FR2702841A1 - Peptide/anti-peptide immunological device and method - Google Patents

Abstract

The invention makes it possible equally well to detect antigens or antibodies with exactly the same device and method. This is made possible by virtue of the technology of peptides immobilised on a support, making it possible to distinguish with very high sensitivity between an antibody complexed with its antigen and a free antibody, whereas the prior art used these peptides only for capturing possible serum antibodies in a sample before detecting them using second, labelled antibodies.

Description

PEPTIDE-ANTI-PEPTIDE IMMUNOLOGICAL DEVICE AND METHOD
The present invention aims at the rapid qualitative and quantitative determination of a ligand in a fluid. It relates to a device and a method based on the technology of peptides and labeled monoclonal antibodies highly specific of these peptides, characterized, among others, by the possibility of detecting antigens or antibodies concerning the same condition, with exactly the same device.

The method which is the subject of this invention consists of a single-stage and rinseless immunofiltration of a drop (associating the test sample with one or more labeled antibodies specific for the antigen sought in this sample). through a thin ribbon support (which will be described later) or any other rigid support (of the Porex type for example), on which are immobilized peptides to trap the antibodies labeled by their specific immunological site only when this site is free of any antigen, ie when the sample is negative in antigen;
And leaving these labeled antibodies through the ribbon or the support to give a fast signal (chromogenic or otherwise) in seconds under this tape (in contact with means of revelation), when the sample is said positive: the antigens then saturating these antibodies brands.

 The detection of antibodies in the sample is possible with exactly the same ribbon: a large part of the desired antibodies (that is to say having the same idiotype as the labeled antibodies) contained in the seropositive sample, compete (unequal). because infinitely more numerous) with the antibodies labeled at the level of the peptides immobilized on the ribbon: the idiotypic sites of a large part of the antibodies sought in the sample saturate rapidly the immobilized peptides which can no longer trap the labeled antibodies: the latter then cross the ribbon and report in seconds the seropositive sample. This process differs from the previous ones (which will be mentioned later) by the fact that peptides, specially prepared and immobilized on a ribbon or other support, go to trap ANY antibodies (whether human or mouse) that present themselves to this membrane or this support, by attaching themselves to the idiotyp sites However, if this antibody is saturated with antigen, its idiotype sites are not free and the peptides will not trap it. In the same way, if serum antibodies saturate the peptides, the labeled antibody will not be trapped and will deliver a signal of seropositivity.

In this process, in contrast to the previous methods, a double selection takes place at the level of the selective ribbon 1 - saturation of the ribbon with the labeled antibody free of any antigen (negative antigen test) or non-saturation with the antibody labeled already bound to its antigen (positive antigen test) 2 - saturation of the ribbon by the desired human antibodies (antibody test positive) or by the labeled antibodies in the absence of serum antibodies (negative antibody test).

In short, three hypotheses arise in the operation of this process
1 - universal negative: the absence of antigen or antibodies in the sample, leads exactly to the same immunological reaction: trap labeled antibodies
2 - antigen positivity: saturation DES
MARKED ANTIBODIES that are no longer recognized by the peptides of the selective ribbon, peptides that remain free.

3 - antibody positivity: saturation DES
SELECTIVE RIBBON PEPTIDES (by serum antibodies) that can no longer afford the antibodies labeled, however, free from any antigen
This process is therefore different from French patent 9012661 (PCT / FR91 / 00891) published under the number 2667943 and concerning a different immunological process: immunoglobulins anti-enzyme or anti-Fab, immobilized on a support, piegent labeled antibodies when their enzyme is not obscured by an antigen or when their Fab site is not saturated with an antigen, but allowing these labeled antibodies to pass through when saturated with antigens.These immunoglobulins fix only the antibodies used for the test and not serum antibodies. For example: goat immunoglobulins, anti mouse Fab, will trap only the labeled mouse antibodies used for the test, and not the serum antibodies of the sample to be tested.

The first fundamental difference is that each peptide used is highly specific (by definition) for each labeled antibody that arrives on the membrane: for example an anti-Interleukin 2 monoclonal antibody arriving on the membrane, has idiotypic sites highly specific for the peptide of interest. Interleukin 2 chosen to be immobilized, these are the idiotypic anti sites
Interleukin 2 that peptides immobilized on the membrane will trap if they are free of any
Interleukin 2. Each membrane will therefore be prepared for a precise diagnosis, as for example here, the dosage of interleukin 2. On the other hand, the immunoglobulins anti
Fab or anti-enzymes described in the French patent 9012661 reported above, have no diagnostic specifity: someoit the antigen sought, the labeled antibody unsaturated antigen, is piégé anti-Fab immunoglobulins: these immunoglobulins anti -Fab or anti-enzymes were in fact a necessarily polyclonal serum since they trap all antigen-free antibodies, for example: the product Sigma referenced A4185 is a goat polyclonal serum recognizing all mouse Fabs, this serum will therefore trap all mouse antibodies whose Fab sites will be free of any antigen.The advantage of this method was therefore the universal character of the antigen detection device: the same could be used for any diagnosis, only the labeled antibody associated with the antigen. sample was specific to the diagnosis sought.

