WO2009040364A1

WO2009040364A1 - Multiparameter assay

Info

Publication number: WO2009040364A1
Application number: PCT/EP2008/062731
Authority: WO
Inventors: France Jacqueline H. Fannes
Original assignee: Bio A.R.T. Sa
Priority date: 2007-09-24
Filing date: 2008-09-24
Publication date: 2009-04-02
Also published as: GB0718606D0

Abstract

This invention relates to a rapid and simple determination of small amounts of substances in a solution, by a novel solid phase flow through assay device. The assay may be adapted to rapidly and qualitatively determine the presence of antigens, antibodies, proteins, lipids, allergens, organic molecules and nucleic acid oligomers in a solution, in particular in a sample such as for example a bodily fluid for human or animal diagnostic testing, an environmental sample or a food sample. The invention further provides diagnostic kits to perform the assay of the present invention as well as novel components of said kits such as an insoluble porous material for use in the novel assay.

Description

MULTIPARAMETER ASSAY

Field of the Invention

This invention relates to a rapid and simple determination of small amounts of substances in a solution, by a novel solid phase flow through, i.e. filtration assay device. The assay may be adapted to rapidly and qualitatively determine the presence of antigens, antibodies, proteins, lipids, allergens, organic molecules and nucleic acid oligomers in a solution, in particular in a sample such as for example a bodily fluid for human or animal diagnostic testing, an environmental sample or a food sample. The invention further provides diagnostic kits to perform the assay of the present invention as well as novel components of said kits such as an insoluble porous material (membrane) for use in the novel assay.

Background to the Invention

Assay systems which are both rapid and sensitive have been developed to determine the concentration of a substance, generally referred to as the analyte, present in low concentration in a fluid sample, and typically include an analyte binding compound, such as an analyte specific antibody (binding compound) immobilized on a support. Immunoassays depend on the binding of an antigen or hapten to a specific antibody and have been particularly useful because they give high levels of specificity and sensitivity. These assays employ one of the above reagents in labeled form, the labeled reagent being referred to as the tracer. A convenient format for an Enzyme Immunoassay (EIA) is solid phase immunoassay in which one of the assay reagents is immobilized on a solid support. The solid support may be in the form of a dipstick, the inside wall of a test tube or cuvette or the well of a microtiter plate. A particularly useful solid support is a microporous membrane .

A membrane immunoassay based on a microporous support is often referred to as flow-through assay. Examples of flow- through EIA wherein flow is generated by capillary action are the assays described in U.S. Patent No. 3,888,629 to Bagshaw, U.S. Patent No. 4,246,339 to Cole et al . , U.S. Patent No. 4,632,901 to Valkirs et al . , U.S. Patent No. 4,277,560 to Grey, U.S. Patent No. 4,812,293 to McLaurin et al., European Patent No. 0 458 231 to Stewart et al . and European Patent No. 1 344 059 to Fannes.

In membrane EIA, any number of liquids may be caused to flow through the membrane to effect binding, separation and washing of assay components. The final step in most membrane EIA procedures is contacting a color developing reagent, such as a chromogen or labeled immunochemical reagent, with the membrane. For example, in a typical sandwich assay for the detection of unknown antigen in a sample, antibody for the unknown antigen is bound to the membrane and reacted with the antigen. After a washing step, the membrane is reacted with a second labeled antibody and the amount of bound labeled antibody is measured as an indication of the antigen quantity in the sample. In the competitive technique, antibody bound to a solid surface may be contacted with the sample containing an unknown quantity of the antigen to be determined, and with labeled antigen of the same type.

After washing, the amount of labeled antigen bound on the surface is measured, providing an indirect indication of the amount of unknown antigen in the sample.

Known labels are of the radioactive or fluorometric type, which are detected by instrumentation; colorimetric labels, typically an enzyme label which causes the conversion of a corresponding substrate to colored form; as well as coloured particulate labels, such as coloured latex particles, or gold or silver particles.

In case a chromogen is used, such as Tetra Methyl Benzidine (TMB) , said chromogen reacts with enzyme, such as Horse-Radish Peroxidase (HRP) , captured on the membrane to produce a colored product that may be detected as evidence of the presence of analyte or measured as evidence of the concentration of analyte. The colored product may be soluble, in which case it will pass through the membrane and be detected in the filtrate, or it may be insoluble and form a colored spot on the membrane. As exemplified in the examples hereinafter, in a flow-through assay of the present invention, the colored product will form a colored spot on the membrane.

These conventional immuno assay methods require a multitude of individual experimental steps in order to test a large number of analytes, (e.g. to determine the presence of different allergens in a sample) , and are accordingly not useful to test for example, a patient with respect to the presence of allergies since the amount of different allergens has mounted to a few hundred and is increasing steadily. These conventional assays also require;

• a separate control, which amounts to more sample material, consumables and time;

• one or more washing steps, e.g. when using gold particles as label that can bind to or be absorbed onto the membrane, and which amounts to more consumables and time.

In an effort to simplify the determination of a plurality of different analytes, the aforementioned immuno assays have been miniaturized into micro-array assays wherein the analyte binding compounds (capture proteins) are arranged as array elements consisting of discrete spots with a density of up to 100 spots / cm². When performed in a multiwell format, these micro-array assays, such as for example described in US 2003/0153013, US 2003/0113713 and GB 2401942, allow parallel processing and analysis of a multitude of individual immuno assays. All of these array methods are characterized in that the array elements constitute an assay domain, i.e. a discrete location on a surface where an assay reaction occurs. In view of the miniaturization these micro array assays require sophisticated apparatus, in particular to check the performance and detecting the binding of an analyte such as CCD cameras or laserscanners .

It is accordingly an object of the present invention to provide an assay for the detection of one or more analytes in a sample, particularly including an internal control, which can be carried out in a short time with only a small amount of sample in a simple device and, which is reliable, sensitive, and can easily be used at home or in field locations Without the need of sophisticated lab equipment like CCD cameras or laserscanners .

Compared to the conventional methods, the assay (s) of the present invention allow to combine screening and identification in a single test. In the detection of for example allergens, auto-immune antigens this will significantly reduce the response time. A further advantage of the assay (s) according to the invention is that it can be adapted in for example follow-up for immunotherapy. This and further details of the assay of the present invention are provided hereinafter.

Summary of the Invention

This invention is based on the finding that a porous membrane, spotted, at discrete location (s) (in at least one discrete location) , with a single analyte binding compound and subsequently coated with a plurality of analyte binding compounds, allows a flow-trough assay for the simultaneous detection of a plurality of analytes in a sample eventually with an incorporated control as to the quality of the assay performance.

The membrane is in particular useful in a (vertical) filtration assay and is homogenously coated with the plurality of analyte binding compounds and fully occupied, after application of the blocking agent, prior to its application in the assay (s) of the present invention.

Given the complexity of the sample, i.e. comprising a plurality of analytes and the particular coating of the porous membrane with a plurality of analyte binding compounds, it was not to be expected that pre-coated spotting with one or more of single analyte binding compounds, in particular with one internal control, would yield a reproducible assay with a clear and easily interpretable read-out.

It is accordingly a first objective of the present invention to provide a membrane, hereinafter also referred to as an insoluble porous material, homogenously coated and occupied with one or more analyte binding compounds within a single test zone, in particular with at least two analyte binding compounds and characterized in having at least one zone (spot) pre-coated (pre-spotted) with only one analyte binding compound; in particular pre-coated (pre-spotted) with an auto control.

The insoluble material as used herein, is typically occupied within said single test zone with from 2 to 20, in particular from 2 to 10, more in particular from 2 to 5 different analyte binding compounds and spotted with from 1 to 20, in particular from 1 to 10, more in particular from 1 to 5 zones (spots) each independently spotted with only one analyte binding compound, even more in particular with only one zone (spot) spotted with an analyte binding compound. In a further embodiment, the insoluble porous material according to any one of the aforementioned embodiments is further characterized in that at least one of the zones (spots) pre-coated (pre-spotted) with only one analyte binding compound is characterized in that it differs from the analyte binding compounds used in the homogenous coating of the insoluble porous membrane.

