FR2725023A1 - DIRECT DIAGNOSIS OF HAPTENES - Google Patents

Abstract

A device for rapidly assaying and characterising the presence of a molecule with a molecular weight of less than 10,000 daltons, i.e. a haptene, in a liquid sample, said device consisting of a hydrophilic support enabling capillary migration of a liquid therein, and having a plurality of separate regions in which the reagents are deposited and/or dried. The device consists of at least the following portions: (a) a receiving portion for the sample to be tested; (b) a reservoir portion containing a conjugate which is both marked with a marker enabling direct detection thereof, and also is a ligand for the molecule to be assayed, portions (a) and (b) being superimposed or sequential; (c) two successive barriers consisting of affinity pair moieties of which one, known as a negative reading barrier, retains said conjugate during migration thereof over the support when the desired molecule is not present in the sample, while the other retains said conjugate when the desired molecule is present, in which case said molecule is directly or indirectly bound to said conjugate, and said second barrier is known as a positive reaction reading zone; and (d) an absorptive portion arranged adjacent to the hydrophilic support for promoting liquid flow from the sample receiving portion (a) through the other portions that make up the device.

Description

DIRECT DIAGNOSIS OF HAPTENES
The present invention relates to a solid phase method for characterizing and assaying molecules with a molecular weight of less than 10,000 daltons in a liquid sample, by using a system combining dry chemistry and chromatography, the use of pairs antigen-antibody or streptavidin-biotin affinities, a simple labeling system, the.

all allowing direct reading of the presence or absence of the molecule sought in the sample and making it possible to directly link the signal observed to the concentration of the substance sought.

Haptens are small molecules, of diverse chemical nature and with a molecular weight of less than 10,000 daltons. They have the distinction of not being immunogenic. Thus, to have anti-hapten antibodies, it is necessary to couple them beforehand to a macromolecule called carrier protein which is usually chosen from the following proteins: serum albumin, thyroglobulin, ovalbumin, fibrinogen and Keyhole Lympet
Hemocyanin (KLH).

 Haptens are substances with a wide variety of reactive groups. These groups constitute not only the elements serving as a basis for their coupling but also their immunodeterminers which distinguish them from molecules which are structurally analogous to them.

 Generally, the use of these groups in the couplings decreases the specificity of the anti-hapten antibodies.

 Even if in certain situations (in particular drug abuse) the recognition of a wide range of haptens is recommended, the search for an excellent specificity remains however of rigor.

This objective of optimizing the specificity of anti-hapten antibodies is achieved from a process including:
- the choice of a reactive group common to the molecules close on the structural level (structural analogues),
- the use of a spacer between the hapten and the carrier protein.

 The determination of haptens in a sample and in particular in biological fluids requires obtaining antibodies of very specific quality.

The haptenation of macromolecules is a crucial step that must be well mastered; in particular, the trap of anti-carrier protein antibody formation should be avoided. In obtaining antiCaptenes antibodies,
Van Weemans and Schuurs (1), (2), (3) have long demonstrated the existence of three types of heterology:
- the use of different coupling agents makes it possible to distinguish a bridge heterology,
- the choice of different coupling sites on the hapten molecules allows the development of a heterology of sites,
- a third heterology concerns the case where different structurally close haptens are coupled from the same site using the same coupling agent.

 The protein-hapten bond which determines the quality of the immunogen and, subsequently, that of the antibody, takes place at the level of the most reactive groups.

The groups E and a amino (pKa between 8 and 10), hydrogen sulfides, phenolics (pKa of 9), imidazoles (pKa of 7) and carboxyls (pKa between 2 and 4) are generally used for these protein-hapten bonds.

 The pKa, that is to say the pH at which half of the groups is protonated, determines the change in reactivity as a function of the pH; the non-protonated forms of the nucleophilic groups are those which are reactive.

 Finally, the reactivity of the hapten linkage groups also depends on the micro-environment of the residue to be coupled.

The hapten dosage requires at least several elements:
- an antibody of excellent quality,
- an efficient dosing system comprising a solid phase and a very sensitive revelation system.

 As small molecules, haptens are preferably dosed by a competition system. It is in reality the competition between the hapten to be assayed and the labeled hapten for limited sites of antihapten antibodies. The signal observed is therefore, by definition, inversely proportional to the concentration of hapten in the fluid to be assayed: the more hapten to be assayed, the weaker the measured signal and vice versa, the higher the measured signal, the weaker is the concentration of the hapten to be measured.

 As in all competition systems, the measurement of the substance to be dosed is made indirectly. This poses problems in particular in so-called rapid direct reading tests without special instrumentation, and whose interpretation must therefore be very easy.

 Numerous rapid diagnostic tests of the strip or similar type using the migration by chromatography or by capillarity of substances capable of forming affinity pairs with ligands and the observation of the reaction or non-reaction with said ligand have been described. . Both competitive methods or sandwich methods have been developed extensively and numerous patents or patent applications describe such systems; among the closest to the system of the invention we can cite patent EP 306,772 describing a device for rapid analysis based on a chromatography system and in which a means of absorption of the downstream liquids makes it possible to absorb the reaction liquids; this system includes a reagent at the reading window to detect the presence of the sample. However, this system is suitable for the direct determination of a large protein-type molecule.

Patent application WO 91/12528 relates to a rapid diagnostic device using the principle of chromatography combined with dry chemistry and in which a capture system of avidin-biotin or streptavidin-biotin type makes it possible to retain during the chromatographic migration of a ligand previously coupled to biotin. The device described in this patent application makes it possible to implement sandwich type reactions between two immunologically reactive substances of antigen-antibody type or even reactions of competition type. Nothing is said in this patent application on how one could directly either directly or by a sandwich method assay small molecules with molecular weight less than 10,000 daltons
In all of the various publications describing diagnostic tests, few are devoted to the detection of small molecules. Among these, mention may be made of US Pat. No. 4,358,535, in which Falkow et al; describe a methodology for the detection of DNA sequence associated with diseases and in which the DNA sequence is fixed in denatured, single chain form on a nitrocellulose type support. A labeled polynucleotide probe specific for a particular DNA sequence is then brought into contact with the sample under hybridization conditions; this device requires a washing step to remove the non-hybridized probes.

