TECHNOLOGICAL FIELD

This invention relates to diagnostic devices, methods and kits. In particular, the present invention provides novel devices and techniques for flexible detection of target molecules based on a universal lateral flow device.

PRIOR ART

1. Raphael C. Wong, Harley Y. Tse, in Lateral Flow Immunoassay, Human press 2008.

2. William James. Aptamers. In Encyclopedia of Analytical Chemistry. R.A. Meyers (Ed.) pp. 4848-4871 O John Wiley & Sons Ltd, Chichester, 2000.

3. Pai SS, Ellington AD. Using RNA aptamers and the proximity ligation assay for the detection of cell surface antigens. Methods Mol Biol. 2009;504:385-98

4. M Sassanfar and JW Szostak, "An RNA Motif That Binds ATP," Nature 364 (1993): 550-553.

BACKGROUND

There is an increasing need for accurate, fast and low cost assays in the fields of in-vitro diagnostics (IVD), Point of Care (POC) diagnostics and research laboratories detection assays. Currently, lateral flow tests also known as Lateral Flow Immunochromatographic Assays (LFIA) ¹ are widely used as a detection and diagnosis tool. Lateral flow tests are based on a simple device intended to detect the presence (or absence) of a target analyte in a sample.

Often produced in a dipstick format, Lateral flow tests are a form of immunoassay in which the test sample flows along a solid porous matrix via capillary action. After the sample is applied to the test device it encounters a colored or fluorescent particulated reagent which mixes with the sample and flows downstream along the strip, encountering lines or zones at which an antibody or antigen are fixed. Depending upon the presence of the target analytes in the sample, the colored particulated reagent is bound at the test line or test zone. The time required to obtain the test result is a key driver for these products. Test results can be usually obtained in a few minutes. Generally there is a tradeoff between time and sensitivity and therefore tests that require higher sensitivity may take longer to develop.

The LFIA strips are designed for each assay specifically in order to be able to detect the specific requested target molecule. For each assay a unique strip should be designed and constructed, having at least three variable reagents, a first target specific antibody conjugated to the reporter particles, a second, target specific antibody having different epitope specificity fixed at the test line for capture and a secondary antibody specific to the antibody conjugated to the particles fixed at the control line.

SUMMARY OF THE INNOVATION

According to its first aspect the present invention provides a strip for the detection of at least one target molecule in a sample, the strip comprises a sample pad, a reagent pad, a base membrane and an absorbent pad; wherein the reagent pad comprises at least one member of an affinity couple; and wherein the base membrane comprises a results zone comprising at least two distinct regions, a first of the at least two regions comprises a member of an affinity couple and the second of the at least two regions comprises a member of an affinity couple which is complementary to the member comprised in the reagent pad, wherein the member of an affinity couple in the first region is different from the member of an affinity couple in the second region.

According to its second aspect the present invention provides an assay for the detection of at least one target molecule in a sample, comprising:

a. Incubating the sample with at least two target recognition agents, each target recognition agent is connected to a member of an affinity couple; and

b. contacting the incubated sample with a strip, the strip comprising a sample pad, a reagent pad, a base membrane and an absorbent pad; wherein the reagent pad comprises at least one member of an affinity couple; and wherein the base membrane comprises a results zone comprising at least two distinct regions, a first of the at least two regions comprises a member of an affinity couple and the second of the at least two regions comprises a member of an affinity couple which is complementary to the member comprised in the reagent pad, wherein the member of an affinity couple in the first region is different from the member of an affinity couple in the second region;

whereby appearance of a signal in both results regions in the results zone on the base membrane indicates the presence of the target molecule in the sample; and whereby appearance of a signal only in the second results region in the results zone on the base membrane indicates the absence of the target molecule in the sample.

According to its third aspect the present invention provides a device comprising a strip of the invention contained in a housing, wherein the housing comprises at least two windows: a sample window and a results window.

According to its fourth aspect the present invention provides a method for detecting at least one target molecule in a sample comprising contacting the sample with a diagnostic device of the invention.

According to its fifth aspect the present invention provides a kit for the detection of a target molecule in a sample comprising (a) at least two labeled target recognition agents capable of specifically binding to the target molecule, and (b) a strip and/or device according to the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to understand the invention and to see how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

Figures 1A and IB are representative examples of the universal lateral flow strip (Figure 1A) and a device comprising the strip (Figure IB).

Figure 2 a schematic representation of the "Two steps-All in one" assay device, using an external unit having a cylinder shape and a valve.

Figure 3 a schematic representation of the "One step-All in one " assay device, using as an external unit a pad.

Figures 4A to 4F are pictures of the universal strip showing detection of recombinant Platelet-Derived Growth Factor (PDFG)-BB using two specific PDFG aptamers at different concentrations (Figures 4A, 4D), using one aptamer (Figures 4B, 4C) and negative controls (Figures 4E, 4F), the upper line corresponds to a control line and the lower line corresponds to a test line. Each experiment is shown in duplicates.

Figures 5A and 5B are pictures of universal strips showing detection of various human IgG (hlgG) concentrations on strips with polystyrene particles coated with avidin (Figure 5A) or with gold particles coated with streptavidin (Figure 5B). Lane 1= No hlgG (buffer only), lane 2=hIgG 1 μg/μl, lane 3=hIgG 100 ng/ml, lane 4= hlgG 10 ng/ml, the upper lane corresponds to a control line and the lower line corresponds to a test line.

DETAILED DESCRIPTION

The present disclosure provides a novel universal strip and device for the detection of target molecules in a sample, methods of using the strip and device and kits comprising the same. Specifically, the present disclosure provides a lateral flow strip and a device comprising the strip that is suitable for detection of a variety of target molecules.

In commonly used diagnostic lateral flow devices, the strip includes components such as antibodies that are specific for the target molecule; thus, the strip is target specific and enables the detection of one target for a designed strip. As such, different strips should be designed, manufactured and used for each target molecule. For example, different strips were used for pregnancy tests, tests for detecting infectious agents, biomarkers tests etc.

The inventors have developed a strip that was designed such that it does not contain any target specific component. This unique feature of the strip and the device including the strip enables the design, manufacture and use of one type of a strip that suits all target molecules and is hence target-independent. Accordingly, the strip and the device according to the invention are suitable for detecting and determining the presence, absence of a variety of target molecules without having the need to modify the strip according to the tested target molecule. In addition, the strip and the device described here are suitable for detecting different amounts of target molecule.

Thus in accordance with its first aspect, the present invention provides a strip for the detection of at least one target molecule in a sample, wherein the strip (10) comprises a sample pad (12), a reagent pad (14) a base membrane (16) and an absorbent pad (18); wherein the reagent pad comprises at least one member of an affinity couple; and wherein the base membrane comprises a results zone comprising at least two distinct regions, a first of the at least two region (22)comprises a member of an affinity couple and the second region (24) of the at least two regions comprises a member of an affinity couple which is complementary to the member comprised in the reagent pad, wherein the member of an affinity couple in the first region is different from the member of an affinity couple in the second region.

In accordance with its second aspect, the present invention provides an assay for the detection of at least one target molecule in a sample comprising:

a. Incubating the sample with at least two target recognition agents, each target recognition agent is connected to a member of an affinity couple; and

b. Contacting the incubated sample with a strip, the strip comprising a sample pad, a reagent pad a base membrane and an absorbent pad; wherein the reagent pad comprises at least one member of an affinity couple; and wherein the base membrane comprises a results zone comprising at least two distinct regions, a first of the at least two regions comprises a member of an affinity couple and the second of the at least two regions comprises a member of an affinity couple which is complementary to the member comprised in the reagent pad, wherein the member of an affinity couple in the first region is different from the member of an affinity couple in the second region;

whereby appearance of a signal in both regions in the results zone on the base membrane indicates the presence of the target molecule in the sample; and whereby appearance of a signal only in the second region in the results zone on the base membrane indicates the absence of the target molecule in the sample.

It is to be understood that the term "absence" in the context of the assay also encompasses presence of the target molecule in an amount that is lower than the detection limit of the assay.

The strip according to the present disclosure is a lateral flow analytical apparatus. In accordance with the present disclosure, the strip is used to determine the absence or presence of a target molecule in a sample suspected to include the target molecule. As used herein the term "strip" denotes a narrow, elongated piece of porous or fibrous material capable of absorbing and promoting liquid flow.