However, the Fab site of an antibody (mouse for example) is very large and comprises several sites (including the idiotype site which binds the antigen): the serum (goat for example) polyclonal anti-Fab (mouse by example) will therefore contain a large majority of immunoglobulins, each specific to a different site on the Fab branch of the mouse antibodies, to which it will bind whether or not there is antigen, ie, only a small part of these anti-Fab immunoglobulins will recognize on the Fab branch the specific sites of the desired antigen, whereas the rest of these anti-Fab immunoglobulins will bind to all their respective sites on the mouse Fab branch, whether there are or not, an antigen attached to the idiotypic site of the labeled antibody.Where a very poor performance: only very rare mouse antibodies, labeled, saturated with antigens, will escape the membrane supporting anti-Fab immunoglobulin; Hence, if the entire membrane is treated with peptides, the least labeled antibody whose idiotype site will be saturated with antigen, will cross this membrane to deliver an antigen. signal, from which a sensitivity infinitely higher than in the process cited above.Certes, the device is more universal, everyone will be especially
prepared for a precise diagnosis.

 Second fundamental difference: the French patent 90/12661 quoted above, describes only antigen assays, and not antibodies. This is logical: anti-mouse Fab immunoglobulins will trap only mouse antibodies and will not react, like peptides, to the presence of serum antibodies.

 Third fundamental diference: the specificity of the process object of the present invention is infinitely more important: it has just proved that a polyclonal serum (of goat for example) anti-mouse Fab recognized a significant percentage of human antibodies; however, in a human sample (blood or plasma or serum) to be tested, there are very large amounts of antibodies, and they will enter the membrane, in competition with the labeled antibodies of mice intended to complex at the antigen sought ie immunoglobulins (goat) immobilized on the membrane, will be completely saturated by the Fab branches of human antibodies and let slip the mouse labeled antibodies, yet free of any antigen, which will result in a false positive signal under the membrane, while there are no antigens in the sample.

Fourth fundamental difference: peptides do not
recognize that the idiotype site of an antibody that comes into contact with them: whatever the origin of this antibody: whether it is a monoclonal labeled antibody (mouse for example) used to be associated with the sample, or all the antibodies polyclonal (human seriques, for example) having the same idiotype.Example: a peptide of the HIV p24 antigen, will recognize and trap both the idiotype site of a mouse monoclonal antibody anti-p24 specific for this peptide, that the idiotype site of a circulating antibody seric human anti p24
recognizing said peptide.

 However, the same antigen has several different epitopes and therefore causes in the host, the appearance of an antibody (having a specific idiotype site of this epitope) against each epitope. This explains why the method that is the subject of the present invention can use several different peptides (equivalents of epitopes of the antigen), immobilized on the same support.

membrane, and several labeled monoclonal antibodies each specific for an epitope of the antigen, for the same diagnosis.

 Example 1 (FIG. 4 / Plate III) an anti-GP120 antibody detection device will use, immobilized on the membrane support, a whole series of peptides each recognizing an anti-epitope antibody, thus the very numerous seric antibodies. anti-GP120 (micrograms), although each having a different idiotype, will all be trapped by the peptides on the membrane support which will be fully saturated the anti-GP1-labeled mouse antibody associated with the sample, will not be trapped by the peptides (totally saturated) of the ribbon that it passes through to signal the seropositivity.

Example 2: The p24-HIV antigen circulates in seropositive patients at the beginning, in infinitesimal quantities (sometimes less than 50 picograms / ml) indisputable by current techniques, which constitutes the enormous danger
seropositive blood donors at the beginning.This p24 antigen has many epitopes and there are many anti-p24 mouse monoclonal antibodies (each specific for a p24 epitope). Using not one, but several monoclonal antibodies of mice, labeled, anti p-24 (each being
directed against a different epitope), it is possible to detect infinitesimal amounts of p24 because each molecule of antigen will be surrounded by several monoclonal hypermarket antibodies making the signal visible. On the ribbon, will be immobilized peptides each corresponding to the idiotype of each of the labeled monoclonal antibodies used: in the absence of p24, the latter will all be trapped: no signal, while a quantity of 10 picograms / ml of p24 in the sample will mask a sufficient number of their idiotype sites to allow them to cross the membrane support and cause a signal.This explains the extreme sensitivity of the process.

But this also explains the extreme specificity of this technique: the signal will be visible only if each of the monoclonal antibodies labeled antip24 was saturated by its corresponding epitope of the p24. This problem of specificity is the cause of many false results because the usual techniques use a single monoclonal antibody specific for a single epitope of p24 for example. Or, it happens that this epitope exists on another protein not concerned by AIDS and the result is
then a false positive.

REVIEW OF THE PRIOR ART
Previous methods for determining the presence or concentration of antigenic substances in biological fluids, by immunological means, are now well known and are based on the formation of a complex between the antigenic substance to be determined and one or more antibody, one of the components of the complex capable of being labeled with a radioactive element (such as, for example, Iodine 125), to allow its detection and / or quantitative analysis after separation of the antigen or antibody complexed labeled antigen or uncomplexed labeled antibody.

In competition immunoassay methods, the antigenic substance contained in the sample of the test liquid competes with a known amount of labeled antigen for a limited amount of antibody binding sites: the amount of The labeled antigen bound to the antibody is inversely proportional to the amount of antigen contained in the sample.