 In US Patent 3,893,808, Campbell describes a strip-type test for detecting lead contamination in automotive fuels.

The strip thus described comprises three zones: the first is impregnated with iodine, the second is treated with a mixture of iodine and potassium iodide; after application of a fuel sample on the strip there is migration by capillarity through the two zones to a third zone where an indicator is added; the presence of lead is revealed by a coloration in the third zone by the formation of a complex.

 In US Patent 3,895,914, Alberty et al. describe a test strip for the detection of barbituric acid and their derivatives in biological fluids. The principle of this method is the formation of a mercury-barbiturate complex and the determination of mercury in the complex. The strip comprises a hydrophilic paper having three zones: the first is impregnated with an acid to globally acidify the sample, the second is impregnated with a buffered mercury acetate capable of reacting to form a mercury barbiturate complex; the third zone is impregnated with a compound indicating the presence of mercury such as diphenyl carbazone. This system which seems to be the closest state of the art for the determination of small molecules is nevertheless very far from the proposed system of the invention in which direct reading can be done by labeling a conjugate capable of binding with the molecule sought when it is present, the complex then being capable of being captured by a ligand specific for the complex formed between the conjugate and the molecule to search for. Other systems exist and form the technological background.

 Patent EP 262 328 also describes a strip for the analysis of an analyte in a biological sample by means of a sequential series of reactions, the strip being made up of a capillary material for chromatography and capable of rapidly transporting reagents from the sample not immobilized at the start by means of a chromatographic solvent; this strip consists of three parts: a starting reservoir, a reservoir on arrival and between the two a series of zones impregnated with mobile or immobilized reagents allowing specific reactions with the analyte. This system is however incapable of functioning in dry chemistry (without addition of solvent) and in addition it involves a series of manipulations to possibly modify the chromatography buffers, manipulation incompatible with a rapid and inexpensive test.

 To our knowledge, no rapid diagnostic test has been described for a simple application which is the analysis and determination of a hapten in a sample by direct reading and without manipulation other than bringing the device into contact with the sample to be analyzed, which is the object of the present invention. It makes it possible to overcome the difficulties of interpretation inherent in the tests by competition which exist today to measure the haptens, and to propose to the users a simple means of detecting the presence of a hapten, for example of a drug in a sample. biological. A particularly awaited application is that of a rapid assay of the existence of a drug in the blood or urine of an individual in an emergency department, where precise information on the drug used is necessary. other types of applications can be envisaged in particular the monitoring of a drug treatment for example with opiates, uses of the forensic medicine type possibly making it possible to make tests on sites etc ...

 The system proposed by the present invention and which makes it possible to carry out a direct reading making it possible to link the concentration of the substance to be dosed to the signal observed, consists of a device consisting of a hydrophilic support in which a liquid is liable to migrate by capillarity , having a plurality of distinct regions in which the reagents are deposited and / or dried, and allowing rapid analysis and characterization of the presence of a molecule with a molecular weight of less than 10,000 daltons, (or hapten), and present in a sample, this device being characterized in that it consists of at least the following zones: a) a zone for receiving the sample to be tested, b) a reservoir zone containing a conjugate having the double characteristic of being marked by a marker allowing its direct detection and being a ligand for the molecule to be analyzed, these two zones a) and b) being superimposed or s equivalents, c) two successive barriers made up of halves of affinity pairs, one of which retains said conjugate during its migration on the support in the absence of the molecule sought in the sample, called negative reading barrier and l the other retains said conjugate in the presence of the desired molecule, the latter then being linked directly or indirectly to said conjugate, this second barrier then being a zone for positive reading of the reaction, d) an absorbent means and contiguous to the hydrophilic support making it possible to promote the flow of liquid from the zone a) for receiving the sample through the other constitutive zones of said device, the hydrophilic support and the absorbent means being fixed on a polyethylene film of Mylar type, the assembly being positioned in a box equipped with a receptacle plumb with zone a) accepting 0.05 to 0.5 ml of liquid to be tested and two windows plumb two successive barriers, allowing the reading of the positive or negative result of the reaction by observing the labeling linked to the conjugate.

 In the device thus described, the reservoir consists of a material chosen from glass fiber, cellulose, cellulose derivatives, nylon, fibrous or microporous polymers of high density polyester type, fibers of vegetable origin, animal or synthetic in the form of tablet or multi-layer powders or cellulose wadding.

 In a simplified embodiment, the zones a) and b) form a single zone, and the sample is deposited in a receptacle directly above the reservoir.

 The labeling of the conjugate must be a simple labeling of direct reading and in which the intensity of the revelation is directly linked to the amount of product present in the sample.

 In the device of the invention, the conjugate is marked either with colloidal particles containing a metal, preferably gold, or with an organometallic marker. The labeling of a reagent with particles of colloidal gold has been described and used in numerous diagnostic tests, in particular patent applications WO / 9112528 or US patent 4,859,612 or patent applications EP 291194 and EP 560 411 Finally, the patents EP 250 137 and EP 258963 must be cited.

 The invention does not consist in using labeling with colloidal particles but in using this labeling in a combination involving a reservoir zone containing a dehydrated conjugate, the two successive capture barriers making it possible to have a signal that the response either positive or negative, and great stability of the product by the fact that all of the constituent elements of the device are dehydrated after their deposition allowing storage of said devices at room temperature for a period of several weeks to several months.

 One of the capture systems used which is particularly effective is the avidin-biotin or streptavidin-biotin system.

 It is one of the most important systems for diagnostic testing.

Avidin has an exceptionally high affinity for biotin (10-15 M).

Biotin is easily coupled to macromolecules, in particular proteins, without loss of activity.

Avidin is very stable and has several binding sites and could serve as a bridging molecule between other biotinylated molecules.

These properties allow it a considerable increase in detectability (due to low background noise) in immunological tests.

Biotin is one of the twelve water-soluble factors of the vitamin B complex and also constitutes a coenzyme for the enzymes involved in carboxylation.

Avidin is very stable over a wide range of pH and temperature and the avidin-biotin complex itself has great stability with respect to heat and proteolytic enzymes.

Avidin is a basic glycoprotein with a pI of 10.5; it can be crystallized from saline solutions with a pH between 5 and 7.

