WO2013105090A1 - Dispositif à bandelette d'écoulement latéral versatile - Google Patents

Dispositif à bandelette d'écoulement latéral versatile Download PDF

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Publication number
WO2013105090A1
WO2013105090A1 PCT/IL2013/050023 IL2013050023W WO2013105090A1 WO 2013105090 A1 WO2013105090 A1 WO 2013105090A1 IL 2013050023 W IL2013050023 W IL 2013050023W WO 2013105090 A1 WO2013105090 A1 WO 2013105090A1
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WO
WIPO (PCT)
Prior art keywords
sample
strip
labeled
recognition
target
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PCT/IL2013/050023
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English (en)
Inventor
Ofer Nussbaum
Rafhael LEVI
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Aptateck Bio Ltd.
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Application filed by Aptateck Bio Ltd. filed Critical Aptateck Bio Ltd.
Publication of WO2013105090A1 publication Critical patent/WO2013105090A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow

Definitions

  • This invention relates to diagnostic devices, methods and kits.
  • the present invention provides novel devices and techniques for flexible detection of target molecules based on a universal lateral flow device.
  • LFIA Lateral Flow Immunochromatographic Assays
  • 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.
  • 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.
  • 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.
  • the present invention provides an assay for the detection of at least one target molecule in a sample, comprising:
  • each target recognition agent is connected to a member of an affinity couple
  • reagent pad comprises at least one member of an affinity couple
  • 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;
  • 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.
  • 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.
  • 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.
  • 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.
  • PDFG Platelet-Derived Growth Factor
  • 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 ⁇ g/ ⁇ l
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the strip and the device described here are suitable for detecting different amounts of target molecule.
  • 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.
  • the present invention provides an assay for the detection of at least one target molecule in a sample comprising:
  • each target recognition agent is connected to a member of an affinity couple
  • 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;
  • 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.
  • the strip is used to determine the absence or presence of a target molecule in a sample suspected to include the target molecule.
  • the term "strip” denotes a narrow, elongated piece of porous or fibrous material capable of absorbing and promoting liquid flow.
  • sample 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.
  • 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.
  • the sample is a liquid sample.
  • the liquid sample is liquid in its natural state.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • the population of cells comprises cancer cells. In another embodiment the population of cells is an in vitro cultured cell population.
  • 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.
  • target molecule denotes a molecule which may be found in a tested sample and which is capable of binding to a recognition agent.
  • 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.
  • the term "antigen" refers to a target molecule capable of binding to a recognition agent.
  • 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.
  • the antigen is a micro-organism associated antigen.
  • micro-organism associated antigen is to be understood as a protein or fragment thereof encoded by the viral, bacterial or fungal genome.
  • the antigen is a cancer (or tumor) marker.
  • 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.
  • recognition agent and “target recognition agent” are used interchangeably and denote any molecule capable of specifically recognizing the target molecule.
  • recognition agent also encompasses a binding agent.
  • binding agent refers to any molecule capable of specifically binding to the target molecule.
  • the recognition agent is an aptamer, an antibody (including any antigen binding portion thereof) or a molecular imprinted polymer.
  • the binding agent is an aptamer or an antibody.
  • 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 .
  • 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 .
  • aptamers are produced by chemical synthesis and purified to a very high degree.
  • 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.
  • a reactive group examples include primary amines (NH 2 ), thiol (SH), carboxy group (COOH), phosphates (P0 4 ), Tosyl, and a photo- reactive group.
  • 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.
  • 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.
  • the term antibody also refers to antigen binding portions thereof.
  • antigen-binding portion of an antibody 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.
  • 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.
  • the recognition agents are modified, possibly but not only for enhancing the stability, affinity and specificity of the molecules.
  • 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”.
  • affinity couple 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.
  • DIG Digoxigenin
  • FITC Fluorescein isothiocyanate
  • a member of an affinity couple 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.
  • 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).
  • the recognition agents may be biotinylated. In accordance with some other embodiments, the recognition agents may be attached to a fluorescent moiety.
  • conjugation of the recognition agent and the member of an affinity couple may be by covalent bonding.
  • a strip 10 there is illustrated a strip 10.
  • the strip in accordance with the present disclosure may be prepared in different sizes.
  • 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
  • each two adjacent regions partially overlap.
  • 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.
  • 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.
  • the backing card is made from Polyvinyl chloride (PVC), a combination of PVC/Polystyrene (PS) or polyester.
  • PVC Polyvinyl chloride
  • PS Polystyrene
  • pressure-sensitive adhesive is coating the surface of the backing card, covered by a release liner.
  • 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.
  • sample pads There are two types of materials that are commonly used as sample pads: woven meshes and cellulose filters.
  • the sample pad is made of a paper or other cellulosic materials.
  • the sample pad is made of cotton fiber material, cellulose fiber, glass fiber or combinations thereof.