Immunometric tests particularly suitable for the detection of polyvalent antigens, ie antigenic substances capable of complexing with at least two antibodies at the same time, consist in using an amount of unlabeled antibody bound to an insoluble solid support. in the liquid to be analyzed, and a quantity of soluble antibody carrying an indicator, such as a radioactive isotope, which allows the detection or quantitative determination of the ternary complex formed between the solid phase antibody, the antigen and the These methods involve first contacting the antibody bound to the solid phase with the sample to be analyzed, to extract the antigen from the sample, by forming a binary complex between the antibody. solid phase and antigen.After an incubation period, the solid support is washed to remove the liquid sample residue, including any antigen that has not reacted, and then with a solution containing a known amount of labeled antibody.After a second incubation period to allow the labeled antibody to complex with the solid support-bound antigen via the unlabeled antibody, the solid support is washed a second time to remove the labeled antibody that has not reacted, then the presence of labeled antibody on the washed solid support is detected (for example by measuring the radiation emitted, if the indicator is a radioactive radiation ) and possibly subject to a quantitative determination process.

These methods are known as "sandwich" or "bi-site" methods, since the antigen carries two antibodies bound to its surface at two different sites. These are described by WIDE in "Radioimmunoassay Methods" (Kirkham et al. Hunter, E and S. Livingstone, Ed.

 Edinburgh, 1970, pp. 199-206).

Specific applications (test for the detection of hepatitis antigen) are described in the Patent of
United States of America No. 3,867,517; variants of these methods ("simultaneous" or "inverse") are described by JEONG et al. in "Comparison of RIA with a single incubation two-site immunoradiometric assy (IRMA) as applied to the determination of human thyroid stimulating hormone (HTSH). ), Bio-Rad Laboratories, 1979; in U.S. Patent No. 4,174,384 (labeling two antibodies with a fluorescent chromophore (fluorescein) and a chromophore that absorbs fluorescein-emitted light); in U.S. Patent No. 4,098,876. These methods initially used polyclonal antibodies, the sensitivity of which is insufficient due to the existence of cross-reactivity with other antigens. French Patent Hybridtech NO 81 15030 published in No. 2,487,983, proposed to replace in these immunometric tests, the polyclonal antibodies with monoclonal antibodies, to significantly increase the sensitivity of these methods.

The French patent LIOTTA NO 82 16973, published under the NO 2,514,511, describes a device and a method for determining the presence of antigens in biological fluids, by the ELISA method, while eliminating the washing and dilution operations that It requires and reducing the duration of incubation. The proposed device comprises a matrix consisting of a ribbon paper nitrocellulose or diazobenzyloxymethyl (DBM), porous gel such as polyacrylamide, agarose or collagen, in the pores of which The antigen can be trapped or it can be crosslinked with the gel through the amine groups of the ligand and the carboxyl groups carried by the matrix, or a ribbon of cellulose or plastic fibers inside. from which are trapped particles or beads which contain the ligand.According to this LIOTTA patent, the device comprises - a first zone which contains antigens and antibodies bound to an enzyme, these ant wherein the antibodies are located in said first zone such that they are removed from said first zone when they have reacted with unbound antigens contained in the test sample passing through said first zone, but that they are not not eliminated in the absence of such antigens, and a second zone containing a substance capable of reacting with said enzyme-bound antibodies, to produce a colored reaction revealing the presence of said antibodies.

Specifically, the immunoassay device described by the Liotta patent comprises a porous matrix three-layered laminate, the first of which is impregnated with a specific antibody bound to an enzyme, the second porous layer contains an immobilized reference antigen ( bound), and the third layer contains a substrate producing a color, which reacts with the enzyme bound to the antibody. The free antigen to be assayed present in the sample to be analyzed, put in contact with the first layer, diffuse in the second, then in the third layer. The free antigen present in the sample competes with the bound reference antigen, immobilized in the second layer, to combine with the antibody bound to an enzyme.If enzyme-bound antibody combines with the free antigen, it diffuses freely towards the third layer and produces a colored reaction. On the other hand, if the sample to be analyzed does not contain antigen, all the antibodies The enzyme-linked antibody, which is linked to an enzyme, has free binding sites and combines with the immobilized antigen present in the second layer, whereas the enzyme-linked antibody, which combines with the immobilized antigen in the second layer, does not diffuse into the second layer. the third layer and no color reaction occurs.The French bervet
LIOTTA 87 08007, published under No. 2,599,845 describes a variant of this device, which comprises only two layers, the upper layer carries the antigen with the two reactive antibodies and the lower layer contains a chromogenic substrate, both zones (trapping zone and substrate zone) can also be juxtaposed.

There are very important differences between Liotta and the present invention 1 - The Liotta process uses the principle of antibodies trapped by whole antigens immobilized on a membrane support. Or, if the HCG molecule is used as an antigen (example described by Liotta), it weighs 40,000 daltons.The sites recognized by the antibodies weigh only a few tens of daltons.What makes the membrane support is inefficiently encumbered by the rest of the HCG molecule immunologically neutral, with a ratio active sites / neutral sites of 1 / 1000.Therefore, the labeled antibodies have only a minute chance of being trapped in case of absence of antigens in the sample tested. This is the reason why Liotta uses more than ten superimposed membranes to stop the labeled antibody.

Whereas if we treat a single membrane with only highly specific peptides of the labeled antibody used, there will be on this membrane infinitely more active sites and theoretically no neutral site. By simplifying, we can say that the report active sites Neutral sites is greater than 1000/1, so that the membrane resulting from the present invention is a million times more efficient than those of Liotta.