The "sample" according to the present invention may be any sample including, but not limited to, biological samples obtained from biological systems (including cell cultures, micro-organism cultures), biological samples obtained from subjects (including humans and animals as detailed below), samples obtained from the environment for example soil samples, water samples, agriculture samples (including plant and crop samples), or food samples.

In some embodiments, the sample is a liquid sample. In some embodiments, the liquid sample is liquid in its natural state. In some further embodiments, the liquid sample is pre-treated to be in a liquid state. Pre-treatment may be by any method that changes a sample that is not liquid in its natural state into a liquid state. In some embodiments, pre-treatment is by extraction. In some other embodiments, the sample comprises at least one liquid fraction.

The term "subject" in accordance with the invention includes but is not limited to a human, an animal, in particular, a primate, a household animal or an animal used in agriculture.

Furthermore, the term subject encompasses healthy subjects, subjects suspected of having a condition, subjects suffering from various diseases, subjects receiving various treatments, as well as deceased subjects (e.g. for forensic analysis).

In some embodiments, the biological sample may be a bodily fluid, a tissue, a tissue biopsy, a swab with tissue or body fluid sample, an isolated cell population or a cell preparation.

In certain embodiments the cell in the population of cells or cell preparation is selected from an animal cell, a plant cell, a viral cell, a bacterial cell and a fungal cell.

In some specific embodiments, the population of cells comprises cancer cells. In another embodiment the population of cells is an in vitro cultured cell population.

In some embodiments, the biological sample may be a bodily fluid selected from the group consisting of blood, serum, plasma, urine, cerebrospinal fluid, amniotic fluid, tear fluid, nasal wash, mucus, saliva, sputum, broncheoalveolar fluid, throat wash, vaginal fluid and semen. Samples according to the invention may be samples obtained from the environment for example soil samples or water samples. Water sample may be obtained for example but not limited to from drinking water, sewage, sea water, lakes, and rivers. The method disclosed in the present invention may be applied for research use, home use, municipal use, or governmental use.

Agriculture samples may also be used, for example plant samples and crop samples. Plant samples refer to any plant or pare thereof being for example seeds, fruit, or leaves and include but are not limited to field crops or greenhouse-grown plants. The invention also encompasses plant samples obtained from wild plants (i.e. plants which are not grown by men).

Food samples may be obtained for example from raw materials used for food production, food production line, fresh food, cooled food or frozen food.

The term "target molecule" as used herein denotes a molecule which may be found in a tested sample and which is capable of binding to a recognition agent.

In accordance with some embodiments, the target molecule is an organic molecule. In some further embodiments, the target molecule is a soluble antigen, a cell-surface antigen, or an antigen associated with a micelle, a liposome or a particle. In one embodiment, the target molecule is an antigen.

As used herein the term "antigen" refers to a target molecule capable of binding to a recognition agent.

In some embodiments, the target molecule may be a protein, a polypeptide, a peptide, a ganglioside, a lipid, a phospholipid, a carbohydrate, a small molecule or a nucleic acid.

Non limiting examples of a soluble antigen in accordance with the invention are proteins, enzymes, soluble cancer markers, inflammation-associated markers, hormones, cytokines, drugs, and soluble molecules derived from a virus, a bacteria or a fungus for example, toxins or allergens.

In some embodiments, the antigen is a micro-organism associated antigen. In the context of the invention the term "micro-organism associated antigen" is to be understood as a protein or fragment thereof encoded by the viral, bacterial or fungal genome. In some other embodiments, the antigen is a cancer (or tumor) marker. In general, a tumor marker may be found in the body fluids such as in blood or urine, or in body tissues. Tumor markers may be expressed or over expressed in cancer and are generally indicative of a particular disease process.

Non limiting examples of a cell surface antigen in accordance with the invention are a receptor, a cell surface marker, a micro-organism associated antigen, or a receptor ligand.

The terms "recognition agent" and "target recognition agent" are used interchangeably and denote any molecule capable of specifically recognizing the target molecule. In the context of the invention the term "recognition agent" also encompasses a binding agent.

The term "binding agent" as used herein refers to any molecule capable of specifically binding to the target molecule.

In some embodiments, the recognition agent is an aptamer, an antibody (including any antigen binding portion thereof) or a molecular imprinted polymer. In some specific embodiments, the binding agent is an aptamer or an antibody.

As used herein, the term "aptamers" denotes single-stranded nucleic acid (DNA or RNA) molecules which specifically recognizes and binds to a target molecule.

The aptamers according to the invention may fold into a defined tertiary structure and can bind a specific target molecule with high specificities and affinities ² ' ³ .

Aptamers are usually obtained by selection from a large random sequence library, using methods well known in the art, such as SELEX and/or Molinex ² ' ³ .

According to the present invention and as appreciated in the art, the recognition between the aptamer and the target molecule (antigen) is specific. Aptamers have been successfully generated against a large diversity of targets including proteins, small molecules and RNA ² ' ³ ' ⁴ .

After selection, aptamers are produced by chemical synthesis and purified to a very high degree. In addition, during the chemical synthesis, aptamers modifications can be introduced, enhancing the stability, affinity and specificity of the molecules.

The aptamers may optionally include a chemically reactive group at the 3 and/or 5 termini. The term reactive group is used herein to denote any functional group comprising a group of atoms which is found in a molecule and is involved in chemical reactions.

Some non-limiting examples for a reactive group include primary amines (NH ₂ ), thiol (SH), carboxy group (COOH), phosphates (P0 ₄ ), Tosyl, and a photo- reactive group.

In some embodiments, the aptamer as used herein may optionally comprise a spacer between the nucleic acid sequence and the reactive group. The spacer may be an alkyl chain such as (CH ₂ )6/i2, namely comprising six to twelve carbon atoms.

As used herein the term "antibodies" denotes immunoglobulin molecules comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. In the context of the present invention the term antibody also refers to antigen binding portions thereof.

The term "antigen-binding portion " of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab') ₂ fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody or (iv) single chain antibodies (scFv), a monovalent molecule comprising VL and VH regions linked by a synthetic linker. In accordance with some embodiments, the recognition agents are modified, possibly but not only for enhancing the stability, affinity and specificity of the molecules.

In accordance with the invention the recognition agents may be labeled by association (connection /interaction/conjugation) with a member of an affinity couple, and as such are referred to as "labeled recognition agents".

The term "affinity couple" as used herein denotes any two molecules having a high affinity of interaction to each other namely, binding.

Non -limiting examples for affinity couples are biotin/ biotin binding protein, antigen/antibody, Molecular Imprinted Polymers/target ligand, protein-A/IgG, enzyme/inhibitor, aptamer/target molecule, ligand/receptor, Digoxigenin (DIG)/anti-DIG antibody, Fluorescein isothiocyanate (FITC)/anti-FITC antibody, carbohydrate/lectin and a nucleic acids sequence/complementary nucleic acids sequence.

It is to be understood that the term "a member of an affinity couple" as used herein denotes one member of the affinity couples exemplified above. It is further to be understood that the term "a complementary member" as used herein denotes the complementary member within each couples.

In some embodiments, the member of an affinity couple is biotin and the complementary member of said member is any anti-biotin molecule such as for example streptavidin, avidin or modifications or derivatives thereof (e.g. NeutrAvidin and Extr Avidin).

In some embodiments, the recognition agents may be biotinylated. In accordance with some other embodiments, the recognition agents may be attached to a fluorescent moiety.

As appreciated, conjugation of the recognition agent and the member of an affinity couple may be by covalent bonding.

The terms "conjugation" , "association" , "connection" , "interactions" are used interchangeably to denote a bonding between two chemical entities. The bonding may be for example covalent binding, hydrogen binding, electrostatic binding, hydrophobic interactions and the like. Reverence is now made to Figure 1A showing a schematic illustration of the strip according to the invention.

In Figure 1A, there is illustrated a strip 10. The strip in accordance with the present disclosure may be prepared in different sizes. In some embodiments, the strip has the dimensions of length about 50mm to 80mm and width about 3mm to 5mm. At times, the strip is of 60mm length and 4mm width.

