WO2004081528A2 - Dispositif et procede de dosage - Google Patents

Dispositif et procede de dosage Download PDF

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Publication number
WO2004081528A2
WO2004081528A2 PCT/US2004/007231 US2004007231W WO2004081528A2 WO 2004081528 A2 WO2004081528 A2 WO 2004081528A2 US 2004007231 W US2004007231 W US 2004007231W WO 2004081528 A2 WO2004081528 A2 WO 2004081528A2
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WO
WIPO (PCT)
Prior art keywords
test device
labeled
specific binding
reagent
binding reagent
Prior art date
Application number
PCT/US2004/007231
Other languages
English (en)
Other versions
WO2004081528A3 (fr
Inventor
Joseph R. Robinson
Benedict L. Zin
Original Assignee
Robinson Joseph R
Zin Benedict L
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 Robinson Joseph R, Zin Benedict L filed Critical Robinson Joseph R
Publication of WO2004081528A2 publication Critical patent/WO2004081528A2/fr
Publication of WO2004081528A3 publication Critical patent/WO2004081528A3/fr

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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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/76—Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors

Definitions

  • the present invention relates to assays involving specific binding, especially i munoassays.
  • the test devices and methods of the present invention are suitable for quantitative assays as well as for qualitative assays.
  • the invention particularly relates to analytical devices which are suitable for use in the home, clinic or doctor's office, which are intended to give an analytical result rapidly, and which require the minimum degree of skill and involvement from the user.
  • the use of test devices in the home to test for pregnancy and fertile period (ovulation) is now commonplace. BACKGROUND OF THE INVENTION
  • Another earlier immunoassay device utilizes marking elements and particles which do not affect evaluation. Each of these include bispecific antibodies for the suspected analyte.
  • the device includes a porous catching section that has a pore diameter that is smaller than the diameter of the particles. When the analyte is present, a biochemical reaction takes place between the marking elements and the particles producing a reaction product.
  • the pore size of the catching section is smaller than the size of the particles and smaller than the size of the reaction product. The particles and the reaction product are caught by the catching section, but the marking element can pass through the catching section. Therefore, a visual inspection of the catching section will indicate the presence or the absence of the analyte in the sample. See European Patent Application No. EP 0962771.
  • Figure 1 is an isometric view of a test device according to the present invention.
  • Figure 2 is a cross-sectional side elevation of a test device according to the present invention.
  • Figure 3 is a side view of a test device according to the present invention.
  • the present invention provides a test device for detecting the presence or absence of a selected analyte in a liquid sample.
  • the test device includes a reagent member, a porous carrier, and a detection zone.
  • the reagent member includes a body, a first labeled binding reagent specific for a first binding site of the analyte and a second labeled binding reagent specific for a second binding site of the analyte.
  • the first specific binding site and second binding sites are different.
  • the reagent body is adapted to retain the first and second labeled specific binding reagents when the body and the first and second labeled binding reagents are dry, and to release them when the body and they are moist.
  • the first and second labeled specific binding reagents are capable of forming a first labeled complex with the analyte with the complex including the first labeled specific binding reagent, the analyte, and the second labeled specific binding reagent.
  • the detection zone includes a first porous barrier having an average pore size larger than the diameter of the larger diameter of the first or second labeled specific binding reagent, but smaller than the diameter of the first labeled complex.
  • the detection zone may be a section of the porous carrier or may be separate from and in fluid communication with the porous carrier.
  • the reagent member, porous carrier, and detection zone are arranged so that a fluid applied to the test device would travel sequentially from the reagent member to the porous carrier and to the detection zone.
  • the test device is typically dry before use and moist during use.
  • the test device may further include a sample receiving member arranged so that a fluid applied to the test device would travel sequentially from the sample receiving member to the reagent member
  • the test device may also include a control zone arranged so that a fluid applied to said test device would travel sequentially from the reagent member to the porous carrier, to the detection zone, and to the control zone.
  • the control zone may include a second porous barrier having an average pore size smaller than the diameter of the smaller diameter of the first or second labeled specific binding reagent or an average pore size smaller than the diameter of the larger diameter of the first or second labeled specific binding reagent.
  • Another type of control zone could include an immobilized binding reagent capable of binding to the first labeled specific binding reagent, the second specific binding reagent, or the first and the second specific binding reagents.
  • the control zone may be a section of said porous carrier or may be separate from and in fluid communication with the porous carrier.
