EP3055694A1 - Dispositif et procédé d'analyse d'analytes multiples - Google Patents

Dispositif et procédé d'analyse d'analytes multiples

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Publication number
EP3055694A1
EP3055694A1 EP14755161.8A EP14755161A EP3055694A1 EP 3055694 A1 EP3055694 A1 EP 3055694A1 EP 14755161 A EP14755161 A EP 14755161A EP 3055694 A1 EP3055694 A1 EP 3055694A1
Authority
EP
European Patent Office
Prior art keywords
spots
sample
array
binding
complex
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP14755161.8A
Other languages
German (de)
English (en)
Inventor
Yury Yuzefovitch VENGEROV
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
TATAKOTO COMPANY LTD.
Original Assignee
Tatakoto Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tatakoto Co Ltd filed Critical Tatakoto Co Ltd
Publication of EP3055694A1 publication Critical patent/EP3055694A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • 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
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances

Definitions

  • the present invention relates to diagnosis and analysis of multiple analytes in a liquid sample.
  • the present invention provides a planar, porous matrix for use in the detection of a plurality of analytes in a liquid sample.
  • Other aspects of the invention comprise devices, a diagnostic system and methods of using such device.
  • the invention provides a planar, porous matrix, comprising: at least one first array of first spots and at least one counterpart second array of second spots spaced apart along a proximally-distally extending axis, wherein the second array being distal to said first array, each first spot having a counterpart second spot, each first and corresponding second spots being situated on a line that links the spots and is parallel to said axis; each of said first spots comprises a recognition element capable of specifically binding to an analyte in a sample, thereby forming a complex with (i) said specific analyte or (ii) an analyte-comprising species, at least some of the first spots comprise a recognition element that differs from recognition elements in other first spots; the complex being capable of migrating along said line with a liquid advancing in the direction of said axis; and each of said second spots comprises an immobilized capturing agent capable of binding said complex.
  • Another aspect of the invention provides a planar, porous matrix, comprising at least one first array of first spots and at least one counterpart second array of second spots spaced apart along a proximally-distally extending axis, wherein the second array being distal to said first array, each first spot having a counterpart second spot, each pair of first spot and corresponding second spot being situated on a line that links the spots and is parallel to said axis; each of said first spots comprises a conjugate, the conjugate comprising a label conjugated to a binding moiety for binding to a specific analyte in a sample thereby forming a complex with said analyte, at least some of the first spots comprise conjugates that differ from those in other first spots; the complex being capable of migrating along said line with a liquid advancing in the direction of said axis; each of said second spots comprises an immobilized capturing agent capable of binding (i) to the analyte in the complex, or (ii) to the binding moiety in the complex.
  • a further aspect of the invention provides a planar, porous matrix, comprising at least one first array of first spots and at least one counterpart second array of second spots, the arrays being spaced apart along a proximally-distally extending axis, wherein the second array being distal to said first array, each first spot having a counterpart second spot, each pair of first spot and corresponding second spot being situated on a line that links the spots and is parallel to said axis; each of said first spots comprises an antigen associated with a first member of an affinity couple, the antigen being capable of specifically binding to a labeled analyte -comprising species, thereby forming a complex capable of migrating along said line with a liquid advancing in the direction of said axis; and each of said second spots comprises an immobilized second member of said affinity couple for binding to the first member of the affinity couple.
  • the invention provides a device for detection of analytes in a liquid sample, comprising a sample -receiving zone for receiving the liquid sample and a planar, porous matrix having a reaction zone, the reaction zone comprising at least one first array of first spots and at least one counterpart second array of second spots, the arrays being spaced apart along a proximally-distally extending axis, said first array being distal to the sample -receiving zone and the second array being distal to said first array, each first spot having a counterpart second spot, each first and corresponding second spots being situated on a line that links the spots and is parallel to said axis; each of said first spots comprises a conjugate, the conjugate comprises a label conjugated to a binding moiety for binding a specific analyte in the sample thereby forming a complex, at least some of the first spots comprise conjugates with a binding moiety that differs from that in other first spots, the complex being capable of migrating along said line with
  • the invention provides a device for detecting immunoglobulins in a liquid sample, a sample-receiving zone for receiving the liquid sample and a planar, porous matrix having a reaction zone, the sample-receiving zone comprising a conjugate of a label and a binding moiety, the binding moiety capable of binding an immunoglobulin analyte in said sample, to thereby form a labeled analyte - comprising species; and a reaction zone comprising: at least one first array of first spots and at least one counterpart second array of second spots, the arrays being spaced apart along a proximally-distally extending axis, said first array being distal to the sample- receiving zone and the second array being distal to said first array, each first spot having a counterpart second spot, each first and corresponding second spots being situated on a line that links the spots and is parallel to said axis; each of said first spots comprises an antigen for binding a specific species of immunoglobulins, to
  • a diagnostic system for detecting analytes in a sample comprising a device of the invention as herein described, and an image capturing device for capturing an image formed in said reaction zone.
  • the invention provides a method for detecting a plurality of analytes in a liquid sample, the method comprising: (a) bringing the liquid sample into contact with a sample receiving zone associated with a planar porous matrix, such that the sample transfers from the sample receiving zone to the planar porous matrix, (b) allowing the sample to migrate in a direction parallel to said axis, and (c) detecting the label in the spots of said second array, presence of the label being indicative of presence of an analyte in the sample.
  • the matrix comprises a reaction zone that is spaced apart and distal to the sample receiving zone along a proximally-distally extending axis.
