other end 65 of porous membrane 61 of said selective phase and being supplied with liquid reaction medium by said porous membrane 61. In the embodiment of FIG. 6 , the multimembrane device 60 is advantageously housed inside a substantially monobloc enclosure after assembly, consisting of two assemblies 167 and 169 complementary to one another and integral with one another, during the assembly in particular by means of a latching system, for example of the tenon / moπaise type not shown, and which is provided with a well 66 and an opening or fen be read 68. The crossing of the porous membrane 61 which constitutes the selective phase and which is substantially horizontal, is carried out here longitudinally.

When the conjugate coupled to an enzyme according to the multimembrane device 60 is the anti (X) -E * product, a complex of formula X-anti (X) -E * is formed in the presence of the antigenic substance X which does not is not retained by the porous membrane 61 of the selective phase, which is entrained by the liquid flow and which reaches by capillary action the porous revealing membrane (s) 62 where the labeling enzyme is revealed. The porous membrane 61 here contains in its mass the immobilized immunological reagent which is in this case either X or anti (X) as indicated above.

When the conjugate coupled to an enzyme is the anti [anti (anti) (X)] - E * product, the porous membrane 61 of the selective phase, which contains the immobilized immunological reagent of formula anti (X), fixes said anti [anti (anti (anti) X)] - E if the antigenic substance X is not present in the test sample, and does not fix said anti [anti (X)] product - E * if the antigenic substance X is present in said sample. In the latter case, the product of the X-anti [anti (X)] - E reaction not retained reaches by capillary action the porous revelation membrane (s) 62 where the labeling enzyme is revealed.

Reference is made to FIG. 7 which illustrates a multimembrane device 70 according to the invention similar to that of FIG. 6, with the difference that the porous membrane 71 is no longer substantially horizontal but is rather substantially vertical and is traversed by gravity and capillarity. The other references in FIG. 7 are those described above with regard to FIG. 6. Reference is made to FIG. 8 which illustrates a multimembrane device 80 according to the invention comprising two wells 86a and 86b on its upper part. This device includes:

- A well 86a formed in a covering sheet 67 and into which one to several drops (20 to 250 μl) of the sample to be analyzed is introduced. a first selective phase in capillary connection via one of its ends 83a with the well 86a and supplied by the latter when it has been loaded with liquid (here the aqueous sample to be tested), said first selective phase consisting of a first porous membrane 81a comprises in its mass a first immobilized immunological reagent, e.g. anti (X) - * -, \ _a quantity of said immobilized first immunological reagent being such that it corresponds to the setting of a predetermined amount of antigenic substance X,

a second well 86b formed in the covering sheet, longitudinally offset by comparison with the first well 86a, in capillary connection with the other end 83b of the porous membrane 81a of the first selective phase, supplied with liquid flow by said first selective phase , and comprising in its bottom a conjugate coupled to a non-immobilized labeling enzyme, for example anti (X) ² - E *. said conjugate coupled to a non-immobilized labeling enzyme which may be in particular in the form of a microporous pellet (i) whose upper surface is protected by a glass fiber membrane and (ii) which is disposed in well 86b before dosing,

a second selective phase in capillary connection via one of its ends 85a with the well 86b, supplied by the latter in liquid flow (reaction medium having passed through the first selective phase and having been brought into contact with said conjugate coupled to a non-immobilized labeling enzyme present in said well 86b) and consisting of a second porous membrane 81b comprising in its mass a second immobilized immunological reagent, for example X, said second porous membrane 81b fixing said conjugate coupled to an enzyme not immobilized in the absence of antigenic substance X in the reaction medium leaving the first selective phase, but not fixing said conjugate coupled to an enzyme which reacted in the presence of the antigenic substance X present in said reaction medium leaving said first selective phase, and

one or more porous membranes 62 for revealing the labeling enzyme, this or these porous revealing membranes being observable in an opening 68 formed on the opposite side 69 to the cover sheet 67 and longitudinally offset by comparison with the wells 86a and 86b, the porous revelation membrane (s) 62 being in capillary relation with the other end 85b of the second porous membrane 81b and being supplied with liquid reaction medium by said second porous membrane 81b.

The multimembrane device 80 of FIG. 8, as well as that of FIG. 7, can consist of two sets 167 and 169 which are complementary and can be joined together, as indicated above in the description of the multimembrane device 60 of Figure 6.

