WO1998023958A1 - Dispositifs d'analyse competitive chromatographique a flux lateral utilisant des liaisons specifiques, kits et procedes correspondants - Google Patents

Dispositifs d'analyse competitive chromatographique a flux lateral utilisant des liaisons specifiques, kits et procedes correspondants Download PDF

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Publication number
WO1998023958A1
WO1998023958A1 PCT/US1997/021831 US9721831W WO9823958A1 WO 1998023958 A1 WO1998023958 A1 WO 1998023958A1 US 9721831 W US9721831 W US 9721831W WO 9823958 A1 WO9823958 A1 WO 9823958A1
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Prior art keywords
zone
reagent
reaction zone
capture reagent
comprised
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PCT/US1997/021831
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English (en)
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Schleicher & Schuell, Inc.
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Priority to AU56883/98A priority Critical patent/AU5688398A/en
Publication of WO1998023958A1 publication Critical patent/WO1998023958A1/fr

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

Definitions

  • the present invention relates to a competitive lateral flow chromatographic assay device that uses a number of specific binding reactions for determining the presence or amount of an analyte in a test sample.
  • the present device affords greater readability for a user, allowing even inexperienced users to identify correctly positive or negative results. There is less chance of error.
  • the device comprises four serially connected zones with diffusible and attached reagents disposed in at least two of the four zones. There are four main embodiments, each with at least one diffusible reagent, at least one attached specific binding reagent that serves as an analyte test area, and at least one attached specific binding reagent that serves as a device control area. Also disclosed are methods for using such devices and kits employing such devices.
  • Immunochromatographic assay devices are known to the art.
  • U.S. 4,168,146 discloses a basic sandwich immunochromatographic assay.
  • a porous support has antibodies covalently bound thereto.
  • An antigen bearing sample is applied to the support and migrates to the reaction site. After a specific binding reaction has occurred, the strip is wet with labeled antibodies that form a sandwich with the bound antibody/ antigen complex.
  • the label can be either a fluorescent component or an enzyme.
  • U.S. 4,235,601 and U.S. 4,361,537 both to Deutsch et al., disclose a competitive immunochromatographic test device having multiple reaction zones.
  • the first zone is a sample application area.
  • the second zone is a first reagent area wherein the first reagent can move along with sample into a third zone.
  • a second reagent interacts with the first reagent to form a product which is measured.
  • Competition between the first reagent and the sample reduces the signal.
  • the end of the strip must be immersed into a developing liquid which serves to transport the sample and the first reagent into the reaction zone.
  • U.S. 4,366,241 to Tom et al. discloses an immunochromatographic test device having a concentrating zone for heterogeneous sandwich immunoassays.
  • An immunosorbing zone characterizes the device. This zone comprises either an antigen or an antibody non- diffusively bound to an area that serves as the inlet port for a bibulous support.
  • a sample is deposited in the zone, followed by a solution of a labeled specific binding partner to the non-diffusively bound antigen or antibody.
  • U.S. 4,435,504 to Syva Co. discloses an rajmunochromatographic test device having a two enzyme indication system for determining an analyte wherein a specific binding partner and a second enzyme are non-diffusively bound to a bibulous support in a reaction zone.
  • a first enzyme-labeled specific binding partner is applied to the reaction zone along with the sample.
  • the unbound enzyme-labeled partner binds in the reaction zone in relation to the analyte bound in the reaction zone.
  • the first enzyme and second enzyme are related in that the substrate of one is the product from the reaction of another.
  • a signal is generated by applying a substrate.
  • U.S. 4,446,232 to Liotta discloses an immunochromatographic ELISA test device having two zones with bound antigens or antibodies.
  • the first zone has bound antigens and enzyme labeled antibodies which can react with the bound antigens.
  • the antibodies are positioned within the zone such that they will be removed from the first zone into a second zone if unbound antigens from a sample react, and sample fluids transport the resulting unbound complexes into the second zone, but will not move in the absence of unbound antigens. This means that the antibodies are placed upstream of the bound antigens and react with them. If no antigen is present in the sample, then the bound antigen captures all of the labeled antibodies.
  • the second zone contains enzyme substrate.
  • a labeled reagent with its own detectable property is immobilized in a detection zone without generating a separately migrating species.
  • a reagent zone comprises a solid, porous matrix with an immobilized form of either the analyte, an analog thereof, or the analyte specific binding partner.
  • the labeled reagent may be either applied to the device at the reagent zone or, optionally, two reagent zones may be used, the labeled reagent being deposited in the first reagent zone and the immobilized binding partner in a second. Labeled reagent is complementary to the immobilized component.
  • the reagent zone has immobilized analyte or analog.
