FR2726913A3 - Appts. for immuno-chromatographic analysis of liq. samples - Google Patents

Abstract

Appts. (1) for qualitative and/or quantitative immuno-chromatographic analysis comprises \-2 different porous carrier strips (10, 10a) within the same housing and in parallel, so that the marked (16, 16a) and unmarked (11, 11a) bonding agents at the porous carriers (10, 10a) are the same or different. Also claimed is an application where a different liq. sample contg. the same analyte is applied to each of the different porous carriers (10, 10a).

Description

 The present invention relates to a qualitative and / or quantitative analysis device by immunochromatography.

 This principle is described in several documents, and in particular in the patent applications published under the numbers EP-A-0 149 168, WO-A-86/04683 and EP-A-0 186 799.

The definitions given below of terms used in the description and the claims will facilitate understanding of the invention and the prior art.
- ANALYTE: the compound, body, or composition whose presence is to be determined quantitatively or qualitatively, and which can be a mono- or polyepitopic, antigenic or haptic ligand, a molecule or a plurality of molecules all having at least one epitopic site or common receiving site
- SPECIFIC BINDING AGENT: a molecule whose three-dimensional structure has one or more zones or cavities which bind to a specific spatial or polar location of another molecule. The binding agent is also known by the name "ligand", and the molecule with which it specifically binds is called "receptor" or even "anti-ligand". Mention will be made, for example, of antibodies, "anti" antibodies, antigens, and fragments of antibodies or antigens
- LIGAND: any compound for which a natural receptor exists or for which a receptor can be prepared;
- RECEPTOR (ANTI-LIGAND): any compound or any composition capable of recognizing a specific spatial or polar organization of a molecule
- MARKER: system, comprising one or more components, at least one of which is conjugated or associated with a specific binding agent. The marker provides a signal which can be detected by external means, usually by determination of electromagnetic radiation, preferably by visual examination. Such systems often include chromophores, such as dyes, and enzymes or even colored particles, such as colloidal gold
- IMMUNOCHROMATOGRAPHY: immunochromatography comprises at least one binding agent adsorbed or immobilized in one or more zones in a porous support. The latter allows the displacement of liquid in a sorption direction, as well as the transport of an analyte through said zones. The binding agents can be free to move in the direction of chromatographic sorption, or else immobilized permanently, for example by covalent bonds.

 Document EP-AO 149 168 describes, for example, an immuno-chromatographic device of the "sandwich" type, comprising a hollow body (capillary tube) in which there are one or more porous supports connected together and arranged along the longitudinal axis of the body. hollow, that is to say in series; these porous supports contain at least one binding agent, specific for an analyte, labeled with an enzyme, in a first zone upstream, relative to the direction of the chromatographic sorption, of at least a second zone; this second zone comprises at least one binding agent, specific for the same analyte, unlabeled, and immobilized in the porous support.

The labeled binding agent can move freely by wet chromatography, but the unlabeled binding agent immobilized in the second zone cannot move.

 When the tube is brought into contact with a sample likely to contain the analyte sought, the porous support soaks the sample. The latter diffuses along the porous support, dissolving and carrying away the labeled binding agent. If the specific analyte is present in the sample, an immune complex forms and migrates to the second area in which the unlabelled binding agent is found. When the complex arrives in this zone, it is immobilized by the "sandwich" capture of the unlabeled binding agent, and / or of the analyte. A visible coloration is then developed by adding substrate for the enzyme or by incorporating one or more chromogenic agents and / or substrates also in the second zone.

 Markers other than enzymatic can be used, in particular markers which do not require any additional operation, and / or visible to the naked eye in concentrated form, for example colloidal gold.

 The disadvantage of this type of device lies in the fact that the analyzes are carried out, in practice, with a single porous support. This is problematic when it is desired to use several different liquid samples from the same individual or patient, such as saliva and blood, as their respective analytes can interfere, obscuring or even distorting the true result. The same problem arises when it is desired to detect two or more different analytes present in a single sample.

 Thus, to overcome these drawbacks, the object of the present invention relates to an analysis device, as well as its use, either with two or more different liquid samples for the detection of the same analyte, or with the same liquid sample for the detection of two or more different analytes, while retaining a reliable result.

Indeed, the invention relates to a device as previously described, the following practical modality having been retained
- The device comprises at least two porous band supports, distinct from each other, contained in the same case, and arranged in parallel
- The labeled and unlabeled binding agents, respectively of the two porous supports are the same or different.

The invention will be better understood by referring to the appended drawing in which
- Figure 1 shows a perspective view from above of a qualitative and / or quantitative analysis device by immunochromatography according to the invention
- Figure 2 shows an exploded perspective view of the device of Figure 1.