In said embodiments wherein the insoluble porous material is pre-coated (pre-spotted) with only one zone (spot) of a single analyte binding compound said zone typically constitutes an assay control (hereinafter also referred to as internal control or auto control) . The controls allows to check the proper functioning / performance of the assay (that is, e.g. as to the activity and reactivity of the different assay components) as well as to check whether the assay is properly performed) . In case the control zone (spot) does not produce the expected read-out, the assay is either malfunctioning or was not properly executed.

Optionally, this control constitutes an additional pre- coated zone on the porous insoluble material according to the invention. Accordingly in a further embodiment the present invention provides an insoluble porous material coated with one or more analyte binding compounds, typically coated with from 2 to 20, in particular from 2 to 10, more in particular from 2 to 5 different analyte binding compounds within a single test zone and spotted with from 1 to 21, in particular from 1 to 11, more in particular from 1 to 6 zones (spots) each independently containing only one analyte binding compound, wherein said zone(s) include one control zone (spot) .

In one embodiment of the present invention the pre-coated (pre-spotted) zone(s) (spot(s)) are also present within the single test zone of the insoluble porous membranes of the present invention.

As is evident from the examples hereinafter, homogenously coating of the insoluble porous material with a plurality of analyte binding compounds could be achieved without cross-interference of the analyte/analyte-binding compound combinations (supra) and retaining a reproducible, easy to interpret read-out.

In general, the insoluble porous material is obtainable by;

• homogenously coating the insoluble porous material with the analyte binding compound (s) via immersion of the layer in coating and post-coating solutions;

• followed by a drying procedure; and characterized in that no washing steps are used.

As a result of this procedure, the membrane is fully occupied, i.e. all binding sites on the membrane are either bound to one of the analyte binding compound (s) or blocking agent (s) present in the post-coating solutions (infra) .

When comprising one or more pre-coated (pre-spotted) zone(s), said zone(s) spotted with only one analyte binding compound, the insoluble porous material is obtainable by;

• spotting of the layer at one or more discrete locations, with a solution containing only one analyte binding compound; in particular spotted at only one discrete location with a solution containing only one analyte binding compound; more in particular spotted at only one discrete location with an auto control;

• homogenously coating the insoluble porous pre-spotted material with the analyte binding compound (s) via immersion of the layer in coating and post-coating solutions;

• followed by a drying procedure and characterized in that no washing steps are used.

In a particular embodiment, when comprising only one zone (spot) pre-spotted with only one analyte binding compound, the insoluble porous material is obtainable by;

• spotting of the insoluble porous material at only one discrete location with a solution containing only one analyte binding compound; more in particular spotted at only one discrete location with an auto control;

• homogenously coating the insoluble porous pre-spotted material with the analyte binding compound (s) via immersion of the layer in coating and post-coating solutions; in particular with only one analyte binding compound;

Alternatively, in the different methods to obtain an insoluble porous material containing one ore more pre- spotted zones, an additional drying step is introduced between the pre-spotting and the coating of the insoluble porous material. In said alternative embodiment, the insoluble porous material is obtainable by;

• spotting of the layer, i.e. at one or more discrete locations, with a solution containing only one analyte binding compound;

• drying the thus spotted layer;

• homogenously coating of the insoluble porous pre- spotted material with the analyte binding compound (s) via immersion of the layer in coating and post- coating solutions;

In the coating step, the insoluble porous material (membrane) is immersed under gentle agitation for 1 to 5 hours, in a bath brought at room temperature and containing the coating (application) buffer and the analyte binding compound (s). In particular under gentle agitation for 3 hours with a direct contact of the reactive zone with the application (coating) buffer. In other words, the coating solution as used in the methods of the present invention may comprise only one analyte binding compound or a plurality of different analyte binding compounds, in particular 1, 2, 3, 4, 5, 10 or 20 analyte binding compounds.

In case nitrocellulose membranes are used, the coating buffer (application buffer) features very low salinity and basic pH character (pH in the range of 9.0 to 11.0); such as for example TrIs (1.2 g/L) / NaCl (8.8 g/L) .

The post-coating or blocking procedures for flow-through assays are widely used and known to the skilled artisan. However, and as emphasized in the examples of the present invention, there are no washing steps after the spotting and immersion of the insoluble porous material. Such washing steps would lead to Mesorption' of the analyte binding compounds due to the presence of surfactant agents in the washing solutions typically used in the art known coating or blotting procedures. As for example provided in the examples hereinafter, the remaining free sites can be blocked by the use of a commercially available polypeptide fraction of collagen, i.e. Prionex® (Polysciences Inc., Pennsylvania). Other blocking agents (proteins or synthetic components) may also be used. In case of allergen screening the typical blocking agents isolated from milk (e.g.: casein, beta lactoglobulin, ...) or Bovine Serum Albumin A should be avoided as these agents could be considered allergens and interfere with the actual screening.

In a further embodiment, the present invention provides a method to obtain an insoluble porous material for use in a method to determine the presence of at least two analytes of interest in a sample, said method comprising; a. spot at discrete locations, the insoluble porous material with at least two solutions each independently containing only one analyte binding compound; optionally including one control spot; alternatively spot with only one solution containing only one analyte binding compound, in particular said spot being a control spot; b. immerse the material obtained in step a in a solution comprising at least two analyte binding compounds; c. immerse the material obtained in step b in one or more post-coating solutions; and d. dry the thus obtained material. Again as for the methods described hereinbefore, this method is performed without washing steps.

In an alternative embodiment of the aforementioned method, an additional drying step is present after spotting the insoluble porous material with at least two solutions each containing only one analyte binding compound, i . e . after step a) and prior to immersion of the thus pre-spotted insoluble porous material, i.e. prior to step b) .

As already mentioned hereinbefore, and using the aforementioned coating procedures, the membranes are further characterized in that the membranes are fully occupied and allows a simple filtration assay without the needs of intermediate incubation/washing steps as typically found in the art.

The combination of a single test zone comprising a plurality of analyte binding compounds with one or more zones pre-coated (pre-spotted) with a single analyte binding compound allows to detect the presence of said analytes in a sample (and even semiquantify said analytes by a homogenous staining of the test zone) , with simultaneous identification of said analytes through the pre-spotted zones.

Also the presence of a control spot (zone) , as shown in a particular embodiment of the present invention, provides a further advantage to the membranes of the present invention. This internal control allows in a simple filtration assay, not only to determine the presence of a plurality of analytes of interest in a single assay, but also and simultaneaously to get a readout as to the good (see above) performance of said assay.

Thus, compared to the typical assays to determine the presence of a plurality of analytes of interest in a sample, the membranes of the present invention allow this to be achieved in a simple filtration assay without the need of sophisticated laboratoty equipment for test performance and/or interpretation of the assay results.

In a further objective, the present invention provides a multilayer support comprising the insoluble porous material, in any one of its different embodiments, as defined hereinbefore. In a particular embodiment said multilayer support comprises;

• An upper cover layer of a water-impermeable material having at least one hole, whereby said hole overlays a test zone;

• An intermediate porous layer comprising the insoluble porous material as provided hereinbefore; and

• A lower absorbent layer comprising at least one layer of a hydrophilic material.

In a fourth objective, the present invention provides an assay (method) as well as an assay device for testing the presence of at least two analytes of interest in a sample, said assay and assay device comprising an insoluble porous material or a multilayer support as defined in any one of the different embodiments of the present invention. The assay (method) typically comprises the steps of; • Passing the sample through the insoluble porous material of the present invention; • Exposing the sorbed porous material with at least two labeled second analyte-binding compounds specific for the analytes of interest, i.e. passing said at least two labeled second analyte-binding compounds through the insoluble porous material; and • Determine the binding of the labeled second analyte- binding compounds to the membrane.