  • the reagent zone has immobilized antibody specific for the analyte or analog.
  • the second zone (or in some cases third zone), is a detection zone. This zone also has a solid, porous matrix, as well as an immobilized form of a binding substance for the labeled reagent, such as an ion exchange material, or a specific binder, such as immobilized anti-antibody.
  • U.S. 4,956,302 to Gordon et al. discloses a lateral flow immunochromatographic device.
  • a chromatographic medium has a pre-filtering zone for particulate matter in samples such as with whole blood, a reaction zone, and a downstream zone.
  • the reaction zone has two components therein.
  • the upstream component is a diffusible, labeled specific binding material.
  • the downstream component is an immobilized reagent capable of binding either analyte or the labeled specific binding material. One detects labeled specific binding material at the downstream site in the reaction zone.
  • Upstream from the pre-filtering zone of the medium is a sample application means, either a well or an absorbent pad.
  • Downstream from the medium is a liquid absorption means.
  • U.S. 5,030,558 to Litman et al. discloses a competitive immunochromatographic assay with a reaction zone separate from the detection zone.
  • a test solution is made containing sample and predetermined amounts of two or more first specific binding partners which are analogous to the analyte and are labeled.
  • This solution is deposited onto a reaction section of bibulous material, on which there already is immobilized predetermined amounts of two or more second specific binding partners capable of binding either analyte or first specific binding partners.
  • First specific binding partners not bound by the second specific binding partners move downstream and are detected there, increasing with increased analyte.
  • U.S. 5,104,793 to Buck discloses a multi-zone sandwich immunochromatographic assay and devices therefor.
  • the method requires the use of a three reaction zone strip.
  • the first zone has labeled antibody deposited thereon which can form a complex with the analyte.
  • the second zone contains a mobile inhibitor for the label on the complex.
  • the third zone has a bound capture antibody which forms a sandwich with the labeled complex.
  • the label can be enzymatic or fluorescent.
  • U.S. 5,1 6,952 also to Litman et al. discloses a multi-zone competitive immunochromatographic assay and devices therefor which have predetermined threshold analyte levels.
  • a test solution is made containing sample and predetermined amounts of two or more labeled first specific binding partners which are analogous to the analyte. This solution is deposited onto a contact section. Either in the contact section or downstream there is immobilized predetermined amounts of two or more second specific binding partners capable of binding either analyte or first specific binding partners. In the presence of analyte, first specific binding partners not bound by the second specific binding partners move downstream and are detected there, increasing with increased analyte.
  • U.S. 5,229,073 to Lao et al. discloses a one-step competitive immunochromatographic assay using multiple reaction stripes to quantify lipoprotein analyte amounts.
  • the strip has a depositing zone, with labeled lipoprotein present, and multiple captures zones spaced apart downstream, each with immobilized anti-lipoprotein antibody. The more analyte present, the more stripes having labeled analyte appear.
  • a competitive lateral flow, specific binding chromatographic assay device for determining the presence or amount of an analyte in a test sample comprises four serially connected zones with particular specific binding reagents disposed in particular zones.
  • the present device affords greater readability for a user, allowing even inexperienced users to correctly identify positive or negative results. There is less chance of error.
  • Analytes suitable for the present invention include small organic compounds and other antigenic substances such as herbicides, hormones, microorganisms, nucleic acids, organic compounds, pesticides, proteins, peptides, steroids, therapeutics, toxins, and vitamins.
  • specific binding reagents refer to molecules or complexes that specifically bind to another molecule or complex.
  • the term includes reagents involved in antigen/antibody reactions, biotin/avidin reactions, carbohydrate/lectin reactions, complementary nucleic acid sequence reactions, effector/receptor reactions, enzyme cofactor/enzyme reactions, and the like.
  • the first zone is an application zone for receiving a test sample solution.
  • the application zone is comprised of a fluid transport material, such as cellulosics, glass fibers, polyesters, woven or non-woven rayons, as well as bibulous materials such as filter paper. These materials may contain binders for added strength, as known to the art. Reagents that help the release or diffusibility of analyte can be added to this zone. These include blocking agents, sample stabilizers, or solubilizing agents such as proteins, polysaccharides, and surfactants, (such as, anionic, ionic, non-ionic, or zwitterio ic surfactants).
  • blocking agents can aid in the resolubilization and movement of immunoreagents from one zone into another zone within the device. Also, they can discourage non-specific binding of reagents to the chromatographic material in the reaction zone. Blocking agents present in the application zone can move along with analyte or analyte complexes through the chromatographic material.
  • a "fluid transport material" includes materials that support capillary movement of liquids placed thereon, but does not necessarily have a pore structure or surface chemistry that separates proteins chromatographically.