It has four subunits of 15,600 KD each and is also sensitive to strong light.

It is absorbed on glass, like generally all basic solutes.

On the other hand, a low absorption on the polyethylene is also observed.

Each avidin subunit is capable of binding to biotin in a pH range between 2 and 13.

The biotin binding sites by avidin are very deep. Each atom of biotin contributes strongly to this interaction. Thus, several analogs or small fragments of biotin (urea, glycol, tetrahydrofuran, caproic acid) are capable of inhibiting the avidin-biotin interaction.

The relatively deep burial of biotin is responsible for the very strong affinity. However, the four binding sites are not distributed in the same way: two sites are found very close to each other and, thus each pair of binding sites would only bind a biotinylated protein. Thus, contrary to what is commonly accepted, avidin with its pairs of binding sites, would behave more as a bridging agent between two biotinylated proteins rather than as an amplification agent.

A protein similar to avidin, streptavidin from Streptomyces avidiini is used with a big advantage which is that of having a lower background noise.

Streptavidin is an extracellular protein of Streptomyces avidiini and its functional part has a PM of 47 KD and comprises 4 subunits, each having a biotin binding site.

The binding of avidin to biotin is not disturbed by extreme pH, salts or even chaotropic agents such as guanidine hydrochloride (up to 3M), urea, guanidine chloride, etc. ...

 Besides the antigen-antibody, or avidin, or streptavidin / biotin affinity pairs, other affinity pair systems can be recalled here and used in the device of the invention.

This is the Alimmunoglobulin protein system (lg):
Protein A is extracted from the cell wall of Staphylococcus aureus.

It has a strong affinity for the Fc fragment of most lg. Its MP is between 42 and 56 KD and its pl is 5.1. The equilibrium constant of the
Protein A with IgG varies considerably depending on the species and the pH, but the binding of the antigen to the Fab increases the affinity between the Fc fragment of lg and Protein A.

 The Protein A-Ig complex is not affected by EDTA or by low concentrations of detergents (Tween, Triton X-100, Brij) often used in immunological tests.

 This system can be extended to other specific immunoglobulin binding proteins, for example, protein G. Another affinity system which can be used in the determination of haptens is that of lectins and carbohydrates; concanavalin A (Con A) consists of 26 KD subunits which would form a tetramer. Each subunit contains an Mn2 and a Ca2 + which are necessary for the activity of Con A. Likewise, each subunit has a carbohydrate binding site. However, excess metal prevents this activity.

 In the same way as the conjugate can be marked by colloidal particles and in particular by particles of colloidal gold, it can be marked by an organometallic marker making it possible to reveal the presence of the product by infrared detection, the wavelength of observation being dependent on the marker used; The advantage of using this type of organometallic marker is that by the simultaneous use of several different markers, namely those which can be revealed at different wavelengths in infrared, it is possible on the same device to carry out research and characterization of different molecules present in the same sample or in different samples. To be read, the device of the invention in which the conjugate is marked with a marker of organometallic type, requires the use of a simple reader in which said device is arranged after deposit and migration of the sample.

 In the device of the invention, the absorbent means which makes it possible to drain the liquid from the sample deposited at the receptacle provided for this purpose consists of a hygroscopic material, in particular cellulose derivatives or cellulose wadding, nylon, fibrous or microporous polymers of high density polyester type, fibers of vegetable, animal or synthetic origin in the form of powders of tablets or of multiple layers.

 The invention also relates to a method for rapid analysis and characterization of the presence of a molecule with a molecular weight of less than 10,000 datums, or hapten, in a liquid sample, said method being characterized in that it uses a device as described in its various embodiments above and in the examples below and consisting of a hydrophilic support in which a liquid is capable of migrating by capillarity, having a plurality of distinct regions in which the reagents are deposited by impregnation and dried, this device consisting of at least the following zones: a) a zone for receiving the sample to be tested, b) a reservoir zone containing a conjugate having the double characteristic of being marked by a marker allowing its detection direct and to be a ligand for the molecule to be analyzed, these two zones being superimposed or sequential, c) of two barr successive iers made up of halves of affinity pairs, one of which retains said conjugate during its migration on the support in the absence of the molecule sought in the sample, called a negative reading barrier and the other retains said conjugate in presence of the molecule sought, the latter then being linked directly or indirectly to said conjugate, this second barrier then being a zone for positive reading of the reaction d) of an absorbent means and contiguous to the hydrophilic support making it possible to promote the flow of liquid from zone a) for receiving the sample through the other constituent zones of said device, the hydrophilic support and the absorbent means being fixed on a polyethylene film of Mylar type, the assembly being positioned in a box provided with a receptacle level with area a) accepting 0.05 to 0.5 ml of liquid to be tested and two windows level with the two successive barriers allowing the reading of the positive or negative result of the reaction by observing the labeling linked to the conjugate, said method comprising depositing 0.05 to 0.5 ml of sample to be tested in the reception zone provided for this purpose , followed by direct or indirect observation by any appropriate optical means of the result obtained at the level of the positive and negative reading barriers.

 The use of a device as described above for detecting and characterizing the presence of a hapten in a liquid sample is an integral part of the invention. Any type of molecule having a weight of less than 10,000 daltons can be analyzed and assayed by a device as described above; but the invention is particularly advantageous when hapten is a drug which it is sought to detect in a biological fluid, this fluid possibly being blood, urine or saliva.

 The existence of a rapid diagnosis seems particularly advantageous when the drug use in a patient is suspected (drus abuse) or during drug treatment with drugs with side effects.

The drugs whose characterization or monitoring of their presence in a biological fluid can be particularly precious are, without limiting nature but to be the most important, included in one of the following classes: - opiates, or alkaloids including morphine, heroin, codeine, dextromethorphan, or their derivatives and metabolites - synthetic opiates, including methadone, propoxyphene, phenylcyclidine, derivatives and metabolites,
- cannabinoids including 9-tetrahydrocannabiol (or cannabis), cocaine,
LSD, benzoïl of ecgonine, marijuana, their derivatives and metabolites, - amphetamines, MDM (ecstazy), 2,5 imethyoxy-4Methamphetamine, catecholamines including ephedrine, L-Dopa, epinephrine, narcine, papaverine or their derivatives and metabolites, - barbituric acid derivatives or metabolites including phenobarbital, secobarbital, primidone, ethosuccimide, diphenylhydantoin, - benzodiazepines, their derivatives and metabolites, - steroids including estrogens, androgens, adrenocortical steroids, their derivatives and metabolites, - glycosides and aglycones including digoxin, digoxigenin, saponins and sapogenins, their derivatives and metabolites,
- substances mimicking steroids, such as diethylsilbestrol.