2 -The new concept of peptide-anti-peptide pair
Liotta could use HCG and polyclonal anti-HCG antibodies at any manufacturer. Here, a rigorous step in the preparation of the reagents is required: a monoclonal antibody must be selected after immunization of the mouse with HCG, for example. antibodies must not cross with any hormone similar to HCG such as LH or FSH, so it must be highly specific. Then cut HCG antigen in large peptides, identify the one that is recognized by this monoclonal antibody. this large peptide selected in smaller peptides to determine the ultra specific peptide of the monoclonal antibody. The production in large quantities of this peptide will be provided by genetic engineering by bacteria when the messenger RNA then the DNA will have been reconstituted from of the amino acid sequence of this peptide.It is also possible to use the latest automatic techniques of in vitro production of peptides if their length does not exceed a number of amino acids.

U.S. Patent No. 4,632,901 (and corresponding International PCT Application No. 85/05451) describes a method and device for making immunoassays that do not require the use of long incubation steps associated with multiple washes. , and allow by their simplicity, the implementation by a doctor, at its consultation, and even by the patient at home.This device comprises a first porous element consisting of a membrane or a filter to which is bound an antibody, monoclonal or polyclonal which recognizes the antigen to be identified, and a second element which is absorbent and has capillary passages perpendicular to its upper and lower surfaces; this second element is in capillary communication with the membrane, or the like, porous, which forms the first element of the device, and has a pore size such that it induces the passage of liquid through the first element without applicatio n external means.The filter membrane may be nylon bearing NH 2 groups to which the antibodies are covalently bound, by coupling with glutaraldehyde and the absorbent element may be in fibrous filter material such as cellulose acetate fibers. cellulose, polyester, polyolefin, etc. These two elements can be separated from each other by another porous element made of polyethylene, for example) which does not bind antibodies in a non-specific manner and prevents the labeled antibody binds non-specifically to the upper surface of the absorbent element.

Also, European Patent Application ABBOTT Laboratories No. 186,100 discloses a device for determining the presence or amount of a substance in a test sample, and which comprises a non-absorbing porous fibrous matrix, such as glass, impregnated with a hydrophobic polymer which forms a surface coating on at least a portion of the matrix and to which is attached a reagent, for example an antibody or an antiene, capable of reacting with the desired substance in the sample, this device being able to be associated with a layer of underlying absorbent material and / or a layer of fibrous material such as glass fibers or cellulose filter paper, which covers the fibrous matrix and can act as a pre-filter.

The European patent application MUREX Corp. No. 206,561 also discloses a diagnostic device which comprises a filter element comprising at least one reaction zone associated with at least one peripheral zone, an absorbing element associated only with the peripheral zone of the first element and a filter holding element The "reaction" that occurs in the reaction zone may as well be a separation by filtration that an immunological coupling and it is in the same zone, that appear the read signals of the reaction. liquid sample reaches the filter through a funnel whose discharge port is calculated so that its diameter is sufficient, in combination with the pressure of the liquid in the funnel, so that the liquid discharges on the upper surface of the filter but does not cross the latter, the hydrostatic pressure being subsequently adjusted in such a way that the liquid enters the filter by gravity and is diffuse through the latter by capillary action, without crossing it perpendicularly from one side to the other.

The KONICA Corp. Patent No. EP-A-328106 is also a superposition of three compartments which have the same function as in the patent object of the present invention but it is still here a trapping free labeled antibodies with a corresponding antigen immobilized in the compartment median.

The European patent Boehringer Biochemica Robin (0 303 110) relates rather to a horizontal filtration on strips with again immobilized antigens on the selective zone, to trap the labeled antibodies in case of absence of antigens in the sample.

All these processes have in common a filtering element, through which flows the liquid sample to be tested. The filtration is made selective by the presence at this level, immobilized, of an identical antigen to that sought while in the device. object of the present invention, the filtration is made hyperselective by the presence at the level of the filter element, peptides on the one hand hyperspecific of the labeled antibody used, and on the other hand in infinitely greater number than the antigens; moreover, all this prior art is intended exclusively for the determination of antigens and not of antibodies.

Mattiasson, Danielson, Hermansson and Mosbach (FEBS Letters.

85: p 203,1978) have highlighted the changes that occur in the structures of proteins (antigens for example) when they are no longer in their physiological conditions, and propose a series of interesting solutions.

On the other hand, the literature is abundant on the high qualities of resistance, conservation and stability of the peptides.

As regards the devices of the prior art using peptides immobilized on membranes, they serve exclusively to trap the corresponding serum antibodies which will be revealed later after several complex steps of washing and revelation.They do not solve the problem the coexistence of antigen + antibody complexes and an obligatory stage of complex treatment of the sample with solutions of TRIS-HCl and then of glycine to separate the antigens from their antibody, is indispensable. The most typical case being the screening of HIV in neonates of seropositive mothers: the presence of p24 antigen associated with the antibodies of the mother, condemns the infant whereas if it possesses only the maternal antibodies, it will be spared.

It has therefore been the object of the present invention to provide a device and a method for the rapid qualitative and quantitative determination of the presence of a reactive ligand in a fluid, better meeting the requirements of the practice than devices and methods. processes for the same purposes proposed in the prior art.

The present invention firstly relates to a device for the rapid qualitative and quantitative determination of the presence of a reactive ligand in a fluid of the type comprising (FIG. 1 / Plate I) two separate elements - a container (A) (sample pear, tube or any other) containing a solution of labeled antibody and intended to receive and dilute, a drop of the test sample (whole blood or plasma or serum or saliva or urine or tears or cephalo-rachidian liquid or other biological fluid). The labeled antibody may be freeze-dried if the sample does not need to be diluted.