  • the second zone is a reagent zone comprised of a fluid transport material having a proximal end and a distal end.
  • the proximal end is connected to the application zone.
  • Diffusible specific binding reagents that participate in competitive specific binding reactions are disposed in this zone. These include labeled conjugates, i.e., a signal component attached to an analyte analog by conventional means, (such as covalent or non-covalent forces), and competitive specific binding reagents (such as antibody/antigens), i.e., a reagent that can form a complex with either the analyte or the labeled conjugate.
  • Suitable signal components or labels include, chromagens, colloidal metallic particles, colloidal non-metallic particles, colored latex particles, enzymes, fluorescers, luminescence, stains, and the like.
  • the third zone is a reaction zone comprised of a chromatographic material, having a proximal end and a distal end. The proximal end is in contact with the distal end of the reagent zone.
  • chromatographic materials include porous materials capable of transporting the sample solution and reagents from the reagent zone and supporting an irreversible specific binding reaction, either directly or through chemical modifications known to the art.
  • suitable materials include, but are not limited to, cellulosics (such as nitrocellulose), nylon membranes, and polyvinylidene fluoride membranes which are porous, preferably from about 0.05 microns to 15 microns, and wettable, as known to the art.
  • At least two capture reagents are attached to the chromatographic material in the reaction zone.
  • a first capture reagent can bind specifically either to the analyte, a labeled conjugate, to a complex of a labeled conjugate and a competitive specific binding reagent, or (in some versions of the present invention, if analyte is present in the test sample), to a complex of the competitive specific binding reagent and analyte. The presence of analyte will diminish this signal.
  • a second capture reagent can bind specifically to the labeled conjugate, at a site different than that of the first capture reagent.
  • the capture reagents can be disposed in many formats, including being disposed as a series of lateral lines across the advancing fluid front or as a series of non- lateral dots, symbols, et cetera, which are equidistant from the sample application zone.
  • a third capture reagent is used in a control area or line to aid in interpretation of the assay result. The inclusion of a third capture reagent enables one to incorporate both a positive control line as well as a negative control line into a single assay device.
  • control lines greatly enhances the readability of the assay by a lay person because one can compare a test result with both a relatively weak and a relatively strong signal, reducing errors in reading the result.
  • the placement of these capture reagents does not have to be in any necessary order, although preferred orders are described in detail in a following section.
  • the fourth zone in the device is an absorption zone comprised of a bibulous material that is in contact with the distal end of the reaction zone.
  • the bibulous material is capable of drawing solution from the reaction zone.
  • the volume of bibulous material present in the zone is sufiBcient to draw solution through the chromatographic material in the reaction zone.
  • Suitable bibulous materials include blotting papers, filter papers, non- woven natural polymers, and non-woven synthetic polymers. The dimensions of the materials will vary depending upon sample size and absorptive capacity, as known to the art.
  • the present invention including these four zones can be modified to include elements known to the art, such as pre-filters and sample flow control layers.
  • the general method for using the present specific binding chromatographic assay device is as follows. First, a test sample solution is applied to an application zone which contains buffer and a wetting agent, as described above. The volume of the sample solution is sufficient to wet the application zone, a reagent zone, a reaction zone, and be drawn into an absorption zone for at least one minute, the four zones being serially connected for fluid transport. Typically, these times will range from one minute to twenty minutes. Samples having a thicker viscosity, such as blood, will take longer times than more aqueous solutions. Sufficient time is allowed to pass for the sample solution to traverse the reagent zone and the reaction zone, and go into the absorption zone, all as described above.
  • the solution is drawn from the chromatographic material in the reaction zone, typically for at least one minute.
  • the reaction zone produces a first detectable signal related to the presence or amount of analyte.
  • a solution containing enzyme substrate must be added to the reaction zone in order to create the detectable signal.
  • the control area produces a second detectable signal, (and in some cases, a third detectable signal), which indicates a successful operation of the device, regardless of the presence of analyte.
  • a kit for determining the presence or amount of an analyte in a test sample comprises the specific binding chromatographic assay device as described above and a wetting solution which can be applied to the application zone of the specific binding chromatographic device, either alone or in combination with the test sample.
  • wetting solutions include solutions which can extract or resolubilize an analyte from a sample source.
  • This wetting solution is capable of wetting the application zone, the reagent zone, the reaction zone, and being absorbed into the absorption zone.
  • suitable wetting solutions include aqueous buffered solutions suitable for physiological samples as well as organic solvents for the extraction of non-aqueous samples.
  • Suitable analytes for the present invention may be found in physiological fluids, environmental fluids, soil samples, food processing fluids, or foodstuffs.