  • the present invention also provides a test device as described above for detecting the presence or absence of human chorionic gonadotrophin (hCG) in urine.
  • hCG human chorionic gonadotrophin
  • Specific components of one preferred embodiment of a test device for detecting hCG include a first colored latex particle labeled anti-hCG monoclonal antibody for a first antibody binding site of hCG and a second colored latex particle labeled different anti-hCG antibody specific for a second antibody binding site of hCG, which together with hCG form a first colored latex particle labeled complex.
  • the porous carrier may be a nitrocellulose porous carrier.
  • the detection zone may be a first porous agarose barrier having an average pore size larger than the diameter of the larger diameter of the first or second colored latex particle labeled anti-hCG antibody, but smaller than the diameter of said first labeled complex.
  • the colors of the latex particles are visually distinguishable from white.
  • the present invention also provides a method of detecting the presence or absence of an analyte in a liquid sample by applying the liquid sample to the test devices described herein, whereby at least a portion of any first complexes formed are retained at the detection zone, and at least a portion of the uncomplexed first and second labeled specific binding reagents pass through the detection zone; and (b) detecting the presence or absence of the first complexes at the detection zone.
  • the presence of the first complexes in the detection zone indicates the presence of the said analyte in the sample.
  • the assay devices and methods of the present invention may be to detect a wide variety of analytes by choosing appropriate specific binding reagents.
  • the analytes can be, for example, proteins, haptens, inimunoglobuliiis, hormones, polynucleotides, steroids, drugs, or infectious disease agents (e.g. of biological, chemical, or biochemical origin).
  • infectious disease agents e.g. of biological, chemical, or biochemical origin.
  • infectious disease agents e.g. of biological, chemical, or biochemical origin.
  • examples of analytes include, but are not limited to, hCG (human chorionic gonadofrophin) (a hormone whose presence indicates pregnancy), LH (lutinizing hormone) (a hormone whose presence indicates ovulation), infectious disease agents, E- 3-G (estrone-3-glucoronide) and P-3-G (pregnanediol-3-glucoronide).
  • the former three are suitably analyzed with the present device in a sandwich
  • the present devices and assays can also be adapted, based upon the detailed description herein, to detect simultaneously or sequentially more than one analyte in a sample, which can also have significant clinical utility.
  • the ratio of the levels of apolipoproteins Ai and B can indicate susceptibility to coronary heart disease, while the ratio of the levels of glycated hemoglobin (HbA) to unglycated (HbAo) or total (Hb) hemoglobin can aid in the management of diabetes.
  • Test devices may also be configured, based upon the detailed disclosure herein, to measure two steroids simultaneously, such as, for example, E-3-G and P-3-G.
  • a test could also be configured, based upon the detailed disclosure herein, to detect the presence of various different sereotypes of one bacterium, or to detect the presence of soluble serological markers in humans.
  • a multiple analyte test for the detection of different serotypes of Streptococcus can be prepared for groups A, B, C and D.
  • a cocktail of monoclonal antibodies labeled with labels of different diameters, each specific for various pathologically important group serotypes, or a polyclonal antiserum raised against a particular Streptococcal group, could be placed onto a reagent member and one or more detection zones properly sized, as described herein, to detect each could be placed on the device.
  • the reagent member may be one individual reagent sub-member or more than one reagent sub-members each containing one or more specific binding reagents.
  • the reagent member may be composed of, for example, a fibrous material or pad-like structure that may be woven or non-woven, such as, for example, a fiberglass pad, with openings or interstitial spaces which act as pores, and may be termed pores herein.
  • the reagent member may be a natural or synthetic porous material such as a macroporous body.
  • the reagent member may be a section of the porous carrier or may be a separate component.
  • a separate component reagent member may facilitate the ease with which the labeled components are taken up by the liquid sample as compared to if the labeled components are inco ⁇ orated as pre-dosed reagents on the porous carrier. Manufacturing is also facilitated by having the reagent member as a separate component that is placed next to and slightly overlapping the porous carrier during manufacture of the test device.
  • the reagent member is in direct moisture-conductive contact with the porous carrier, and the detection zone, whether on the porous carrier or is a separate component.
  • the reagent member may be spaced away from the region of contact between the porous carrier and the reagent member.
  • the quantity of liquid sample required to saturate the reagent member is not less than the quantity of liquid sample capable of being absorbed by the mass of porous carrier linking the reagent member and the detection zone.