  • 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.
  • the reagent pad is made of glass fibers.
  • the reagent pad comprises particulated material conjugated to at least one member of an affinity couple.
  • 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.
  • 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.
  • 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.
  • the particles are colored particles.
  • the particles are polystyrene particles. In some further embodiments, the particles are gold particles.
  • 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.
  • 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.
  • 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.
  • 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.
  • the base membrane enables the flow (migration) of the sample along the strip.
  • 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.
  • 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.
  • test line (22) 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).
  • 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).
  • 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).
  • 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.
  • the member or complementary member comprised in the different regions of the base membrane are conjugated to a carrier.
  • the carrier is a nucleic acid or protein.
  • the carrier further comprise a cross linker.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the base membrane is treated before use with materials, such as proteins, for example BSA (blocking) to avoid non-specific binding.
  • materials such as proteins, for example BSA (blocking) to avoid non-specific binding.
  • the strip further comprises an absorbing pad.
  • the absorbing pad is composed of cellulose fibers.
  • 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.
  • 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.
  • the sample is applied onto the sample pad.
  • the sample is incubated with at least two target recognition agents.
  • the sample is incubated with two different target recognition agents before application onto the sample pad of the strip.
  • 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.
  • 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.
  • the different labeled recognition agents are from the same type of recognition agents, such as aptamers, antibodies or MIP.
  • the different labeled recognition agents are different types of recognition agents.
  • the first labeled recognition agent is an aptamer, antibody or MIP and the second labeled recognition agent is an antibody.
  • the first labeled recognition agent is an aptamer, antibody or MIP and the second labeled recognition agent is an aptamer.
  • the at least two labeled recognition agents recognize and bind to different regions (epitopes) of the target molecule.
  • 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.
  • 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.
  • binding affinity refers to the strength of binding of one molecule to another as known in the art.
  • 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.
  • each labeled target recognition agent is connected to a different member of a different affinity couple.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the sample (incubated with at least two labeled target recognition agents) is applied onto the sample pad of the strip.
  • the strip is a dipstick and is dipped into a sample such that the only the sample pad is in contact with the sample.
  • 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.
  • the sample interacts with the particles in the reagent pad through interactions provided by the members of the affinity couple.
  • the sample further flows to the base membrane.
  • 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.
  • 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").
  • one binding agent is associated with biotin ⁇ associated-recognition agent i"
  • the second recognition agent is associated with DIG ( ⁇ 'associated-recognition agent 2").
  • the sample comprising a target molecule is pre-incubated with associated-recognition agent 1 and associated-recognition agent 2.
  • 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.
  • 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.
  • the sandwich like complexes migrate to the base membrane without interacting with the coated particles at the reagent pad together with unbound anti-DIG coated particles.
  • 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.
  • 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.
  • 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.
  • 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.
  • biotin-binding agent 1 -target molecule-associated- DIG-antiDIG coated particles 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.
  • 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.
  • 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 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.
  • test Line 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 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.
  • the signal is due to the presence of colored particles.
  • the results in the results zone on the base membrane can be determined by either the naked eye.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • the strip is located within the housing.
  • 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.
  • the housing comprises windows (openings) that expose areas of the strip.
  • the housing comprises a results window.
  • the results window exposes areas in the strip for visualization.
  • the results window is aligned with the results zone on the base membrane to allow observation of the results on the strip.
  • the housing comprises more than one results window.
  • the dimensions of the results opening are 3mm wide and 13mm long.
  • 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.
  • 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).
  • the device comprises a sample window.
  • the dimensions of the window opening are 3mm wide and 6mm long.
  • 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.
  • the sample window allows application of the liquid sample to the sample window.
  • 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.
  • 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.
  • 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.
  • the sample is applied to the sample window with a dropper or a similar device to the sample window.
  • the device comprises at least one additional unit.
  • the additional unit is an external unit.
  • 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.
  • 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.
  • 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.
  • the external unit has a uniform shape.
  • the external unit has different shapes in different locations of the unit.