  • Physiological fluids include blood, lymphatic fluids, saliva, spinal fluid, sweat, urine, and the like.
  • conventional processing of these fluids may be needed to prepare a sample.
  • the sample may require filtration, extraction, or concentration.
  • interfering components present in the sample may need to be removed or inactivated.
  • FIGURE 1 is a isometric view of a preferred embodiment of the device illustrating the housing.
  • FIGURE 2 is an overhead view of the device with the upper portion of the housing removed.
  • FIGURE 3 is a diagrammatic view of a preferred embodiment of the present invention using one diffusible reagent and three reaction lines.
  • FIGURE 4 is an isometric view of both positive and negative results from the assay formats shown in FIGURES 3 and 5.
  • FIGURE 5 is a diagrammatic view of a preferred embodiment of the present invention using two diffusible reagents and three reaction lines.
  • FIGURE 6 is a diagrammatic view of a first and a second preferred embodiment of the present invention using two diffusible reagents and two reaction lines.
  • FIGURE 7 is an isometric view of both positive and negative results from the assay format shown in FIGURE 6.
  • Preferred embodiments of the present invention comprise enzyme-linked, competitive, multi-zone, lateral flow immunoassays that use horseradish peroxidase (HRP).
  • the assays are performed on a lateral flow strip (10) having four zones — a sample application zone (12), a reagent zone (14), an indicating reaction zone (16), and an excess absorbency zone (18).
  • the zones can be assembled onto a common structural backing material (20) that does not readily allow lateral flow of a solution (such as ARcare 7823, made by Adhesives Research, Inc. of Glen Rock, Pennsylvania, U. S. A.).
  • this assembly can then be placed inside a structural housing (22) having a first port (24) over the application zone which forms a well for placing the sample, and a second port (26) over the reaction zone so as to allow the user to view the signal from reagents thereon.
  • this device can be quite small, having a laminating strip of about 6 cm by 20 cm.
  • the assembly can be exposed for use as a dipstick.
  • the sample application zone is comprised of a glass fiber impregnated with a Tris buffered, (pH 7.5), saline solution (TBS) containing a surfactant, such as Triton® X-100 (Rohm & Haas), (0.01%-0.5%(w/v)), deposited thereon.
  • a surfactant such as Triton® X-100 (Rohm & Haas)
  • this glass fiber is No. 9254, made by Lydall, Inc. of Rochester, New Hampshire, U. S. A.
  • the reagent deposit zone also comprised of glass fiber, but not having any surfactant or TBS present. Diffusible reagents are deposited on or about the glass fiber.
  • reagents may be placed on the glass fiber through conventional dispensing means, preferably as a lateral line having an even concentration of reagent.
  • the indicating reaction zone is comprised of a nitrocellulose paper (NC) having a pore size of about 3 to 15 microns, (preferably SSLUTM made by Schleicher & Schuell, Inc. of Dassel, Germany), backed by a plastic for structural support.
  • Capture reagents are attached or immobilized here.
  • These reagents can be either antibodies which bind to diffusible reagents, analyte, or complexes of both.
  • the most distal zone, the excess absorbency zone is comprised of an absorbent material, such as 900 cellulose paper, (made by Schleicher & Schuell, Inc. of Keene, New Hampshire, U.S.A.).
  • the following examples of preferred embodiments of the present invention all involve assays for atrazine, a triazine pesticide, as an analyte.
  • the atrazine sample can come from water or be absorbed onto non-liquid materials, such as food stuffs, soil, or packaging.
  • non-liquid materials such as food stuffs, soil, or packaging.
  • conventional sample processing which is known to those of ordinary skill in the art must occur in order to solubilize any atrazine which may be present.
  • the ordinarily skilled artisan can also appreciate that the present invention is applicable to many analytes, as described above.
  • a diffusible reagent (32) comprised of an atrazine-horseradish peroxidase conjugate (AHRP) solution, is applied laterally across the reagent zone as a stripe.
  • the AHRP solution comprises 0.01 to 10 ug/ml, (preferably 8 ug/ml), of an AHRP conjugate, in a pH 7.5 Tris buffered solution also containing 0.05% to 10% (w/v), (preferably 1%) bovine serum albumen (BSA), 0.5% to 10% (w/v), (preferably 5%), sucrose, and 0.5% to 50% (w/v), (preferably 30%), trehalose.
  • a first capture reagent (34) is a rabbit anti-atrazine antibody (RAAA) solution in phosphate buffered saline (PBS), pH 7.2.
  • RAAA rabbit anti-atrazine antibody
  • PBS phosphate buffered saline
  • the RAAA solution can be made with an antibody available from Biostride, Inc. of Palo Alto, California.