 Those skilled in the art will of course be able to adapt the principle of the device and of the assay method to the molecule which it wishes to assay while respecting the very principles of this device as briefly described above and detailed below in the examples. This diagnostic test therefore allows in a single step, without washing step or enzymatic revelation, neither use of complex apparatus, nor of particular technical knowledge to detect and measure the presence of a hapten in a sample: the manipulator directly applies the sample to be dosed in the receptacle provided for this purpose, for example directly above the reservoir containing the conjugate and allows the migration to continue for a few minutes. Whether the samples contain or do not contain the desired molecule, one or more the other of the two capture bands will capture the labeled conjugate; The existence of these two positive and negative capture bands gives the device internal control over the proper functioning of the reaction. In addition, having a capture zone for both a positive and a negative reaction makes it possible to concentrate the reagents at the level of a band whatever the result, and therefore to assay small quantities of molecules sought. with great reliability. In addition, this test can be quantitative thanks to the intrinsic characteristics of the reagents, in fact a coloration appears from a predetermined concentration of the hapten to be assayed, linked to a balance between the conjugate and the labeling of this conjugate. Finally, this type of test has a very low unit cost and therefore allows it to be used extensively on a site either as a diagnosis or as a pre-diagnosis and as a guide for possible treatment.

 Finally, a person skilled in the art will be able to extrapolate the operating principles of this device to any test making it possible to simultaneously assay several molecules either in the form of multistrip strips or in the form of discs comprising quarters whose barriers are impregnated with specific reagents of a hapten given and different from one district to another, or any other geometry allowing a simple way to discriminate between the different molecules sought within the same sample or a mixture of samples.

The examples below, which relate to embodiments of possible devices for the determination of amphetamine, will enable readers to grasp all the advantages of this test as regards its ease of implementation and its reliability. They are obviously not limiting and are there only by way of illustration. FIGS. 1 to 4 represent the different devices as described in examples 1 to 4.

 FIG. 1 represents a device in which the reservoir (11) contains a conjugate consisting of a labeled anti-hapten antibody and coupled to biotin with downstream, a negative reading barrier (21) consisting of haptens fixed on the support , capable of fixing the anti-hapten of (11) when that has not reacted with the hapten of the sample, and downstream a positive reading barrier consists of streptavidin (31) capable of capturing the antihaptene labeled and coupled on the one hand to biotin and on the other hand to the hapten of the sample.

 Figure 1a shows the device in its housing, which housing is provided with a receptacle (5) for depositing the sample; FIG. 1b represents a longitudinal sectional profile view of the various elements of the device comprising a filter (6), the reservoir (11), the nitrocellulose membrane (40), the hydroscopic blotting zone (50), all placed on a plastic support of Mylar type (60); Figure îc shows the principle of the test. The arrow indicates the direction of migration.

 FIG. 2 represents a device in which the reservoir (12) is filled with labeled anti-hapten antibodies, upstream is present a sample of haptens coupled to biotin (15), deposited and dehydrated; the first reading barrier (22) consists of streptavidin and the second reading barrier consists of an antibody directed against the anti-hapten antibody present in the reservoir (12).

 FIG. 2a represents the device in its housing, which housing is provided with a receptacle (5) allowing the deposit of the sample; FIG. 2b represents a longitudinal sectional profile view of the various elements of the device comprising a filter (6), the reservoir (12), the nitrocellulose membrane (40), the hydroscopic blotting area (50), all placed on a plastic support of Mylar type (60); Figure 2c shows the principle of the test. The arrow indicates the direction of migration.

FIG. 3 represents an embodiment in which the reservoir (13) contains a specific protein of the hapten to be assayed, the first barrier (23) consists of an anti-hapten antibody and the second barrier (33) consists an antibody against the specific protein as deposited in the reservoir (13);
FIG. 3a shows the device in its case, which case is provided with a receptacle (5) allowing the deposit of the sample; FIG. 3b represents a longitudinal sectional profile view of the various elements of the device comprising a filter (6), the reservoir (13), the nitrocellulose membrane (40), the hydroscopic blotting area (50), all placed on a plastic support of Mylar type (60); Figure 3c shows the principle of the test. The arrow indicates the direction of migration.

 FIG. 4 represents an embodiment in which the reservoir (14) is filled with labeled haptens, the first negative reading barrier (24) consists of a first anti-hapten antibody and the second reading barrier (34) consisting of a second anti-hapten antibody, one and the other of the two antibodies having a difference in affinities of 1 to 3 logs.

 FIG. 4a represents the device in its housing, which housing is provided with a receptacle (5) allowing the deposit of the sample; FIG. 4b represents a longitudinal sectional profile view of the various elements of the device comprising the filter (6), the reservoir (14), the nitrocellulose membrane (40), the hydroscopic blotting area (50), all placed on a plastic support of Mylar type (60); Figure 4c shows the principle of the test. The arrow indicates the direction of migration.

 In FIGS. 1 to 4, the biological material contained in the reservoir is either plumb, or slightly shifted downstream of the sample receptacle (5).

EXAMPLE 1 dosage of amphetamine according to a first embodiment.

This first embodiment uses the streptavidinbiotin system as a positive reaction reading barrier.

 FIG. 1 shows schematically the device of this embodiment, the reservoir zone containing a conjugate consisting of an anti-hapten antibody labeled with colloidal gold, the anti-hapten being covalently coupled to biotin.