Strip 10 comprises a sample pad (12), a reagent pad (14) a base membrane (16) and an absorbent pad (18). The strip further comprises a backing card (20), wherein the sample pad, the reagent pad, the base membrane and the absorbent pad are attached to the backing card and positioned parallel to the backing card

As represented in Figure 1A, each two adjacent regions partially overlap. Moving from left end to right end of the strip, the right end of the sample pad overlaps with a first (left) end of the reagent pad. The second (right) end of the reagent pad overlaps with a first (left) end of the base membrane. The first (left) end of the absorbent pad overlaps with the second (right) end of the base membrane.

In some embodiments, the overlap between the regions is about 2mm to 10mm. In some other embodiments, the overlap between the regions a region is 5 mm. It should be noted that the overlap of the regions enables each region to be in direct contact with its adjacent region.

The backing card according to the present disclosure is prepared from a material capable of providing a support for the strip. In some embodiments, the backing card is made from Polyvinyl chloride (PVC), a combination of PVC/Polystyrene (PS) or polyester. Typically, pressure-sensitive adhesive is coating the surface of the backing card, covered by a release liner. Upon use, the backing card may be attached to the different components of the strip as described herein. For example, by removing the release liner and attaching the components thereto the backing card by the pressure- sensitive adhesive.

The sample pad of the strip according to the disclosure is an absorbing pad made of bibulous, porous or fibrous material capable of absorbing liquid rapidly.

There are two types of materials that are commonly used as sample pads: woven meshes and cellulose filters. In some embodiments, the sample pad is made of a paper or other cellulosic materials. Is some further embodiments, the sample pad is made of cotton fiber material, cellulose fiber, glass fiber or combinations thereof.

At times, pre-treatment with a surface-active agent during manufacture may be used to reduce any inherent hydrophobicity and improve the ability to take up and transport a liquid sample rapidly and efficiently.

The reagent pad according to the present disclosure is made of an inert material. For example the reagent pad is made of glass fibers.

The reagent pad comprises particulated material conjugated to at least one member of an affinity couple.

In some embodiments, the at least one member of an affinity couple is associated with (coated/conjugated to) particles and as such may also be referred to as "particulated material". The particles according to the invention may be considered as a solid matrix providing support for the at least one member of an affinity couple.

Typically, the particles have the size of 20nm to 500nm. At times, the particles have the size from 20nm to lOOnm, preferably from 20nm to 40nm. At times, the particles have the size from lOOnm to 500nm, preferably from 200nm to 500nm, more preferably about 300nm.

Non limiting examples include beads, e.g. magnetic beads, metallic beads, particles, colored particles (such as gold and latex sols), fluorescent particles, agarose beads, sephadex beads, glass beads, quantum dots, resins, plastic paper, particle, microsphere.

In some embodiments, the particles are colored particles.

In some embodiments, the particles are polystyrene particles. In some further embodiments, the particles are gold particles.

As appreciated, if fluorescent labeled particles or magnetic labeled particles are used, designated readers to assess the test results are needed such as a fluorescence reader or magnetic strip reader. Designated strip readers may be used for quantitative determination of the results.

In some embodiments, the reagent pad comprises dried particles coated conjugated with at least one member of an affinity couple. Association between the particles and the member of an affinity couple can be through covalent bonds, adsorption processes, hydrophobic or electrostatic interactions or combinations thereof.

In some embodiments, the reagent pad comprises polystyrene particles (e.g. blue polystyrene) coated with avidin. In some further embodiments, the reagent pad comprises gold particles coated with streptavidin.

As described herein, the reagent pad is prepared by drying thereto a suspension of buffer comprising coated particles.

The base membrane (16) according to the present disclosure herein has a porous structure that facilitates liquid advancement along the strip by capillary forces. The base membrane is preferably characterized by having good binding capabilities for proteins.

Without being bound by theory, the base membrane enables the flow (migration) of the sample along the strip.

In some further embodiments, the base membrane is selected from porous material, porous membrane, a granular membrane and an absorbent material. In some embodiments, the base membrane is made of porous material. In some embodiments, the base membrane is made of NitrocellulosePolyvinylidene fluoride, (Charge- modified) nylon or Polyethersulfone. In some further embodiments, the base membrane has a width of about 20 to 40 mm. In some embodiments and the base membrane has a width of 25mm.

The base membrane (16) comprises a result zone comprising at least two distinct regions, a first of the at least two regions (22) comprises a member of an affinity couple and the second of the at least two regions (24) comprises a member of an affinity couple which is complementary ^complementary member") to the member conjugated to the particulate material comprised in the reagent pad. The member of an affinity couple in the first region is different from the member of an affinity couple in the second region. It should be noted that the strip includes at least two regions in the results zone on the base membrane which are applied during the manufacture of the strip. The at least two regions are capable of producing a signal in each region depending for example on the presence of a sample on the strip, of the presence of the target molecule in a sample and quality of the assay as detailed below. The detection can be in any form and is not limited to a line shape.

The absence of a signal in the first region of the results zone may also indicate that the sample comprises a target molecule but the concentration of the target molecule in the sample is lower than the detection capability of the assay.

For simplicity, if a signal is present in the first region, it is referred to as the test line (22) and if a signal is present in the second region, it is referred to as the control line (24).

For simplicity, Figure 1A shows only two such distinct regions showing two signals referred to by 22 and 24 but more such regions may be included (to show more signals in lines). As detailed below, the base membrane may include several regions (in the form of lines). In some embodiments, the at least two regions are striped as two parallel lines.

Non-limiting example of the strip includes the following components: the member conjugated to the particulated material comprised in the reagent pad is anti- DIG antibody (particles coated/associated with anti-DIG antibody). Following in this non-limiting example, the base membrane comprises in the second region a member of an affinity couple which is complementary ^complementary member") to the member comprised in the reagent pad, namely, DIG.

In some embodiments, the member or complementary member comprised in the different regions of the base membrane are conjugated to a carrier. In some further embodiments, the carrier is a nucleic acid or protein. In some further embodiments, the carrier further comprise a cross linker.

As used herein the term "crosslinkers" refers to crosslinking reagents which contain two or more reactive ends capable of chemically attaching to specific functional groups (primary amines, sulfhydryls, etc.) on proteins or other molecules.

In some embodiments, the member of an affinity couple may be bound to the recognition agent, particles or the at least two regions of the results zone on the base membrane via a reactive group. Non limiting examples of a reactive group are primary amines (NH ₂ ), Sulfhydryls (SH), Carboxyls (COOH), Carbonyls (-CHO) or any other reactive group which is capable of binding a member of an affinity couple. Generalizing the above non-limiting example, member of an affinity couple is conjugated to a particulated material comprised in the reagent pad and the complementary member of the member of the same affinity couple is comprised in the second region of the at least two regions in the results zone in the base membrane. This region in the base membrane provides a possible signal referred to as a control line.

As described, the base membrane also comprise a first region (22) comprises a member of an affinity couple. This region in the base membrane provides a possible signal referred to as a test line.

At times, the base membrane is treated before use with materials, such as proteins, for example BSA (blocking) to avoid non-specific binding.

The strip further comprises an absorbing pad. In some embodiments, the absorbing pad is composed of cellulose fibers.

Without being bound by theory, the size and porosity of the absorbing pad ensures proper flow of the sample along the strip and may also be used to control the amount of sample that flows within the strip.

In some embodiments, the strip according to the present disclosure is used in an assay based on lateral flow principle. Accordingly, the structure of the strip allows a sample to move along or through the strip via the different components and regions in the path of liquid flow by capillary action.

In some embodiments, the sample is applied onto the sample pad.

In accordance with the assay described herein, the sample is incubated with at least two target recognition agents. In some further embodiments, the sample is incubated with two different target recognition agents before application onto the sample pad of the strip. In some embodiments, the sample is incubated with two different target recognition agents; each recognition agent is connected to (i.e. labeled by) a different member of a different affinity couple.

The target recognition agent used herein may be labeled with a member of an affinity couple during its preparation and provided to the user as a labeled target recognition agent. Alternatively, the target recognition agent may be selected by the user and labeled by the user employing the members of the affinity couples provided in the kit of the invention as described below.

In some other embodiments, the different labeled recognition agents are from the same type of recognition agents, such as aptamers, antibodies or MIP.

In some other embodiments, the different labeled recognition agents are different types of recognition agents. According to some specific embodiments, the first labeled recognition agent is an aptamer, antibody or MIP and the second labeled recognition agent is an antibody. In some further specific embodiments, the first labeled recognition agent is an aptamer, antibody or MIP and the second labeled recognition agent is an aptamer.