In general, to produce a one-piece multimembrane device (in particular made of plastic), it suffices that a conduit is formed at the interface of the assemblies 167 and 169 for housing each of the porous membranes 61, 71, and 81b between a well 66 or 86b and an opening 68 or even between two wells 86a and 86b. In addition, the housings intended for the substantially horizontal porous membranes 61, 81a and 81b can be inclined so as to allow passage by capillarity and gravity through said membranes. Of even the housing of the porous membrane 71 of Figure 7 can be inclined by rappoπ to the veπicale.

Of course, the material of the assemblies 167 and 169 must not give rise to parasitic immobilizations by covalence or adsorption. If necessary, it will be treated at the level of the wells, reading openings and housings of the porous membranes of the selective phases using an appropriate means such as milk powder.

REGILAIT ^R above.

The multimembrane device 80 of FIG. 8 is particularly effective for the determination of DDi which may be present in the plasma at a concentration greater than normal. The plasma sample to be analyzed is deposited in well 86a; the sample migrates longitudinally through the membrane 81a containing in its mass an anti-monoclonal anticoφs (DDi), here the anti-monoclonal 9C3, to retain a given quantity of DDi; the resulting reaction medium reaches well 86b where there is an anti-monoclonal anti (DDi) coupled to a labeling enzyme, here the anti-monoclonal 2F7 coupled to β-galactosidase; the resulting reaction medium migrates longitudinally through the membrane 81b containing in its mass the immobilized DDi antigen, this membrane 81b (i) retaining the conjugate 2F7- (β-galactosidase) when all of the DDi present in the plasma sample has been fixed to the membrane 81a and (ii) not retaining the product DDi-2F7- (β-galactosidase) resulting from the reaction of said conjugate 2F7- (β-galactosidase) with the free DDi having crossed said membrane 81a; the resulting reaction mixture then reaches the development membrane (s) 62 containing the BEST MODE MPB The best mode of carrying out the invention consists in using a device according to FIGS. 1-4 and 6-7. The mixture of the liquid sample to be analyzed which may contain the immunological substance X with a conjugate coupled to a labeling enzyme is prepared outside the multimembrane system and then distributed on a thin membrane constituted by a nonwoven web of fibers of glass, this sheet being located above the underlying selective phase containing the immunological reagent immobilized in its mass, one or more porous revelation membranes being underlying this selective phase.

The best mode also includes the implementation of a multimembrane device according to FIG. 5 and better still FIG. 8 for determining of an immunological substance content likely to be higher than the normal content. In this case, the liquid sample to be analyzed is distributed on a membrane made up of a non-woven sheet of glass fibers situated above the first selective phase. Other advantages and characteristics of the invention will be better understood on reading which will follow comparative tests and exemplary embodiments. Of course, all of these elements are in no way limiting but are provided by way of illustration. In said embodiments, the active sites which are still free from the membranes used have been saturated, in particular by means of REGILAIT ^R milk powder.

IMMOBILIZATION TESTS ON POROUS MEMBRANE

A porous membrane of 20 cm x 20 cm, or of nitrocellulose (porosity 0.45 μm) is immersed in an aqueous buffer of 100 ml containing the immunological material to be immobilized by covalence on a hydrophilic polyfluorocarbon membrane (IMMOBILON ^R : porosity 0.45 μm), polyamide (IMMUNODYNE ^R : porosity 0.65 μm), or polysulfone (ULTRABIND ^R : porosity 0.45 μm), or by adsorption on a nitrocellulose membrane (porosity 0.45 μm) after treatment with an aqueous solution 0.1 M CH3COOH activation - 0.5M, leaving a height of liquid of about 2 mm above said porous membrane. Incubation takes place for 18 h at a temperature between 4 ° C and RT.

Increasing concentrations (5 to 500 μg / ml) of immunological material are used for this purpose in the buffer and measurement, after drying at 40 ° C. if necessary under reduced pressure, the quantity of material fixed by each membrane. The same series of tests was carried out by varying the buffer.