In the assays (methods) of the present invention, both the exposure step as well as the sample application step consist of a filtration step, i.e. the material (sample or solution comprising the labeled second analyte-binding copound(s)) is passed from one side of the insoluble porous material (membrane) to the opposite side according to a perpendicular axis on the surface of the insoluble porous material.

As already mentioned hereinbefore the assays (methods) of the present invention, are particularly useful to determine the presence of a plurality of analytes in a sample, in particular in a sample chosen from the group consisting of cell fractions, serum, whole blood, urine, plasma for human or animal diagnostic testing; soil, mud, minerals, water, air for environmental testing; any food materials for food testing; or any other medium/suspension/hard material which can be used for one of these purposes. It is accordingly a fifth objective of the present invention to provide the use of any of the above, to test the presence of at least two analytes of interest in a sample. In particular, to test the presence or absence of a compound in a sample, said compound being selected from the group comprising antigens, antibodies, peptides, proteins, lipids, allergens, organic molecules and nucleic acid oligomers; more in particular to test the presence or absence of allergens; even more in particular to test the presence of absence of allergens from grasses, weeds, moulds, foods, trees, epidermals and dust.

These and other objectives of the present invention are provided in more detail hereinafter.

Brief Description of the Drawings

Figure 1 Schematic representation of the components present in the auto control assay. TMB (Tetra

Methyl Benzidine) exemplifies a chromogen typically used in an EIA. HRP (Horse-Radish Peroxidase) exemplifies the enzyme labelled second antibody used in a typical sandwich EIA. CRP (C-Reactive Protein) exemplifies the analyte to be determined.

Figure 2 Schematic representation of the different readouts in the auto control assay. A. Valid result (auto control is blue) Sample is interpret as CRP negative (< 10 mg/L) . B. Valid result (auto control is blue) Sample is interpret as CRP medium positive (75 mg/L) . C. Valid result (auto control is blue) Sample is interpret as CRP highly positive (100-150 mg/L). D. Invalid result (auto control is not stained) Sample cannot be interpreted even if a blue colouration is present.

Figure 3 Pictures of the CRP auto control assay for a Blanco sample and samples comprising respectively 10, 75 and 150 mg CRP/L.

Figure 4 Schematic representation of the components present in the multiparameter assay. TMB (Tetra Methyl Benzidine) exemplifies a chromogen typically used in an EIA. HRP (Horse-

Radish Peroxidase) exemplifies the enzyme labelled second antibody used in a typical sandwich EIA. food allergen or casein and/or ovalbumine exemplifies the analytes to be determined.

Figure 5 Schematic representation of the different readouts in the multiparameter assay. A. sample contains both analytes . B. sample contains only one of the analytes, i.e. only casein. C. sample contains only one of the analytes, i.e. only ovalbumine D. negative sample, neither of the analytes is present

Figure 6 Pictures of the multiparameter assay described in Example 2. Figure 7 Measurement of the Lightness Chroma & Hue (LCH) values of the coloration of the test zone in the membranes of the CRP assay. The LCH values were determined on 13 locations of the coloured spot (test zone) using a CCD camera. The total measured surface equaled 7000 pixels. 3 sample loads have been tested : low load: 62 ng/ml (series 1) with a mean LCH = 11,57 ± 0,37 (SD) and a Coefficient of Variation (CV) of 3,20%; medium load: 1250 ng/ml (series 2) with a mean LCH = 19,94 ± 0,25 (SD) and a (CV) of 1,20%; high load: 5000 ng/ml (series 3) with a mean LCH = 25,84 ± 0,54 (SD) and a (CV) of 2,01%. The low SD and CV values across the spot and consistent for the different sample loads demonstrate the homogenous coating of the membranes using the procedures of the present invention.

Description of the Invention

As already mentioned hereinbefore, the present invention is based on the finding that a porous membrane, spotted, at discrete location (s), with a single analyte binding compound and subsequently coated with a plurality of analyte binding compounds allows an assay to determine the simultaneous detection (semiquantification) and identification of a plurality of analytes in a sample.

In the assay one of the single analyte compounds could serve as an internal control to monitor the performance of the assay, i.e. including a control as to determine whether the assay was carried out according to the protocol/instructions provided therewith. The particular combination of immersion, i.e. homogenous coating of the porous membrane with a plurality of analyte binding compounds, and spotting, provides a membrane suitable to provide a single assay for the detection of a plurality of analytes in a sample which can be carried out in a short time with only a small amount of sample and, which is reliable, sensitive and can easily be used at home or in field locations.

Membrane

It is accordingly a first objective of the present invention to provide an insoluble porous material homogenously coated with one or more different analyte binding compounds in a single test zone, in particular with at least two different analyte binding compounds and characterized in having at least one zone (spot) pre- coated (pre-spotted) with only one analyte binding compound, in particular including a control spot. In a further embodiment, pre-coated (pre-spotted) with only one analyte binding compound, whit. In a particular embodiment the insoluble porous material homogenously coated with one or more different analyte binding compounds in a single test zone embodiment is characterized in having only one zone (spot) pre-coated (pre-spotted) with only one analyte binding compound, in particular with a control spot. In a further embodiment the at least one pre-coated spot is present within the single test zone.

Pre-coating of the insoluble porous material, is typically done using spotting or other methods for immobilizing an analyte binding compound at discrete locations to an insoluble porous material; particularly said pre-coated spotting is done within the single test zone. As used herein, the zone(s) pre-coated (spotted) with only one analyte binding compound has/have a diameter of up to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8 or 2.0 mm; in particular up to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 mm; more in particular have a diameter of up to 0.1, 0.2, 0.3, 0.4 or 0.5 mm. Depending on the number of analytes to be determined, the porous membrane may comprise from 1 to 20, in particular from 1 to 10, more in particular from 1 to 5 of said zones (spots) .

In one embodiment of the present invention, at least one of said zones is pre-coated (spotted) with an analyte binding compound that differs from the analyte binding compounds used in the homogenous coating of the membrane, i.e. an analyte binding compound that selectively binds to an analyte which is not recognised by any one of the analyte binding compounds used in the homogenous coating of the membrane. In this latter case, the zone pre-coated (pre-spotted) with such an analyte binding compound, that selectively binds to an analyte which is not recognised by any one of the analyte binding compounds used in the homogenous coating of the membrane, will serve as an internal control, hereinafter also referred to as auto control, as to the performance, implementation of the methods (assays) of the present invention. For example, in case of a sandwich assay, the internal control will consist of an analyte binding compound that selectively binds to the labelled second analyte-binding compound.

As already mentioned hereinbefore, in one embodiment the porous membrane may comprise an additional pre-coated zone (spot, dot) and depending on the number of analytes to be determined the membrane may comprise from 1 to 21, in particular from 1 to 11, more in particular from 1 to 6 of said zones (spots) . In a further embodiment the invention provides an insoluble porous material homogenously coated with one or more different analyte binding compounds in a single test zone, in particular with at least two different analyte binding compounds and characterized in having one zone (spot) pre-coated (pre-spotted) with an auto control.

In the flow-through methods (assays) of the present invention, the characteristics of the porous material with regard to pore size, thickness and type, are influenced by the sample analysed. As is known to the skilled artisan from, for example U.S. Patent No. 3,888,629 to Bagshaw,

U.S. Patent No. 4,246,339 to Cole et al . , U.S. Patent No. 4,632,901 to Valkirs et al . , U.S. Patent No. 4,277,560 to Grey, U.S. Patent No. 4,812,293 to McLaurin et al . , European Patent No. 0 458 231 to Stewart et al . and European Patent No. 1 344 059 to Fannes, the membrane is selected to have a pore size which permits filtration (flow-through) of the sample and is made of a type that allows binding of biological substances.