In some embodiments, the at least two labeled recognition agents recognize and bind to different regions (epitopes) of the target molecule. In some further embodiments, the at least two labeled recognition agents are the same type of recognition agent but having different structures and are targeted to different epitopes on the target molecule. In some other embodiments, the at least two labeled recognition agents are two antibodies or two aptamers but having different structures and being capable of binding to different regions of the target molecule.

As appreciated, the binding of each of the labeled recognition agents to a target molecule is characterized by a specific binding affinity. The term binding affinity refers to the strength of binding of one molecule to another as known in the art.

In some embodiments, the at least two labeled target recognition agents are provided in liquid form. In some further embodiments, the at least two labeled target recognition agents are provided in a lyophilized form. In some further embodiments, the lyophilized labeled recognition agents are rehydrated with a suitable buffer to form a liquid form.

The tested sample is incubated with at least two target recognition agents each target recognition agent is connected to a member of an affinity couple ("labeled target recognition agents"). Incubation can be inside a reaction tube comprising the at least two labeled target recognition agents or the at least two labeled target recognition agents added at a tube containing the sample.

In some embodiments, each labeled target recognition agent is connected to a different member of a different affinity couple. In some further embodiments, a first of the at least two labeled recognition agents is associated (connected, bound) to a member of an affinity couple and the second of the at least two labeled recognition agents is associated to a member of an affinity couple which is complementary to the member comprised in the reagent pad, wherein the member of an affinity couple associated with the first labeled recognition agent is different from the member of an affinity couple associated to the second labeled recognition agent.

The incubation of the sample with the liquid comprising the at least two labeled target recognition agents connected (associated) to two different members of a different affinity couple is under conditions allowing the binding of the at least two labeled recognition agents to the target molecule and the formation of a complex between the target molecule and the at least two labeled recognition agents. Thus, the conditions of time and temperature may vary depending on the target molecule and the labeled recognition agents and/or the binding affinity between the two.

In some embodiments, incubation may be for 10 min to 1 hour at specified or room temperature, or for about 30 min at room temperature. In some further embodiments, incubation is at a temperature of 4°C. In some further embodiments, incubation is at a temperature of 37°C. In some other embodiments, mixing may be added by manually mixing or instrumental mixing.

The assay according to the present disclosure comprises contacting the sample incubated with the at least two labeled recognition agents with the sample pad region of the strip.

As appreciated, if a target molecule is present in the sample, the at least two labeled target recognition agents are capable of binding to the target molecule, thereby forming a complex of member 1 -target recognition agentl-target molecule- target recognition agent2-member2. The complex is formed by the specific recognition and interactions between the target molecule and the at least two labeled recognition agents.

As used herein the term "complex" denotes an entity comprising more than one molecule which is bound or is in association with at least one other molecule, for example by a hydrogen bonds, adsorption processes, hydrophobic or electrostatic interactions or combinations thereof. Hence the term "member 1 -target recognition agentl-target molecule-target recognition agent2-member2" relates to an association between the labeled recognition agents and the target molecule. The complex may be considered as a "sandwich" like complex, having the target molecule in between the two labeled recognition agents. One labeled recognition agent is connected to a member of an affinity couple and one labeled recognition agent is connected to a different member of a different affinity couple.

In some embodiments, the sample (incubated with at least two labeled target recognition agents) is applied onto the sample pad of the strip.

In some other embodiments, the strip is a dipstick and is dipped into a sample such that the only the sample pad is in contact with the sample.

In some further embodiments, the sample (incubated with at least two labeled target recognition agents) is applied onto the sample pad and flows (migrates) along or through the strip along the flow path of the liquid by capillary forces to the reagent pad. It should be noted that when referring to a sample applied onto the strip, the sample is the pre-incubated sample. Application of the sample is by direct application using any known means in the art.

In some other embodiments, the sample interacts with the particles in the reagent pad through interactions provided by the members of the affinity couple.

In some embodiments, the sample further flows to the base membrane. Depending on the presence or absence of a target molecule in a sample, a signal is detected in the at least two regions in the results zone of the base membrane, for example in the form of line.

The following non-limiting example is provided in order to simplify the different components involved in the strip and assay using the strip:

Each of the two target labeled recognition agents is connected (associated) with a different member of an affinity couple, for example one binding agent is associated with biotin ^associated-recognition agent i") and the second recognition agent is associated with DIG (^'associated-recognition agent 2"). In accordance with the assay described here, the sample comprising a target molecule is pre-incubated with associated-recognition agent 1 and associated-recognition agent 2. For simplicity, it is assumed that the target molecule is present in the sample; therefore a complex between the target molecule and associated-recognition agent 1 and associated-recognition agent 2 is formed in a form of a sandwich-like complex associated-recognition agent 1-target molecule-associated-recognition agent 2 (herein "sandwich like complex").

The pre-incubated sample comprising the sandwich like complex flows downstream the strip along the flow path of the liquid by capillary forces, reaching the reagent pad. The reagent pad comprises in this non-limiting example particles coated with anti-DIG.

Assuming that the sample comprised the target molecules and a sandwich like complex was formed, one possibility is that the particles coated with anti-DIG (from the reagent pad) interact with the sandwich like complex via the DIG-anti DIG interactions forming a new complex between sandwich like complex (associated- recognition agent 1-target molecule-associated-recognition agent 2) and anti-DIG as follows:

biotin-recognition agent 1-target molecule-recognition agent 2-DIG-antiDIG coated particles ( "complex at reagent pad").

As appreciated, the anti-DIG coated particles are dried on the reagent pad in excess relative to possible concentrations of target molecules in a sample; the flow towards the base membrane includes the complex at reagent pad and unbound anti- DIG coated particles.

Alternatively, at least some of the sandwich like complexes (associated- recognition agent 1-target molecule-associated-recognition agent 2) migrate to the base membrane without interacting with the coated particles at the reagent pad together with unbound anti-DIG coated particles.

Hence, the conjugation (association/connection) between the free end of the "sandwich like complex" and the particulated material may take place either at the reagent pad or at the first region of the results zone on the base membrane. As described, the base membrane (16) comprises a results zone comprising at least two distinct regions, a first of the at least two regions (22 - when present corresponds to test line) comprises a member of an affinity couple and the second of the at least two regions (24- when present corresponds to control line) comprises a member of an affinity couple which is complementary (^'complementary member") to the member conjugated to the particulated material comprised in the reagent pad.

The member of an affinity couple in the first region is different from the member of an affinity couple in the second region. The member of an affinity couple in the first region comprise a member of an affinity couple which is complementary ("complementary member") to the member connected to at least one of the labeled target recognition agents.

Further to the non-limiting example described here, the first of the at least two regions in the results zone on the base membrane comprise avidin (test line) and the second region of the at least two regions (control line) comprises DIG.

Therefore, according to some embodiments, the complex may be formed at the reagent pad and referred to as biotin-binding agent 1 -target molecule-associated- DIG-antiDIG coated particles ("complex at reagent pad"), then migrates to the base membrane. The free biotin end of the sample is captured by the avidin in the first region and a signal appearing as a line in the test line and the unbound anti-DIG coated particles are captured by the DIG in the in the second region and a signal appearing as a line in the control line. The presence of particles in the two lines is then detected.

In some other embodiments, the conjugation between the sandwich like complex and the coated particles takes place not on the reagent pad, but in the results zone. This occurs, for example, when anti-DIG coated particles flow through the strip and are captured by the free DIG end of the sandwich like complex which is bound through the free biotin end to the avidin in the first region of the results zone on the base membrane and a signal appears at the test line.

In a different non-limiting example, if the sample does not include a target molecule than after pre-incubation with associated-recognition agent 1 and associated-recognition agent 2 no complex is formed and the sample comprising the associated-recognition agent 1 and associated-recognition agent 2 is flowing to the regent pad. Part of the associated-recognition agent 2, namely associated-DIG binds to anti-DIG coated particles whereas some of the anti-DIG coated particles remain unbound. The liquid sample then migrates to the base membrane, where the unbound anti-DIG coated particles are captured by the DIG comprised in the in the second region and a signal appearing as a line in the control line. A signal is detected due to the presence of the particles in the control line. No signal is detected in the test line as no particles capable of producing a signal were captured at this region.