  • the reaction zone has at least one first array of first spots and at least one counterpart second array of second spots.
  • the at least one first array of spots comprise each a recognition element capable of specifically binding to a specific analyte in the sample, to thereby form a labeled complex with said specific analyte; the labeled complex comprising a label-comprising species originally present in (i) the sample-receiving zone, or (ii) each of said first spots.
  • the at least one second array of second spots is spaced apart along said axis from and is distal to said first array.
  • Each first spot in the first array has a counterpart second spot in the second array, each first and corresponding second spots being situated on a line that links the spots and is parallel to said axis.
  • Each of the second spots comprises an immobilized capturing agent for binding the labeled complex.
  • the labeled complex is capable of migrating along said line with a liquid advancing in the direction of said axis.
  • the matrix comprises a reaction zone spaced apart and distal to the sample receiving zone along a proximally- distally extending axis
  • the reaction zone comprises at least one first array of first spots and at least one counterpart second array of second spots.
  • At least some of the first spots comprise a conjugate that has a label and a binding moiety for binding a specific analyte in the sample to thereby form a labeled complex.
  • At least some of the first spots comprise a conjugate with a binding moiety that differs from that in other first spots.
  • the at least one counterpart second array of second spots is spaced apart along said axis and is distal to said first array.
  • Each first spot in the first array has a counterpart second spot in the second array, each first and corresponding second spots being situated on a line that links the spots and is parallel to said axis.
  • Each of said second spots comprises an immobilized capturing agent for binding to (i) the analyte in the labeled complex, or (ii) the binding moiety of the labeled complex.
  • the labeled complex is capable of migrating along said line with a liquid that advances in the direction of said axis;
  • the sample receiving zone comprises a conjugate of a label and a binding moiety, the binding moiety being capable of binding an immunoglobulin analyte in said sample, to thereby form a labeled analyte-comprising species.
  • the matrix comprises a reaction zone spaced apart and distal to the sample receiving zone along a proximally-distally extending axis.
  • the reaction zone has at least one first array of first spots and at least one counterpart second array of second spots, the arrays being spaced apart along a proximally-distally extending axis. Said first array is distal to the sample-receiving zone and proximal to said second array.
  • Each first spot in the first array has a counterpart second spot in the second array, each first and corresponding second spots being situated on a line that links the spots and is parallel to said axis.
  • Each of said first spots comprises an antigen for binding a specific species of immunoglobulins in the sample to form a labeled complex with said labeled analyte- comprising species.
  • the antigens are associated with a first member of an affinity couple.
  • the labeled complex is capable of migrating along said line with a liquid advancing in the direction of said axis.
  • Each of said second spots comprises an immobilized second member of said affinity couple for binding to the first member of the affinity couple.
  • the arrangement of arrays on the matrices of the invention comprises discrete spots for binding and capturing different analytes, enabling analysis of multiple analytes in a single sample and a single assay run (i.e. multiplex detection).
  • analyte is used herein to denote a chemical or biological species, which may be present in a tested sample.
  • chemical or biological species are infectious disease markers, diagnostic serum markers, biomarkers of animal or plant diseases, drugs and substances of abuse, hormones, immunoglobulins, environmental analytes, toxins, and others.
  • the analyte is composed of amino acids (i.e. is a peptide, polypeptide or protein). In other embodiments, the analyte is composed of nucleic acids.
  • Infectious diseases markers are molecular determinants of a host or a pathogen, usually proteins.
  • Non-limiting examples of such markers are Hepatitis B virus (HBV) surface antigen (HBs), antibodies to HBV, Human Immunodeficiency virus (HIV), Hepatitis C virus (HCV), Herpes Simplex viruses (HSV) and other virus antigens.
  • HBV Hepatitis B virus
  • HBV Human Immunodeficiency virus
  • HCV Hepatitis C virus
  • HSV Herpes Simplex viruses
  • PCT Pro- calcitonin
  • Prognostic or diagnostic serum markers of mammalian diseases are molecular indicators that reflect the presence and/or severity of a disease state. Examples include troponin I, MB-type creatine kinase (CK-MB), brain natriuretic peptide (BNP) and myoglobin as markers of cardiac disease; prostate specific antigen (PSA); cancer antigen 125 (CA-125 or MUC16); cancer-embryonic antigen (CEA); D-dimer (a marker of coagulation and thrombocytopenia) and others.
  • PSA prostate specific antigen
  • CA-125 or MUC16 cancer antigen 125
  • CEA cancer-embryonic antigen
  • D-dimer a marker of coagulation and thrombocytopenia
  • Drugs and substances of abuse are smaller molecules having mood or mind altering effects, usually consumed for non-medicinal purposes, and may lead to physical and mental damage, dependence and addiction. Examples include marijuana, cocaine, amphetamine and others, the presence of which is usually tested in urine. Hormones are protein or steroid molecules that partake in intercellular communication and signal transduction in a multicellular organism. The levels of specific hormones in the blood or urine are indicative of certain physiological states, such as human chorionic gonadotropin (hCG) used in pregnancy testing, Luteinizing (LH) and Follicle-stimulating (FSH) hormones used in determination of maturation and function of the reproductive system, and others.
  • hCG human chorionic gonadotropin
  • LH Luteinizing
  • FSH Follicle-stimulating
  • Immunoglobulins generally refer to antibodies of various types which may be indicative of various abnormal physiological conditions, such as allergies associated with elevated levels of IgE-type or IgG-type antibodies targeted to specific allergens or elevated levels of specific IgG-type antibodies in autoimmune diseases.