Any variety of membranes may be used, including various synthetic or natural materials. In one embodiment of the present invention, the material is made from nylon, nitrocellulose (charged or neutral nitrocellulose) , cellulose, fiberglass, polysulfofone, polyvinylidene, difluoride, polyester or any other polymeric material whereon a biological substance may bind; in a particular embodiment the material is made of nitrocellulose.

In a particular embodiment as provided in the examples hereinafter, the membranes are further characterized in that one face of said membrane consist of an inert material; in particular a paper cast. As used herein, this inert face is opposite to the face of the membrane where the sample is applied, i.e. the inert face is not exposed to the loading of the sample.

The pore size should be such to allow filtration (flow- through) of the sample to be analysed and as typically pores with a diameter of 0.1 to 12 Dm; in particular the material has pore sizes selected from the group consisting of 0.1, 0.2, 0.45, 0.8, 1.2, 3.0, 5.0, 8.0 and 12 Dm; more in particular the material has a pore size of 0.45 Dm.

In flow-through assays driven by hydrostatic pressure it is known that the thickness may influence the flow rate of the sample through the membrane (porous material) . Accordingly, in one embodiment of the present invention, the porous material has a thickness up to 2500 Dm; in particular up to 1500, 1000 or 500 Dm; more in particular up to 500 Dm.

In one embodiment, as provided in more detail in the examples hereinafter, the porous material has a pore size of 0.1 to 12 Dm and a thickness up to 2500 Dm; in particular a pore size selected from the group consisting of 0.1, 0.2, 0.45, 0.8, 1.2, 3.0, 5.0, 8.0 and 12 Dm, and a thickness of up to 500 Dm; more in particular a pore size of 0.45 Dm and a thickness of 500 Dm.

Sample

As used herein, the "sample" is meant to include any liquid composition comprising the analytes of interest. In case the analytes of interest are present in solid materials such as for example in mud, dust, soil, minerals, or food materials; or are present in extracts such as obtainable from plants, tissues or other organisms; it may be required to suspend or to dilute said material in water or an aqueous liquid. This liquid may contain suspending agents, if necessary to achieve good suspension of the target moiety. Adaptations of the diluent solutions, that may be necessary to prevent clogging of the system or to optimize the condition wherein the analytes of interest can bind to the analyte- binding compounds, are known to the skilled artisan, see for example "Sample preparation techniques in analytical chemistry, Somenath Mitra, Wiley-Interscience Press, September 12, 2003".

For example, if the sample to be tested is isolated from food, a sonication process may be required in order to dissolve aggregates; if the sample to be tested is isolated from urine, adjustment of pH may be needed; if the sample contains lipids i.e. "fat sample", delipidation may be requested. A hemolytic solution may be requested in order to hemolyse red blood cells, for samples derived from blood. In some cases the salinity needs to be adapted. For microbiological application: a sample from food for the detection of contaminants have sometimes to be enriched through the addition of some nutriments or lysed by the addition of lytic agents into the diluent solution. The diluent solution may also comprise a preservative such as thimerosal or sodium azide

In one aspect the sample can be chosen from a group comprising cell fractions, serum, whole blood, urine, plasma for human or animal diagnostic and/or allergen testing; soil, mud, minerals, water, plant material or air for environmental and/or allergen testing; any food materials for food testing; or any other tissue/ medium/ suspension/ hard material which can be used for one of these purposes.

Analyte and Allergens

The analyte can be any one of the group including, but which is not limited to; small molecules including both organic and inorganic molecules with a molecular weight of less than 2000 daltons; proteins; peptides; antisense oligonucleotides; siRNAs; antibodies, including both monoclonal and polyclonal antibodies; ribozymes; etc.

In a particular embodiment, the analyte is an antibody or an antigen. Whereby said antigen may be chosen from the group comprising allergens or any biological agent such as bacteria, viruses, molds, mycobacteria, parasites or pathogens; and the antibody may be chosen from any class of immunoglobulins such as IgE, IgM, IgA or IgD. The presence of a specific antibody in a sample might give indications on stage, location and nature of a disease. An increase of IgE is a measure for allergic reactions and presence of helminthic parasites; the increase of IgG confirms the presence of infections which is already in a extended stage ; IgM indicates that infections are present in an early state. IgA can be especially detected in secretions and IgD are present on membranes of B cells.

In a particular embodiment the materials and methods of the present invention are used to determine the presence or absence of allergens in a sample. ^λAllergens' include biological or chemical substances that trigger the immune system, i.e. causing an allergic reaction. An allergy is a reaction produced by the body's immune system to a substance (the allergen) that would normally be thought of as harmless. It is this response that causes the symptoms that are classed as allergic reactions. Allergy is therefore not a failure of the immune system, but its over activity. The response of an allergic person to an allergen can produce a wide range of symptoms. Some people suffer symptoms such as asthma, eczema, rashes, itchy eyes, sinusitis, blocked or runny nose and hay fever, however, more serious symptoms can occur. With allergies such as those to venoms, nuts and shellfish, for example, a potentially life threatening condition called anaphylactic shock can occur. This happens when the body produces a reaction so severe that the throat swells, blood pressure drops and the person has difficulty in breathing. In some cases this type of reaction can be fatal. The incidence of allergies is increasing in the developed countries (i.e. in Europe, Northern America and Japan). Estimates range from 20-50% of the population being affected. It is now known that allergies are the result of an unbalance in the T-cell compartment of the immune- system. More precisely, allergies are accompanied by an increased activity of so-called T-helper 2 (Th2) cells relative to T-helper 1 (ThI) cells, giving rise to increased IgE (immunoglobulin E) production. IgE specific for an allergen is not normally detected in the blood and is only produced when a person becomes sensitized to a substance .

An allergen can be almost anything which acts as an antigen to stimulate such an immune response.

Common allergens

Food . The most common are milk , fruit , fish, eggs and nuts . Pollen , especially ragweed, which causes hay fever.

Mould from plants and food, which are most likely to cause asthma . House dust , which contains mites as well as dander from house pets. Venom from insects (such as bees, wasps and mosquitoes) or scorpions . Plant Oils, especially poison ivy, oak or sumac.

Additionally, feathers, wool, dyes, cosmetics and perfumes may also act as allergens.

Biological sources of allergens include pets, insects, dust, mites, plants, bacteria, and mold. Chemical sources are often gases or particles released by items such as building materials, fabrics, glues, paints, solvents, dyes, food materials and perfumes. Hence, in this embodiment the sample is taken from any of the aforementioned sources using standard procedures such as for example provided by Janusz Pawliszyn (Sampling and Sample Preparation for Field and Laboratory, 2002, Elsevier Press) .

Analyte binding compounds

As used herein, the "analyte-binding compound" is a compound which either specifically binds the analyte; which is capable to react with the analyte; or which is capable to react with a reaction product obtained by reaction of the analyte. It is typically chosen from a group comprising peptides, proteins, lipids, nucleic acids and organic molecules.

Accordingly, in first instance, the analyte-binding compound is selected for its ability to selectively bind directly with the analyte. For example, if the analyte is an antigen, the analyte-binding compound may be an antibody, monoclonal or polyclonal which specifically binds the analyte. For screening purposes, i . e . determining the presence of an analyte of interest in a sample, the analyte binding compound will typically exist of a polyclonal antibody. If the analyte is an antibody, the analyte-binding compound may be an antigen, hapten or anti-antibody. If the analyte is an enzyme, the analyte- binding compound may be a receptor or a substrate for the enzyme. If the analyte is a nucleic acid, for example, RNA or DNA, the receptor may be a complementary oligomer of DNA or RNA or a nucleic acid binding protein. Alternatively, and in particular if the analyte is an enzyme, the analyte-binding compound may be a substrate or a compound capable to react with the reaction product obtained by reaction of the enzyme with its substrate. These further reactive compounds could be any revealing agent such as compounds that produce a colored readout based on for example change in oxidation status or pH.