A first negative reading barrier of the reaction consists of amphetamine fixed on the support
If the sample whose presence of hapten is sought contains amphetamine, the latter will then form an affinity complex with the anti-hapten-biotin-colloidal gold conjugate and, during the migration by capillarity along the support not to be retained by the hapten barrier (fixed amphetamine), the paratopes of the conjugate then being saturated by the corresponding epitopes of the hapten; on the contrary, it will be captured at the streptavidin barrier by the formation of the streptavidin / biotin-conjugate complex; if, on the contrary, the sample does not contain amphetamine, then the migration of the conjugate will, at the level of the first fixed amphetamine barrier, lead to the formation of an amphetamine / conjugate complex and the coloring then will be revealed at the level of this first barrier.

 In other words, the marking at the first window corresponding to the hapten barrier indicates the absence of hapten in the sample, the coloring at the second reading window, at the level of fixed streptavidin, indicates the presence hapten in the sample.

 But in any case, a coloration must appear at the level of one or the other barrier.

 Preparation of the device.

 a) Preparation of the anti-amphetamine-biotin conjugate.

NHS biotin is used for coupling to the anti-amphetamine antibody. The N-hydroxysuccinimide esters react with primary amines to form amide bonds. The reaction usually consists of a nucleophilic attack on an amine (E in most cases). The result is the formation of a stable amide bond with production of N-hydroxysuccinimide.

This reaction is favored by the alkaline pHs favorable to the unprotonated state of the amides. Certain precautions must be taken for the use of this type of esters which are not very soluble in water; thus pre-solubilization in dimethyl sulfoxide (DMSO) or in dimethyl formamide (DMF) is often recommended. The use of buffers containing amines such as Tris and azides is formally discouraged. Hydrolysis of the NHS ester is usually the only reaction that could interfere with the coupling itself. This hydrolysis reaction is favored by very dilute protein solutions; it does not take place with concentrated protein solutions.

Biotinvlation reagents
Equipment
Antibody solution at 10 mg / ml in PBS
Biotin NHS (CALBIOCHEM Ref. 203188)
0.1 M carbonate-bicarbonate buffer pH 9.5
Dimethysulfoxide (DMSO) (Prolabo Ref. 23486297).

 Protocol 1. Dialyse the antibody solution against 0.1 M carbonatebicarbonate buffer pH 9.5.

2. Prepare a 100 mM biotin solution in DMSO.

3. Mix the biotin with the antibody solution in order to be at 10 mM final in biotin.

4. Bring the protein concentration to 4 mg / ml by adding 100 mM carbonate-bicarbonate buffer pH 9.5.

5. Incubate 1 hour at room temperature with shaking.

6. Dialyze the solution against 2 mM borate buffer pH 9.0.

7. Store at 4 "C. Do not freeze.

b) Preparation of the anti-amphetamine-biotin-colloidal gold tracer
The tracer has a crucial role: it allows the reading of the test result.

In addition, the choice of the conditions for manufacturing the gold particles is very important since their size determines the migration performance of the test.

 bl) Protocol for the production of colloidal gold particles 1. The containers used must be washed carefully, rinsed with demineralized water and siliconized.

2. Prepare a 0.01% solution of chloroauric acid in demineralized water.

3. Prepare a 1% solution of sodium citrate in deionized water.

4. Bring the required volume of chloroauric acid solution to the boil.

5. Boil for 10 minutes.

6. Add the sodium citrate solution to obtain a final sodium citrate concentration of 0.7%.

7. Leave to stir for 20 minutes (the solution turns blue in 10 minutes and then red in 15 minutes after adding the sodium citrate solution).

b2) Protocol for manufacturing the conjugate
In the case where a monoclonal antibody is used, the isoelectric point is determined beforehand by isoelectric focusing electrophoresis (IEF).

The antibody is dialyzed for 2 hours at room temperature against a 2 mM sodium borate solution buffered to a pH greater than 0.5 unit at the isoelectric point of the antibody considered.

The concentration of the antibody solution is adjusted to 0.1 mg / ml with the 2 mM sodium borate solution buffered to pH9.

To 2 ml of the antibody solution are added 20 ml of a suspension of micro particles of colloidal gold whose average diameter is between 10 and 30 nm.

The mixture is stirred for 5 minutes at room temperature.

 It is centrifuged at 15,000 g for 30 minutes and at 4 "C.

The supernatant is removed and the residue is taken up in 5 ml of a 20 mM Tris solution buffered to pH 7.4 and containing 1% bovine serum albumin 0.1%
Tween 20 0.5% polyethylene glycol 3000.

The reaction mixture is again centrifuged 2 times under the same conditions. At the end of the third centrifugation, the pellet is taken up in 1 ml of a 20 mM Tris solution buffered to pH 7.4 and containing 1% albumin serum, 0.1% Tween 20 and 0.5% of polyethylene glycol 3000.

The optical density of the solution is measured at 595 nm.

The tracer solution thus obtained is stable for 6 months at 4 "C.

c) Preparation of the migration support
c,) Preparation of Amshetamine BSA
As a small molecule, amphetamine cannot be directly attached to a solid support. Prior coupling to a carrier macromolecule is necessary. BSA was chosen as the model.

The mixed anhydride method can be used to couple amphetamine to BSA (Erlanger et al 1959), (4).

To 7.94 moles of BSA, add 15.9 moles of tri-n-butylamine and 8 parts of isobutylchlorocarbonate. BSA 1125 (200,000 dpm, 300pg) is added at the start of the reaction to serve as a marker.

After 30 min of stirring at 10 "C., add 0.3 moles of amphetamine. The mixture, the pH of which is adjusted to 9.5 with sodium hydroxide, is kept for 3 hours at 4" C. It is then dialyzed against distilled water overnight at 4 "C. The pH of the dialysate adjusted to 4.5 with 10.1 N HCl and after overnight at 4" C. the suspension obtained is centrifuged for 20 min. at 3000 rpm. The precipitate is dissolved in a minimum volume of water (<1 ml) then it is lyophilized and kept at -20 C.

By this method, we manage to couple 25 +3 moles of amphetamine per mole of
BSA.

Coatinq of streptavidin, amshetamine and anti-mouse antibody
Place 1 l of 10 mg / ml streptavidin in PBS
Place 1 µl of 4mg / ml BSA-amphetamine in PBS
Place 1 µl of 10 mg / ml anti-mouse antibody in 0.1 µM Acetate buffer pH 4.7.

Wait 15 minutes at room temperature.

Immerse in PBS Sucrose 5% Milk 0.5%
Leave to stir for 30 minutes at room temperature (RT).