  • Environmental analytes are chemical or biological substances indicative of environmental pollution in food, water, etc, which may include antibiotics, toxins and other pollutants.
  • nucleic acids will refer to nuclear and mitochondrial DNA, as well as RNA, which by the rule of homologous complementation may be used for the detection of infectious pathogens (bacterial or viral or other microorganism) in the host.
  • infectious pathogens bacterial or viral or other microorganism
  • nucleic acid- based diagnostics may be used to detect a specific gene variant or expression levels of a specific gene that may be indicative of certain diseases or disease conditions, such as genetic aberrations associated with congenital disorders, Philadelphia chromosomal translocation associated with chronic myeloid leukemia (CML) and others.
  • CML chronic myeloid leukemia
  • the liquid sample may be any sample for analysis in a liquid form.
  • the sample may be originally in liquid form, or may be a solid sample solubilized in a carrier liquid, typically water or a suitable buffer.
  • the sample may be of a biological source, such as blood, urine, saliva, cerebrospinal fluid or other medical or veterinary samples; a liquid fraction of any of these; or solids from such a source that are solubilized or suspended in a liquid.
  • a biological source such as blood, urine, saliva, cerebrospinal fluid or other medical or veterinary samples; a liquid fraction of any of these; or solids from such a source that are solubilized or suspended in a liquid.
  • non-limiting examples of such samples are water or beverage samples, food samples, plant extracts, cell or bacterial cultural fluids, etc.).
  • One characteristic feature of the matrices of the invention is the presence of at least two arrays of spots on the matrix, that comprise at least one array referred to herein as a first array, and at least one other array referred to herein as a second array.
  • Each spot in the second array (referred to as second spot) corresponds to a spot (i.e. first spot) in the first array.
  • Each of the first spots in the first array comprises a recognition element, which may be analyte-specific conjugates or antigens associated with a member of a binding couple, as will be further explained below.
  • the recognition element is specific to analytes in the sample. Therefore, the recognition element in each of the first spots may differ from that in other first spots of the first array, thereby enabling association with different analytes in the sample.
  • the term complex will denote, in the context of this disclosure, the species formed by the association of the recognition elements in the first spots with the analyte or analyte-comprising species originating from the sample, as will be further explained below.
  • the second array includes capturing agents for capturing the complex, which immobilize the complex to allow its ensued detection.
  • the selection of the capturing agents is consistent with the nature of the complex to be captured, such that complementary couples are formed.
  • the capturing agent may be placed onto the matrix in discrete spots or in a consecutive line. In the specific embodiment wherein the capturing agent is placed in a consecutive line, discrete spots will form (and therefore identified later on) where the complex has been immobilized by the capturing agent.
  • each of the recognition elements is a conjugate.
  • the conjugate comprises a label conjugated to, i.e. chemically associated with, a binding moiety for specifically binding the analyte in the sample to form the complex.
  • the capturing agent may bind either to the analyte in the complex, or to the binding moiety in the complex. Namely, in the so-called "sandwich type assay", the capturing agent may be the same or different binding moiety that binds specifically to the analyte in the complex.
  • the capturing agent may be identical to the analyte, or may be another substance having the same specificity as analyte in which case the complex will be captured in the absence of analyte (so-called “competition assay”).
  • the binding moiety may be a member of a binding couple, capable of specific interaction, consisting of antibody-antigen, complementary nucleic acid sequences, sugar-lectin, and receptor-ligand, where one of the binding couple is the binding moiety and the other is the analyte.
  • a specific example of a binding moiety, as well as of a capturing agent, are molecular entities that comprise binding sites of an antibody, such entities being antibodies or a variety of constructs which include the binding sites as known per se.
  • the capturing agent has the same specificity as the analyte and, therefore, binds to the binding moiety itself. In such a case, there will be an inverse correlation between the capturing of the complex and the detection signal obtained.
  • Non-limiting examples of the capturing agent in accordance with this embodiment are immobilized analytes, or a conjugate of analyte with carrier proteins in case of low molecular weight analytes.
  • the capturing agent may comprise a low molecular weight molecule conjuga ted to a larger molecule, usually a protein (i.e. a carrier molecule) for immobilization.
  • a protein i.e. a carrier molecule
  • Such selection of a capturing agent is typically suitable for capturing low molecular weight analytes, such as drugs of abuse.
  • At least some of the first spots comprise a conjugate with a binding moiety that differs from other first spots and hence capable of binding to a different analyte.
  • the conjugates are loosely associated with the matrix and accordingly are capable of dissociating from the matrix and migrating with liquid that flows along the matrix along the said axis.
  • each of the recognition elements is an antigen associated with a first member of an affinity couple.
  • the recognition elements are capable of forming the complex with analyte-comprising species.
  • the complex is immobilized by an immobilized capturing agent, which is a second member of said affinity couple that binds to the first member of the affinity couple.
  • analyte-comprising species denotes a multi-component molecular entity, which may comprise a label conjugated to an anti-immunoglobulin that binds to a specific immunoglobulin (i.e. the analyte) in the sample.
  • the analyte-comprising species associate with the specific recognition elements in the first spots, to thereby form the complexes.
  • the capturing agent may be avidin, in which case the recognition element will comprise biotinilated antigens (i.e. antigens conjugated to biotin), where the biotin/avidin binding will be the basis for capturing the complex in the second spot.
  • biotinilated antigens i.e. antigens conjugated to biotin
  • a specific example of such an antigen is an allergen.
  • the detection of an analyte in a sample is based on the detection and visualization of the captured complex once immobilized in the second spots.