  • at least part of the external unit is suitable for contact with the sample window.
  • the external unit is attached by the user. In some further embodiments, the external recognition unit is attached by the manufacture.
  • 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.
  • 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.
  • 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.
  • the external unit having 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).
  • the external unit having 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.
  • the external unit is to be fitted to the sample window.
  • Figure 2 further show a valve at the lower bottom end.
  • the external unit comprise the at least two labeled recognition agents in a liquid form (solution).
  • the external recognition unit may also contain additional components such as running buffer with precise amounts of two labeled target specific recognition agents.
  • 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".
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the external unit has a shape of the sample window in the housing.
  • the external unit comprises or is a pad.
  • the pad is made of any porous absorbent material.
  • 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.
  • the strip may be constructed without a sample pad.
  • the external sample pad is placed in the device and serves as a sample pad of the strip.
  • 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 ".
  • 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.
  • the method of using the pad comprises fitting the pad into the sample window and applying the tested sample to the pad.
  • 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.
  • 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.
  • 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.
  • the housing of the diagnostic device comprises additional windows for any modification of the device.
  • 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.
  • 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.
  • 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.
  • a common label such as biotin
  • a differentiating binding partner such as DIG, FITC, lectin, oligonucleotide etc.
  • 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.
  • 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.
  • 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.
  • 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.
  • the user selects the kit according to the desired target molecule.
  • kits 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.
  • the at least two labeled recognition agents, each labeled recognition agent attached to a member of an affinity couple are in a solution.
  • 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.
  • the kit comprises a strip or a device as described here and at least two members of an affinity couple.
  • the kit comprises instructions to connect each of the at least two members with a recognition agent (forming labeled recognition agent).
  • the at least two members of an affinity couple are in a solution.
  • the at least two member of an affinity couple are lyophilized.
  • the kit further comprises a buffer.
  • 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.
  • the kit comprises an external unit.
  • 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.
  • the external unit is in a form of a pad.
  • the pad comprises the at least two labeled recognition agents dried onto.
  • the kit comprises buffers and solutions.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the presence of the target molecule in the sample is indicative of the presence or the absence of a condition in the subject.
  • the absence of the target molecule in the sample is indicative of the presence or the absence of a condition in the subject.
  • disease 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.
  • 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.
  • PBP2 penicillin-binding protein 2
  • KSP kinesin spindle protein
  • the pathological condition is a viral, bacterial or fungal infection.
  • 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.
  • 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.
  • the pathological condition is an autoimmune disease.
  • 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.
  • 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.
  • condition is a non-pathological condition.
  • Non-limiting non-pathological conditions comprise pregnancy.
  • compositions comprising, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”. This term encompasses the terms “consisting of” and “consisting essentially of”.
  • 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.
  • 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.
  • 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:
  • NC nitrocellulose
  • 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
  • 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):
  • 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).
  • a detection element that is labeled with biotin and the other target binding agent is bound to DIG or FITC.
  • 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.
  • a tube container
  • 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.
  • 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.
  • a measured amount of the tested sample is added to the detection unit tube, mixed well and 60-80 ⁇ of the mixture are applied to the sample pad of the universal strip or to the sample opening in the device.
  • 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.
  • Example 2 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • the external unit contains a lyophilized form of the target binding agents, optionally, a buffer is added.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • a common label such as biotin
  • a differentiating binding partner such as DIG, FITC, lectin, oligonucleotide etc.
  • 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 ⁇ 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.
  • 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.
  • PDGF-BB platelet-derived growth factor B-chain homodimer
  • PDGF-BB-2 ( AptaA): (SEQ ID NO:l)
  • PDGF-BB-1 ( AptaB): (SEQ ID NO:2)
  • 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
  • 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.
  • Figure 4 shows the results obtained with the six tested samples listed in Table 1. The interpretation of the results is as follows:
  • 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.
  • 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.
  • results 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.
  • Sample preparation Positive samples- 25 ⁇ 1 Goat anti Human IgG conjugated to FITC (5 ⁇ g/ml) were mixed with 25 ⁇ 1 biotinilated-Goat anti-human IgG (lC ⁇ g/ml) and 25 ⁇ 1 of human IgG at various concentrations (1 ⁇ g/ ⁇ l, 100 or 10 ng/ml).