 In accordance with Figures 1 and 2, which show a preferred embodiment according to the present invention, the device, indicated generally by the reference number (1), comprises a hollow housing (2), made of waterproof material, for example Made of PVC, polypropylene or polyethylene, consisting of an upper part (3) and a lower part (4).

 These two parts (3, 4) can be formed by any suitable method, but preferably by injection molding. The upper part (3) in fact constitutes the cover of the housing (2) and is provided with means (5) for complementary fixing with the lower part (4), for example studs. The lower part (4) comprises housing means (6) complementary to the fixing means (5) of the upper part (3), for example recesses or holes, as well as holding means (6a) of the porous supports. (10, loua), which will be described later. The holding means (6) are well known to those skilled in the art and consist, for example, of longitudinal parallel flanges formed during the molding of the housing (2). The two parts (3, 4) thus formed can be joined to one another by clipping, gluing or welding, advantageously by ultrasonic welding.

 The upper part (3), or cover, comprises communication means (7, 8) connecting the interior of the boot (2) with its external environment, at one of its ends. These means can be a hole provided in the upper part (3), or a membrane, permeable to the passage of a liquid sample, incorporated in or provided on said upper part. In a particularly preferred embodiment of the invention, these means consist of two wells provided side by side on the case (2) and opening onto the interior thereof.

 The upper part also includes means (9) allowing observation of the result of the analysis carried out, arranged between the communication means (7, 8) and the other end of the device.

These means may consist, for example, of at least one window elongated in the longitudinal direction of the housing (2), or else, if it is a plastic housing (2), the means (9) may be made of transparent material, so as to form at least one window in the upper part (3). Advantageously, and as illustrated in the figures, these means consist of two windows (13, 13a), possibly side by side and of essentially elliptical or oval shape.

 The observation means (9) open onto a part of the interior of the housing (2) called the reading area (18). This zone comprises at least one porous support (10, 10a) in the dry state, in the form of a strip. According to a particularly advantageous embodiment of the invention, the device comprises two supports separate from one another and arranged in parallel and the communication means (7, 8) each open onto a porous support (10, 10a). The latter can be any support allowing chromatographic transport, that is to say in a direction of sorption of a liquid applied to it, and in particular is chosen from cellulose, natural or synthetic, and its derivatives. In a preferred embodiment of the device according to the invention, the porous supports (10, loua) consist of nitrocellulose.

 Advantageously, the porous supports (10, 10a) are covered, on their lower faces, with a layer (17, 17a) of impermeable material, for example a plastic material such as polyethylene or polypropylene.

 The porous supports (10, 10a) thus described comprise, in a first zone, upstream in the direction of chromatographic sorption, at least one specific binding agent marked (16, 16a) as defined above, deposited on or in the supports porous (10, 10a), and movable in or on it in the wet state. By "mobile" is meant that the labeled binding agent (16, 16a) can move with the liquid sample by chromatography or sorption. During this transport, the labeled agent (16, 16a) can form an immune complex with an analyte possibly present in the sample. The marker associated or conjugated with this specific binding agent (16, 16a) is a marker chosen preferably. to be visible to the naked eye in concentrated form, essentially insoluble in water, and optionally chosen from the group consisting of colloidal gold, dye soils, and a colored latex.

 As shown in the figures, this first zone can be covered in whole or in part by a layer of absorbent material (14, 14a), for example a sheet of nonwoven fibers, which serves to store the liquid sample applied through the means communication (7, 8). A layer (15, 15a) more absorbent than that of storage (14, 14a) and the porous supports (10, 10a), can optionally be provided at the distal end of the device, partially covering the porous supports (10, 10a ). This distal layer (15, 15a) serves to evacuate the excess of sample possibly applied to the porous supports (10, 10a), while accelerating the chromatographic transport, which has the consequence of reducing the duration of time necessary to obtain a result.

 Downstream of the first zone is the reading zone (18), in which is disposed at least one unlabelled bonding agent (11, pila), permanently immobilized on or in the porous supports (10, 10a). This unlabeled binding agent (11, 11a) conforms to the definition given previously in the description. By "immobilized" is meant that this unlabeled binding agent (11, lla) cannot move freely by chromatography, when a liquid sample is applied to the porous supports (10, 10a). The techniques for immobilizing such agents are in themselves well known to those skilled in the art and do not require a detailed description.

 In a preferred embodiment, the labeled (16, 16a) and unlabeled (11, 11a) binding agents of the two porous supports (10, 10a) respectively are the same, so that one and the same analyte can be analyzed.