The interpretation of the results is as follows:

When only the "control Line" is showing it indicates a negative result, namely, no target molecule was present in the test sample.

If two lines are showing, one at the "test Line" and one at the "control Line" it indicates a positive result, namely the target molecule is presence in the tested sample.

If only one line appears at the "test Line" or if no lines appear at all it indicates that the test is not valid (due to some technical failure).

According to some embodiments and as appreciated in the art, the interaction between the recognition agent and the target molecule is specific and may be detected by the appearance of a detectable signal by using a colorimetric sensor, a fluorimetric/lumination sensor or a magnetic sensor. In some embodiments, the signal is due to the presence of colored particles. In some other embodiments, the results in the results zone on the base membrane can be determined by either the naked eye. In some further embodiments, the results in the results zone on the base membrane are determined by employing a designated reader device, depending on the nature of the particles. A device, such as the ESE Quant can be used if for example, fluorescence particles are used.

The strip according to the present disclosure may be used as a part of a device. To this end, the strip is placed within a housing providing a solid casing for the strip.

In accordance with another aspect, depicted in Figure IB, the present disclosure provides a device (40) comprising a test strip according to the invention contained in a housing (42), the housing includes at least one window: a sample window (44) and/or a results window (46).

In accordance with a further aspect, the present disclosure provides a method for detecting the presence of at least one target molecule in a sample comprising contacting the sample with a diagnostic device as described herein.

According to some embodiments, the method for detecting the presence of at least one target molecule in a sample comprises: a. Incubating the sample with at least two labeled target recognition agents; and b. Contacting the incubated sample with a device of the invention; whereby appearance of a signal in both regions in the results zone of the base membrane as apparent in the results window indicates the presence of the target molecule in the sample.

Reverence is now made to Figure IB showing a schematic representation of a diagnostic device in accordance with the present disclosure.

The device (40) comprises a strip provided within a housing (42). The housing includes at least one opening/window having suitable dimensions. The For example, Figure IB shows two windows (44, 46). However, the housing may include one or even more than two such windows. The housing may include the entire strip within or only part of the strip.

In some embodiments, the housing is a plastic cover or plastic cassette with dimensions of about 20 mm wide and 70 mm long, for example. The housing may be transparent or sealed or any combinations thereof. In some further embodiments and as shown in Figure IB, the strip is located within the housing. In some other embodiment, the plane of the housing is parallel to the plane of the strip.

The strip may be placed within the housing by the manufacture or by the user.

In some embodiments, the housing comprises windows (openings) that expose areas of the strip.

In some embodiments, the housing comprises a results window. The results window exposes areas in the strip for visualization. In some embodiments, the results window is aligned with the results zone on the base membrane to allow observation of the results on the strip.

In some embodiments, the housing comprises more than one results window.

As appreciated, there is no difference in the quality of the visualization of the results when the results are visualized on the strip or on the results window of the diagnostic device.

In some further embodiments, the dimensions of the results opening are 3mm wide and 13mm long. In some embodiments, the results window is parallel to the plane of the results pad region of the strip. In some other embodiments, the results window is above the results zone on the base membrane of the strip.

The results opening expose the results in the results zone on the base membrane of the strip. For simplicity, Figure IB shows two lines in two regions (52, 54) being observed in this window. As detailed above, these lines are indicative of the presence of a target molecule in the sample (52 test line) and of the performance of the assay (54 control line).

In some embodiments, the device comprises a sample window.

In some further embodiments, the dimensions of the window opening are 3mm wide and 6mm long.

In some embodiments, the sample window is parallel to the plane of the sample pad region of the strip. In some other embodiments, the sample window is above the sample pad of the strip.

The sample window defines an area suitable for application of a sample to the device. In some embodiments, the sample window allows application of the liquid sample to the sample window.

Without being bound by theory, application of a sample in the sample window enables the sample to get into close contact with the sample pad of the strip and thereby enabling the sample to be absorbed into the sample pad and flow along the strip as described herein. As appreciated, in terms of strip functionality there is no difference in the quality of the results when the sample is applied onto the sample pad directly on the strip or onto the sample window of the diagnostic device.

In some embodiments, the sample may be directly applied onto the sample window, enabling close contact of the sample with the sample pad and the sample is then allowed to flow along the strip via the reagent pad and to the results zone on the base membrane. Detection of a signal in the results window is performed, wherein the signal in both regions of the results zone on the base membrane is indicative of the presence of target molecule in the sample. It should be noted that the sample directly applied onto the sample window is a sample pre-incubated with at least two target labeled recognition agents. As detailed herein above, the absence of a signal in the test line may suggest that the target molecule is not present in the sample or that the concentration of the target molecule in the sample is below the detection limit of the method.

In some embodiments, the method further comprises comparing the signal intensity in the test line shown in the results window to the signal intensity produced on other strips or devices by samples with known amount of the target.

In some embodiments, the sample is applied to the sample window with a dropper or a similar device to the sample window.

In some embodiments, the device comprises at least one additional unit. In some further embodiments, the additional unit is an external unit. As used herein the term "external unit" indicates that the unit is not an integral part of the lateral flow device. The external unit may be connected to the device at different locations.

In some embodiments, the external unit is connected to the sample window.

The term "connected" denotes any physical communication (contact) between the at least part of the external unit and the sample window. In some embodiments, the external unit has a shape that enables the insertion of at least part of the external unit into the sample opening and allowing physical contact with the sample window and the sample pad of the strip. In some embodiments, the external unit has a uniform shape. In some further embodiments, the external unit has different shapes in different locations of the unit. In some embodiments, at least part of the external unit is suitable for contact with the sample window.

In some embodiments, the external unit is attached by the user. In some further embodiments, the external recognition unit is attached by the manufacture.

As detailed herein, the strip itself and the device including it are not target molecule specific but rather form part of a universal device. Therefore, the external recognition unit if present forms the individualization of the measurement and is prepared for each respective target molecule. The external recognition unit is specific to at least one target molecule.

In some embodiments, the external recognition unit comprises at least two labeled target recognition agents each target recognition agent is connected to a member of an affinity couple. In some further embodiments, each target recognition agent is connected to a member of a different affinity couple.

In some embodiments, the external recognition unit is in the form of a cylinder (tube). In some embodiments, at least part of the external unit is in the form of a cylinder. The external recognition unit having a shape of a cylinder has two ends, a first top end and a second bottom end. The first top end is for sample administration and the second bottom end is to be connected (attached) to the sample window and enabling close contact with the sample pad. The external unit may be connected via it bottom second end directly or through a connecting tube to the sample window. The sample opening described exactly fit the second (bottom) end of the external recognition unit.

In some embodiments, the external unit having a cylinder shape (or at least a cylinder shape) includes a valve located at the second end (bottom end). The valve can be opened manually enabling the contents of the unit to flow downwards to the sample pad (via the sample opening). In some further embodiments, the external unit having a cylinder shape (or at least a cylinder shape) includes at the second bottom end a membrane designed to dissolve at a certain rate upon contacting liquid.

Figure 2 shows a non-limiting representative schematic example of the device and the external unit in a shape of a cylinder and a connecting tube fitting to the sample window. As shown in Figure 2, the external unit is to be fitted to the sample window. Figure 2 further show a valve at the lower bottom end. In some embodiments, the external unit comprise the at least two labeled recognition agents in a liquid form (solution). As appreciated, the external recognition unit may also contain additional components such as running buffer with precise amounts of two labeled target specific recognition agents.

In some embodiments, the external recognition unit contains at least two labeled target specific recognition agents in a lyophilized form.

The diagnostic device may be operated in different modes described in the Examples. The method also referred to as to as "Two step, all in one".

In some embodiments, a measured amount of the sample to be tested is applied to the external recognition unit through the opening in its top (first end) and mixed well. In some embodiments, if the external unit contains a lyophilized form of the target binding agents a buffer is added.

In some embodiments, the sample is incubated in the external unit. Conditions of the incubation of the sample with the external unit comprising the at least two labeled target recognition agents each labeled target recognition agent is connected to a member of an affinity couple, may vary and as detailed above, depend on the association (interaction/binding) of the target molecule within the sample and the at least two labeled target recognition agents.

In some embodiments, the external unit includes a valve. The valve at the bottom end (second end) is then opened, allowing the liquid of the external recognition unit to come in contact with the sample pad of the universal strip below, at the sample window of the device. If the target is present in the sample, a complex is formed between the target and the two labeled recognition elements. This complex migrates along the flow path of the strip and the results are formed and interpreted as described above.