  • Downstream from the first line is a second line.
  • This second line comprises a second capture reagent (36) which is an anti-HRP (A- HRP) solution in PBS, pH 7.2.
  • A-HRP solution can be made with a polyclonal goat antibody from Jackson ImmunoResearch Laboratories, Inc. of West Grove, Pennsylvania, U. S.
  • This second capture reagent is distal to the first capture line by a distance that allows a clear differentiation between signal readouts when read by the user and to provide for no interference between reactions, about 5 mm.
  • the binding capacity of the deposited second capture reagent is balanced so as to capture enough signal component to produce a readable signal at the threshold limit for analyte detection for the assay. While typically for atrazine this would be about 3 ppb, one can adjust the readout to detect either greater or lesser threshold amounts.
  • a third capture reagent (38) is also an A-HRP solution similar to the second capture reagent except that the concentration is higher so as to result in the capture of more signal molecule.
  • the binding capacity of the deposited third capture reagent is balanced so as to be able to capture enough signal component to produce a readable signal that substantially matches the readable signal produced by the first capture reagent when no analyte is present in the sample.
  • This third capture reagent is distal to the second capture line by about 2 mm. All three capture reagents are attached to the nitrocellulose through the particular protein binding surface characteristics of nitrocellulose. If another chromatographic material was selected, then, in certain cases known to the art, conventional immobilization techniques may be needed. Four drops (0.15 to 0.20 ml) of sample (30) are added to the application zone, in these examples it is an aqueous solution having from 0 to 20 ng/ml of atrazine (A).
  • the AHRP As the sample moves into the reagent zone, the AHRP is solubilized into the sample solution. Within about 5 to 7 seconds after sample application, the solution is swept into the reaction zone. The AHRP and A compete to bind to the RAAA in the first line. The amount of AHRP deposited in the reagent zone is sufficient such that if no A is present in the sample, then there is excess AHRP that can sweep past the first line and react and bind with both the second capture reagent and the third capture reagent. The second and third lines in the reaction zone serve as end of assay indicators. After a specified time, (with HRP as the signal component the time is about five minutes to twenty minutes), an HRP substrate such as a tetramethyl benzidine (TMB) solution is added to the strip by means of dispensing into the viewing port (26).
  • TMB tetramethyl benzidine
  • the intensity of this first line will vary, unlike the intensities of the more distal lines.
  • the first capture reagent has more AHRP than A bound to it, making the color intensity closer to that produced by the combination of the third capture reagent and A-HRP than to the color intensity produced by the combination of the second capture reagent and A- HRP.
  • Color intensity is indicated by the width of the lines.
  • a true positive result also is shown in Figure 4. Now, the first and second lines are approximately the same in color intensity, (indicated by the width of the lines), while the third line is noticeably more intense.
  • a first diffusible reagent (31) comprised of an RAAA solution, is impregnated laterally across the reagent zone as a first stripe.
  • the RAAA solution comprises 0.01 mg/ml to 10 mg/ml of RAAA in a PBS solution, pH 7.5, also containing about 5% to 25%, (preferably 20%), w/v glycerol and about 0.5% to 10%, (preferably 5%), w/v sucrose.
  • This solution is applied to the reagent zone at a defined rate, usually dispensing a final volume of 0.1 ul/cm to 10 ul/cm.
  • a second diffusible reagent (32) comprised of an AHRP solution as described above, is applied laterally across the reagent zone as a second stripe, distal to the first stripe by about 3 mm.
  • a first capture reagent (34) is a goat anti-rabbit Ig (GARIG) solution containing about 0.01 mg/ml to 10 mg/ml of GARIG in a PBS solution, pH 7.2. (The GARIG solution is made with an antibody from Schleicher & Schuell, Inc. of Keene, New Hampshire, U.S.A.).
  • a second line is downstream. This second line comprises a second capture reagent (36) is an A-HRP solution similar to that described above. This second capture reagent is distal to the first capture line by a distance that allows a clear differentiation between signal readouts when read by the user and provides for no interference between reactions, about 5 mm..
  • the binding capacity of the deposited second capture reagent is balanced so as to capture enough signal component to produce a readable signal at the threshold limit for analyte detection for the assay. While typically for atrazine this would be about 3 ppb, one can adjust the readout to detect either greater or lesser threshold amounts.
  • a third capture reagent (38) is also an A-HRP solution similar to the second capture reagent except that the concentration is higher so as to result in the capture of more signal molecule.
  • the binding capacity of the deposited third capture reagent is balanced so as to be able to capture enough signal component so as to produce a readable signal that substantially matches the readable signal produced by the first capture reagent when no analyte is present in the sample.