All the results obtained appear in Tables I to IV below, and show (i) the risk of salting out during covalent fixation when the concentration of the immunological material increases from 100 to 200 μg / ml. of a pan, and is greater than 200 μg / ml, of another pan, and (ii) the advantage of KH2PO4 as a buffer. TABLE I

FIXING CAPACITY ON MEMBRANE immunological material: DDi buffer: carbonate pH8

Fixed quantities (μm / cm ² )

Concentration in the bath IM OBILON ^R ULTRABIND ^R IMMUNO- NITROCEL¬

(μg / ml) DYNE ^R LULOSE ¹

(1) (1) (1) (2)

5 1.32 2.5 2.5 2.5

10 1.8 4.89 5 5

20 3.2 9.75 10 10

100 29.32 33 32 50

200 21.5 14 33 96

Notes

(1) porous membrane for covalent immobilization;

(2) porous membrane for immobilization by adsorption.

TABLE II

FIXING CAPACITY ON MEMBRANE immunological material: DDi buffer: carbonate pH8

Fixed quantities (μm / cm ² )

Concentration in the bath ULTRABIND ^R I MUNODYNE ^R NITROCEL-

(μg / ml) LULOSE ^R

(1) (1) (2)

5 2.5 2.5 2.5

10 3 3.8 12.5

20 10 2.9 25

100 12.5 7.5 50

200 22.5 13 100

Notes

(1) porous membrane for covalent immobilization:

(2) porous membrane for immobilization by adsorption.

TABLE III

FIXING CAPACITY ON MEMBRANE ACCORDING TO THE BUFFER porous membrane: ULTRABIND ^R immunological material: 2F7

Concentration Fixed quantity (μg / cm ² - ¹ in the buffer buffer buffer buffer buffer buffer

(μg / ml) P0 ₄ ^{3 "} P0 ₄ ^3" co ₃ ^{2 ~} co ₃ ^2_ KH ₂ P0 ₄ pH 6.5 pH 7.5 pH 8 pH9

50 10.25 8.5 11.1 9.2 12.2

100 15 14.5 18.5 16.75 26.75

500 13 27 40.5 32 54

TABLEAlr iv

CAPACITY OF FIXATI ON ON MEMBRANE ACCORDING TO THE BUFFER

: porous membrane: ULTRABIND ^R

: immunological material: D.D

Concentration Fixed quantity (μg / cm ²⁾ in the buffer buffer buffer buffer buffer buffer

(μg / ml) P0 ₄ ^{3 "} P0 ₄ ^3" co ₃ ^{2 ~} co ₃ ^2_ KH ₂ P0 ₄ pH 6.5 pH 7.5 pH 8 pH9

50 11.52 10.62 8.25 8.75 11.6

100 18.37 20.45 11.62 20.35 20

200 11.52 23.37 18.5 18.25 28.5

500 12 24 5

_* 5 16.25

PREPARATION I

Obtaining the SUDPOΠ membrane

An IMMUNODYNE ^R membrane (porosity: 5 μm; reference "BIAO50HC5) or VERSAPOR ^R (porosity: 3 μm) is immersed for 0.25-0.50 h in a PBS buffer at pH 7 consisting of (i) 16 parts by volume of a mix of NaH ₂ PO ₄ at 13.8 g / 1 and NaCl at 9 g / 1 (solution A) and (ii) 84 parts by volume of a mixture of Na2HPO ₄ at 14.19 g / 1 and NaCl at 9 g / 1 (solution B), said buffer also containing 5% w / v PVP (MW: 10,000) or REGILAIT ^R. Rinse thoroughly with cold distilled water (2-8 ° C) then finally with warm distilled water (37 ° C). It is dried in an oven at 37-40 ° C. A membrane with saturated active sites is obtained intended for the suppoπ of the non-immobilized conjugate coupled to the labeling enzyme, for example the conjugate 14 (FIGS. 1 and 5) or 34 (FIG. 3) of formula anti (X) -E *, conjugate 24 (Figure 2) of formula anti [anti (X)] - E *, or conjugate 44 (Figure 4) of formula anti (X) ¹ -E *. In the present case, the labeling enzyme is peroxidase.

Is deposited on said dried membrane 1 to 5 μl (by pads each materialized by a preferably circular caπouche) of a PBS buffer solution at pH 7 (as defined above) containing 1 μg / ml of said conjugate.

This gives the membrane 12 containing on its upper surface 13 said non-immobilized conjugate. This membrane is cut into strips each containing a non-immobilized conjugate pad. PREPARATION II Obtaining the selective phase

A porous nitrocellulose membrane with a porosity of 0.40-0.50 μm is immersed (for example a membrane sold by the company called SCHLEICHER & SCHUELL with a porosity of 0.45 μm, reference: 439196) for 0.25-0.50 h , in an aqueous activating solution containing 0.1 M CH3COOH and 0.5 M NaHSO Dried at 40 ° C without rinsing.