  • the complex is labeled by a label, which is present either in the first spots (as part of the conjugates) or in the analyte-comprising species, as defined above.
  • the label is typically optically detectable and encompasses any detectable label known in the art, typically optically detectable labels such as, but not limited to, fluorescent labels or colloidal particles.
  • colloidal gold particles are used as a label which can act as an indicator of presence or absence of an analyte in a liquid sample.
  • the conjugate may be labeled using a fluorescent material, such as, but are not limited to, fluorescent particles, quantum dots, lanthanide chelates, and fluorochromes, such as FITC, Rhodamine, etc.
  • the label is a nanoparticle, typically a gold nanoparticle.
  • nanoparticles are those having at least one of their dimensions in the nanometric range, typically 15 nm to 80 nm in length or diameter.
  • the use of a nanoparticle label further enables control of the flow-migration properties of the conjugate across the matrix.
  • the arrays are located on the matrix in a spaced apart arrangement along a proximally-distally oriented axis.
  • proximal and distal or any lingual variation thereof will be used to denote positions or directions on the matrix relative to the point of placement of the liquid sample (i.e. the point where the sample comes into contact with the matrix).
  • This proximally-distally oriented axis thus defines the direction of migration of the liquid sample placed onto (or that is brought into contact with) the matrix towards the arrays of spots that are located distally to the sample placement point.
  • molecular entities that exist in discrete spots in the first array migrate in a linear direction with the migrating liquid, substantially along said proximally-distally oriented axis.
  • the matrix is formed of a material that permits uniform flow, such that a liquid sample placed on the matrix proximal to the first array, migrates substantially linearly along the proximally-distally oriented axis, whereby molecular entities that exist in the spots of the first array migrate with the flow of liquid, along discrete lines parallel to the axis, to the counterpart spot in the second array.
  • the matrix is formed of a material that permits uniform flow, such that a liquid sample placed on the matrix proximal to the first array, migrates substantially linearly along the proximally-distally oriented axis.
  • the fluid-assisted movement of the molecular entities or particles from the first spot towards the second spot and onwards defines a so-called flow channel.
  • the term "channel" may at times be used herein to denote this linear flow along a substantially straight line virtually linking between counterpart spots.
  • the liquid which forms these flow channels may be focused by the use of flow focusing agents that are added to substances used in the spots in the first array.
  • the arrays are arranged such that the second array is more distal to the first array along said axis (i.e. spaced apart).
  • Each first spot has a counterpart second spot corresponding to (or located on) the same flow channel and the two counterpart spots are arranged in a line parallel to said axis.
  • each spot in the second array lies on a line parallel to the proximally-distally oriented axis that passes through the corresponding first spot, thereby linking each first spot with its counterpart second spot. Therefore, the complexes formed at the first spots migrate along these lines to the counterpart second spots, forming virtual flow channels.
  • the matrix may also comprise at least one array of spots that serves for control, which will be referred to herein as a third array.
  • the third array comprises third spots, in which each of the third spots counterparts a first and a second spot in the first and second arrays, respectively, and comprising a control capturing agent for binding the recognition element.
  • control spots are used to verify the viability of the conjugate or the analyte- comprising species, and consists of capturing agents that are specific to the conjugate or the analyte-comprising species. In cases when the conjugate is not intact, no label will be detected at the control spots, indicating that the test is invalid. In cases where the same control capturing agent may be used for providing control signals for all relevant species on the matrix, the control capturing agent may be placed onto the matrix in discrete spots or in a consecutive line. In the specific embodiment wherein the control capturing agent is placed in a consecutive line, discrete spots will form (and therefore identified later on) where the relevant species have been immobilized by the capturing agent.
  • Each spot in the third array lies on the line parallel to the proximally-distally oriented axis that passes through its corresponding first and second spots.
  • the spots may have a variety of shapes which may typically be circular, elongated, or rectangular.
  • the spots may be mini-spots having a largest diameter not exceeding 2 mm, typically not exceeding 0.5 mm and at times even not exceeding 0.1 mm.
  • the invention is not limited by the size, shape or manner of formation of such spots.
  • the first spots may be formed by a plurality of closely located or converged smaller spots.
  • the spots in the arrays are arranged such that each of said first spots is substantially equally distant from its counterpart second and third spots.
  • the spots in each of the first, second and third array are arranged along a first, second and third lines, respectively, the first, second and third lines being perpendicular to said axis.
  • the matrix is a lateral flow assay strip.
  • the strip may have any elongated shape, provided that longitudinal capillary flow is enabled, e.g. a rectangular shape.
  • the thickness of the porous matrix e.g. the strip
  • the thickness of the porous matrix is typically 0.1 to 2 mm, more typically 0.15 to 1 mm, preferably 0.2 to 0.7 mm, although any strip that allows capillary flow is suitable for use in accordance with the invention.
  • the width of the strip is typically less than 20 mm, preferably less than 10 mm, e.g. in the range of about 3 mm and 8 mm.
  • the width of the strip can be determined depending on the amount of analytes to be tested and the type of housing encasing the strip. However, the dimensions of the strip may vary and should not be considered as limiting the invention in any manner.
  • the strip may have any desired length provided that the capillary flow is enabled.
  • the length is therefore determined depending on various requirements, e.g. the amount of analytes to be tested, the type of housing encasing the strip, the presence and number of control spots, preferred speed of reaction etc.
  • the length of the strip is typically between about 2 cm and 10 cm, e.g. in the range of about 5 cm and 8 cm.
  • agents are added or conditions are provided to ensure that the flow of the liquid comprising the complex is focused, i.e. directed, in order to facilitate migration along the line in the direction of the axis.