In some embodiments, the external recognition unit comprises a membrane at its second bottom end, the liquid from the recognition unit flows through the membrane and comes in contact with the sample pad of the universal strip at the sample window.

Without being bound by theory, the use of an external unit having a valve or a membrane at the second bottom end enables to control the incubation time of the sample with the at least two target molecules. Thus, by increasing the incubation time, lower amounts of target molecules in a sample can be detected.

In some further embodiments, the external unit has a shape of the sample window in the housing. In accordance with some further embodiments, the external unit comprises or is a pad. The pad is made of any porous absorbent material. In some embodiments, the pad is made a glass fiber or cellulose fibers.

Figure 3 shows a non-limiting representative schematic example of the device and the external unit that is a pad. The pad has a form fitting to the shape of the sample window. In some specific embodiments, the strip may be constructed without a sample pad. In some further specific embodiments, the external sample pad is placed in the device and serves as a sample pad of the strip.

In some embodiments, the external recognition pad is soaked with precise amounts of at least two labeled target specific recognition agents. The recognition agents are associated with members of an affinity couple.

The diagnostic device may be operated in different modes described in the Examples. The methods are also referred to as to as "One step, all in one ".

In some embodiments, the external recognition pad is fitted into the sample window either by the manufacturer or the user, according to the nature of the test. The external pad is placed such that it has a direct contact with the sample pad of the strip or in the absence of a sample pad it has a direct contact with the reagent pad.

In some embodiments, the method of using the pad comprises fitting the pad into the sample window and applying the tested sample to the pad. In some other embodiments, the tested sample is diluted in the running buffer before being applied onto the pad.

The liquid of the sample rehydrates the labeled recognition agents and if the target is present in the sample a complex is formed between the target and the two labeled recognition agents. This complex migrates along the flow path of the strip and the results are formed and interpreted as described herein. In some further embodiments, appearance of a signal in both regions in the results zone on the base membrane as apparent in the results window indicates the presence of the target molecule in the sample.

Without being bound by theory, the use of an external unit in a form of a pad does not enable control of the incubation time of the sample with the at least two labeled recognition agents. Since the sample is provided in its natural form (with no previous incubation) onto the pad in the sample window, the time of incubation is determined by the time of the flow.

It should be noted that the housing of the diagnostic device comprises additional windows for any modification of the device. In some embodiments, one target recognition agent is connected (associated/labeled) with biotin and the other target recognition agent is connected (associated/labeled) with DIG or FITC.

The strip and the device are not limited for the detection of only one target molecule. The number of the target molecules can vary for example being 2, 3, 4, 5 or more, allowing the performance of simultaneous different assays on the same strip. In some embodiments, the strip may be prepared such that more than one test line is present in the strip, whereby each of the test lines is composed of a member from a different member of an affinity couple. Non limiting examples for the multiple lines include: biotin/biotin binding proteins; polypeptide/related antibody; ligand/receptor; complementary oligonucleotide pairs etc.

In some embodiments, the external unit contains a mixture of labeled target specific recognition agents, one labeled with a common label, such as biotin, and the other with a differentiating binding partner, such as DIG, FITC, lectin, oligonucleotide etc., whereby its complementary member is fixed at one of the test lines.

As appreciated, the strip and/or the device described herein may form part of a kit to be used in the detection of the presence or absence of a target molecule in a sample.

As described herein, one of the significant advantages of the strip and the device of the invention is that they are not target specific. Namely, they may be used by the end user to test any desired molecule.

Accordingly, in certain embodiments the kit of the invention comprises the strip and/or the device and different members of an affinity couple that can be employed by the end user to label any desired target recognition agent.

In other embodiments the kit of the invention comprises the strip and/or the device and at least two target recognition agents labeled with different members of an affinity couple. In such embodiments, the user selects the kit according to the desired target molecule.

This provides flexibility in using the kits according to the invention. Thus, the present disclosure provides in accordance with a further aspect, a kit comprising at least two labeled recognition agents, each labeled recognition agent being attached to a member of an affinity couple, and a test strip or device as described herein. In some embodiments, the at least two labeled recognition agents, each labeled recognition agent attached to a member of an affinity couple, are in a solution. In some further embodiments, the at least two labeled recognition agents, each labeled recognition agent attached to a member of an affinity couple, are lyophilized. The kit further comprises a buffer.

In some embodiments, the kit comprises a strip or a device as described here and at least two members of an affinity couple. In some further embodiments, the kit comprises instructions to connect each of the at least two members with a recognition agent (forming labeled recognition agent). In some embodiments, the at least two members of an affinity couple are in a solution. In some embodiments, the at least two member of an affinity couple are lyophilized. The kit further comprises a buffer.

In some further embodiments, a kit comprises at least two labeled recognition agents, each labeled recognition agent attached to a member of an affinity couple, and a device as described herein.

In some embodiments, the kit comprises an external unit.

In some embodiments, the external unit has a cylinder (tube), having a valve or a membrane at one end. The external unit in the cylinder shape comprises the at least two labeled recognition agents in a solution or in a lyophilized form.

In some embodiments, the external unit is in a form of a pad. The pad comprises the at least two labeled recognition agents dried onto.

In some embodiments, the kit comprises buffers and solutions.

In some embodiments, the kit comprises references to determine the amount of the target molecule in the sample and optionally a calibration curve, wherein the calibration curve being specific to the target molecule.

In some other aspects, there is provided the use of the strip and/or the device in accordance with the invention for diagnosis of a condition in a subject. In yet some a further aspects, there is provided the use of the strip and/or the device in accordance with the invention for monitoring the efficiency of a therapeutic regimen in a subject suffering from a condition.

Is some other aspects, there is provided the use of the strip and/or the device in accordance with the invention for research purposes. Non limiting examples include laboratory use, scientific experiments and the like.

In some other aspect, there is provided a method for diagnosis of a condition in a subject or monitoring the efficiency of a therapeutic regimen in a subject suffering from a condition, comprising using an assay or a method in accordance to the invention, wherein the presence or the absence of the target molecule is associated with the condition and wherein the presence of the target molecule in sample is indicative of the level of the condition and thereby of the efficiency of the therapeutic regimen in the subject.

In some embodiments, a sample is obtained from a subject and the sample is subjected to an assay or a method according to the invention. The sample is suspected to comprise the target molecule. In some embodiments, the presence of the target molecule in the sample is indicative of the presence or the absence of a condition in the subject. In some further embodiments, the absence of the target molecule in the sample is indicative of the presence or the absence of a condition in the subject.

As used herein, "disease", "disorder", "condition" and the like, as they relate to a subject's health, are used interchangeably and have meanings ascribed to each and all of such terms.

In some embodiments, the condition is a pathological condition.

The "pathological condition" according to the present invention may be selected from but not limited to cancer, inflammation, blood coagulation disorders, and autoimmunity. Accordingly, the method of the invention may be used in the detection of known cancer markers, markers of inflammation, such as Procalcitonin which is a known marker for sepsis, peptides such as penicillin-binding protein 2 (PBP2), kinesin spindle protein (KSP), toxins and allergens.

In some other embodiments, the pathological condition is a viral, bacterial or fungal infection. Non-limiting examples of viral infection comprises Hepatitis B virus (HBV), hepatitis C virus (HCV), Cytomegalovirus (CMV), Human immunodeficiency virus (HIV), Epstein-Barr virus (EBV), HERPES virus, Polio virus, and influenza virus.

Non-limiting examples of bacterial infection comprises Listeria, Diphtheria, E.coli, Group B streptococcus (GBS), Group A streptococcus, Tuberculosis (TB), Salmonella, Vibrio Cholerae, Campylobacter, Brucellosis, meningococcus, Streptococcus pneumonia and Candida.

In some embodiments, the pathological condition is cancer. Cancer is interchangeably used with the terms malignancy, tumor and is referred to herein as a class of diseases in which a group of cells display uncontrolled growth and invasion that may destroy adjacent tissues, and sometimes leads to metastasis (spreading to other locations in the body). Cancer may be a solid cancer or a non-solid cancer and may be classified as carcinoma, sarcoma, lymphoma, leukemia, germ cell tumor, or blastoma.