The immunological reagent referenced 5 (Figures 1 and 5), 15 (Figures 2 and 4) or 25 (Figure 3) is fixed, immersing the nitrocellulose membrane (thus activated) for at least 1 h and at most 48 h and dried) in a buffer (i) carbonate at pH 8 (containing 0.012 M CO3 ² - and 0.15 M NaCl) or (ii) PBS at pH 7 (as in Preparation I above), said buffer containing 50 to 150 mg / ml of said immunological reagent. The immobilization of the immunological reagent is thus obtained in the mass of the membrane according to a surface concentration of 100 μg / cm ² .

The active sites of the membrane which are still free are then saturated, either as indicated in Preparation I with PVP or (preferably) REGILAIT, and fixed by means of distilled water containing 10% w / v of CH3COOH and 25% w / v isopropanol (if necessary). Rinse thoroughly with 0.05 M MES or PBS at pH 7 above. The remaining liquid is absorbed by placing the membrane between two sheets of filter paper and then tapping carefully. It is then dried in an oven at a temperature of 30-37 ° C, for at least 1 night.

The membrane thus obtained can be kept as it is or cut into strips of dimensions identical to those of the strips of the membrane suppoπant the non-immobilized conjugate of Preparation I above, for several months at

2-8 ° C.

PREPARATION III

Obtaining a membrane comprising the D-glucose / glucose oxidase mixture (a) Immersed for 0.25-0.50 h, in an aqueous solution of TWEEN ^R at 5% w / v, a cellulose membrane (designated " filter No 122 ") sold by the company known as MEDIAS FILTRANTS, reference 011220013, with a porosity of 0.5 μm). It is dried overnight in an oven at 40 ° C. The membrane is then immersed in an aqueous bath containing 10% w / v of D-glucose, ecaπe the supernatant liquid and then dried in an oven at 40 ° C. for 1 night.

(b) 1 μl of purified glucose oxidase (titer: 500 IU / ml) is placed on the membrane and then dried at 37 ° C. in an oven.

If necessary, the active sites of the membrane which are still free are saturated as indicated in Preparation I. This membrane is cut into strips of dimensions identical to those of the strips of Preparation I above.

PREPARATION Illbis

Obtaining a membrane comprising D-glucose and a membrane comprising glucose oxidase. A membrane comprising D-glucose is prepared as indicated in step (a) of Preparation III above.

A membrane comprising glucose oxidase is prepared by using the aforementioned cellulose membrane ("filter No. 122") which is saturated with an aqueous solution of TWEEN ^R at 5% w / v and on which we then deposit 1 μl of glucose oxidase as indicated in step (b) of preparation III. Then dried at 37 ° C in an oven.

PREPARATION IV

Obtaining a membrane comprising TMB

Depositing on an IMMUNODYNE ^R membrane (porosity 0.45 μm; reference: BIA045HC5) at the level of pads materialized by a layer of preferably circular, an amount of 2.5 μl per pad of an alcoholic composition (ethanol) containing 5 g / 1 of TMB. The membrane is kept as it is or cut into strips of dimensions identical to those of the strips of Preparation I above, protected from light. PREPARATION V

(a) According to a process similar to that of preparation I, a porous membrane suppoπ of the non-immobilized conjugate is prepared in which the peroxidase is replaced by an alkaline phosphatase or respectively β-galactosidase.

(b) According to a process similar to that of Preparation IV, a porous membrane is prepared in which the TMB is replaced by the mixture BCIP / NBT or respectively the methoxynaphthyl-galactoside. In this case, the multimembrane device according to the invention does not comprise the membrane 2 which has become superfluous having regard to the choice of the labeling enzyme for the non-immobilized conjugate. EXAMPLE 1

DDi identification

A multi-membrane device is prepared to prepare pre-cut strips, in accordance with FIG. 1 or in FIG. 6, comprising an assembly of:

a selective phase according to preparation II above, comprising as immobilized immunological reagent an antigenic substance X,

a porous membrane 2 according to preparation III above, comprising a D-glucose / glucose oxidase mixture (as a variant, the porous membrane 2 can be replaced by a membrane comprising D-glucose and a membrane comprising glucose oxidase obtained according to preparation Illbis), and - a porous membrane 3 according to preparation IV above comprising the TMB.