  • the flow focusing agents may be used for minimizing the thickness (i.e. the dimension perpendicular to the flow direction) of the flow channel, thereby preventing interaction between individual flow channels and minimizing side diffusion.
  • the flow focusing agents may be agents that increase the viscosity or slow down dissolving of reagents in the spots by the migrating liquid.
  • these may be agents that do not dissolve in the liquid, thereby forming a physical barrier in the first spot, forcing the liquid to flow circumferentially around the perimeter of the first spot, thereby slowing-down and focusing the flow of the liquid.
  • the flow focusing agent is added to the reagents which form first spots. Examples of flow focusing agents are sucrose, glucose, glycerol, and others.
  • each of the first spots comprises a detergent that facilitates dissociation between the conjugates and matrix to permit their migration with the migrating liquid.
  • the invention provides a planar, porous matrix, comprising: at least one first array of first spots and at least one counterpart second array of second spots spaced apart along a proximally-distally extending axis, wherein the second array being distal to said first array, each first spot having a counterpart second spot, each first and corresponding second spots being situated on a line that links the spots and is parallel to said axis;
  • each of said first spots comprises a recognition element capable of specifically binding to an analyte in a sample, thereby forming a complex with (i) said specific analyte or (ii) an analyte-comprising conjugate,
  • the first spots comprise a recognition element that differ from recognition elements in other first spots; the complex being capable of migrating along said line with a liquid advancing in the direction of said axis;
  • each of said second spots comprises an immobilized capturing agent capable of binding said complex.
  • the invention provides a planar, porous matrix, comprising: at least one first array of first spots and at least one counterpart second array of second spots spaced apart along a proximally-distally extending axis, wherein the second array being distal to said first array, each first spot having a counterpart second spot, each first and corresponding second spots being situated on a line that links the spots and is parallel to said axis;
  • each of said first spots comprises a conjugate, the conjugate comprising a label conjugated to a binding moiety for binding to a specific analyte in a sample thereby forming a complex with said analyte, at least some of the first spots comprise conjugates that differ from those in other first spots; the complex being capable of migrating along said line with a liquid advancing in the direction of said axis;
  • each of said second spots comprises an immobilized capturing agent capable of binding (i) to the analyte in the complex, or (ii) to the binding moiety in the complex, as described above.
  • the invention provides a planar, porous matrix, comprising: at least one first array of first spots and at least one counterpart second array of second spots spaced apart along a proximally-distally extending axis, wherein the second array being distal to said first array, each first spot having a counterpart second spot, each first and corresponding second spots being situated on a line that links the spots and is parallel to said axis;
  • each of said first spots comprises an antigen associated with a first member of an affinity couple, the antigen being capable of specifically binding to a labeled analyte - comprising conjugate, thereby forming a complex capable of migrating along said line with a liquid advancing in the direction of said axis;
  • each of said second spots comprises an immobilized second member of said affinity couple for binding to the first member of the affinity couple.
  • the invention further provides a device comprising the matrix of the invention for detection of analytes in a liquid sample.
  • a specific embodiment of the matrix and of a device comprising it is one intended to be used for the detection of immunoglobulins in a liquid sample.
  • a device for detection of analytes in a liquid sample comprising:
  • a sample-receiving zone for receiving the liquid sample and a planar, porous matrix having a reaction zone, the reaction zone comprising:
  • each first array of first spots and at least one counterpart second array of second spots are spaced apart along a proximally-distally extending axis, said first array being distal to the sample-receiving zone and the second array being distal to said first array, each first spot having a counterpart second spot, each first and corresponding second spots being situated on a line that links the spots and is parallel to said axis,
  • each of said first spots comprises a conjugate
  • the conjugate comprises a label conjugated to a binding moiety for binding a specific analyte in the sample thereby forming a complex
  • at least some of the first spots comprise conjugates with a binding moiety that differs from that in other first spots, the complex being capable of migrating along said line with a liquid advancing in the direction of said axis;
  • each of said second spots comprises an immobilized capturing agent for binding (i) to the analyte in the complex, or (ii) to the binding moiety in the complex.
  • the invention provides a device for detecting immunoglobulins in a liquid sample, a sample-receiving zone for receiving the liquid sample and a planar, porous matrix having a reaction zone,
  • the sample -receiving zone comprising a conjugate of a label and a binding moiety, the binding moiety capable of binding an immunoglobulin analyte in said sample, to thereby form a labeled analyte-comprising conjugate;
  • reaction zone comprising:
  • each first array of first spots and at least one counterpart second array of second spots are spaced apart along a proximally-distally extending axis, said first array being distal to the sample-receiving zone and the second array being distal to said first array, each first spot having a counterpart second spot, each first and corresponding second spots being situated on a line that links the spots and is parallel to said axis,
  • each of said first spots comprises an antigen for binding a specific species of immunoglobulins, to thereby form a complex with said labeled analyte- comprising conjugate, the antigens being associated with a first member of an affinity couple, the complex being capable of migrating along said line with a liquid advancing in the direction of said axis;
  • each of said second spots comprises an immobilized second member of said affinity couple for binding to the first member of the affinity couple.
  • the area in which the arrays are located on the matrix is defined as the reaction zone, while the area in which the sample is placed is defined as the sample-receiving zone.
  • the sample receiving zone may constitute a part of the matrix on which the liquid sample is placed, or may be a separate matrix or pad, which is associated with the matrix via physical contact.
  • the arrangement of the device is such that proximally-distally oriented axis extends from the sample-receiving zone to the reaction zone.