  • This third capture reagent is distal to the second capture line by about 2 mm. All three capture reagents are attached to the nitrocellulose through the particular protein binding surface characteristics of nitrocellulose. If another chromatographic material was selected, then, in certain cases known to the art, conventional immobilization techniques may be needed.
  • sample (30) Four drops (0.15 ml to 0.20 ml) are added to the application zone, in these examples it is an aqueous solution having 0 ng/ml to 20 ng/ml of atrazine (A).
  • A atrazine
  • the RAAA and the AHRP are solubilized into the sample solution.
  • a competition starts between AHRP and A for binding with the RAAA so as to form either RAAA- A complex or RAAA- AHRP complex.
  • the solution is swept into the reaction zone.
  • the amount of AHRP deposited in the reagent zone is sufficient such that if no A is present in the sample, then there is excess AHRP complex that can sweep past the first line and react and bind with both the second capture reagent and the third capture reagent.
  • the second and third lines in the reaction zone serve as end of assay indicators.
  • TMB is added to the strip as drops into the viewing port (26).
  • the first and second lines are approximately the same in color intensity, (indicated by the width of the lines), while the third line is noticeably more intense.
  • increasing the amount of analyte present in the sample intensifies the end-of-assay line and diminishes the intensity of color development in the first line.
  • the readability of the assay is enhanced by the comparative function. The user simply has to decide whether the first line looks closer to the second or the third line.
  • FIG. 6 A first version of the present invention having two diffusible reagents and two reaction lines is illustrated in Figure 6.
  • a first capture reagent (34) is a goat anti-rabbit Ig (GARIG) solution as described above. Downstream from the first line is a second line. This second line comprises a second capture reagent (36) which is an A-HRP solution similar to that described above. This second capture reagent is distal to the first capture line by a distance that allows a clear differentiation between signal readouts when read by the user and provides for no interference between reactions, about 5 mm. The binding capacity of the deposited second capture reagent is balanced so as to capture enough signal component to produce a readable signal that substantially matches the readable signal produced by the first capture reagent when no analyte is present. Both capture reagents are attached to the nitrocellulose through the particular protein binding surface characteristics of nitrocellulose. If another chromatographic material was selected, then, in certain cases known to the art, conventional immobilization techniques may be needed.
  • GARIG goat anti-rabbit Ig
  • sample (30) Four drops (0.15 ml to 0.20 ml) are added to the application zone, in these examples it is an aqueous solution having 0 ng/ml to 20 ng/ml of atrazine (A).
  • A atrazine
  • the RAAA and the AHRP are solubilized into the sample solution.
  • a competition starts between AHRP and A for binding with the RAAA so as to form either RAAA-A complex or RAAA-AHRP complex.
  • the solution is swept into the reaction zone.
  • the amount of AHRP deposited in the reagent zone is sufficient such that if no A is present in the sample, then there is excess AHRP complex that can sweep past the first line and react and bind with the second capture reagent.
  • the second line in the reaction zone serves as an end of assay indicator. After a specified time, TMB is added to the strip as drops into the viewing port (26).
  • a first capture reagent (34) is a goat anti-rabbit Ig (GARIG) solution as described above. Downstream from the first line is a second line. This second line comprises a second capture reagent (36) which is an RAAA solution similar to that described above. This second capture reagent is distal to the first capture line by a distance that allows a clear differentiation between signal readouts when read by the user and provides for no interference between reactions, about 5 mm. The binding capacity of the deposited second capture reagent is balanced so as to capture enough signal component to produce a readable signal that substantially matches the readable signal produced by the first capture reagent when no analyte is present. Both capture reagents are attached to the nitrocellulose through the particular protein binding surface characteristics of nitrocellulose. If another chromatographic material was selected, then, in certain cases known to the art, conventional immobilization techniques may be needed.
  • GARIG goat anti-rabbit Ig
  • sample (30) Four drops (0.15 ml to 0.20 ml) are added to the application zone, in these examples it is an aqueous solution having 0 to 20 ng/ml of atrazine (A).
  • A atrazine
  • the RAAA and the AHRP are solubilized into the sample solution.
  • a competition starts between AHRP and A for binding with the RAAA so as to form either RAAA-A complex or RAAA-AHRP complex.
  • the solution is swept into the reaction zone.
  • the amount of AHRP deposited in the reagent zone is sufficient such that if no A is present in the sample, then there is excess AHRP complex that can sweep past the first line and react and bind with the second capture reagent.
  • the second line in the reaction zone serves as an end of assay indicator. After a specified time, TMB is added to the strip as drops into the viewing port (26).
  • the intensity of this first line will vary, unlike the intensity of the more distal line.