Wash with PBS, shaking for 15 minutes.

Remove the PBS.

Immerse in a 2% solution of PVA (Polyvinyl alcohol).

Leave for 30 minutes with stirring.

Remove the PVA solution and dry the support at room temperature.

Before use, keep the support under vacuum in aluminum foil, at room temperature.

Preparation of the conjugate reservoir
Immerse the fiberglass sheet (GF) (200X250 mm) in the saturation solution (PBS / Milk 1% Sucrose 5%).

Leave for 15 minutes at room temperature.

Leave to dry for 1 night at 37 "C.

Deposit of conjugate by spot of 10, ul
Let dry at least one hour at room temperature.

Test protocol
Place 200 μl of sample at the receptacle. Wait 5 minutes for migration at room temperature.

Read the result in the reading windows.

Positive sample
Signal appearance in the second reading window marked Positive.

This signal corresponds to the stopping of the Amphetamine-Anti-Amphetamineor colloidal-Biotin complex by streptavidin.

Negative sample
Signal appearance at the first marked reading window
Negative. This signal corresponds to the stopping of the conjugate by the amphetamine barrier.

EXAMPLE 2 Determination of amphetamine using embodiment # 2.

FIG. 2 is a schematic representation of this embodiment in which the conjugate consists of an anti-amphetamine antibody bound to colloidal gold deposited in the reservoir and dried, and upstream of said conjugate or contiguous in the reservoir but not mixed with the conjugate, a sample of amphetamine coupled to biotin is also deposited and then dehydrated.

The strip consists downstream of the reservoir of a negative reading barrier consisting of fixed streptavidin then of a positive reading barrier consisting of an antibody directed against the anti-amphetamine antibody; the latter being a mouse antibody, it is therefore a mouse anti-immunoglobulin antibody.

Preparation of the strip
Place 1 µl of 10 mg / ml streptavidin in PBS.

Deposit 1 µl of mouse anti-Ig antibody to 10 ml in 0.1M acetate buffer pH 4.7.

Wait 15 minutes at room temperature.

Immerse in a PBS mixture, Sucrose 5% Milk 0.5%.

Leave to stir for 30 minutes at room temperature.

Wash with PBS, shaking for 15 minutes.

Remove the PBS.

Immerse in 0.2% PVA (Polyvinyl alcohol) solution.

Leave for 30 minutes with stirring.

Remove the PVA solution and dry the support at room temperature.

Before use, keep the support under vacuum in aluminum foil, at room temperature.

Preparation of the conjugate reservoir
Immerse the fiberglass sheet (GF) (200X250mm) in saturation solution 2 (PBS / Milk 1% / Sucrose 5%),
Leave for 15 minutes at room temperature,
Let dry 1 night at 37 "C,
Deposit of conjugate by spot of 10 pI,
Let dry at least one hour at room temperature.

Deposit of the Amphetamine-Biotin complex.

The Amphetamine-Biotin complex is prepared according to the method described above. Biotin-X-NHS is used for coupling to the amine function of amphetamine.

10 μl of amphetamine-biotin are deposited on the conjugate reservoir.

Test protocol
Deposit 200 μl of sample. Wait 5 minutes for migration at room temperature.

Read the result in the reading windows.

Positive sample
The amphetamine contained in the sample competes with amphetamine coupled to biotin (and deposited on its trajectory) for limited sites of anti-amphetamine labeled with colloidal gold (tracer).

The tracer is complexed by the amphetamine to be dosed. The complex migrates; it is then stopped by the anti-mouse Ig antibody barrier.

Negative sample
Place 200 µl of sample at the sample well. Amphetaminebiotin complexes the whole anti-amphetamine-colloidal gold tracer. This molecular complex is completely stopped at the streptavidin window. The second window should remain colorless.

EXAMPLE 3: Determination of amphetamine by embodiment No. 3.

The embodiment n "3 is shown schematically in Figure 3:
The conjugate consists of a specific protein capable of coupling with amphetamine, in this case alphafoetoprotein or AFP, labeled with colloidal gold. Downstream of the reservoir in the direction of liquid migration is the first positive reading barrier made up of an amphetamine-bound anb antibody, then downstream the negative reading barrier made up of an antibody directed against AFP. This system is a sandwich type assay because if the amphetamine is in the sample it will find itself sandwiched between AFP on the one hand and the anti-amphetamine antibody fixed at the level of the positive reading barrier d 'somewhere else.

On the contrary, in the absence of amphetamine in the sample, the conjugate will migrate to the negative reading barrier where it will be captured by the anti-AFP antibody.

Preparing the migration medium
Simultaneously deposit 3 µl of anti-amphetamine antibody and 1 µl of anti-AFP antibody. These two solutions are 10 mg / ml in PBS.

Wait 15 minutes at room temperature.

Immerse in PBS Sucrose 5% buffer, Milk 0.5%.

Leave to stir for 30 minutes at room temperature.

Wash with PBS, shaking for 15 minutes.

Remove the PBS.

Immerse in a 2% solution of PVA (Polyvinyl alcohol).

Leave for 30 minutes with stirring.

Remove the PVA solution and dry the support at room temperature.

Before use, keep the support under vacuum in aluminum foil, at room temperature.

Preparation of the coniuciue tank
Immerse the fiberglass sheet (GF) (200X250 mm) in saturation solution 2 (PBS / Milk 1% / Sucrose 5%).

Leave for 15 minutes at room temperature.

Leave to dry for 1 night at 37 "C.

Deposit of the conjugate by spot of the Opi.

Let dry at least one hour at room temperature.

Test protocol
Deposit 200p1 of sample. Wait 5 minutes for migration at room temperature.

Read the result in the reading windows.

Positive sample
Place 200 pI of sample at the receptacle. The amphetamine contained in the sample is complexed by the tracer which is colloidal gold labeled AFP. This complex is stopped by the anti-amphetamine antibody: there is an appearance of a signal in the reading window.

Negative sample
Place 200 pI of sample at the receptacle. The plotter stops only at the level of the second reading window where it is immobilized by the anti-AFP.