  • the liquid capacity of the reagent member is at least equal to the liquid capacity of the working portion of the porous carrier.
  • the reagent member has openings, spaces, or pores (all included in the term Pores herein) that are at least 10 times greater than the maximum size of the largest label used in the device. Larger pore sizes or openings give better release of the labeled reagent.
  • the reagent member preferably is not protein-binding or is easily blockable by means of reagents such as BSA or PVA, to minimize non-specific binding and to facilitate free movement of the labeled reagents after the reagent member has become moistened with the liquid sample.
  • the reagent member can also include surface active agents, such as for example, Tween 20 preferably at about 1%, or solvents, if necessary, to render it more hydrophilic, to promote rapid uptake of the liquid sample, and/or to facilitate the travel of the sample and the reagents though the porous carrier, and/or to minimize or to eliminate any interaction of the reagents or complexes with the porous carrier.
  • Surface active agents can be inco ⁇ orated in the solution containing the labeled reagent when this solution is applied to the reagent member during manufacture of the device or the component.
  • the reagent member can inco ⁇ orate several labeled specific binding reagents each carrying a different label, e.g., having different colors or fluorescent properties. This will facilitate the manufacture of a multiple analyte testing device.
  • the labeled reagents are preferably inco ⁇ orated in the reagent member in bulk, e.g., large sheet, form before being subdivided into individual members for use in a test device of the present invention. This is the case whether the reagent member is a separate component or a section of the porous carrier.
  • the reagent member may be dried, e.g., by vacuum or air-drying, or preferably by freeze- drying.
  • the solution can also contain a surface active agent, such as a detergent such as, for example, Tween 20 1% solution, and/or a glazing material, such as, for example, a sugar, e.g., sucrose.
  • the labeled reagents may enhance release of the labeled reagents and may promote stability of certain specific binding reagents. Inco ⁇ orating the labeled reagents in a separate reagent member(s), rather than pre-dosing onto the porous carrier that also inco ⁇ orates the detection zone, enhances sensitivity of the test because a substantial quantity of the liquid sample is able to take up the labeled reagents before migrating through the porous carrier to the detection zone. This enhances potential reaction time without significantly increasing overall test time. Also, the liquid which permeates the carrier has a more uniform and consistent composition.
  • the labeled reagents are specific binding partners for the analyte. More preferably, the first and second labeled reagents bind specifically to different sites of the analyte.
  • the labeled reagents and the analyte cooperate together in a "sandwich" reaction. This results in the labeled reagents being retained in the detection zone if analyte is present in the sample.
  • the two binding reagents should have specificities for different epitopes on the analyte.
  • the binding reagents are preferably highly specific antibodies, and more preferably, are monoclonal antibodies, including, but not limited to non-human, chimeric, and humanized monoclonal antibodies.
  • the two reagents specific for the analyte, hCG preferably are monoclonal anti-hCG - clone "MIH 9816" from Seradyn and monoclonal anti-beta hCG - clone "057-10043" from OEM Concepts.
  • the label can be any entity the presence of which can be detected and, preferably, readily detected. Concentration of the label into a small zone or volume should give rise to a readily detectable signal such as, for example, a strongly-colored area. This can be evaluated by eye or by instruments if desired.
  • the label is a direct label, i.e., a label that can be used to produce an instant analytical result without the need to add further reagents in order to develop a detectable signal and/or, in its natural state, is readily visible either to the naked eye when it accumulates, or with the aid of an optical filter and/or applied stimulation, e.g. UV light to promote fluorescence.
  • the labels are particulate labels. Direct particulate labels including, but not limited to, colored latex particles and particularly those colors that are visually distinguishable from white, gold sols, non-metallic colloids, and dye sols. They are robust and stable and can, therefore, be used readily in a analytical device which is stored in the dry state.
  • the particulate label is a latex particle, such as a colored latex particle which can be readily visible to the eye if it complexes and is retained in the detection zone or if it is bound in the control zone.
  • the assay result can be read instrumentally, e.g., by color reflectance or microscopically.
  • the labels can inco ⁇ orate a fluorescent compound which can respond to applied electromagnetic energy such as ultraviolet light or visible light, to provide an emitted signal that can be measured instrumentally.