 In this case, a different liquid sample containing the same analyte is applied to each different porous support (10, 10a). These liquid samples can be of very diverse origins, in particular of natural or synthetic origin, and preferably of human, animal, or vegetable origin.

 By way of example, mention may be made in particular of water samples taken from the environment, or from waste water outlets, for the purpose of screening for pollutants or other molecules, or else liquid extracts of plants or of fermentation. , such as hydrolysates, or isolates. In the case of liquid samples of human origin, these are preferably chosen from the group consisting of urine, blood, saliva, mucus, cerebrospinal fluid, synovial fluid, tear fluid, and perspiration.

 According to another variant of the invention, the labeled (16, 16a) and unlabeled (11, 11a) binding agents of the two porous supports (10, 10a) respectively are different. In this case, the analyte to be detected with each porous support (10, 10a) is different.

 Optionally, and as shown in FIGS. 1 and 2, the porous supports (10, 10a) also include at least one other bonding agent (12, 12a), as defined previously, specific for the labeled bonding agent, and immobilized permanently in the reading zone (18) of the porous support (10, 10a), downstream of the unlabeled binding agent (11, 11a) relative to the direction of chromatographic sorption. This agent is used only for monitoring purposes to indicate to the user that the device has worked properly.

Claims (11)

 1. Device (1) for qualitative and / or quantitative analysis by immunochromatography, comprising a hollow case (2) made of waterproof material, at least one porous support (10, 10a) in the form of a strip in the dry state contained in said bottom case, and means of communication (7, 8) with the outside of the bottom case (2), so that one or more liquid samples can come into contact with said support (10, 10a), at least one labeled bond (16), specific for an analyte, deposited on or in the porous support (10, 10a), said labeled binding agent (16) being mobile in or on said porous support (10, 10a) in the wet state , in a direction of chromatographic sorption, and at least one non-labeled binding agent (11, pila) specific for the same analyte, permanently immobilized in a reading zone (18) in said porous support (10, 10a), and which, therefore, is not mobile in the wet state, the relative positioning of the bonding agent ue (16, 16a) and the reading zone (18) being such that a liquid sample brought into contact with said porous support (10, 10a) of the device (1), upstream of the labeled binding agent (16 ) depending on the direction of sorption, can carry the latter and migrate to the reading area (18), and means (9) allowing the observation and possibly the quantification of immobilization of the labeled binding agent complex (16, 16a) / analyte in the reading area (18), characterized in that the device (1) comprises at least two porous supports (10, 10a) in strip separate from one another, contained in the same housing, and arranged in parallel, and in that the labeled (16, 16a) and unlabeled (11, 11a), respectively binding agents of the two porous supports (10, 10a) are the same or different.  2. Device according to claim 1, characterized in that two separate means (7, 8) of communication are assigned to the two porous supports (10, 10a) respectively, and consist respectively of two wells provided on the case (2), possibly side by side, opening onto the two porous supports (10, 10a) respectively  3. Device according to claim 1, characterized in that two windows (13, 13a) provided on the case (2), possibly side by side, open on the two reading areas (18) respectively.  4. Device according to claim 1, characterized in that at least one other binding agent (12), specific for the labeled binding agent, is permanently immobilized in the reading zone (18) of each porous support (10, 10a), downstream of the unlabeled binding agent (11, 11a) with respect to the direction of chromatographic sorption.  5. Device (1) according to any one of the preceding claims, characterized in that the labeled (16, 16a) and unlabeled (11, lla) binding agents of the two porous supports (10, 10a) respectively are the same , so that you can analyze a single analyte.  6. Use of a device (1) according to claim 5, characterized in that a different liquid sample containing the same analyte is applied to each different porous support (10, 10a).  7. Use according to claim 6, characterized in that the different liquid samples applied are of natural or synthetic origin, and preferably of human, animal, or vegetable origin.  8. Use according to claim 6 or 7, characterized in that the different liquid samples applied are of human origin and are chosen from the group consisting of urine, blood, saliva, mucus, synovial fluid, tear fluid, and sweating.  9. Device (1) according to any one of claims 1 to 4, characterized in that the labeled (16, 16a) and unlabeled (11, 11a) bonding agents respectively of the two porous supports (10, 10a) are different.  10. Use of a device (1) according to claim 8, characterized in that the analyte to be detected with each porous support (10, 10a) is different.  11. Device (1) according to claim 1, characterized in that the marker is a marker visible to the naked eye in concentrated form, essentially insoluble in water, optionally chosen from the group consisting of colloidal gold, dye soils, and a colored latex.