EXAMPLE 4 Amphetamine assay using as a conjugate
Amphetamine marked with colloidal gold and as a positive or negative reading barrier for the reaction. two anti-amphetamine antibodies before different affinities for amphetamine as explained below
Choosing anti-amphetamine antibodies II is a crucial step. The affinity of the antibodies for amphetamine is at least 10-9 M. The calculation of the affinity and the choice of the antibodies is made from experiments of competition between the different forms of the hapten.

In the case of amphetamine, the antibody 1 is chosen to have an affinity for free amphetamine of 10 × 10 M. On the other hand, its capacity for fixing the amphetamine-colloidal gold complex is weaker.

Antibody 2 is chosen to have an affinity for amphetamine of the order of 1 to 8 to 109 M. It likewise binds complexed amphetamine to colloidal gold.

Preparation of the Coating migration support for anti-amphetamine
Add 1 µl of 10 mg / ml anti-amphetamine 1 to PBS.

Place 1 µl of 5 mg / ml anti-amphetamine 2 in PBS.

Wait 15 minutes at room temperature.

Immerse in PBS Sucrose 5% Milk 0.5%
Leave to stir for 30 minutes at room temperature.

Wash with PBS, shaking for 15 minutes.

Remove the PBS.

Immerse in a 2% solution of PVA (Polyvinyl alcohol).

Leave for 30 minutes with stirring.

Remove the PVA solution and dry the support at room temperature.

Before use, keep the support under vacuum in aluminum foil, at room temperature.

Coniucca manufacturing protocol
Amphetamine-colloidal gold conjugate is made from BSAamphetamine, but it can also be prepared directly without the use of a carrier protein.

The protocol for preparing BSA-amphetamine is the same as that which was set out in Example 1.

BSA-amphetamine is labeled with colloidal gold following the antibody coupling protocol.

Preparation of the conjugate reservoir
Immerse the fiberglass sheet (GF) (200X250 mm) in the saturation solution (PBS 1 Milk 1% / Sucrose 5%).

Leave for 15 minutes at room temperature.

Leave to dry for 1 night at 37 "C.

Deposit of the conjugate by spot of 10 pI.

Let dry at least one hour at room temperature.

Test protocol
Deposit 200 μl of sample. Wait 5 minutes for migration at room temperature.

Read the result in the reading windows.

Positive sample
The amphetamine in the sample competes with the labeled amphetamine for attachment to the sites of the anti-amphetamine antibody 1. This first barrier which has more affinity for free amphetamine, fixes it and lets through the amphetamine-colloidal gold conjugate. The anti-amphetamine 2 antibody stops the conjugate and displays a signal at the window marked Positive.

Negative sample
In the absence of amphetamine in the sample, the tracer is stopped by the anti-amphetamine 1 antibody barrier. A signal appears at the level of the window marked Negative.

The embodiment of this assay is shown in FIG. 4.

These 4 embodiments are examples allowing the person skilled in the art to choose, on a case-by-case basis, the experimental conditions for preparing and preparing the device and its use as a function of the hapten that he wishes to assay.

In all of the embodiments proposed here, the conjugate must always be in excess in relation to the suspected quantities of haptens in the sample if it is desired to have a yes / no response at the level of the positive reading barrier or the negative reading barrier.

However, if a person skilled in the art wishes to have a quantification of the hapten sought in the sample, it will then be easy for him to prepare several devices, in the geometry he prefers, whether in parallel of multistrip type or in star shape. or any geometry he wishes, and in which the amount of conjugate can be increasing according to a previously established curve, having made it possible to prepare abacuses correlating the amount of hapten with the amount of conjugate.

 It is clear that in Example 1 for example if the amount of conjugate is such that it can only fix 50% of the amphetamine present in the sample the result will be an equivalent coloration at the level of the positive reading barrier or the negative reading barrier; if the conjugate can only bind 75% of the amphetamine present in the sample, the color will be present at the two reading barriers but will be three times more intense at the positive reading barrier (streptavidin) than level of the negative reading barrier (fixed amphetamine).

The production of such types of charts allowing quantitative dosing is therefore easy to carry out for those skilled in the art.

It is also clear to a person skilled in the art that the two positive and negative reading barriers can, depending on the case, be achieved by using a substance having affinity either for the conjugate or for the conjugated-hapten complex, either for hapten whatever its affinity partner; for example if we repeat the embodiment of Example 2 in which the positive reading barrier consisted of an anti-mouse antibody so as to capture the anti-amphetamine labeled with colloidal gold from the conjugate, it is extremely easy to imagine that this anti-mouse Ig antibody could be replaced by a protein A also capable of forming an affinity complex with the immunoglobulins, fixed on the support
The common point and the originality of all these tests is:: - the principle of having a positive and negative double reading, that is to say of systematically checking that the reaction works properly by the existence of '' a coloring whatever the result of the test, - to be very simple in its realization and to require no additional solvent to be added at the time of the test, nor any subsequent washing, - to make available to practitioners a device which is stable over time at room temperature since all of the constituent components of the device are previously dehydrated.

Claims (18)