  • the first capture reagent has more RAAA-AHRP complex than RAAA-A bound to it.
  • Color intensity is indicated by the width of the lines.
  • the first and second lines are approximately the same in color intensity, (indicated by the width of the lines).
  • a true positive result also is shown in Figure 7.
  • the first and second lines are of noticeably different color intensities, (indicated by O
  • the second line being noticeably more intense.
  • increasing the amount of analyte present in the sample intensifies the end-of-assay line and diminishes the intensity of color development in the first line.
  • capture reagents can be changed, and the present assays are still functional.
  • a negative readout would have a less intense line most proximal, followed by two equally more intense lines.
  • a positive readout would also start with a less intense line, followed by a second less intense line, then a more intense, most distal line.

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Abstract

La présente invention se rapporte à un dispositif d'analyse compétitive chromatographique à flux latéral qui utilise un certain nombre de liaisons spécifiques pour établir la présence ou la quantité d'un analyte dans un échantillon d'essai. Le dispositif de la présente invention apporte une plus grande facilité de lecture à l'utilisateur, et il permet même à des utilisateurs sans expérience d'identifier correctement des résultats positifs ou négatifs. Le risque d'erreur est ainsi diminué. Ledit dispositif comporte quatre zones reliées en série, dotées de réactifs fixés et diffusibles, disposés au sein d'au moins deux desdites zones. Il existe quatre réalisations principales de l'invention, chacune comportant au moins un réactif diffusible, au moins un réactif attaché et destiné à une liaison spécifique, qui sert de zone d'analyse de l'analyte, et au moins un réactif attaché et destiné à une liaison spécifique, qui sert de zone de régulation du dispositif. L'invention se rapporte également à des procédés d'utilisation de ces dispositifs et à des kits utilisant de tels dispositifs.
PCT/US1997/021831 1996-11-27 1997-11-26 Dispositifs d'analyse competitive chromatographique a flux lateral utilisant des liaisons specifiques, kits et procedes correspondants WO1998023958A1 (fr)

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AU56883/98A AU5688398A (en) 1996-11-27 1997-11-26 Competitive lateral flow, specific binding chromatographic assay devices, kits , and methods

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6365417B1 (en) 2000-02-09 2002-04-02 A-Fem Medical Corporation Collection device for lateral flow chromatography
US6998273B1 (en) 2000-02-09 2006-02-14 A-Fem Medical Corporation Collection device for lateral flow chromatography
WO2006036212A2 (fr) * 2004-04-22 2006-04-06 Medtox Scientific, Inc. Edulcorants sans sucre a utiliser dans des dispositifs d'essai
WO2012097788A1 (fr) * 2010-12-03 2012-07-26 Ralf Hilfrich Test rapide pour la détermination qualitative et/ou quantitative d'anticorps contre les papillomavirus humains contenus dans un fluide corporel et dispositif pour la mise en œuvre du test rapide
WO2014025715A3 (fr) * 2012-08-08 2015-07-16 Paul Saunders Appareil de chromatographie compact à multiples milieux
CN106526173A (zh) * 2016-10-31 2017-03-22 广州科方生物技术股份有限公司 一种用于免疫层析试纸条的包被液及其制备方法
CN108267603A (zh) * 2018-03-28 2018-07-10 韶关学院 基于信号放大***的双氯芬酸量子点免疫层析检测卡及检测方法
KR20200019234A (ko) * 2017-06-28 2020-02-21 벡톤 디킨슨 앤드 컴퍼니 고농도의 분석물을 포함한 분석물을 측정하기 위한 용량 반응 곡선의 신호 부분을 감소시키는 단계를 사용하는 샌드위치-유형 분석법
US20200348298A1 (en) * 2018-01-27 2020-11-05 Becton, Dickinson And Company Multiplex lateral flow assay for differentiating bacterial infections from viral infections
US11630106B2 (en) 2017-05-19 2023-04-18 Philip Morris Products S.A. Diagnostic test for distinguishing the smoking status of a subject
EP4080212A4 (fr) * 2019-12-16 2024-02-28 Proteometech Inc. Bandelette immunochromatographique et kit, et procédé d'analyse immunochromatographique compétitive les utilisant