  • the device may comprise a sample receptacle in association with the sample- receiving zone such that at least a portion of the liquid sample introduced into the sample receptacle transfers to the sample -receiving zone, and thereafter to the reaction zone.
  • the device may also comprise, by some embodiments, a diluent compartment that is capable of releasing a diluent onto the sample receiving zone (a diluent may, for example, be a buffer such as 0.1M NaCl in 0.05 M phosphate buffer, pH 7.4 (PBS).
  • a diluent may, for example, be a buffer such as 0.1M NaCl in 0.05 M phosphate buffer, pH 7.4 (PBS).
  • PBS phosphate buffer
  • the diluent compartment is typically made to be rupturable, for releasing the diluent onto the matrix upon rupturing of the compartment.
  • the device has two parts, e.g. a main body housing the matrix, and a cap which may house the diluent compartment.
  • the arrangement may be such that, once combined with the matrix-containing body, the diluents compartment is automatically ruptured to thereby release its contents.
  • the device also typically comprises a window over the reaction zone to permit visualization of the reaction.
  • the invention provides a diagnostic system for detecting analytes in a sample that comprises a device of the kind disclosed herein, and an image capturing device for capturing an image formed on said matrix.
  • the system comprises a computerized device linked to said image capturing device for image analysis.
  • the invention provides a method for detecting a plurality of analytes in a liquid sample.
  • the method comprises bringing the liquid sample into contact with a sample receiving zone associated with a planar porous matrix such that the sample transfers from the sample receiving zone to the planar porous matrix, the matrix comprising a reaction zone, being spaced apart and distal to the sample receiving zone along a proximally-distally extending axis.
  • the reaction zone comprises at least one first array of first spots comprising a recognition element capable of specifically binding to an analyte in the sample, thereby forming a labeled complex with said specific analyte, the labeled complex comprising a label-comprising species present in (i) the sample -receiving zone, or (ii) each of said first spots.
  • the reaction zone further comprises at least one counterpart second array of second spots spaced apart along said axis and being distal to said first array such that each first spot in the first array having a counterpart second spot in the second array, each first and corresponding second spots being situated on a line that links the spots and is parallel to said axis, each of said second spots comprises an immobilized capturing agent for binding the labeled complex.
  • the labeled complex is capable of migrating along said line with a liquid advancing in the direction of said axis.
  • the sample is allowed to migrate in a direction parallel to said axis. Then, the label is detected in the spots of the second array, presence of the label being indicative of presence of an analyte in the sample.
  • the label is detected continuously or at discrete time points.
  • the label is visualized through the use of an image capturing module, and the image is optionally further processed by image analysis software.
  • the label-comprising species are said to be located in either (i) the sample- receiving zone, or (ii) each of said first spots.
  • the species of recognition elements is said label-comprising species in the first spots, being a conjugate comprising the label and a binding moiety for specifically binding the analyte in the sample to form the labeled complex; and the immobilized capturing agent binds (i) to the analyte in the complex, or (ii) to the binding moiety in the complex.
  • the label-comprising species is present in the sample-receiving zone, the labeled-comprising species being a conjugate of a label and a binding moiety, the binding moiety capable of binding an immunoglobulin analyte in said sample, to thereby form the labeled analyte-comprising species.
  • the recognition element is an antigen associated with a first member of an affinity couple, the recognition element capable of forming said labeled complex with said label-comprising species, and the immobilized capturing agent is a second member of said affinity couple for binding to the first member of the affinity couple.
  • the method further comprises detecting presence of a label in said third spots.
  • Fig. 1 shows a schematic representation of a device in accordance with an embodiment of the invention.
  • Fig. 2A shows a schematic top view of a matrix in accordance with an embodiment of the invention comprising first and second arrays of spots.
  • Figs. 2B and 2E shows a schematic top view of a matrix in accordance with another embodiment of the invention comprising first, second and third arrays of spots.
  • Figs. 2C and 2D shows a schematic top view of a matrix in accordance with another embodiment of the invention comprising several first and second arrays and one third array of spots.
  • Fig. 3 shows a device in accordance with an embodiment of the invention.
  • Figs. 4A-4E show images of test results taken in time sequence of analysis a sample containing the hepatitis B surface antigen (HBs antigen) at the concentration of 5ng/ml, using immunological reagents as labeled conjugates [Fig. 4A - 15 seconds; Fig. 4B - 20 seconds; Fig. 4C - 30 seconds; Fig. 4D - 40 seconds; Fig. 4E - 50 seconds].
  • HBs antigen hepatitis B surface antigen
  • Figs. 5A-5E show images of test results taken in time sequence of exemplary analysis of two samples: (i) no cocaine; (ii) containing 100 ng/ml of cocaine [Fig. 5A - 15 seconds; Fig. 5B - 20 seconds; Fig. 5C - 30 seconds; Fig. 5D - 40 seconds; Fig. 5E - 50 seconds].
  • Fig. 1 is a schematic representation of an exemplary device in accordance with an embodiment of the invention.
  • the device 100 comprises a planar base 102, typically made from a polymer, on-top of which a planar, porous matrix 104 is placed.
  • the matrix is made of a porous, absorbent material, e.g. a nitrocellulose membrane.
  • the matrix is associated in one of its ends with a sample application pad (forming the sample-receiving zone) 106.
  • the other end of the matrix is associated with a liquid driving pad 108, promoting capillary flow of the liquid through the matrix.