In some further embodiments, the pathological condition is an autoimmune disease. As appreciated in the art, autoimmune diseases arise from an overactive immune response of the body against substances and tissues normally present in the body. Non limiting examples of autoimmune disease are Multiple sclerosis, Arthritis, Autoimmune hepatitis, Crohn's disease, Diabetes mellitus, type 1, Inflammatory bowel disease, Multiple sclerosis, Psoriasis, Rheumatoid arthritis, Wegener's granulomatosis.

Using the detection methods of the present invention the level of target molecules indicative of the pathological state may be determined. Therefore, the measurement of the levels of these target molecules can serve to diagnose the pathological condition, to monitor disease progression and to monitor efficacy of a therapeutic regiment, i.e. monitor the response of the subject to treatment.

In some further embodiments, the condition is a non-pathological condition.

Non-limiting non-pathological conditions comprise pregnancy.

As used herein the term "about" refers to ± 10 %.

The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to". This term encompasses the terms "consisting of" and "consisting essentially of". The phrase "consisting essentially of" means that the composition or method may include additional ingredients and/or steps, but only if the additional ingredients and/or steps do not materially alter the basic and novel characteristics of the claimed composition or method. Throughout this specification and the Examples and claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

The term "about" as used herein indicates values that may deviate up to 1 percent, more specifically 5 percent, more specifically 10 percent, more specifically 15 percent, and in some cases up to 20 percent higher or lower than the value referred to, the deviation range including integer values, and, if applicable, non-integer values as well, constituting a continuous range.

It must be noted that, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise.

NON-LIMITING EXAMPLES:

Example 1: Construction and operation of the Universal Lateral Flow strip, a device comprising the strip and kits comprising the strip and the device

The strip was prepared by using a backing card made of PVC/PS with pressure- sensitive adhesive SM31-25, (Kin Bio Tech), onto which the following components were consecutively attached to the pressure-sensitive adhesive:

(a) a 25 mm wide nitrocellulose (NC) membrane (Laminated - HiFlow Plus HF240, 2.5cm, Cat. No. HF24004S25, Millipore) onto which two different detection elements, diluted in Phosphate buffer 0.1 M pH 7.8, 0.2 % sucrose, were striped in the form of two parallel lines, ("base membrane")

The detection elements were as follows: Mouse anti-DIG antibodies or mouse anti-FITC antibodies were dispensed onto the nitrocellulose membrane at the first line ("test line") and BSA-biotin conjugate was dispensed onto the nitrocellulose membrane at the second line ("control line").T e striped NC membrane was dried at 37 °C for 30 minutes in order to fix the reactive compounds to the membrane and

(b) a 10 mm pad made of glass fiber (Cat. No. SB06 Kin Bio Tech) ("Reagent pad") onto which polystyrene particles coated with avidin were dried.

(c) a 17 mm wide pad prepared from cotton fiber Grade 237 (Anlstrom) ("Sample pad")

(d) a 17 mm wide absorbent pad, made of cellulose fibers (absorbent pad Cat. No. CH37K, Kin Bio Tech)

Assembly: As a first step, lateral flow cards were prepared in the following order, whereby each of the elements (2)-(5) is placed by the following order, slightly overlaps the adjacent elements in order to ensure the lateral flow on top of the backing card (1):

1. Backing card

2. Double striped NC membrane (base membrane)

3. Reagent pad

4. Sample pad

5. Absorbent pad.

The cards were cut into 60 mm long 4 mm wide strips by a guillotine cutter resulting in multiple strips as depicted in Figure 1A.

Each strip was inserted into a plastic cassette (Cat. No KB 1470, Kin Bio Tech) that has openings for sample loading fitting to the sample pad of the strip ("sample window") and for result observations fitting to the results zone on the base membrane of the strip ("results window"), such as shown in Figure IB. The results window of the device was designed to enable detection of the information in the results zone on the base membrane of the strip. The strip is inserted such that it is parallel to the plane of the plastic cassette. The plastic cassettes comprising the strips (herein "devices") were kept desiccated at room temperature until use.

A kit comprising the strip or the device also comprises a specific detection unit prepared for each target molecule.

The specific detection unit of the kit comprises a tube (container) containing running buffer with precise amounts of two target specific recognitions agents, each recognition agent bound to a detection element (labeled recognition agents). One target recognition agent is bound to a detection element that is labeled with biotin and the other target binding agent is bound to DIG or FITC.

Alternatively, the specific detection unit of the kit is comprised from a tube (container) containing precise amounts of two labeled target specific recognition agents in a lyophilized form, each recognition agent bound to a detection element. One target recognition agent is bound to a detection element that is (labeled with) biotin and the other target recognition agent is bound to a DIG or FITC molecule.

Alternatively, the kit comprising the strip or the device further comprises two tubes each containing a specific detection element, one being biotin and the other being DIG or FITC. The kit comprises instructions how to connect (label) each of the two recognition elements to one of specific target recognition agents.

The kit also includes instructions how to use the kit.

For the operation of the assay, a measured amount of the tested sample is added to the detection unit tube, mixed well and 60-80 μΐ of the mixture are applied to the sample pad of the universal strip or to the sample opening in the device.

The results are analyzed by visual inspection of the results zone on the base membrane in the strip or the results opening in the device, as follows: the appearance of only one line at the control line indicates a negative result, while the appearance of two lines, at the test line as well as the control line indicates a positive result for the presence of the target molecule in the tested sample.

Example 2: Construction and operation of a "Two step, all in one" universal assay device and kit.

This example provides a modification of the device describe in Example 1. An external recognition unit having a shape of a cylinder and having two ends, a first top end for sample administration and a second bottom end attached through a connecting tube to an opening in the plastic cassette of the device located above (and perpendicular to the plane defined by the strip and/or the device) the sample pad of the universal strip (Figure 2).

Optionally the connecting tube is part of the cylinder.

The sample opening described in Example 1, is adopted to exactly fit the second (bottom) end of the external recognition unit.

The external unit may include a valve located on its second end (bottom end). The valve can be opened manually enabling the contents of the unit to flow downwards to the sample pad (via the sample opening).

Alternatively, the external unit may be separated from the sample pad at the sample opening by a membrane designed to dissolve at a certain rate upon contacting liquid.

The external recognition unit is specific for each target molecule and comprises the respective labeled target specific recognition agents.

The respective external recognition unit is attached by the user according to the specific target to be detected. Alternatively, the external recognition unit can be attached by the manufacture.

The external recognition unit contains running buffer with precise amounts of two target specific recognition agents, one labeled with biotin and the other with DIG or FITC.

Alternatively, the external recognition unit contains precise amounts of two target specific binding agents, one labeled with biotin and the other with DIG or FITC in a lyophilized form.

For the operation of the device, a measured amount of the sample to be tested is applied to the external recognition unit through the opening in its top and mixed well.

If the external unit contains a lyophilized form of the target binding agents, optionally, a buffer is added.

The valve at the bottom is then opened, allowing the liquid of the external recognition unit to come in contact with the sample pad of the universal strip below, at the sample opening of the device. If the target is present in the sample, a complex is formed between the target and the two labeled recognition elements. This complex migrates along the flow path of the strip and the results are formed and interpreted as described in Example 1 above.

Alternatively, if the external recognition unit comprises a membrane at its second bottom end, the liquid from the recognition unit flows through the membrane and comes in contact with the sample pad of the universal strip at the sample opening.

The kit according to this Example comprises an external recognition unit as described above and the universal lateral flow strip inside a plastic cassette (the device).

Example 3: Construction and operation of a "One step, All in one," universal assay device and kit

This example provides a modification of the device described in Example 1. A glass fiber or cellulose fiber external recognition pad having the shape of the opening in the plastic cassette (sample opening) being above the sample pad and perpendicular to the plane of the device of the strip is used (Figure 3). The external recognition pad is soaked with precise amounts of two target specific recognition agents, one labeled with bio tin and the other with DIG or FITC, diluted in drying buffer (HEPES buffer 25 mM, 1% BSA, 0.05% Tween-20, 5 % sucrose, 1 %.Trehalose and 0.1 mg/ml NaN ₃ ) and dried at 37 °C for 30 minutes.

The external recognition pad can be fitted easily into the opening in the plastic cassette located above the sample pad of the universal strip (the sample opening in the device of Example 1) and brought in direct contact with the sample pad of the strip.