  • the label is a colored (other than white) latex particle of spherical or near- spherical shape and having a maximum diameter of not greater than about 2000 nanometers. More preferably, these particles range in size from about 50 to about 750, and most preferably from about 200 to about 600 nanometers. Especially preferred are latex particle 250 nanometers in diameter and 500 nanometers in diameter.
  • the labels of each specific binding reagent can be of the same or different diameters and of the same or different colors.
  • Indirect labels such as enzymes, e.g. alkaline phosphatase and horseradish peroxidase, can be used but these usually require the addition of one or more developing reagents such as substrates before a visible signal can be detected.
  • additional reagents can be inco ⁇ orated in the reagent member, in the porous carrier, or in the sample receiving member if present, such that they dissolve or disperse in the aqueous liquid sample.
  • the developing reagents can be added to the sample before contact with the test device or the test device can be exposed to the developing reagents after the complexing reaction has taken place.
  • Coupling of the label to the specific binding reagent can be by covalent bonding, hydrophobic bonding, or other techniques known in the art.
  • the labeled reagents migrate with the liquid sample as it progresses to the detection zone. Preferably, the flow of sample continues beyond the detection zone and sufficient sample is applied to test device in order that this may occur. Excess labeled reagents which do not participate in forming any first complexes are flushed through the detection zone by this continuing flow.
  • the porous carrier is nitrocellulose sheet having a pore size of at least about 1 micron, even more preferably of greater than about 5 microns, and yet more preferably about 8-12 microns.
  • Nitrocellulose sheet having a nominal pore size of up to approximately 12 microns, is available commercially from Schleicher and Schuell GmbH.
  • the nitrocellulose sheet is "backed", e.g. with plastics sheet, to increase its handling strength.
  • This can be manufactured easily by forming a thin layer of
  • a pre-formed sheet of nitrocellulose can be tightly sandwiched between two supporting sheets of solid material, e.g. plastics sheets.
  • the flow rate of an aqueous sample through the porous carrier is such that in the untreated material, aqueous liquid migrates at a rate of 1 cm in not more than 2 minutes, but slower flow rates can be used if desired.
  • the spatial separation between the reagent member and the detection zone, and the flow rate characteristics of the porous carrier, can be selected to allow adequate reaction times during which the necessary specific binding can occur. Further control over these parameters can be achieved by the inco ⁇ oration of viscosity modifiers (e.g., sugars, modified celluloses, and surfactants) in the sample to slow down the reagent migration.
  • viscosity modifiers e.g., sugars, modified celluloses, and surfactants
  • porous materials may be used for the first and/or second porous barriers.
  • different grades or concentrations of agarose can be used for different pore sizes.
  • pores can be made in suitable materials by laser, drilling, microdrilling, or photoablation, for example.
  • Porous barriers can be applied to the porous carrier material in a variety of ways including, but not limited to, printing techniques such as, for example, micro-syringes, pens using metered pumps, direct printing and ink-jet printing.
  • the presence or intensity of the signal from the first complexes which are retained in the detection zone can provide a qualitative or quantitative measurement of analyte in the sample.
  • a plurality of detection zones arranged in series, through which the aqueous liquid sample can pass progressively, can also be used to provide a quantitative measurement of the analyte.
  • Different sized labels and or labeled of different colors can be used to label specific binding reagents for different analytes to provide a multi-analyte test.
  • the different detection zones can have different pore sizes to retain different sized complexes at each different porous barrier.
  • the dry porous carrier material comprises a chromatographic strip, such as a strip of nitrocellulose.
  • the nitrocellulose can be backed with moisture impermeable material, such as a polyester sheet.
  • a chromatographic strip such as a strip of nitrocellulose.
  • the nitrocellulose can be backed with moisture impermeable material, such as a polyester sheet.
  • nitrocellulose as the porous carrier material has considerable advantage over more conventional strip materials, such as paper, because nitrocellulose has a natural ability to bind proteins without requiring prior sensitization. No chemical treatment is required which might interfere with the essential specific binding activity of the reagent.
  • Nitrocellulose can be blocked using simple materials, such as polyvinylalcohol.
  • nitrocellulose is readily available in a range of pore sizes and this facilitates the selection of a porous carrier to suit particularly requirements such as sample flow rate.
  • the porous carriers can be manufactured individually, or may be in the form of a longer strip or a sheet during manufacture.
  • the porous carrier can be manufactured in sheet form with the detection zone and the control zone applied to the sheet. The sheet can then be cut into strips.