A sample containing 0 to 10 μg / ml of DDi is reacted in a flask with the monoclonal antibody coupled to peroxidase (9C3 -peroxidase). The resulting reaction mixture is distributed over a sheet of glass fiber placed above the porous membrane of the selective phase. With the device of FIG. 1, according to which the selective phase is passed through perpendicularly, it is observed that

(i) the test is negative for DDi concentrations in the sample less than or equal to 0.2 μg / ml, (ii) the test is doubtful for DDi concentrations in the sample between 0.2 μg / ml and 0.4 μg / ml, and (iii) the test is positive for DDi concentrations in the sample greater than or equal to 0.4 μg / ml.

In short, the device of FIG. 1 is reliable for any concentration of DDi greater than or equal to 0.4 μg / ml. With the device of FIG. 6, according to which the selective phase is passed longitudinally, the results show that the sensitivity is 1 pg / ml (i.e. the minimum detectable concentration reliably in DDi in the sample of 1 picogram per milliliter). EXAMPLE 2 Identification of PAI-1

A multimembrane device is prepared according to FIG. 7 using

- as a selective phase, a porous nitrocellulose membrane (thickness: 0.35 mm; porosity: 0.45 μg) containing in its mass an anti-monoclonal antico PAs (PAI-1), namely 7D4, - as porous revelation membranes 62 a first membrane containing the D-glucose / glucose oxidase mixture (alternatively, a membrane containing D-glucose and a membrane containing glucose oxidase may be used) and a second membrane containing TMB.

The mixture of an aqueous sample containing 0 to 10 μg / ml of PAI-1 and the anti-monoclonal anti-antibodies (PAI-1) coupled to peroxidase, i.e.

7F5-peroxidase.

The results obtained show that said device has a sensitivity of 1 pg / ml for the assay of PAI-1.

EXAMPLE 3 Identification of the DDi

A multimembrane device is used according to FIG. 6 or 7 operating according to the principle of FIGS. 2, 2a and 2b, with

- as a selective phase, a nitrocellulose membrane identical to that of Example 2 and containing in its mass the monoclonal anticoφs 2F7, - as porous revelation membranes, a membrane containing the mixture

D-glucose / glucose oxidase (alternatively, a membrane containing D-glucose and a membrane containing glucose oxidase may be used) and a membrane containing TMB. Via well 66, a mixture consisting of an aqueous sample containing 0 to 1 μg / ml of DDi and a monoclonal or polyclonal anti-idiotype antibody directed against 2F7 and coupled to peroxidase is introduced.

According to the results obtained, it can be seen that the sensitivity threshold for determining the DDi is 0.1 μg / ml. EXAMPLE 4

DDi identification

A multimembrane device is used according to FIG. 8 operating according to the principles of FIGS. 5, 5a and 5b, with: - as a first selective phase, an ULTRABIND ^R membrane (thickness 0.2 mm; porosity: 0.45 μm) containing in its mass a predetermined amount of the monoclonal anticoφs 9C3 [ie anti monoclonal anticoφs (DDi)] - this predetermined amount corresponds to the normal content of DDi in the plasma -,

- as charge placed in well 86b, the monoclonal anticoφs 2F7 coupled to peroxidase or to β-galactosidase,

as a second selective phase, the aforementioned ULTRABIND ^R membrane containing the immobilized DDi in its mass, and

- as porous revelation membranes, either a membrane containing the D-glucose / glucose oxidase mixture (alternatively a membrane containing D-glucose and a membrane containing glucose oxidase can be used) and a membrane containing TMB, or a membrane containing the MPB.

The plasma sample containing DDi (from a concentration below normal to a concentration above normal) is introduced into well 86a. The results obtained show that the sensitivity of the determination of excess DDi by comparison with normal is 0.1 pg / ml. EXAMPLE 5

Identification of PAI-I

The procedure is as indicated in Example 4 above, replacing the products DDi, 2F7-peroxidase (or 2F7-galactosidase) and 9C3 respectively by the products PAI-1, 7D4-peroxidase (or 7D4-galactosidase) and respectively 7F5. The sensitivity threshold is also 0.1 pg / ml. EXAMPLE 6

Identification of _j -antitrypsin It is known that inflammation of the cow's udder results in the presence of tryptic inhibitors in milk, among which the o -antitrypsin is preponderant and characteristic. The determination of the absence of α _j - antitrypsin in milk is therefore particularly advantageous for the health of the livestock and the hygiene of the consumer.