In one object of the present invention, the analyte binding molecules are antibodies or fragments thereof; in particular polyclonal antibodies that selectively bind the analytes of interest.

As provided in the examples hereinafter, in one embodiment of the present invention, the concentration ratio between the analyte binding compounds used for the spotting and the analyte binding compounds used for the homogenous coating is at least 5, and typically in the range from 5 to 15, more in particular the concentration used for the spotting is 10 times higher than the concentration used for the homogenous coating.

For example, in case of polyclonal antibodies, the analyte binding compounds used for the spotting are applied with a concentration ranging from 0.25 mg/ml to 2.0 mg/ml and the analyte binding compounds used for the coating are applied with a concentration ranging from 0.025 mg/ml to 0.4 mg/ml, in particular from 0.05 mg/ml to 0.2 mg/ml.

As used herein, the solutions for spotting the insoluble porous material (membrane) are said to comprise only one analyte binding compound. Based on the aforementioned definition, said solution will selectively detect a particular analyte to be determined using the membranes / assays of the present invention. In a particular embodiment each of said spotting solutions will detect a different analyte of interest, including the auto control.

Those skilled in the art will appreciate from the foregoing that a variety of analyte/analyte-binding compound combination may be employed and that the foregoing examples are not to be construed as limiting the invention to the specific examples described herein.

Assay device

In a second objective, the present invention provides a multilayer support comprising the insoluble porous material as defined hereinbefore. In particular the multilayer support further comprising; - an upper cover layer of a water-impermeable material having at least one hole, whereby said hole overlays a test zone; - an intermediate porous layer comprising at least one insoluble porous material as provided herein; and a lower absorbent layer comprising at least one layer of a hydrophilic material.

According to the present invention the water-impermeable material of said upper cover layer as defined above is chosen from plastic adapted to the sample to be tested comprising polypropylene, polyvinylchloride orstyrene- ethylene/butylene styrene (SEBS) (Rubin (1990), Schouten and van der Vegt (1987)) . This prevents unwanted background signals caused by hydrophobic or hydrophylic interaction of some of the components present in a sample with the plastic.

The assay device of the present invention comprises at least one hole in the water-impermeable material having a diameter of at least 1 mm overlaying a test zone. The hole limits the surface of the porous membrane to which the sample is exposed and, when large volumes are spotted onto this multilayer device, the hole also helps the liquid to be absorbed by the area defined by the hole. The hole is not limited by the size of the test zone and may be between 1 to 20 mm, in particular have a diameter of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 mm, more in particular have a diameter between 1 and 10 mm. In a further embodiment of the present invention, the assay device comprises from 1 to 10, in particular from 1 to 5, more in particular from 1 to 3 of the aforementioned holes in the water-impermeable material, each of said holes overlaying a test zone. Thus in a further embodiment of the present invention, the assay device comprises a plurality of porous membranes according to the invention wherein each of said membranes is an independent test zone, accessible by one or more of the holes above.

Given the homogenous coating of the membrane, the whole area of the insoluble porous membrane could function as test zone. In particular, the test zone consists of the area of the insoluble porous membrane exposed to the sample and comprising the pre-coated zone(s) (spots) with only one analyte binding compound. The test zone(s) may be between 1 to 15 mm wide. A test zone with a size smaller than 1 mm is possible but in such case interpretation, i.e. reading of results may be difficult. A test zone with a size higher than 15 mm is possible but in such case, more volume of reagents is needed. The soaking of larger sample volume, e. g. from 0,1 ml to 2 ml may allow the detection of lower concentration of the analyte to be detected. The test zone(s) may have a diameter of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 mm, in particular have a diameter between 1 and 10 mm.

Preferentially said test zone has a diameter of 3 to 4 mm. For such a zone sample volumes between 5 to 500 Dl are preferentially used.

Both hole and test zone may have any form such as a circle, square, triangle, cross or any regular or irregular surface.

The volume of the sample added is preferentially equal or smaller than the volume of the reagent solution. E.g. if a 20 Dl sample is applied, 25Dl of the reagent solution is used; when a 25-30D1 of sample is used, 50Dl of reagent solution is applied. If the test zone is larger than 4 mm or if sample volumes are larger than 100 Dl the composition of diluent buffer may be modified by the addition of sucrose going from 1 % up to 40%. Possible concentrations are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 and 40%. Sucrose may be added into the diluent buffer in order to slow down the flow rate phenomenon in order to get a lower detection limit and to avoid the appearance of a heterogeneous colored spot.

The lower layer of the device of the present invention is an absorbent member or layer having capillary passage ways generally transverse to the upper and lower surfaces. The lower absorbent layer is assembled with the intermediate porous layer in a manner, which permits direct communication between the pores or interstices of the porous layer and the capillaries of the absorbent layer. Thus, as a liquid is applied onto the intermediate porous layer and is subsequently absorbed by the lower absorbent layer and saturates it, the liquid is drawn through capillary force into the absorbent member. As a result, flow can be induced through the lower absorbent layer when a liquid sample is applied to the surface of the intermediate porous layer even though the hydrostatic pressure of the fluid is so low that unaided it could not flow through the intermediate layer without the application of pressure to force it through or a vacuum to draw it through. The absorbent layer comprises at least one layer of hydrophilic material in contact with and positioned on the side of the insoluble porous layer opposite the side of the cover layer.

A variety of fibrous filter materials can be used for the absorbent layer. A useful material is cellulose acetate fibers oriented as in a cigarette filter. Those skilled in the art will appreciate that other absorbent members made of polyester, polyolefin or other materials may be used in place of cellulose acetate. The present invention suggests that the hydrophilic material of the device allows communication between the porous material and the absorbent layer and is preferentially AP120, provided by the company mdi (Advanced Microdevices (PVT) LTD. 21, Industrial Area, Ambala Cantt, 133 001 India) . Alternatively, equivalent filter pads may be used. These absorbent layers or filter pads can be provided by several companies involved in membrane technology, e. g. Whatman, Sartorius and Millipore.

As is evident to the skilled artisan the insoluble porous material or the multilayer support as defined herein, can be used in an assay device to determine the presence of a plurality of analytes in a sample.

Diagnostic Kit / Assay

It is accordingly a further aspect of the present invention to provide a diagnostic kit as well as an assay comprising any of the above. The diagnostic kit may further comprise; a) Solutions comprising an enzyme-labeled second analyte- binding compound, an enzyme-labeled second analyte- binding complex or an enzyme-labeled detection molecule, for each of the analyte binding compounds coated on the insoluble porous material; b) A solution comprising a precipitating substrate for the enzyme linked to the second analyte-bindig complex or an enzyme-labeled detection molecule able to generate a colored deposit upon reaction with the enzyme used in the solutions of a) ; c) Optionally a color chart for the interpretation of the colored deposit; d) Colour Intensity measurement apparatus, like a reflectance reader e) Optionally an instruction leaflet.

In a further embodiment the diagnostic kit may optionally comprise one or more of a diluent solution, a fixative solution, a solution comprising a capturing molecule, a solution comprising a substrate for the enzyme linked to the capturing molecule, a standard solution or a control solution .

In one embodiment, the assay as provided herein comprises the steps of;

- passing the sample through the insoluble porous membrane as defined herein; - exposing the sorbed porous membrane to at least two labeled second analyte-binding compounds specific for the analytes of interest, i.e. pass through the sorbed porous material at least two labeled second analyte- binding compounds specific for the analytes of interest including an auto control, such as a CRP auto control; and

- determine the binding of the labeled second analyte- binding compounds to the membrane.