- In some places (see below, the mounting examples), a ribbon (B) supports the immobilized peptides on its upper side, and on its lower side, the revealing means (for example chromogenic) indicating that the labeled antibodies originating from A have escaped the peptides of the upper face and crossed this ribbon.

This tape consists of any porous material (paper, nylon, glass, Porex media, etc.) that is transparent, so that it is not necessary to turn the ribbon over to read the coloring of B: the deposit of the Then, a few seconds later, the reading is done on the upper side of the ribbon, which can be simply glued to any opaque medium preferably, or simply inserted into a medical file or pinned to a card.

To perform a semiquantitative assay (FIG. 3 / Plate II), the sample is first brought into contact with a first container (A1) containing NON-MARKED antibodies this time, in an amount only sufficient to bind to certain only amount of antigen; in a second step, the contents of the container Al are transferred to the container (A2) containing antibodies labeled this time
if there is an excess of antigen (Fig 3 + / Plate II), the antigens still free after step A1, will bind in
A2 to the antibodies (labeled this time) and the complexes will cross the selective membrane B to deliver an antigen excess signal.If there is no excess of antigens (Fig 3- / Plate II) , all of which will have been bound in A1 to the unmarked antibodies of the first step, so that the labeled antibodies of A2 will not be saturated with antigen and will be trapped in B.

According to another alternative embodiment of a semiquantitative dosage, a labeled antibody solution is added to the recipient Al in a second time, after incubation of the sample with the Al content (labeled antibody), which avoids using a container A2.

According to the present invention, it is possible to perform quantitative assays, by associating the revelation membrane B with a quantization apparatus known in itself, such as RIA (if the labeling is radioactive), spectrophotometer, etc.

The functional check (fig 2 / plate I)) consists in depositing the first drop of A (sample + conjugate) in a space on the ribbon, well indicated (by printing "CTRL" for example) and which is colored anyway (testifying to the good conservation of the reagents), and a second drop of A in a second space also well signaled ("TEST" for example) in which the signal appears only in case of positive test.

Only the "test" space having been prepared with peptides.

The device object of the present invention differs well from the following devices found in the prior art
These patents all describe membrane overlays, each of which plays a different role, but does not possess the contact step A of the labeled antibody with the sample, before depositing the drop.

They have in common the reading of the result by flipping the device while the device object of the present invention offers a reading of the result seen from above by transparency.

In addition, they are necessarily rigid and opaque instruments, welded cards, etc., whereas here it is a small ribbon (easily inserted in a medical file for example).

None of them incorporate the slightest functional control.

None of them allows the determination of antibodies.

Finally, they all integrate, dry, conjugated antibodies at the level of their upper membrane in order to proceed directly to the assay by deposit of the sample, whereas here there is first the passage of the sample by a container. (A) on the one hand for the compulsory dilution of the samples (which would be too viscous otherwise) and especially to put in contact (by intense mechanical agitation or vortex, notably) the sample with the conjugated antibodies for a sufficient time so that the eventual The antigen-labeled antibody reaction is as complete as possible, which is not the case when the sample is placed directly on the labeled antibodies: the reactions are only partially (if they occur)
Moreover, the declination of this first step A makes it possible to carry out several different tests: by not using a container A but an AX container containing a labeled antibody specific for an X antigen, a second container, AY, containing a specific labeled antibody. of a Y antigen, etc. The ribbon can be unique: if it is treated with peptides of all labeled antibodies used. It will then have a space "TEST X", a space "TEST Y", etc. and a single "CTRL" space
This example can very well be applied to tumor markers: the detection of a debutant digestive cancer is more effective if three or more different markers are in excess, whereas taken separately, they are not very specific: example: colon cancer: the CEA (carcinoembryonic antigen) is increased only in 55% of cases, CA 19-9 only in 60% of cases, CA 15-5 in 45% of cases and AFP (alpha foeto proteibne) In contrast, when at least three markers are in excess, they sign 99% of colon cancers.Thus a device with three ribbons (ACE, CA 19-9, CA 15-5) where only one markers is in excess, should not worry, while if the three tests deliver a signal of excess, there is a very high probability of cancer and extensive explorations should be made, focused on the digestive sphere.

But it is also possible to reduce these three vessels to a single container by associating the three labeled antibodies specific to the three markers: if there is signal, it is that at least one of the three is in excess: at this moment There, you have to test with the three different recipients to know which of the markers is in excess and especially if it is one or all the markers which are in excess. This example is also valid for the different antigens HIV or for any other affection involving several parameters.

The subject of the present invention is also a method for rapid qualitative and quantitative determination of the presence of a reactive ligand in a fluid, which is characterized in that during a first step, a sample of fluid to be analyzed is placed in a container (A), with a labeled antibody, in that during a second step, the product of this first reaction comes into contact with peptides (specific for the idiotype of the labeled antibody). A) immobilized on a support B and able - either to trap the labeled antibody coming from A very quickly if it is not saturated with antigens (negative antigen assay) - or to let it pass through B (to provoke a signal on the underside of B) if it is saturated with antigens (positive antigen assay) - is able to be saturated with the desired serum antibodies and thus let B pass for a seropositivity signal (positive antibody assay).

If the desired ligand is an antigen, the labeled antibody (at A) forms with it a complex that will cross the selective membrane B, while the labeled antibody is trapped at B in the absence of antigen in the sample.