  • the sample receiving member can be made from any bibulous, porous or fibrous material capable of absorbing liquid rapidly.
  • the porosity of the material can be unidirectional (i.e., with pores or fibers running wholly or predominantly parallel to an axis of the member) or multidirectional (i.e., omnidirectional, so that the member has an amo ⁇ hous sponge-like structure).
  • Porous plastics material such as polypropylene, polyethylene (preferably of very high molecular weight), polyvinylidene fluoride, ethylene vinylacetate, acrylonitrile and polytetrafluoro-ethylene can be used.
  • Sample receiving members can also be made from paper or other cellulosic materials, such as nitrocellulose. Materials that are now used in the nibs of fiber-tipped pens are suitable and such materials can be shaped or extruded in a variety of lengths and bross-sections appropriate in the context of the invention.
  • the material comprising the sample receiving member should be chosen such that it and the reagent member can be saturated with aqueous hquid within a matter of seconds.
  • the material remains robust when moist, and for this reason paper and similar materials are less preferred in any embodiment wherein the sample receiving member protrudes from a housing. The liquid must thereafter permeate freely from the sample receiving member into the reagent member.
  • the control zone can be designed to convey an unrelated signal to the user that the device has worked.
  • the control zone can be a second porous barrier having a pore size that will not allow one or more of the uncomplexed labeled specific binding reagents from passing through, an antibody that will bind to one or more of the labeled reagents (e.g., an anti-mouse antibody if the labeled reagent is an antibody that has been derived using a murine hybridoma) to confirm that the sample has permeated the test strip.
  • the control zone can contain an anhydrous reagent that, when moistened, produces a color change or color formation, e.g.
  • control zone could contain immobilized analyte which will react with excess labeled reagent(s).
  • the control zone indicates to the user that the sample has proceeded past the detection zone and should be located downstream from the detection zone in which the desired test result is recorded. A positive control indicator, therefore, tells the user that the sample has permeated the required distance through the test device.
  • an absorbent sink or reservoir can be provided at the distal end of the test device.
  • the absorbent sink or reservoir may comprise, for example, Whatman 3MM chromatography paper, and should provide sufficient abso ⁇ tive capacity.
  • a length of porous test device which may extend beyond the detection zone may act as a sink or reservoir.
  • the reagent member, porous carrier, and detection zone may be contained within a casing or housing, which is preferably composed of a moisture impervious material.
  • the ample receiving member may extend out of the housing through an aperture and can act as a means for permitting a liquid sample to enter the housing and reach the reagent member.
  • the housing should be provided with means, e.g., an appropriately placed aperture or window which may be open or covered, preferably with a transparent or translucent material, which enables the detection zone to be observable from outside the housing so that the result of the assay can be observed.
  • the housing may also be provided with further means as above to enable the control zone to be observed from outside the housing.
  • the housing is provided with a removable cap or shroud which can protect the protruding porous receiving member during storage before use. If desired, the cap or shroud can be replaced over the protruding sample receiving member, after sample application, while the assay procedure is being performed.
  • the casing can be constructed of opaque or translucent material inco ⁇ orating at least one aperture through which the analytical result may be observed, together with a removable and replaceable cover for the protruding bibulous urine receiving member.
  • the present invention also provides an analytical method for detecting the presence or absence of an analyte.
  • a device as set forth above is contacted with a liquid sample suspected of containing the analyte, such that the sample permeates by capillary action via the reagent member through the porous carrier into the detection zone and the labeled reagents migrate therewith to the detection zone, the presence of analyte in the sample being determined by observing the extent (if any) to which complexes of the analyte and the labeled reagents are retained in the detection zone.
  • the present invention can be used as a pregnancy testing device wherein the analyte is hCG and the labeled specific binding reagents are anti-hCG antibodies.
  • a fertile period prediction device essentially as just defined except that the analyte is LH and the labeled specific binding reagents are labeled anti-LH antibodies.
  • kits suitable for home use comprising a plurality (e.g., two or more) devices individually wrapped in moisture impervious wrapping and packaged together with appropriate instructions to the user.
  • a compound device can inco ⁇ orate two or more test devices arranged in parallel and possibly sharing some components, for example, such that a single application of liquid sample to the device initiates sample flow in them simultaneously. Separate analytical results can be determined in this way. If different reagents are used on the test devices, the simultaneous determination of a plurality of analytes in a single sample can be made. Alternatively, multiple samples can be applied individually to an array of test devices and analyzed simultaneously.