The exposure of the sorbed porous membrane can be done either by;

- exposure to (flow-through of) a solution comprising a plurality of labeled second analyte-binding compounds, in particular at least two labeled second analyte- binding compounds, more in particular comprising up to 20 labeled second analyte-binding compounds, even more in particular comprising from 2 to 5 labeled second analyte-binding compounds, specific for the analytes of interest;

- a sequential exposure to (flow-through of) solutions each independently comprising a labeled second analyte- binding compound specific for an analyte of interest, wherein the sorbed porous membrane is sequentially exposed to solutions each independently comprising a labeled second analyte-binding compound specific for an analyte of interest; or - exposure to (flow-through of) a combination of solutions comprising a plurality or only one labeled second analyte-binding compound specific for an analyte of interest as provided hereinbefore.

In a particular embodiment of the present invention, the assay consists of an immunoassay, i.e. the analyte / analyte-binding compound combination is based on the binding of an antigen or hapten with a specific antibody. Any convenient immunoassay format can be used, and would typically comprise steps wherein the addition of the first analyte-binding compound, sample, second analyte-binding compound, substrate solution and fixation solution are performed subsequently, one after the other.

Alternatively, said method may comprise steps wherein the addition of the first analyte-binding compound, sample, second analyte-binding compound, substrate solution and fixation solution are not all performed subsequently, some of these may be premixed in advance before bringing them onto the device. Other assay variants are possible. For example, the labeled antibody and sample may be mixed prior to addition to the porous member. In another embodiment of the invention, the assay device is used to perform competitive assays, i. e., assays in which the first analyte-binding compound is bound to the porous member and for which the analyte in the sample competes with a fixed quantity of labeled analyte added to the sample solution or added following sample addition. Competitive immunoassays are conveniently run in this fashion using an antibody, for example, a monoclonal or polyclonal antibody preparation as first analyte-binding compound bound to the solid phase. Labeled antigen can be added to the sample before the sample is added to the porous layer. Alternatively, it can be added subsequent to addition of the sample or concurrently therewith. When a capturing molecule is used, the sample may be assayed in a variety of ways. For example, in a "sandwich assay", a first analyte-binding compound and a second labeled analyte-binding compound may be combined with the sample to bind the analyte prior to addition to the porous member. Alternatively, a first analyte-binding compound and a sample may be combined prior to addition to the porous member, or added in the sequence of first an analyte-binding compound and then sample, to be followed by addition of a labeled second analyte-binding compound. In such sandwich assays, the capturing molecule is selected to bind the first analyte binding compound and not the labeled second analyte-binding compound. In a competitive assay format, the intensity of the coloration of the deposit is inversely proportional to the concentration of the marker to be detected.

The label of the labeled second analyte-binding compound would be any suitable label which allows the second analyte-binding compound to be detected. Suitable labels include radioisotopes, e.g. ¹²⁵I, fluorochromes, enzymes, antibodies, polynucleotides and linkers such as biotin. In particular the label is selected from a luminescent compound, a fluorescent compound or an enzyme.

More in particular, the assay is based on an enzyme immunoassay (EIA) , wherein the label of the labeled second analyte-binding compound is an enzyme label. Again, any convenient enzyme label can be used in the assays of the present invention, such as for example enzymes that react in one step with a precipitating substrate, e.g. horse radish peroxidase (HRP) , alkaline phosphatase (AP) , and dehydrogenase. Dehydrogenase can be specified as being for instance glucose-6-phosphate dehydrogenase, lactate dehydrogenase or a malate dehydrogenase.

Uses

As already mentioned herein before, the materials and methods (assays) of the present invention are useful to test the presence of a plurality of analytes of interest in a samp1e .

As the analytes of interest include both organic and inorganic molecules with a molecular weight of less than 2000 daltons; proteins; peptides; antisense oligonucleotides; siRNAs; antibodies, including both monoclonal and polyclonal antibodies; ribozymes; antigens, including allergens; etc. - the methods (assays) of the present invention are useful in, for example;

• the diagnosis and/or monitoring of treatment of allergic diseases or intolerance manifestations;

• the diagnosis and/or monitoring of treatment of autoimmune diseases induced by organ or non-organ specific auto-antigens; • the diagnosis and/or monitoring of treatment of infectious diseases induced by amongst others, viruses, bacteria, molds, mycobacteria or parasites;

• the testing of cardiac and/or inflammatory markers, in particular for cardiac markers chosen from the group comprising myoglobin, creatine kinase and troponin and for inflammatory markers selected from the group consisting of c-reactive protein and interleukins;

• testing for the presence of bacteria, viruses, mycotoxins, toxins, residues of pesticides, residues of antibiotics and residues of chemical substances;

• testing for the presence of carcinogenic antigens; or

• testing for the presence of drugs or abuse molecules.

In particular the present invention provides the use of the porous insoluble material, multilayer support, assay devices and methods (assays) of the present invention in determining the presence of a plurality of allergens in a sample, in particular to determine the presence of at least two allergens in a sample. The allergens being selected from the group consisting of food allergens, such as for example milk , fruit , fish, eggs and nuts; Pollen , especially ragweed, which causes hayfever; Mould from plants and food, which are most likely to cause asthma; House dust , which contains mites as well as dander from housepets; Venom from insects (such as bees, wasps and mosquitoes) or scorpions; Plant Oils, especially poison ivy, oak or sumac; feathers; wool; dyes; cosmetics and perfumes.

This invention will be better understood by reference to the Experimental Details that follow, but those skilled in the art will readily appreciate that these are only illustrative of the invention as described more fully in the claims that follow thereafter. Additionally, throughout this application, various publications are cited. The disclosure of these publications is hereby incorporated by reference into this application to describe more fully the state of the art to which this invention pertains .

EXAMPLES

The following examples illustrate the invention. Other embodiments will occur to the person skilled in the art in light of these examples.

EXAMPLE 1 : AUTO CONTROLEASSAY

In this assay, nitrocellulose is preferably used as insoluble porous material , due to its neutral charge and its suitability for the procedure described below.

1° coating procedure (spotting and immersion)

In the coating procedure, membranes are cut into strips of 0.8 to 1,5 wide cm and then cut in squares.

The upper corner of the membrane section is spotted with 0.5 Dl to 2 Dl of the auto control analyte-binding compound with a concentration of 1 mg/ml to 2 mg/ml (e.g.: anti species antibody) . The control analyte-binding compound can eventually be diluted into a buffer (i.e. phosphate buffer saline "PBS") .

For the CRP application of the present example, the control analyte-binding compound is a goat polyclonal anti serum anti -rabbit IgG, applied with a concentration of 2 mg/ml .

The membranes are incubated at room temperature for 15 minutes (may be extended to 2 hours) . When dried, they are kept at 4° till the immersion steps.

Membranes are then slowly immersed under gentle agitation for for 3 hours, with a direct contact of the reactive/test zone with the application (coating) buffer, in a bath brought at room temperature and containing the coating (application) buffer and the analyte binding compound.

For the CRP application of the present example, the analyte binding compound is a goat polyclonal antiserum anti-human CRP with high affinity applied at excess with a binding concentration of 0.2 mg/ml and the coating buffer made of Tris (1.2 g/1) and NaCl (8.8 g/1) .

After the coating (spotting and immersion) , membranes are not washed off in order to avoid "desorption" of the analyte and control binding compounds due to the presence of surfactants agents in the wash buffer. Indeed, substances like Triton or Tween 20 are usually present in the wash buffer.

2° Post coating-blocking procedure.

The aim of such blocking procedure is to block any remaining free sites after coating procedure (see above) .

After the coating (spotting and immersion) procedure, strips are placed under gentle agitation during 3 hours at room temperature (18-25°C) in a second bath containing a blocking agent. In this experiment, the remaining free sites are blocked by the use of very purified BSA (bovine serum albumin) at 1% concentration.