If the desired ligand is an antibody, there will be competition at B level between the serum antibodies (whose idiotype is identical to that of the labeled antibody) and the labeled antibodies (of A) that have been associated with the antibody. sample: the peptides of the selective membrane B are rapidly submerged and saturated by the idiotype of serum antibodies arriving in huge quantities (a seropositive blood in HIV for example contains large amounts of anti-HIV antibodies). encounter more peptide still available on membrane B and cross it to deliver lower, a seropositivity signal.This signal is not due to the passage of an antigen-antibody complex labeled through B but rather to entrapped by B, labeled antibodies; this "non-trapping" being due to the rapid and significant saturation of the selective membrane B by the serum antibodies.

 This "non-trapping labeled antibodies, yet free" is one of the original features of this invention and the big difference with the prior art.

EXAMPLES OF DEVICES
EXAMPLE 1: FIGS. 7 / Plate VI and FIGS. VII
PREPARATION OF HBs ANTIGEN DOSING DEVICE
1 - Preparation of labeled monoclonal antibodies: AH1 and AH2
2 - Preparation of peptides: PH1 and PH2
3 - Preparation of the membrane support B
4 - Operating mode of the assay
1 - The anti-HBs monoclonal antibodies are obtained from immunization of mice with the HBs antigen and then selected from all the antibodies obtained, for their high affinity and specificity for the corresponding peptides.

Marking retained: Fe (CN) 6
Number: 2 numbered AH1 and AH2 each recognizing a different epitope of HBs antigen.

 2 - The peptides (directed against the idiotype site of AH1 and AH2) are obtained by digestion of the HBs antigen and then selected on a column by their corresponding monoclonal antibodies.

3 - The membrane support: A 2cm2 nylon ribbon (Pall), divided in pencil into two squares on which one
print on the left "TEST" and on the right "CTRL" (control). In the center of the square "test" is deposited 10 microliters of a solution containing peptides PH1 and PH2 at a rate of 10 micrograms each per ml of PBS + BSA (Bovine Serum Albumin) While on the square "ctrl" one deposes a solution of PBS + BSA only. After three hours rinsing and drying.

 The membrane is then inverted and 3 μl of an alcoholic solution of benzidine at 1 mg / ml is deposited under each square.

The squares "CTRL" will color anyway at
contact of the sample containing the conjugates because these
with or without antigens sought, will not be
retained on the upper face of the box "ctrl" and will go
stain the substrate.

The "TEST" squares, supporting the peptides, will trap at the
upper surface of B, non-labeled antibodies
saturated with antigens, which will not be able to enter
contact with the substrate of the lower face.
against if the labeled antibodies are saturated in
antigens, they cross B and come into contact with
substrate for producing a signal.

4 - Operative mode
- a drop of whole blood or plasma is taken
or serum of 10 microliters which is poured into the
container A containing 90 microliters of a relief
Conjugated antibody conjugates at 10 micrograms / ml
in a solution containing TRIS 0.1 M, BSA 1%, PEG
1%, Antibiotics (Gentalline and Mycostatin), serum
fetal calf 1%, Antitrypsin, Aprotinin.

 Vortex 30 seconds at least.

- A sample of 50 microliters is taken which is deposited in
B on the square "ctrl": a blue spot appears
immediately by transparency: the reagents are well
canned.

- the remaining 50 microliters are removed
deposit on the square "test" and we get the results
following 0 to +++ depending on the concentration
of HBs antigen in the serum
(1 IU = 1 nanogram / ml)
HBs in IU / ml: 0 0.0001 0.001 0.01 0.1 1 10
Intensity of the spot 0 +/- + + / ++ ++ +++
EXAMPLE 2: FIGS. 7 / Plate VI and FIGS. 9 / PL.VIII
ANTI-HBs ANTIBODY ASSAY
Same assembly, same device. Same sampling.

If 50 microliters of a known seropositive serum are taken as anti-HBs antibodies, the following color intensities are obtained according to the dilutions made on the serum.
Dilution rate: 1 / 100,000 1 / 10,000 1/1000 1/100
Signal obtained: +/- + ++
EXAMPLE 3: HIV 1 Antibodies The antibodies and peptides p24-GP40 and GP120 are kindly offered by Innogenics: two monoclonals for each protein derived from immunization of mice with these peptides.

2 - the chosen marking is triple, to amplify the signal
- ferritin for iron
- haptoglobin to exalt ferritin
- Silver nitrate to amplify the substrate spot 3 - Neutralization of possible seric antibodies
Very high protein binding microbeads (Lab.IBF France) are treated with peptides derived from HIV viruses and placed in solution in a container A0 which will receive first the sample to be tested. The supernatant is then poured into the following different containers (see below) to be in contact with the labeled antibodies.

4 - The peptides of B are prepared from the monoclonal antibodies provided and then fixed on a membrane 4 cm long and 1 cm wide, thus divided into four squares: 1st printed square: "p24" - 2nd square - "GP41 "- 3rd square" GP120 "- 4th square" CTRL "
After rinsing and drying, the membrane is turned over and a spot of 3 μl of benzidine at 1 mg / ml is placed in the center of each square.

OPERATIVE MODE
A container marked with felt: "A-p24" is prepared with 90 microliters of the solution containing this time the two monoclonal antibodies labeled anti p-24.

Then a second container "A-GP41" with 90 microliters of the anti-GP41 labeled antibody solution, then a third container "A-GP120" with also its anti-GP120 labeled antibodies.

10 microliters of serum to be tested from the first container A0 (thus stripped of parasitic antibodies) are poured into each of the containers.

Vortex agitation 30 seconds.