For this purpose we use a device according to Figure 1 or Figure 6 with

a selective phase containing in its mass the α ^ -antitrypsin as an immobilized immunological substance, and

- a porous revelation membrane containing the MPB. Is distributed on the nonwoven sheet of glass fiber, located above the face 11 of the membrane 10 or at the bottom of the well 66, a mixture consisting of a sample of milk and an anticoφs anti (α _j -antitrypsin) coupled with β-galactosidase. The sensitivity of the determination is 0.5 μg / ml with the device in FIG. 1 and 2 μg / ml with that in FIG. 6. EXAMPLE 7

Identification of HBsAg

Use is made of a device according to FIG. 6 or 7 operating on the principle of FIGS. 3, 3a and 3b, with

- a selective phase consisting of an IMMOBILON ^R membrane (thickness: 0.2 mm; porosity: 0.65 μm) containing in its mass, as immobilized immunological reagent, an anti-monoclonal antibody [F (ab ') 2] directed against the F (ab ') 2 fragment of an anti anticoφs (HBsAg), and

a porous membrane containing the D-glucose / glucose oxidase mixture (alternatively a membrane containing D-glucose and a membrane containing glucose oxidase can be used) and a porous membrane containing TMB.

Via well 66 an aqueous mixture is introduced, consisting of a sample containing from 0 to 1 μg / ml of HBsAg and of a monoclonal anticoφs directed against HBsAg and coupled to peroxidase.

The sensitivity of the HBsAg determination is 1 pg / ml. EXAMPLE 8

Identification of HBsAg

A multimembrane device is used according to FIG. 6 or 7 operating according to the principle of FIGS. 4, 4a and 4b, with a selective phase identical to that of Example 7 above and containing in its mass a first monoclonal anticoφs directed against the first epitope of HBsAg, and

- The porous revelation membranes of Example 7 above. An aqueous mixture comprising a sample containing 0 to 1 μg / ml of HBsAg and a second monoclonal antico monocs directed against a second epitope of HBsAg and coupled to peroxidase is introduced into well 66.

The sensitivity of the HBsAg determination is 2 pg / ml. EXAMPLE 9 Identification of anti-HIV antibodies

Multi-membrane devices are prepared according to Example 3 for the determination of anti (HIVl), anti (HIV2) and anti (HIV3) antico l'exemples. The sensitivity of the determination which is 0.5 pg / ml allows the identification of each of these antico cess in saliva. EXAMPLE 10

Identification of Neisseria

A multimembrane device for the identification of Neisseria strains in the liquid of canned vegetables is prepared according to Example 8. The sensitivity is 2 pg / ml. EXAMPLE 11

Identification of PAI-1

The procedure is as indicated in Example 4 above, replacing the products DDi, 2F7-peroxidase (or 2F7-galactosidase) and 9C3 respectively by the products PAI-1, 7D4-colored latex and 7F5 respectively. the colored latex in the form of submicron particles having an average diameter of 40-45 nm replacing the abovementioned labeling enzyme E *, in one section. and using as assembly 62 one or two membranes devoid of enzyme developer, on the other hand. In this case the membrane 81b of the selective phase is not located in the lumen of the window 68. The sensitivity of the determination of the excess of PAI-1 compared to normal is 0.1 pg / ml.

In general, when the marking means E is constituted by colloidal gold, colloidal silver, colloidal gold doped with silver, or a colored latex which is in the form of microbeads with a diameter less than

0.1 μm, it is important that the selective phase 61 (according to FIG. 6) or 81b (according to FIG. 8) does not occupy all the light of the window 68 (FIG. 6 or 8), so that the observation of the possible coloring of the assembly 62 is not disturbed by the coloring of said selective phase. A configuration according to FIG. 7 is suitable in the sense that only a poπion (here the edge) of the membrane 61 of the selective phase is present under the assembly 62. EXAMPLE 12

Identification of HBsAb

Use is made of a device according to FIG. 7 operating according to the principle of FIGS. 3, 3 a and 3b with

- a selective phase consisting of an IMMOBILON ^R membrane (thickness: 0.2 mm; porosity: 0.65 μm) containing in its mass, as immobilized immunological reagent, an anti-monoclonal antibody [F (ab ') 2] directed against the fragment F (ab ') 2 ^ ^{an ant} i ^cor P ^s anti (HBsAb), and

- one or two porous membranes according to preparation IV above but devoid of TMB. Via well 66, an aqueous mixture consisting of a sample containing 0 to 1 μg / ml of HBsAb and a monoclonal anticoφs directed against HBsAb and coupled to colloidal gold is introduced.