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Abstract

La présente invention concerne une bandelette pour la détection d'au moins une molécule cible dans un échantillon, un dispositif comprenant la bandelette, des procédés d'utilisation de la bandelette et/ou du dispositif, et des trousses les comprenant.
PCT/IL2013/050023 2012-01-11 2013-01-10 Dispositif à bandelette d'écoulement latéral versatile WO2013105090A1 (fr)

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WO2015181790A1 (fr) * 2014-05-30 2015-12-03 Csir Procédé et dispositif de détection de bactéries d'organime entier
WO2015182296A1 (fr) * 2014-05-29 2015-12-03 株式会社日立製作所 Kit d'essai clinique et procédé de détection de substance chimique
WO2015153018A3 (fr) * 2014-04-02 2016-02-18 Chembio Diagnostic Systems, Inc. Immunoessai utilisant un conjugué de piégeage
CN106501243A (zh) * 2016-10-01 2017-03-15 桂林理工大学 一种用分子印迹试纸条快速检测三聚氰胺的方法
US9823247B2 (en) 2014-03-07 2017-11-21 The Regents Of The University Of California Methods and devices for integrating analyte extraction, concentration and detection
USD825075S1 (en) 2016-02-23 2018-08-07 Flora Bioscience, Inc. Test strip holding device
WO2019081361A1 (fr) * 2017-10-21 2019-05-02 Expedeon Ltd Dosage immunologique à écoulement latéral universel
CN109839499A (zh) * 2019-01-18 2019-06-04 华侨大学 一种核酸适配体与其靶标相结合的监测装置及其应用
CN110780067A (zh) * 2019-11-04 2020-02-11 南京欧凯生物科技有限公司 一种检测降钙素原的荧光免疫层析试纸及其制备方法
CN111465855A (zh) * 2017-12-11 2020-07-28 由联邦材料研究和检测机构主席所代表的经济与能源部长所代表的德意志联邦共和国 在固定于用于侧流测定的条上捕集区中的无标记光学检测
CN111751525A (zh) * 2020-06-18 2020-10-09 东南大学 一种基于有序微纳结构的侧向流免疫试纸条
EP3816626A1 (fr) * 2019-10-30 2021-05-05 Feral GmbH Agencement de test de flux latéral approprié pour la détection d'un analyte dans la salive
US11209427B2 (en) 2017-03-27 2021-12-28 The Regents Of The University Of California Semi-quantitative lateral-flow immunoassay for the detection of CSF leaks
US11287426B2 (en) 2015-09-04 2022-03-29 The Regents Of The University Of California Methods and devices for analyte collection, extraction, concentration, and detection for clinical applications
US11327075B2 (en) 2016-08-22 2022-05-10 The Regents Of The University Of California Hydrogel platform for aqueous two-phase concentration of a target to enhance its detection
US11828755B2 (en) 2016-06-09 2023-11-28 The Regents Of The University Of California Biomarker concentration and signal amplification for use in paper-based immunoassays and a single platform for extracting, concentrating, and amplifying DNA

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