For CRP application, the blocking buffer is made of; NaCl (8.5 g/1), Na₂HPO₄ (1.25 g/1) , NaH₂PO₄.2HO (0.160 g/1), Thimerosal (1 g/1), and BSA (lOg/1).

It has been observed, that due to high binding of the control and analyte binding compounds, there is no displacement by the BSA which block all the remaining free sites. The blocking protein is added in excess in order to be able to block any remaining free site.

The structure of the analyte and control binding compounds is not affected by the use of the blocking agent.

Again, as for the coating procedure, after the application of the blocking protein the membranes are not washed off in order to avoid "desorption" of the analyte and control binding compounds due to the presence of surfactants agents in the wash buffer. Indeed, substances like Triton or Tween 20 are usually present in the wash buffer.

Strips are dried at 37°C for 1 hour and placed overnight in an incubator at room temperature (18-25°C) in order to protect them from dust. The drying step can also be performed at 37° without affecting results. Strips are then packaged in order to protect them from humidity and stored at 4°C or room temperature (18-25°C)

3° CRP Test procedure

A schematic drawing of the different elements present in the CRP test procedure is provided in Figure 1.

The CRP test procedure comprises the following steps; a) 20 Dl of serum or whole blood (1/50 diluted in a diluent/lysis buffer) are applied onto the membrane and allowed to soak for 30 sec to 1.5 minutes.

Specific binding compound (goat polyclonal immunoglobulins anti human CRP) will bind to CRP protein eventually present in the sample.

There will be no binding of the control analyte-binding compound (goat polyclonal anti serum anti -rabbit IgG) to the CRP protein eventually present in the sample.

b) 25 Dl of so called conjugate (rabbit anti-human CRP immunoglobulins, horse radish peroxidase labelled) are applied onto the membrane and allowed to soak for 30 sec to 1.5 minutes. The conjugate will bind to the CRP eventually present into the sample, forming a sandwich.

The conjugate will bind to the goat anti-rabbit immunoglobulins (auto control binding compound) bound to the membrane irrespective of the presence of CRP in the samp1e .

c) developing the assay by addition of 25 Dl of insoluble substrate (Tetra Methyl Benzidine) onto the membrane that is allowed to soak for 30 sec to 1.5 minutes. Incubation with the substrate must induce the appearance of a dark blue colouration on the spotting zone (auto control zone) . If there is no colouration onto the spotting zone, the enzymatic reaction failed and the result cannot be interpreted. Onto the remaining area of the test zone, the intensity of colouration is proportional to the quantity of CRP present into the sample.

d) The addition of a fixative solution will stop the enzymatic reaction and fix the colouration of both spots.

The interpretation of results is performed visually by comparison of the colour intensity (Figure 2) with a colour chart or by the use of a rapid reader.

EXAMPLE2 : MULTIPARAMETERASSAY

In this assay, nitrocellulose is preferably used as insoluble porous material , due to its neutral charge and its suitability for the procedure described below. 1° coating procedure (spotting and immersion)

In the coating procedure, membranes are cut into strips of 1,2 to 1,5 wide cm and then cut in squares.

The upper left and upper right corner of the membrane section is spotted with 0.5 Dl to 2 Dl of purified analyte binding compounds, in particular with antibodies specific for the analytes of interest, in a concentration ranging from 0.25 mg/ml to 1 mg/ml .

In the present example;

• the first analyte binding compound (upper left corner) is an anti-casein antibody, more in particular a rabbit anti-casein antibody; and

• the second analyte binding compound (upper right corner) is an anti-ovalbumin antibody, more in particular a rabbit anti-ovalbumin antibody.

Membranes are then slowly immersed under gentle agitation for 1 to 5 hours, in a bath brought at room temperature and containing the coating (application) buffer and the analyte binding compounds.

In the present example; • the analyte hinging compounds are purified antibodies, in particular rabbit antibodies, against the allergens casein and ovalbumin, applied in excess and at a binding concentration of 0.1 mg /ml; and

• the coating buffer is made of Tris (1.2 g/L) and NaCl (8.8 g/L) .

After the coating (spotting and immersion) , membranes are not washed off in order to avoid "desorption" of the analyte binding compounds due to the presence of surfactants agents in the wash buffer.

Indeed, substances like Triton or Tween 20 are usually present in the wash buffer.

2° Post coating-blocking procedure.

After the coating (spotting and immersion) procedure, strips are placed under gentle agitation during 3 hours at room temperature (18-25°C) in a second bath containing the blocking agent.

In this experiment, the remaining free sites are blocked by the use of a polypeptide fraction of collagen protein at 10% concentration (Prionex ^R) .

It has been observed, that due to high binding of the analyte binding compounds, there is no displacement by the Prionex ^R protein, which block all the remaining free sites . As for the auto control assay, the structure of the analyte binding compounds is not affected by the use of the blocking agent. Again as for the coating procedure, after the application, membranes are not washed off in order to avoid "desorption" of the analytes and control binding compounds due to the presence of surfactants agents in the wash buffer. Indeed, substances like Triton or Tween 20 are usually present in the wash buffer.

Strips are dried at 37°C for 1 hour and placed overnight in an incubator at 20-35⁰C in order to protect them from dust.

Strips are then packaged in order to protect them from humidity and stored at 4°C or room temperature (18-25°C)

3° Allergens Test procedure

A schematic drawing of the different elements present in the allergens procedure is provided in Fig.4. Results of the present example are provided in Fig.6.

The procedure comprises the following steps; a) 20 Dl of sample are applied onto the membrane and allowed to soak for 30 sec to 1.5 minutes. Specific binding compounds (purified anti Allergen 1 and/or 2 antibodies) will bind to casein and/or ovalbumin proteins eventually present into the sample.

b) 25 Dl of so called conjugate (anti-casein and anti- ovalbumin antibodies horse radish peroxidase labelled) are applied onto the membrane and allowed to soak for 30 sec. to 1.5 minutes. Alternatively, the conjugates are applied sequentially onto the membrane, i.e. one after the other. The conjugate will bind to the casein and/or ovalbumin eventually present in the sample.

c) Developing the assay by addition of 25 Dl of insoluble substrate (Tetra Methyl benzidine) onto the membrane that is allowed to soak for 30 sec. to 1.5 minutes.

If sample contains casein protein, incubation with the substrate will induce the appearance of a dark blue colouration on the left spotted zone.

If sample contains ovalbumin protein, incubation with the substrate will induce the appearance of a dark blue colouration on the right spotted zone.

Onto the remaining area of the test zone, the incubation with the substrate will induce the appearance of a blue colouration if casein and / or ovalbumin are present into the sample.

The interpretation of results is performed visually by comparison of the colour intensity with a colour chart or by the use of a rapid reader.

Claims

1. A membrane for use in a vertical filtration method to determine the presence of two or more analytes of interest in a sample, characterized in that said membrane is homogeneously occupied with two or more different analyte binding compounds in a single test zone; typically with from 2 to 20, in particular from 2 to 10, more in particular from 2 to 5 different analyte binding compounds .

2. A membrane according to claim 1, further comprising at least one zone pre-coated (pre-spotted) with a single analyte binding compound; typically from 1 to 21, in particular from 1 to 11, more in particular from 2 to 6 zones each independently coated with only one analyte binding compound; even more in particular comprising only one zone pre-coated (pre- spotted) with a single analyte binding compound.

3. A membrane for use in a vertical filtration method to determine the presence of an analyte of interest in a sample, characterized in that said membrane is homogeneously occupied with one analyte binding compound in a single test zone and comprises at least one zone pre-coated (pre-spotted) with a single analyte binding compound; typically from 1 to 21, in particular from 1 to 11, more in particular from 2 to 6 zones each independently coated with only one analyte binding compound; even more in particular comprising only one zone pre-coated (pre- spotted) with a single analyte binding compound. .