This recognition pad is not an integral part of the lateral flow device and a separate recognition pad is prepared for each respective target.

For the operation of the test, the recognition pad is fitted into the device at the sample opening, either by the manufacturer or the user, according to the nature of the test. The tested sample is either applied directly or diluted in the running buffer and applied to the recognition pad. The liquid of the sample rehydrates the labeled recognition agents and if the target is present in the sample a complex is formed between the target and the two labeled recognition agents. This complex migrates along the flow path of the strip and the results are formed and interpreted as described in Example 1 above.

The kit of this example comprises a dry recognition pad, running buffer and the universal lateral flow strip inside a plastic cassette (the device).

Example 4: Construction and operation of a Multi assays universal device.

In order to enable detection of more than one target molecule in a sample, the universal strip may be prepared such that more than one test line is present in the strip, whereby each of the test lines is composed of a member from a different target specific binding pair.

The multiple lines may include: biotin/Avidin; polypeptide/related antibody; ligand/receptor; complementary oligonucleotide pairs etc. The number of the target molecules can vary for example being 2, 3, 4, 5 or more, allowing the performance of simultaneous different assays on the same strip.

The recognition tube in this case will contain a mixture of target specific recognition agents, one labeled with a common label, such as biotin, and the others with a differentiating binding partner, such as DIG, FITC, lectin, oligonucleotide etc., whereby its member of an affinity couple is fixed at one of the test lines.

For the operation of the test, a measured amount of the sample to be tested is applied to the recognition tube and mixed well. If one of the targets in question is present in the sample it forms a complex with both labeled, target specific recognition elements. 60-80 μΐ from this mixture is then applied to the sample pad of the strip and the liquid flows along the flow path of the strip. If the sample contains the respective target molecule(s) the labeled target- recognition elements complex or complexes bind to the respective binding partner at the respective test line, where a line then appears. The presence of a target in the sample is determined by appearance of a visual result line at the respective test line.

Example 5: Validation of the assay using labeled aptamers

The performance of the strip was tested using recombinant PDGF-BB that was added to a sample at a known concentration and incubated in the presence of two PDGF specific aptamers, one of the aptamers was conjugated to biotin and the second to DIG. Materials:

• Recombinant platelet-derived growth factor B-chain homodimer (PDGF-BB) was obtained from Sigma.

• The aptamers for PDGF-BB were designed as follows

PDGF-BB-2 ( AptaA): (SEQ ID NO:l)

biotin-5'-GCGATACTCCACAGGCTACGGCACGTAGAGCATCACCATGATCCTG-3'

PDGF-BB-1 ( AptaB): (SEQ ID NO:2)

DIG-5 -GCAGTTACTCAGGGCACTTGCAAGCAATTGTGGTCCCAATGGGCTGAGTAT-3'

• Running buffer - PBS, 0.4% BSA, 0.05 % Tween- 20, 0.1 mg/ml NaN ₃

• Polystyrene particles coated with avidin, 0.01 % solids suspension in running buffer.

• Universal strips striped with anti-DIG antibodies at the test line and BSA- biotin at the control line and cut at the bottom of the NC membrane, in order to allow wet run.

Methods:

The samples were prepared in running buffer and contained recombinant PDGF-BB (10 pg), the two specific PDGF aptamers and relevant controls, according to the following Table 1

Table 1 - The different PDGF-BB samples that were tested with the universal strip

No. of Sample AptaA AptaB PDGFBB comments

1 + + +

2 + +

3 + + -

4 + + + half of the amount of aptamers

5 + + - In the presence of non-relevant control protein

6 + + - Buffer control The samples were incubated at room temperature for 30 minutes. After the incubation 45 μΐ running buffer were added for a final volume of 100 μΐ and distributed into micro-tubes, 50 μΐ per tube.

The strips were inserted into each sample tube and the liquid advance was noted. Once the liquid reached the absorbent pad the strip was transferred into a micro-tube containing the micro-particles suspension and the results were read after about 10-15 minutes from insertion of the strips into the micro-particles suspension.

Results:

Figure 4 shows the results obtained with the six tested samples listed in Table 1. The interpretation of the results is as follows:

When only the "Control Line" is apparent it indicates a negative result, namely, no analyte was present in the test sample.

If two lines are apparent, one at the "Test Line" and one at the "Control Line" it indicates a positive result, namely the target analyte is present in the tested sample.

If only one line appears at the "Test Line" or if no lines appear at all it indicates that the test is not valid (due to some technical failure).

The results can be determined by either the naked eye (as shown clearly in Figure 4) or can be read using a designated reader device, depending on the nature of the detection moiety.

The results, which are shown in Figure 4A clearly demonstrate that the system is capable of detecting the presence of PDGF-BB in a sample, as indicated by the signal appearing at the test line.

When lower aptamer concentrations were present in the sample (see Figure 4D), the intensity was weaker than that obtained in the presence of higher aptamer amounts.

The accuracy of the assay was tested using several negative controls:

Figures 4B and 4C show results obtained by incubating a sample containing PDGF- BB with only one PDGF specific aptamer (either conjugated to biotin (sample 2) or to DIG (sample 3). As can be seen, when the sample of PDGF-BB was incubated in the presence of only one aptamer no signal was detected in the test line, indicating that the assay requires the presence of two target specific aptamers.

The results also show that the assay is based on the specific recognition between the aptamers and their specific target and no nonspecific binding occurs as shown in the results obtained in sample 6 that does not contain any protein and no signal was detected in the test line. The specificity of the assay is further emphasized when the sample was incubated with the two PDGF-BB specific aptamers and with a non- relevant control protein instead of PDGF-BB. No signal was detected in the test line, indicating that the test is specific only for detection of PDGF-BB when specific PDGF-BB aptamers are used.

In all the six tested samples, a clear signal was detected in the control line, indicating that the assay was correctly performed.

Example 6: Validation of the assay using labeled antibodies

Materials:

• Human IgG (Sigma, I 2511).

• Goat anti-human IgG, polyclonal antibodies (Jackson, 109-006-088) biotinilated using Biotin-SE (Thermo Scientific, 21435), according to the manufacturer instructions.

• Goat anti Human IgG, conjugated to FITC (Jackson, 109-095-088)

• Running buffer - PBS, 0.4% BSA, 0.05 % Tween- 20, 0.1 mg/ml NaN3

• Universal strips striped with mouse anti-FITC antibodies (Jackson, 200-002- 037) at the test line and BSA-biotin at the control line and comprising avidin coated blue polystyrene particles at the reagent pad.

• Universal strips striped with mouse anti-FITC antibodies (Jackson, 200-002- 037) at the test line and BSA-biotin at the control line and comprising Streptavidin coated gold particles (Arista Biologicals) at the reagent pad.

Methods:

Sample preparation: Positive samples- 25μ1 Goat anti Human IgG conjugated to FITC (5μg/ml) were mixed with 25μ1 biotinilated-Goat anti-human IgG (lC^g/ml) and 25μ1 of human IgG at various concentrations (1 μg/μl, 100 or 10 ng/ml).

Negative samples- same as positive samples but without any human IgG.

Experimental procedure:

50μ1 from the sample was applied on the sample pad of a universal strip and allowed to flow. When liquid was absorbed, the remaining sample was slowly added until absorbed (a total of 75μ1 sample). The results were determined visually 10-15 minutes after the sample application.

Results:

The results, depicted in Figure 5, clearly demonstrate the ability of the assay and the kit, based on labeled anti-human antibodies and the universal strip, to effectively detect the presence of human IgG antibodies in a sample. IgG amounts as low as 2.5 ng were detected by the system as can be seen by the appearance of a line at the Test line (strips No. 3). This experiment also demonstrates the flexibility of the system, when strips containing two different kinds of particles (polystyrene coated with avidin - A or gold coated with streptavidin - B) both successfully detected the presence of hlgG in the sample.

Detection of various human IgG (hlgG) concentrations on universal strips with polystyrene particles coated with avidin are shown in Figure 5A, while universal strips with gold particles coated with streptavidin are shown in Figure 5B. 1) No hlgG (only buffer) 2) hlgG 1 μg/μl 3) hlgG 100 ng/ml 4) hlgG 10 ng/ml.

Taken together, these results show that the strip of the invention can be used for detection of a variety of target molecules, since the strip itself does not include any target specific components. This clearly provides an advantage for diagnosis and detection of target molecules.