Claims 1. Device for rapid analysis and characterization of the presence of a molecule with a molecular weight of less than 10,000 daltons, or hapten, in a liquid sample, said device consisting of a hydrophilic support in which a liquid is capable of migrate by capillarity, having a plurality of distinct regions in which the reagents are deposited and / or dried, this device being characterized in that it consists of at least the following zones: a) a zone for receiving the sample to be tested, b) a reservoir zone containing a conjugate having the double characteristic of being marked by a marker allowing its direct detection and of being a ligand for the molecule to be analyzed, the zones in a) and b) possibly being superimposed or sequential. c) two successive barriers made up of halves of affinity pairs, one of which retains said conjugate during its migration on the support in the absence of the molecule sought in the sample, called a negative reading barrier and the other retains said conjugate in the presence of the desired molecule, the latter then being linked directly or indirectly to said conjugate, this second barrier then being a zone for positive reading of the reaction, d) an absorbent means and contiguous to the hydrophilic support making it possible to promote the flow of liquid from zone a) for receiving the sample through the other constitutive zones of said device, the hydrophilic support and the absorbent means being fixed on a polyethylene film of Mylar type, the whole being positioned in a box provided with '' a receptacle level with zone a) accepting 0.05 to 0.5 ml of liquid to be tested and two windows level with the two barrels successive res, allowing the reading of the positive or negative result of the reaction by observing the marking linked to the conjugate 2 Device according to claim 1 characterized in that the reservoir consists of a material chosen from fiberglass, cellulose , cellulose derivatives, nylon, fibrous or microporous polymers of the high density polyester type, fibers of vegetable, animal or synthetic origin in the form of powders of tablets or of multiple layers, or of cellulose wadding. 3. Device according to claim 1 or 2 characterized in that the conjugate marker consists of colloidal particles containing a metal, preferably gold. 4. Device according to claim 1 or 2 characterized in that the conjugate marker is an organometallic marker. 5. Device according to one of claims 1 to 4 characterized in that all of the elements which constitute it are dehydrated. 6. Device according to one of claims 1 to 5 characterized in that the affinity pairs are chosen from the groups consisting of biotin with avidin or streptavidin, antibodies with antigens, immoglobulins with an affine protein such as protein A or protein G, carbohydrates with lectins. 7. Device according to claim 1 to 6 characterized in that the labeled conjugate contained in the reservoir consists of an antihapten antibody labeled in particular with colloidal gold and coupled to biotin, and in that the reaction reading barrier negative consists of a fixed quantity of fixed hapten and in that the positive reading barrier consists of fixed avidin or streptavidin. 8. Device according to claim 1 characterized in that the reservoir contains upstream of the labeled conjugate with respect to the direction of migration of the liquid by capillary action, another conjugate consisting of a determined quantity of the molecule whose presence is desired coupled directly or indirectly to a ligand of the substance constituting the reading barrier of negative reaction. 9. Device according to claim 8 characterized in that the labeled conjugate consists of an anti-hapten antibody labeled in particular with colloidal gold, in that the second conjugate consists of the desired hapten coupled to biotin, that the negative reading barrier consists of streptavidin and that the positive reading barrier consists of an antibody directed against the labeled conjugate. 10. Device according to claim 1 characterized in that the labeled conjugate contained in the reservoir consists of a specific substance having an affinity for hapten marked in particular with colloidal gold, and in that the reaction reading barrier positive consists of a determined quantity of fixed anti-hapten antibody and in that the negative reading barrier consists of a fixed substance having an affinity for said specific substance, in particular an antibody. 11. Device according to claim 10 characterized in that the specific substance having an affinity for hapten is alpha-fetoprotein. 12. Device according to claim 1 characterized in that the absorbent means consists of a hygroscopic material, in particular cellulose derivatives or cellulose wadding, nylon, fibrous or microporous polymers of high density polyester type, fibers of vegetable, animal or synthetic origin in the form of powders of tablets or of multiple layers. 13. A set of devices according to claims 1 to 12 characterized in that each device contains an increasing amount of conjugate allowing a quantitative assay of the hapten by correlation with a pre-established chart. 14. Method for rapid analysis and characterization of the presence of a molecule with a molecular weight of less than 10,000 daltons, or hapten, in a liquid sample, said method characterized in that it uses a device or a set of devices according to one of claims 1 to 1 1 and consisting of a hydrophilic support in which a liquid is capable of migrating by set of devices according to one of claims 1 to 1 1 and consisting of a hydrophilic support in which a liquid is capable of migrating by capillarity, having a plurality of distinct regions in which the reagents are deposited by impregnation and dried, this device being characterized consisting of at least the following zones: a) a zone for receiving the sample to be tested, b ) a reservoir zone containing a conjugate having the double characteristic of being marked by a marker allowing its direct detection and of being a l igand for the molecule to be analyzed, c) two successive barriers made up of halves of affinity pairs, one of which retains said conjugate during its migration on the support in the absence of the molecule sought in the sample, called a barrier negative reading and the other retains said conjugate in the presence of the molecule sought, the latter then being linked directly or indirectly to said conjugate, this second barrier then being a zone for positive reading of reaction d) of an absorbent and contiguous means to the hydrophilic support making it possible to promote the flow of liquid from the area a) for receiving the sample through the other constituent areas of said device, the hydrophilic support and the absorbent means being fixed on a polyethylene film of Mylar type, L ' assembly being positioned in a box provided with a receptacle directly above zone a) accepting 0.05 to 0.5 ml of liquid to be tested and two x windows perpendicular to the two successive barriers allowing the reading of the positive or negative result of the reaction by observing the labeling linked to the conjugate, said process comprising the deposit of 0.05 to 0.5 ml of sample to be tested in the reception area provided for this purpose, followed by direct or indirect observation by any appropriate optical means of the result obtained at the positive and negative reading barriers. 15. Use of a device according to one of claims 1 to 13 to detect and characterize the presence of a molecule with a molecular weight of less than 10,000 daltons, or hapten, in a liquid sample. 16. Use according to claim 15 associating devices according to one of claims 1 to 12 characterized in that they contain increasing amounts of conjugate, allowing a quantitative dosage of hapten by correlation with a pre-established chart. 17. Use according to claim 15 or 16 characterized in that hapten is a drug which one seeks to detect in a biological fluid. 18. Use according to one of claims 15 to 17 characterized in that the drug is in particular included in one of the following classes: - opiates, or alkaloids including morphine, heroin, codeine, dextromethorphan, or their derivatives and metabolites - synthetic opiates, including methadone, propoxyphene, phenylcyclidine, - cannabinoids including 9-tetrahydrocanabinol (or cannabis), cocaine, LSD, benzoïl of ecgonine, their derivatives and metabolites - amphetamines, MDM (ecstazy), 2,5 imethyoxy4Methamphetamine, catecholamines including ephedrine, L-Dopa, epinephrine, narcine, papaverine or their derivatives and metabolites - the derivatives or metabolites of barbituric acid including phenobarbital, secobarbital, primidone, ethosuccimide, diphenylhydantoin - benzodiazepines, their derivatives and metabolites. 19. Use according to one of claims 15 to 17 characterized in that the drug is in particular included in one of the following classes: - steroids including estrogens, androgens,, adrenocortical steroids, their derivatives and metabolites - glycosides and aglycones including digoxin, digoxigenin, saponins and sapogenins, their derivatives and metabolites  - substances mimicking steroids, such as diethylsilbestrol.