Take 50 microliters from any container and place it on the "control" square, wait for staining before carrying out the 3 tests.

Then 50 microliters of "A-p24" are deposited on the square marked p-24.50 microliters of "A-GP41" on the noted square.
GP41 and 50 microliters of the container "A-GPI 20" on the square noted Gel 20.

Depending on the presence or absence of these antigens, we obtain the results schematized in Figure 4 - Plate III
The sensitivity limits obtained are 10 picograms / ml.

EXAMPLE 4: FIGURE 5
SEMI-QUANTITATIVE ASSAY OF D-DIMERE IN PLASMA
The determination of D-dimer and other precursors of fibrinogen is timely and very promising to detect clot formation before embolism occurs.It is possible to make a diagnosis of pre-infarction or pre-infarction pulmonary embolism or pre
Stroke or pre-phlebitis. It is of course more interesting to intervene on a patient before he has an infarct: the treatments and the consequences are much less serious.

D-Dimer circulates in the blood in all human beings at a rate lower than 0.4 micrograms / ml of plasma. Beyond this, the prognosis of embolism is very high but is not 100%: all people with a
D-Dimer elevated do not make embolism.By cons, 100% of people with less than 0.4 micrograms of D
Dimer does not make embolism.It is thus a diagnosis of exclusion, very useful in emergencies, in cardiology, or in follow-up of reanimation after a surgical operation or in front of any suspicion of imminent accident:: it allows to direct the diagnosis and the treatment and especially to avoid this high rate of postoperative pulmonary embolism or in the cardiac arrhythmias very generators of embolies. Just as it avoids unnecessary constraints and care for patients in whom there is certainty that there will be no embolic accident.

Anti-D-Dimer antibodies are 2F7 and 9C3
The peptides directed against them are P2F7 and P9C3
D-Dimer is supplied freeze-dried in vials of 1 mg to be reconstituted with 1 ml of sterile distilled water. The molecular weight of D-Dimere is identical to the PM of its antibodies.

All these products were kindly offered by the company Stago-Diagnostica (Asnières-France).

The principle of the semi-quantitative determination retained, will consist in neutralizing 0.4 micrograms / ml of D-Dimere in a sample by NON-MARKED antibodies. For this one operates as follows 1 - In a container Al containing a solution at 0.4 micrograms / ml of NON-MARKED 2F7 antibody and 0.4 mg / ml of NON-MARKED 9C3 antibody (Molecular weight parity), an equal volume of plasma to be tested and vigorous vortexed for 30 seconds.

The equivalent of 0.4 micrograms / ml of D-Dimer is sandwiched between the unlabeled 9C3 and the unlabeled 2F7. This complex will cross the selective membrane B without giving any coloration.

2 - Then pour the contents of Al into a container
A2 containing the 2F7 and 9C3 antibodies labeled with mercury salts (weak labeling) this time and vortexed for 30 seconds.If the D-Dimere level is greater than 0.4 μg / ml, D-dimer complexes and labeled antibodies are form to deliver an excess signal through B.

3 - Deposit 50 microliters on the control square before depositing 50 microliters on the test square.Results DD (μg / ml): 0 0.1 0.2 0.3 0.4 0.45 1
Signal: 0 0 0 0 +/- ++
EXAMPLE 4 Figure 6 / Plate V
SEMI-QUANTITATIVE ASSAY OF THE SPECIFIC ANTIGEN
PROSTATIC (PSA)
This example is interesting because it is actually a semi-semi-quantitative assay, that is to say that within the semiquantitative dosage itself, there is a limit between adenoma and carcinoma.

PSA does not exist in young subjects and is produced between 0 and 4 nanograms / ml of serum in case of benin adenoma.It greatly exceeds this rate in adeno-carcinoma of the prostate to rise up to sometimes 100 nanograms / ml. Benin adenomas of the prostate may become cancerous.

It would therefore be very interesting to regularly monitor men with adenoma to report early onset of an adeno-carcinoma that can be well treated with new hormone-antagonistic molecules, without resorting to major surgery. and avoid the fatal metastases vertebras.

The device will be a ribbon 3 cm long (3 squares) and 1 cm wide and will include a printed square "PSA" for the qualitative assay: the PSA exists or not, a second printed square "> 4ng / ml" for the semi-quantitative determination with 4 nanogr / ml as limit: the coloration occurs if the rate is higher, and finally the control square.

Procedure 1 - In a container A containing a labeled anti-PSA antibody, 50 microliters of serum to be tested are poured according to the modalities of the preceding examples.Vortex.

2 - In another container Al, containing 4 nanograms / ml (parity of molecular weight) of a non-labeled anti-PSA antibody, another 50 microliters of the same serum to be tested are added and vortexed.

3 - In an A2 container containing an antibody labeled this time, the A1 content is poured. If the PSA level is greater than 4 ng / ml, complexes are formed with the labeled antibody to deliver the excess signal.

Membrane B will be prepared with peptides directed against anti-PSA labeled antibodies
First of all, 50 microliters of A are deposited on the square "CTRL". If coloring is used, the operations are continued.

50 microliters of A are placed on the "PSA" square to detect only the presence or absence of PSA.

Then 50 microliters of A2 are deposited on the "> 4 ng / ml" square to detect the excess of PSA.

The results obtained are shown in Figure 6 / pl V.