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US4959307A (en) * 1986-09-05 1990-09-25 Syntex (U.S.A.) Inc. Immunoseparating strip
US5156953A (en) * 1979-12-26 1992-10-20 Syntex (U.S.A.) Inc. Simultaneous calibration heterogeneous immunoassay

Patent Citations (2)

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US5156953A (en) * 1979-12-26 1992-10-20 Syntex (U.S.A.) Inc. Simultaneous calibration heterogeneous immunoassay
US4959307A (en) * 1986-09-05 1990-09-25 Syntex (U.S.A.) Inc. Immunoseparating strip

Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6365417B1 (en) 2000-02-09 2002-04-02 A-Fem Medical Corporation Collection device for lateral flow chromatography
US6998273B1 (en) 2000-02-09 2006-02-14 A-Fem Medical Corporation Collection device for lateral flow chromatography
US7488606B2 (en) 2000-02-09 2009-02-10 Quantrx Biomedical Corporation Collection device for lateral flow chromatography
WO2006036212A2 (fr) * 2004-04-22 2006-04-06 Medtox Scientific, Inc. Edulcorants sans sucre a utiliser dans des dispositifs d'essai
WO2006036212A3 (fr) * 2004-04-22 2006-10-12 Medtox Scient Inc Edulcorants sans sucre a utiliser dans des dispositifs d'essai
US10605809B2 (en) 2010-12-03 2020-03-31 Abviris Deutschland Gmbh Rapid test for the qualitative and/or quantitative analysis of antibodies against human papilloma viruses (HPV) present in body fluid, and device for carrying out the rapid test
WO2012097788A1 (fr) * 2010-12-03 2012-07-26 Ralf Hilfrich Test rapide pour la détermination qualitative et/ou quantitative d'anticorps contre les papillomavirus humains contenus dans un fluide corporel et dispositif pour la mise en œuvre du test rapide
US9568480B2 (en) 2010-12-03 2017-02-14 Ralf Hilfrich Rapid test for the qualitative and/or quantitative analysis of antibodies against Human Papilloma Viruses (HPV) present in body fluid, and device for carrying out the rapid test
US10006914B2 (en) 2010-12-03 2018-06-26 Abviris Deutschland Gmbh Rapid test for the qualitative and/or quantitative analysis of antibodies against human papilloma viruses (HPV) present in body fluid, and device for carrying out the rapid test
EP3413048A1 (fr) * 2010-12-03 2018-12-12 Abviris Deutschland GmbH Essai rapide de détermination qualitative et / ou quantitative des anticorps contre les papillomavirus humain contenus dans le fluide corporel ainsi que dispositif de mise en uvre dudit essai rapide
CN104823046A (zh) * 2012-08-08 2015-08-05 保罗·桑德斯 紧凑型多介质色谱
WO2014025715A3 (fr) * 2012-08-08 2015-07-16 Paul Saunders Appareil de chromatographie compact à multiples milieux
CN106526173A (zh) * 2016-10-31 2017-03-22 广州科方生物技术股份有限公司 一种用于免疫层析试纸条的包被液及其制备方法
CN106526173B (zh) * 2016-10-31 2018-04-17 广州科方生物技术股份有限公司 一种用于免疫层析试纸条的包被液及其制备方法
US11630106B2 (en) 2017-05-19 2023-04-18 Philip Morris Products S.A. Diagnostic test for distinguishing the smoking status of a subject
KR20200019234A (ko) * 2017-06-28 2020-02-21 벡톤 디킨슨 앤드 컴퍼니 고농도의 분석물을 포함한 분석물을 측정하기 위한 용량 반응 곡선의 신호 부분을 감소시키는 단계를 사용하는 샌드위치-유형 분석법
JP2020526742A (ja) * 2017-06-28 2020-08-31 ベクトン・ディキンソン・アンド・カンパニーＢｅｃｔｏｎ， Ｄｉｃｋｉｎｓｏｎ Ａｎｄ Ｃｏｍｐａｎｙ 高濃度の分析物等、分析物を測定するための用量反応曲線の減少信号部分を使用したサンドイッチ型アッセイ
US20210405044A1 (en) * 2017-06-28 2021-12-30 Becton, Dickinson And Company Sandwich-type assays using decreasing signal portions of dose response curve to measure analytes, including analytes at high concentration
KR102660904B1 (ko) * 2017-06-28 2024-04-25 벡톤 디킨슨 앤드 컴퍼니 고농도의 분석물을 포함한 분석물을 측정하기 위한 용량 반응 곡선의 신호 부분을 감소시키는 단계를 사용하는 샌드위치-유형 분석법
US20200348298A1 (en) * 2018-01-27 2020-11-05 Becton, Dickinson And Company Multiplex lateral flow assay for differentiating bacterial infections from viral infections
CN108267603A (zh) * 2018-03-28 2018-07-10 韶关学院 基于信号放大***的双氯芬酸量子点免疫层析检测卡及检测方法
EP4080212A4 (fr) * 2019-12-16 2024-02-28 Proteometech Inc. Bandelette immunochromatographique et kit, et procédé d'analyse immunochromatographique compétitive les utilisant

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