  • a directional axis extending between the sample application pad and the liquid driving pad defines the proximally-distally oriented flow axis (represented by arrow 110) of the sample in the matrix, namely, a liquid sample will migrate along this axis from the sample application pad to the liquid driving pad through the matrix.
  • the liquid migration through the matrix is typically caused by capillary forces.
  • FIG. 1 A top view of the device of Fig. 1 is presented in Figs. 2A-2E, each representing a device in accordance with an embodiment of the invention.
  • the matrix carries two parallel arrays of spots.
  • a first array 112 comprises a plurality of first, discrete spots.
  • the array comprises 4 spots.
  • Each of the spots in the first array comprises a conjugate, which may be different in each spot of the first array, thereby allowing the analysis of several different analytes on a single matrix using a single sample.
  • the conjugates are associated with the porous matrix, in a manner that enables their dissociation and migration upon contact with a liquid across the matrix along the axis 110. Such dissociation may be assisted by detergent molecules embedded in each of the first spots.
  • a second array 114 positioned distal to the first array along the axis 110, comprises a plurality of second spots.
  • Each of the first spots of array 112 has a counterpart second spot on a line parallel to the axis 110 in the second array 114.
  • a so-called "channel" is formed between each pair of first and second spots, parallel to the direction of the axis 110 and further along the axis.
  • Each of said second spots comprises an immobilized capturing agent.
  • a liquid sample placed on sample receiving pad 106 migrates on the matrix along direction 110. Once the sample reaches array 112, analytes to be captured bind to the conjugate via the binding-moiety of the conjugate, and the so-formed analyte- conjugate complex migrates with the liquid flow in the direction 110.
  • the immobilized capturing agents in array 114 then bind to the analyte-conjugate complex via either the analyte itself or via the binding moiety of the conjugate.
  • the channeled movement of the analyte-conjugate complex assures the formation of appropriate spot couples (i.e. first spot and corresponding second spot), whereby in each "channel" a single analyte is tested and the results for different analytes are thereby isolated one from the other.
  • the matrix may carry a third array 116 of third spots, as shown in Figs. 2B-2C, which are used as control spots.
  • the third spots typically comprise a capture agent capable of capturing the conjugates and thereby demonstrate their intact state.
  • the third spots comprise secondary anti-species antibodies directed to the antibodies used as primary binding moieties in the conjugates.
  • the matrix may carry a plurality of first arrays and second arrays.
  • first arrays 112, 212 and two second arrays 114, 214 are shown, as well as a third array 116.
  • first arrays 112 and 212 are identical to each other, and similarly second arrays 114 and 214 are identical.
  • the third array 116 therefore, provides a common control for both sets of arrays. This allows for repetitive analysis of the analyte while using a single sample.
  • first arrays 112, 212 each comprise different conjugates, and correspondingly, second arrays 114, 214 each comprise different capturing agents.
  • the pair of arrays 114, 214 is perpendicularly shifted from the pair 112, 212, thereby allowing 8 distinct channels to form (rather then the 4 channels of Fig. 2C).
  • the number of control points in array 116 has been doubled to correspond to the new number of channels.
  • the sample receiving zone 220 comprises conjugates of label and anti-immunoglobulin.
  • labeled analyte-comprising species are formed between the conjugates and the immunoglobulins in the sample.
  • the sample then flows along the axis 110 towards the first array 222, in which each of the first spots comprise a different antigen. All of the antigens are biotinilated by a biotin moiety. The antigens bind to the immunoglobulin in the labeled analyte-comprising species to form the labeled complexes.
  • the second array 224 positioned distal to the first array along the axis 110, comprises a plurality of second spots, each spot comprises avidin to enable immobilization of the complexes by association of the biotin-avidin pair.
  • the matrix may carry a third array 226 of third spots, which are used as control spots.
  • the device comprises a housing 302 and a cap 304.
  • the housing houses the matrix, and comprises a clear window 306 positioned above the reaction zone of the matrix for ease of inspection of the test results.
  • the housing also comprises a sample collection pad 308, protruding out of the housing, and associated with the sample receiving zone of the matrix.
  • the cap 304 may house a diluents chamber (not shown) for providing a diluent, typically a buffer, to facilitate flow of the sample through the matrix.
  • the diluent chamber may be made of a rupturable material, such as a thin polymeric sheet.
  • a user places a drop of liquid sample, which may be (although not limited to) a bodily fluid such as blood, urine or saliva, onto the sample collection pad.
  • a bodily fluid such as blood, urine or saliva
  • the housing 302 and the cap 304 are then coupled, causing rupture of the diluent chamber and release of the diluent onto the sample collection pad.
  • the diluted sample flows from the sample collection pad to the matrix for analysis. Once the analysis is complete, the results may be viewed through window 306 and further analyzed.
  • the analysis was carried out using an arrangement of arrays of spots as in Fig. 2B.
  • 100 ⁇ L ⁇ of a serum sample containing HBs antigen at a concentration of 5ng/ml was tested by the device of the invention.
  • the test strip used has a width of 5mm, prepared of a plastic support, a working nitrocellulose membrane CNPF with a 10 ⁇ pore size, GFB-R4 separation membrane (sample receiving zone), and AP045 adsorption- membrane (liquid driving pad). All components of the strip were manufactured by company Advanced Microdevices, Ambala Cantt, (Mdi Easypack kits).
  • colloidal gold conjugates with mouse monoclonal antibodies were synthesized according to a known procedure [Hermanson GT (2008), Bioconjugate Techniques, Elsevier, Amsterdam, 2nd ed., pp. 596, 924-931].