Claims (5)

1 - Immunological peptides-anti-peptides method for dosing antigens or antibodies with the same device, based on the extreme amplification of the immunological reaction between a labeled antibody and its corresponding peptide immobilized on a support, reaction one million greater and faster than with whole antigen molecules immobilized on this support and characterized in that during a first step, a sample is contacted in a container A with one or more labeled antibodies (s). ) specific for the antigen of the desired affection, then in that in a second step, the product of this first reaction is brought into contact with a porous element B supporting, immobilized, specific peptides of (or) the labeled antibody (s). If the sample contains the desired antigen, the latter binds to the labeled antibody which is no longer trapped by the B peptides, and the complexes thus formed (antigen + labeled antibody) pass through B to enter the lower side. B in contact with a means of revealing the presence of this complex. If the sample contains the desired antibodies, the antibodies bind to the B peptides, leaving no room for the labeled antibody to attach to it, even though it is not bound to its antigen, and leaving it there. therefore cross B to reveal the presence of antibodies in the sample. If the sample contains no antigen or antibody, the labeled antibodies from A will be trapped by the B peptides, and no signal will appear on the underside of B. 2 - Process according to claim 1, characterized in that it makes it possible to achieve semi-quantitative doses of antigen by neutralization during a first step in a container Ai, a certain amount of only antigens (considered as physiological) by unlabeled antibodies and especially not having the same idiotype as that recognized by the peptides of B, in that in a second step, the product of this first reaction is contacted in a second container A2 with one or more labeled antibodies before being contacted with the peptides in B. If the antigen level in the sample is higher than normal, antigens still free after step Ai, form complexes with the labeled antibodies (which are no longer pie in B) which will reveal an excess of antigen on the lower side of B. If the antigen level is below or equal to normal, there is no free antigen after step A1 and the labeled antibodies will present in B, which will trap them, free idiotype sites. labeled will not cross B and there will be no signal 3 - Method according to claim 1 and 2, characterized in that it is able to detect x different antigens at the same time from the same sample, in that During a first step, the sample divided into x parts is placed in x different recipipents each containing a labeled antibody specific for one of the x antigens sought; and in that during a second step, the product of this first reaction is brought into contact with x identical porous elements supporting on their upper side an association of peptides able to trap all the labeled antibodies used if the sites of these The latter are free of any antigen, and able to let them pass through this porous element B, if their idiotype site is saturated with the corresponding antigen, to come into contact with means of revealing the presence of this antigen. 4 - A method according to claim 1 and 2, characterized in that it allows for semi-semiquantitative dosages by revealing during a first stage the pathological presence or the absence of an antigen, then revealing during a second step if the level of antigen, already abnormally present (as in several benign chronic diseases), is higher than the limit rate generally accepted, involving a malignant transformation of this benign condition.The process will consist in a simple qualitative dosage as described in claim 1, in a first step, then in a semiquantitative dosage as described in claim 2, in a second time. The two assays using exactly the same element B.Three hypotheses can then be presented  * 1 - First and second step: negative result: absence of pathology  * 2 - First stage positive but the second is negative: Presence of the pathology but at a benin level  * 3 - The two steps are positive malignant pathology 5 - Device for determining indifferently the presence of antigen or antibodies in a sample, characterized in that it comprises  a container A containing the labeled antibody or antibodies specific for the desired affection, intended to receive and possibly dilute a sample of biological fluid, to bring into contact during this first step of the method, a possible ligand contained in the sample; with the labeled antibody (s) specific for the desired ligand,  a porous B-tape of the paper, nylon, fiberglass, microbead, any polymer, preferably transparent or translucent, and supporting type  - on the upper surface of CERTAINES ZONES only called "TEST" zones, peptides (specific for the labeled antibody (s)) able to trap any labeled antibody whose idiotype is free of any antigen (antigen and negative antibody assays), and able to let it pass through this ribbon B  * if the labeled antibody is saturated with antigens (positive antigen assay)  * if the serum antibodies still present in the sample completely saturate the B peptides (positive antibody assay). Areas left empty, without peptides, called "CTRL" (control) zones are used to evaluate the validity of all reagents (before proceeding to the actual test) by staining the substrate spot with the sample containing the labeled antibody, and this regardless of the presence or absence of ligand sought in the sample, because in these areas "CTRL" there are no immobilized peptides.  and on all its lower side of the means for revealing the passage of the labeled antibodies through B, these means possibly being, but not only, a spot or strokes of chromogenic substrates. 6 - Device according to claim 5, characterized in that it allows for semiquantitative dosages of antigens, and comprising  a first container Al in which the sample is brought into contact with non-labeled antibodies capable of neutralizing a certain amount, considered as physiological, of antigens  a second container A2 receiving, after a certain time, the contents of A1 and containing antibodies labeled this time, intended to bind to the possible excess antigens, which remained free after step A1  a porous support B identical to that previously described, which contains on its upper side peptides specific for the labeled antibodies (but not specific for the non-labeled antibodies of the first step A1) and intended to trap the labeled antibodies when the antigen level is below the normal limit value, ie to let these labeled antibodies (complexed to excess antigens) to the lower face of B to bring them into contact with the means of revelation. 7 - Device according to claim 6, characterized in that it allows to detect several antigens at the same time, and comprising  x containers each containing a labeled antibody specific for one of the x antigens, these containers being intended to collect each, during a first step, a part of the sample to be tested.  a porous support B presenting (FIG. 3, plate II):  x x reactive squares (supporting the peptides specific for all the labeled antibodies used), each of these squares receiving a drop of the corresponding container  - 1 non-reactive square (without peptides) for functional control  - and means of revelation to the lower face