  • the device comprises a housing or casing 100 of elongate rectangular form having at one end 101 a portion 102 of reduced cross-sectional area.
  • a cap 103 can be fitted onto portion 102 and can abut against the shoulder 104 at end 101 of the housing.
  • Cap 103 is shown separated from housing 100.
  • Extending beyond end 105 of portion 102 is a porous sample collector 106.
  • Upper face 107 of housing 100 inco ⁇ orates two apertures 108 and 109.
  • the housing is constructed of an upper half 110 and a lower half 111.
  • Housing 100 is of hollow construction. Sample receiving member 106 extends into housing 100. The inner end 112 of sample receiving member 106 is recessed to accommodate a reagent member 113. Aqueous liquid sample applied to sample receiving member 106 can pass freely into reagent member 113, rapidly saturating it. h turn, reagent member 113 is in liquid permeable contact with porous carrier 114.
  • the housing is constructed of an upper half 110 and a lower half 111 and porous carrier 114 overlaps reagent member 113 to ensure that there is adequate contact between these two components and that a liquid sample applied to sample receiving member 106 can permeate via reagent member 113 and into porous carier 114. Porous carrier 114 extends further into housing 100.
  • a gap 115 can be left in the housing 100 by arranging for porous carrier 114 to overlap reagent member 113 only partially.
  • Porous carrier 114 is backed by a supporting strip 116 formed of transparent moisture- impermeable plastics material. Porous carrier 114 extends beyond apertures 108 and 109. Means 117 and 118 are provided within housing 100 to hold porous carrier 114 firmly in place. The transparent backing strip 116 can act as a seal against undesired ingress of moisture from outside the housing 100.
  • the residual space 119 within the housing can contain moisture-absorbant material, such as silica gel, to help maintain the strip 114 in the dry state during storage.
  • moisture-absorbant material such as silica gel
  • the detection zone will he in the region exposed through aperture 108 in order that when the device has been used, the result can be observed through aperture 108.
  • Aperture 109 provides means through which a control zone containing may be observed.
  • the protective cap 103 is removed from the holder and sample receiving member 106 is exposed to a liquid sample e.g. by being placed in a urine stream in the case of a pregnancy test. After exposing sample receiving member 106 to the liquid sample for a time sufficient to ensure that the sample receiving member 106 is saturated with the sample, the cap 103 can be replaced and the device placed aside by the user for an appropriate period lime (e.g. two or three minutes) while the sample permeates porous carrier 114 and the detection zone to provide the analytical result.
  • an appropriate period lime e.g. two or three minutes
  • the user can observe the result through apertures 108 and 109 and can ascertain whether the assay has been completed by observing the control zone 120 through aperture 109, and can ascertain the result of the assay by observing the detection zone 119 through aperture 108.
  • a device can be produced which is suitable for use as a pregnancy test kit or fertile period test kit for use in the home or clinic.
  • the user merely needs to apply a urine sample to the exposed sample receiving member and then (after optionally replacing the cap) can observe the test result through aperture 108 within a matter of a few minutes.
  • the device as just described, can be used to determine the presence of a very wide variety of analytes if appropriate reagents are used.
  • aperture 109 is redundant and may be omitted if the test strip does not contain any control means.
  • the general shape of the housing and cap both in terms of their length, cross- section and other physical features, can be the subject of considerable variation.
  • EXAMPLE 1 Construction of a Test Device
  • a test device is constructed as follows: Monoclonal anti-hCG - clone "MIH 9816" from Seradyn and monoclonal anti-beta hCG - clone "057-10043" from OEM Concepts are respectively conjugated onto red latex beads having diameters of about 250 nm. A solution of these labeled antibodies and Tween 20 1% solution are applied to a reagent member. A detection zone of an agarose matrix is applied to a section of a nitrocellulose carrier strip. A control zone of an agarose matrix is applied to the carrier strip downstream of the detection zone. The pore size of the agarose matrix in the detection zone is about 375 nm.
  • the pre size of the agarose matrix in the control zone is about 175 nm.
  • a sample receiving member and a sink of absorbent material are provided. These components are assembled as in Figures 1-3 so that urine applied to the test device will flow sequentially form the sample receiving member to the reagent member to the porous carrier, through the detection zone and through the control zone to the sink.
  • the sample receiving member is saturated with urine containing hCG.