RECTIFIEDSHEET(RULEgI) ISA/EP

4. A membrane according to any one of claims 2 to 3, wherein the zone(s) pre-coated (pre-spotted) with only one analyte binding compound are coated (spotted) within the single test zone.

5. A membrane according to any one of claims 2 to 4 wherein at least one of said zones coated (spotted) with only one analyte compound is characterized in that, said analyte binding compound differs from the analyte binding compounds used in the single test zone, i.e. in that it constitutes an auto control.

6. A membrane according to any one of claims 1 to 5, wherein the zone(s) pre-coated (spotted) with only one analyte binding compound has/have a diameter of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1-0, 1.2, 1.4, 1.6, 1.8 or 2.0 mm.

7. A membrane according to any one of claims 1 to 6, further characterized in that the membrane has pores with a diameter between 0.1 and 12 μm and has a thickness up to 2500 μm; in particular the material has pore sizes of 0.1, 0.2, 0.45, 0.8, 1.2, 3.0, 5.0, 8.0 and 12 μm and a thickness of 500 μm.

8. A membrane according to claim 7, wherein the material has pore sizes of 0.45, 0.8 and 1.2 μm; in particular pores of 0.45 μm.

9. A membrane according to any one of claims 1 to 8, further characterized in that the material is made from nylon, nitrocellulose, cellulose, fiberglass,

RECTIFIED SHEET (RULE 91) ISA/EP polysulfofone, polyvinylidene, difluoridβ/ polyester or any other polymeric material whereon a biological substance may bind

10. A membrane according to claim 9, wherein the material consists of nitrocellulose.

11. A membrane according to any one of claims 1 to 10, further characterized in that one face of said membrane/ i.e. the one not exposed to the loading of the sample/ consist of an inert material; in particular a paper cast.

12. A membrane according to any one of claims 1 to 11 wherein the analyte binding compounds are antibodies or fragments thereof.

13. A multilayer support comprising;

- An upper cover layer of a water-impermeable material having at least one hole, whereby said hole overlays a test zone;

- An intermediate porous layer comprising a membrane as defined in any one of claims 1 to 14; and

- A lower absorbent layer comprising at least one layer of a hydrophilic material.

14. A multilayer support according to claim 13 wherein the water-impermeable material of said upper cover layer is a plastic; in particular selected from the group consisting of polypropylene/ polyvinylchloride or styrene-ethylene/butylene styrene (SEBS) .

RECTIFIED SHEET (RULE 91) ISA/EP

15. A multilayer support according to claim 14 wherein the test zone has a diameter. of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mm (check met de tekst) ; in particular a diameter of 3 - 4 mm.

16. A multilayer support according to claim 13 wherein the lower absorbent layer is a absorbent member or layer having capillary passage ways generally transverse to the upper and lower surfaces and comprises at least one hydrophilic material in contact with and positioned on the side of the insoluble porous layer opposite the side of the cover layer.

17. A multilayer support according to claim 16, wherein the absorbent member is made of cellulose acetate, polyester or polyolefin.

18. A multilayer support according to claims 16 or 17 wherein the hydrophilic material consists of AP120 or any equivalent absorbent pad.

19. An assay device for testing the presence of an analyte of interest in a sample, comprising a membrane according to any one of claims 1 to 14.

20. An assay device according to claim 19, comprising a multilayer support according to any one of claims 13 to 18.

21. A diagnostic kit for testing the presence of at least two analytes of interest in a sample comprising an assay device according to any one of claims 15 to

RECTIFIED SHEET (RULE 91) ISA/ EP

20.

22. A diagnostic kit according to claim 21, further comprising/ a) Solutions comprising an enzyme-labeled second analyte- binding compound, an enzyme-labeled second analyte- binding complex or an enzyme-labeled detection molecule, for each of the analyte binding compounds coated on the membrane; b) A solution comprising a precipitating substrate for the enzyme linked to the second analyte-bindig complex or an enzyme-labeled detection molecule able to generate a colored deposit upon reaction with the enzyme used in the solutions of a) ; c) Optionally a color chart for the interpretation of the colored deposit; d) Optionally a Colour Intensity measurement apparatus, like a reflectance reader e) Optionally an instruction leaflet.

23. A diagnostic kit according to claims 21 or 22, optionally comprising one or more of a diluent solution, a fixative solution, a solution comprising a capturing molecule, a solution comprising a substrate for the enzyme linked to the capturing molecule, a standard solution or a control solution.

24. An assay to determine the presence of at least two analytes of interest in a sample, comprising the steps of

- passing the sample through the membrane as defined in any one of claims 1 to 14;

- passing at least two labeled second analyte-

RECTIFIED SHEET (RULE 91) ISA/EP binding compounds specific for the analytes of interest through the membrane; and - determine the binding of the labeled second analyte-binding compounds to the membrane.

25. An assay according to claim 24, wherein a solution comprising said labeled second analyte- binding compounds is passed through the membrane or wherein solutions each independently comprising a labeled second analyte-binding compound are sequentially passed through the membrane.

26. An assay according to claims 24 or 25, wherein the label of the labeled second analyte-binding compound is selected from a luminescent compound, a fluorescent compound, an enzyme, as well as metal containing substances; in particular the label is an enzyme label .

27. Use of an assay device according to any one of claims 19 to 20; of a diagnostic kit as claimed in any one of claims 21 to 23/ or of an assay as claimed in any one of claims 24 to 26, to test the presence of at least two analytes of interest in a given sample.

28. Use according to claim 27, wherein the sample can be chosen from a group comprising cell fractions/ serum, whole blood, urine, plasma for human or animal diagnostic and/or allergen testing; soil, mud, minerals, water, plant material or air for environmental and/or allergen testing; any food materials for food testing; or any other medium/suspension/hard material which can be used for

RECTIFIED SHEET (RULE 91) ISA/EP one of these purposes.

29. Use according to claim 27, wherein said analytes are compounds present or absent in the sample; said compounds being selected from the group comprising antigens, antibodies* peptides, proteins, lipids, allergens, organic molecules and nucleic acid oligomers.

30. Use according to claim 29 wherein the analyte is an antigen chosen from the group consisting of any biological agent such as bacteria, viruses, molds, mycobacteria, parasites and pathogens -

31. Use according to claim 27 wherein the diagnostic kit or the assay device is applied for the diagnosis and/or monitoring of treatment of allergic diseases or intolerance manifestations .

32. Use according to claim 31 wherein the allergic disease or intolerance manifestation is induced by allergens selected from the group consisting of food allergens, such as for example milk, fruit , fish, eggs and nuts; Pollen, from grasses, weeds and trees, especially ragweed, which causes hay fever; Mould from plants and food, which are most likely to cause asthma; House dust, which contains mites as well as dander from house pets; Venom from- insects (such as bees, wasps and mosquitoes) or scorpions; Plant Oils, especially poison ivy, oak or sumac; feathers; wool; dyes; cosmetics and perfumes .

33. Use according to claim 27 for the diagnosis

RECTIFIED SHEET (RULE 91) ISA/ EP and/or monitoring of treatment of auto-iπunune diseases induced by organ or non-organ specific auto-antigens .

34. Use according to claim 27 for the diagnosis and/or monitoring of treatment of infectious diseases included by viruses, bacteria, molds, mycobacteria or parasites.

35. Use according to claim 27 in the testing of cardiac and/or inflammatory markers.

36. Use according to claim 31 wherein said cardiac markers are chosen from the group comprising of myoglobin, creatine kinase and troponin and inflammatory markers are chosen from the group comprising C-reactive protein and interleukins.

37. Use according to claim 27 in testing for the presence of bacteria, viruses, mycotoxins, toxins, residues of pesticides, residues of antibiotics, and residues of chemical substances.

38. Use according to claim 27 in testing of carcinogenic antigens.

39. Use according to claim 27 in testing of drugs of abuse molecules.

RECTIFIED SHEET (RULE 91) ISA/ EP