  • the conjugates in concentration corresponding to OD 520 8.0 in PBS buffer with 0.25% BSA, 0.1% Tween 20 and 2% glucose, were applied in the first array of spots by touching the membrane with a steel pin driven by a program-controlled manipulator.
  • the diameter of the pin was 200 ⁇ , the drop volume was 20 nL.
  • each spot was obtained by 10 successive applications of conjugate droplets by the pin.
  • the second array comprised of second spots formed by single pin application of a solution of anti-HBs antigen goat antibodies, with concentration of 1 mg/ml in Tris buffer pH 7.5.
  • An array of anti-mouse IgG rabbit antibodies applied in same way as the third array was used as control.
  • the spots had a diameter of 0.15-0.2 mm and were spaced apart across a distance of 1.5-2 mm along the proximal-distal axis (total distance of about 5 mm for the whole run).
  • the selection of species was based upon desired formation of a so-called "sandwich" complex at the second array, i.e. the analyte is sandwiched at the capturing point and immobilized between the two anti-HBs antibodies.
  • the assay was performed at room temperature. 100 ⁇ . of a liquid serum sample containing HBs antigen at a concentration of 5ng/ml was applied to the sample receiving zone of the strip. The strip was immediately placed in the image capturing device, employed for capturing sequential images of the nitrocellulose matrix. The contact of the sample receiving zone of the strip with the sample initiates the movement of reactants along the matrix, followed by immunochemical reactions and the formation of immune complexes in the discrete spots.
  • the image capturing device allows following the process of antigen detection in a real-time.
  • Figs. 4A-4E show images of the matrix taken during a period of analysis of 1 minute, starting from placement of the sample onto the sample -receiving zone.
  • each part of matrix along the proximally-distally extending axis could be considered as an individual test of a distinct analyte, thus enabling to perform a plurality of analyses in a single liquid sample (multiplex assay).
  • the first array comprised colloidal gold conjugate, prepared and applied to the device as in example 1, with mouse monoclonal antibodies specific to the analyte (in this case being cocaine), but without addition of glucose.
  • the second array comprised immobilized capturing agent-analyte (cocaine) conjugates with bovine serum albumin (BSA) as a carrier protein applied in concentration of 0.5 mg/ml in Tris buffer pH 7.5. Both monoclonal antibodies and BSA-cocaine conjugates used were manufactured by Arista Biologicals (Allentown, PA, USA).
  • the analyte in a liquid sample competes with the capturing agent (present in the spots of the second array) for binding to the antibodies of the conjugates in the first array.
  • This is known as the "competition method”. This method may be used for analysis of low molecular weight analytes, such as drugs.
  • the assay was performed at room temperature. 100 ⁇ L ⁇ of a sample was applied to the sample receiving zone of the strip. The strip was immediately placed in the image capturing device, employed for capturing sequential images of the nitrocellulose matrix. The contact of the sample receiving zone of the strip with the sample initiates the movement of reactants along membrane followed by immunochemical reactions and the formation of immune complexes in specific zones.
  • Figs. 5A-5E show analysis of two samples: (i) PBS not containing cocaine (left column), and (ii) a sample containing lOOng/ml of cocaine in PBS (images in the right column).
  • the configuration used was that of Fig. 2B, with a distance between spots within the arrays of 1.5 mm and distance between channels of 1 mm.
  • the results demonstrate that the antibody conjugate contained in first spots was captured by the immobilized capturing agent of the spots in the second array with production of discrete visible spots within less than 1 minute.
  • the captured images may, in turn, be subjected to further image analysis for comparing spot intensities to known standard controls.
  • Example 1 Despite not using flow focusing agent there was no significant traversal diffusion observed without any converging of channels suggesting that plurality of analytes could be detected on the same strip.

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Abstract

L'invention concerne des dispositifs mobiles et des methodes de diagnostic et d'analyse simultanées d'analytes multiples dans un échantillon de liquide.
EP14755161.8A 2013-07-18 2014-07-17 Dispositif et procédé d'analyse d'analytes multiples Withdrawn EP3055694A1 (fr)

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IL227524A IL227524A0 (en) 2013-07-18 2013-07-18 A device and method for the analysis of multiple anal
PCT/IL2014/050647 WO2015008287A1 (fr) 2013-07-18 2014-07-17 Dispositif et procédé d'analyse d'analytes multiples

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WO2017066645A1 (fr) 2015-10-15 2017-04-20 Inbios International, Inc. Systèmes de dosage à écoulement latéral multiplexé et leurs procédés d'utilisation
CN109477798A (zh) * 2016-07-18 2019-03-15 西门子医疗保健诊断公司 改善的低样品体积尿分析测定条、分析试剂盒以及与其相关的使用方法

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US6136610A (en) 1998-11-23 2000-10-24 Praxsys Biosystems, Inc. Method and apparatus for performing a lateral flow assay
BR0215327A (pt) * 2001-12-24 2006-06-06 Kimberly Clark Co sistema de calibração interna para ensaios de circulação de fluxo
US7371582B2 (en) 2002-01-23 2008-05-13 Boditechmed Inc. Lateral flow quantitative assay method and strip and laser-induced fluorescence detection device therefor
CN1667418B (zh) 2004-03-10 2010-10-06 马杰 多功能测量、分析和诊断便携式装置
US7858396B2 (en) 2005-10-31 2010-12-28 Orasure Technologies, Inc. Lateral flow assay device with multiple equidistant capture zones
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US8486717B2 (en) * 2011-01-18 2013-07-16 Symbolics, Llc Lateral flow assays using two dimensional features

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