  • the urine passes to the reagent member and solubilizes the red latex bead labeled antibodies and the surfactant.
  • hCG complexes with some of the two red latex bead labeled antibodies to form complexes having hCG and the two different red latex bead labeled antibodies. These complexes have a diameter of about 500 nm.
  • the complexes and excess uncomplexed red latex bead labeled antibodies travel in the urine to the porous carrier and to the detection zone. The complexes are retained at the detection zone and a red line appears.
  • the uncomplexed red latex bead labeled antibodies pass through and to the control zone.
  • the uncomplexed red latex bead labeled antibodies are retained at the control zone and a red line appears. This test indicates the presence of hCG in the urine sample.
  • the sample receiving member is saturated with urine without hCG.
  • the urine passes to the reagent member and solubilizes the red latex bead labeled antibodies and the surfactant.
  • the uncomplexed red latex bead labeled antibodies travel in the urine to the porous carrier and to the detection zone.
  • the uncomplexed red latex bead labeled antibodies pass through and to the control zone.
  • the uncomplexed red latex bead labeled antibodies are retained at the control zone and a red line appears. This test indicates the absence of hCG in the urine sample.

Abstract

La présente invention se rapporte à un dispositif d'essai permettant de détecter la présence ou l'absence d'une substance à analyser sélectionnée dans un échantillon liquide. Le dispositif d'essai contient un élément réactif, un support poreux et une zone de détection. L'élément réactif comprend un corps, un premier réactif de liaison marqué, spécifique d'un premier site de liaison de ladite substance à analyser, et un second réactif de liaison marqué, spécifique d'un seconde site de liaison de ladite substance à analyser. Lesdits premier et second sites de liaison spécifiques sont différents. Ledit corps de réactif est adapté pour retenir lesdits premier et second réactifs de liaison spécifiques marqués lorsque le corps et les premier et second réactifs de liaison sont secs, et pour les libérer lorsqu'ils sont humides. Les premier et second réactifs de liaison spécifiques marqués peuvent former un premier complexe marqué avec ladite substance à analyser, ledit complexe contenant le premier réactif de liaison spécifique marqué, la substance à analyser et le second réactif de liaison spécifique marqué. La zone de détection contient une première barrière poreuse présentant une taille de pore moyenne supérieure au diamètre le plus important du premier ou second réactif de liaison spécifique marqué, mais inférieure au diamètre dudit premier complexe marqué. La zone de détection peut être une section du support poreux ou peut être séparé du support poreux tout en étant en communication fluidique avec ce dernier. L'élément réactif, le support poreux et la zone de détection sont disposés de manière qu'un fluide appliqué au dispositif d'essai circule séquentiellement de l'élément réactif au support poreux puis à la zone de détection. Le dispositif d'essai est généralement sec avant utilisation et humide en cours d'utilisation.
PCT/US2004/007231 2003-03-10 2004-03-09 Dispositif et procede de dosage WO2004081528A2 (fr)

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US45502903P 2003-03-10 2003-03-10
US60/455,029 2003-03-10
US45374103P 2003-03-11 2003-03-11
US60/453,741 2003-03-11
US45397703P 2003-03-12 2003-03-12
US60/453,977 2003-03-12

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EP2623982A4 (fr) * 2010-09-30 2014-03-26 Sekisui Medical Co Ltd Bâtonnet diagnostique pour immunochromatographie, et procédé pour sa production
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US10006911B2 (en) 2014-03-07 2018-06-26 The Regents Of The University Of California Methods and devices for integrating analyte extraction, concentration and detection
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
US10578616B2 (en) 2014-03-07 2020-03-03 The Regents Of The University Of California Methods and devices for integrating analyte extraction, concentration and detection
US11635432B2 (en) 2014-03-07 2023-04-25 The Regents Of The University Of California Methods and devices for integrating analyte extraction, concentration and detection
US10359423B2 (en) 2014-03-07 2019-07-23 The Regents Of The University Of California Methods and devices for integrating analyte extraction, concentration and detection
US11885803B2 (en) 2015-09-04 2024-01-30 The Regents Of The University Of California Methods and devices for analyte collection, extraction, concentration, and detection for clinical applications
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
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
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
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
US11867693B2 (en) 2017-03-27 2024-01-09 The Regents Of The University Of California Semi-quantitative lateral-flow immunoassay for the detection of CSF leaks

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