The sensitivity of the HBsAb determination is 2 pg / ml. EXAMPLE 13 Identification of the DDi

A multimembrane device is used according to FIG. 6 or 7 operating according to the principle of FIGS. 2, 2a and 2b, with

- As a selective phase, a nitrocellulose membrane identical to that of Example 2 and containing in its mass the monoclonal anticoφs 2F7. - as porous revelation membranes a membrane containing the mixture

BCIP / NTB.

Through well 66, a mixture consisting of an aqueous sample containing 0 to 1 μg / ml of DDi and a monoclonal anti-kappa chain anticoφs directed against 2F7 and coupled to alkaline phosphatase is introduced. According to the results obtained, it can be seen that the sensitivity threshold for determining the DDi is 0.1 μg / ml.

Claims

1. Method for identifying an immunological substance (X) appearing to all of the antigens and anticoφs and capable of being present in a liquid sample to be analyzed, said method, which implements the reaction of said immunological substance with one of its conjugates [anti (X)] and the use of a multimembrane system, being characterized in that it comprises the steps consisting in

(1 °) bringing into contact the liquid sample capable of containing said immunological substance (X) to be identified with one of its conjugates coupled to an enzyme of formula anti (X) -E * where anti (X) represents a conjugate bifunctional and E * a labeling enzyme, so as to obtain the reaction product of formula

X-anti (X) -E * when said immunological substance is present; and, (2 °) passing the reaction medium thus obtained over a multimembrane device comprising - a first zone comprising a porous membrane provided with an immobilized immunological reagent which is chosen from the group consisting of

(a) immunological materials which react specifically with the conjugate (anti (X)) and the conjugate coupled to an enzyme (anti (X) -E *), but which do not react with the product of the above-mentioned reaction (X-anti (X) -E *), or

(b) immunological materials which react specifically with said immunological substance and the aforementioned reaction product (X-anti (X) -E), but do not react with said conjugate (anti (X)) and said conjugate coupled to a enzyme (anti (X) -E), the weight ratio of said immobilized immunological reagent / surface of the porous membrane provided with said immunological reagent being between 10 and 150 μg / cm ² , and the active sites of said membrane not used to immobilize said immunological reagent being blocked; and, - A second zone comprising at least one porous membrane, which is underlying the membrane containing said immobilized immunological reagent and which contains a specific substrate for the labeling enzyme (E *) for revealing the presence of said enzyme.

2. Method according to claim 1. characterized in that one prepares in step (1 °) a mixture of the sample to be analyzed capable of containing the immunological substance X to be identified with a conjugate directed against anti (X) and coupled to the labeling enzyme, this conjugate being a complex corresponding to the formula anti [anti (X)] - E * and such that (a) in the absence of X, it reacts with anti (X), and

(b) in the presence of X, it can react with anti (X) only after the complete fixation of X by anti (X), then reacts in step (2 °) said mixture with the anti compound (X ) immobilized at the level of a porous membrane, before proceeding to reveal the anti [anti (anti (X)] - E * complex conjugate not retained by the immobilized anti (X) reagent due to the presence of X.

3. Method according to claim 2, characterized in that it comprises the steps consisting in:

(1 °) bringing the liquid sample capable of containing said immunological substance (X) to be identified into contact with a complex conjugate of formula anti [anti (X)] - E * where E * and anti (X) are defined as indicated above, said complex conjugate reacting with the conjugate [anti (X)] directed against said immunological substance (X) in the absence of said substance (X) but not reacting with said conjugate in the presence of said substance (X) free; and, (2 °) passing said mixture of the sample to be analyzed with said complex anti (X) over a multimembrane device comprising - a first zone comprising a porous membrane comprising the conjugate [anti (X)] as an immunological reagent , the weight ratio of said immobilized immunological reagent / surface of the porous membrane provided with said immunological reagent being between 10 and 150 μg / cm ² , and the active sites of said membrane not used to immobilize said immunological reagent being blocked: and,

a second zone comprising at least one porous membrane, which is underlying the membrane containing said immobilized immunological reagent and which contains a specific substrate for the labeling enzyme

(E *) for there revelation of the presence of said enzyme.

4. Method according to claim 1 or 3, characterized in that step (1 °) is carried out outside the multimembrane system.

5. Method according to claim 1 or 3, characterized in that step (1 °) is carried out in the multimembrane system, the sample capable of containing said immunological substance being distributed on a deposit of conjugate coupled to an enzyme arranged not immobilized on a porous suppoπ membrane located above the membrane comprising on its upper face said immobilized immunological reagent.

6. Method according to claim 5, characterized in that the porous membrane supporting the conjugate coupled to an enzyme is surmounted by one or more membranes devoid of any immunological material.

7. Method according to claim 1 or 3, characterized in that the free active sites of the membrane of said second zone at which the immunological reagent is immobilized are substantially blocked.

8. Method according to any one of claims 1 and 3 to 7, characterized in that the free active sites of each of the membranes are blocked.

9. Method according to claim 1 or 3, characterized in that said immunological reagent immobilized at its membrane has a concentration approximated to the surface of said membrane, between 20 and 100 μg / cm ² .

10. The method as claimed in claim 1 or 3, characterized in that the porous membrane comprising the immobilized immunological reagent has a thickness greater than or equal to 0.1 mm, in particular between 0.1 and 10 mm and preferably between 0, 2 and 1 mm.

11. Method according to claim 1, characterized in that the porous membrane comprising the immobilized immunological reagent is crossed by the liquid flow peφendicululaire to its plane.

12. Method according to claim 3, characterized in that the porous membrane comprising the immobilized immunological reagent is traversed by the liquid flow longitudinally.

13. The method of claim 1 or 3, characterized in that it comprises in step (1 °) the use of two porous membranes each comprising an immunological reagent immobilized in its mass and the use of a non-conjugate immobilized with formula anti (X) -E * or anti [anti (X)] - E * disposed on a porous suppoπ between the two said porous membranes.

14. Method according to claim 1 or 3, characterized in that the immobilized immunological reagent is chosen from the group consisting of:

- the immunological substance (X) itself, and - a conjugate of said conjugate.

15. Method according to claim 14, characterized in that the conjugate of the conjugate is chosen from the group consisting of:

(i) an anticoφs [anti (anti (X))] directed against the anticoφs [anti (X)], and (ii) a protein or peptide reacting specifically with respect to said [anti (X)] conjugate .

16. Method according to claim 15, characterized in that the anti [anti (anti (X)] product is chosen from the group consisting of anti [F (ab ') 2] antico ,s, anticoφs anti-kappa chain and anticoφs anti-idiotype.

17. Method according to any one of claims 1-6 and 13, characterized in that the labeling enzyme E * is replaced by a marker chosen from colloidal gold, colloidal silver, colloidal gold doped with silver and a colored latex.

18. The method of claim 17, characterized in that the colored latex is in the form of particles having a particle size less than 0.1 microns and preferably between 25 and 60 nm.

19. Multimembrane system for the identification of an immunological substance (X) capable of being contained in a liquid sample to be analyzed, said system being characterized in that it comprises

a first zone comprising a porous membrane comprising an immobilized immunological reagent which is chosen from the group consisting of

(a) immunological materials which react specifically with the conjugate (anti (X)) and the conjugate coupled to an enzyme (anti (X) -E), but do not react with the product of the above-mentioned reaction (X-anti ( X) -E), or

(b) immunological materials which react specifically with said immunological substance and the aforementioned reaction product (X-anti (X) -E *), but not reacting with said conjugate (anti (X)) and said conjugate coupled to an enzyme (anti (X) -E *), the surface concentration of said immobilized immunological reagent being between 10 and 150 μg / cm ² , and the active sites of said membrane not used to immobilize said immunological reagent being blocked; and,

- A second zone comprising at least one porous membrane, which is underlying the membrane containing said immobilized immunological reagent and which contains a specific substrate for the development of the labeling enzyme.

20. Multimembrane system according to claim 19, characterized in that the labeling enzyme E * is replaced by a marker chosen from colloidal gold, colloidal silver, colloidal gold doped with silver and a colored latex and in that said second zone is devoid of enzyme substrate.

21. Kit for assaying an immunological substance (X), characterized in that it comprises (a) a multimembrane system according to claim 19 or 20 and (b) at least one anti-complex conjugate (X) -E * or anti [anti (X)] - E * or E * is a labeling enzyme for colloidal gold, colloidal silver, colloidal gold doped with silver or a colored latex.