WO1997034150A1 - Elements de liaison prenant naissance sur des particules et destines a des dosages immunologiques - Google Patents

Elements de liaison prenant naissance sur des particules et destines a des dosages immunologiques Download PDF

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Publication number
WO1997034150A1
WO1997034150A1 PCT/US1997/003886 US9703886W WO9734150A1 WO 1997034150 A1 WO1997034150 A1 WO 1997034150A1 US 9703886 W US9703886 W US 9703886W WO 9734150 A1 WO9734150 A1 WO 9734150A1
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WO
WIPO (PCT)
Prior art keywords
label
strip
antibody
specific binding
assay
Prior art date
Application number
PCT/US1997/003886
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English (en)
Inventor
Chi D. Chang
Mary K. Joseph
Michael J. Kaplan
Original Assignee
Abbott Laboratories
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Publication date
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Publication of WO1997034150A1 publication Critical patent/WO1997034150A1/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
    • G01N33/558—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
    • 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
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex

Definitions

  • This invention relates to colloidal particles that are useful for immunoassays, more particularly, immunochromatographic assays.
  • diagnostic assays can be used in diagnostic assays to determine the presence and/or amount of substances of interest or clinical significance in test samples, such as body fluids. These interesting or clinically significant substances are commonly referred to as analytes. Diagnostic assays have become an indispensable means for detecting analytes in test samples by using the reaction between the analyte and a specific binding member, as typified by the immunoreaction between an antigen and the antibody to that antigen.
  • tags or labels composed of a traceable substance that is attached to a specific binding member, such as an antibody, which, in turn, binds to the analyte to form a labeled antibody/analyte complex. The detection of the labeled antibody/analyte complex, or of the labeled antibody that remains unbound, indicates the presence and/or amount of the analyte in the test sample.
  • Assay techniques using metallic sol particles as labels have been developed.
  • particles of a metal e.g., gold, silver, platinum
  • a metal compound e.g., gold, silver, platinum
  • a metal compound e.g., gold, silver, platinum
  • a nonmetallic substance coated with a metal or a metal compound e.g., gold, silver, platinum
  • the specific binding member to be labeled is coated onto the metallic sol particles by adsorption.
  • Particulate labels in immunoassay reagents have also been formed from polymeric dyes.
  • Dye molecules i.e., chromogenic monomers, are polymerized to form colored polymeric particles.
  • the colored polymeric particles can then be linked to a specific binding member for use in an assay. Examples of such dyes include Congo red, Trypan blue, and Lissamine blue.
  • E1 G estrone glucuronide
  • An immunochromatographic assay strip consisting of E1 G immobilized on nitrocellulose and anti-E1 G antibody immobilized on colloidal particles can be used to quantify E1 G concentration in a competitive binding assay. Assay strips are described, for example, in U. S. Patent Nos.
  • the first three techniques have not resolved the problem resulting from temperature variation.
  • the fourth technique is feasible but excessively costly.
  • the fifth technique is workable only over a narrow temperature range. Therefore, it would be desirable to provide a simple technique for stabilizing the dose- response curve of E1 G for an assay for E1 G.
  • This invention provides a label comprising a colloidal particle having a spacer arm attached thereto and a specific binding member attached to the spacer arm.
  • the label is particularly useful as an indicator reagent in immunoassays.
  • the colloidal particle is preferably formed from a polymeric material or from a metallic sol.
  • a preferred material for the colloidal particle is polypyrrole.
  • the spacer arm is preferably a biological linking agent or a synthetic polymeric linking agent.
  • the spacer arm preferably has a high molecular weight, e. g., greater than about 1 ,000.
  • the specific binding member is preferably an antibody or an antigen.
  • An example of a preferred material for the specific binding member is anti-E1 G antibody.
  • the label of this invention exhibits a dose-response curve that varies only slightly over a wide temperature range, e.g., 13 °C to 37 °C when used in immunochromatographic assay strips.
  • FIG. 1 schematically illustrates anti-E1 G antibody attached to a colloidal particle via a spacer arm.
  • FIG. 2 illustrates one configuration of a strip suitable for use in the present invention.
  • FIG. 3 is a graph illustrating how assay results for a conventional antibody vary as a function of temperature.
  • FIG. 4 is a graph illustrating how assay results for an extended antibody of this invention vary as a function of temperature.
  • FIG. 5 is a graph illustrating how assay results for an extended antibody of this invention vary as a function of temperature.
  • FIG. 6 is a graph illustrating how assay results for an extended antibody of this invention vary as a function of temperature.
  • the assay used for preparing this figure employed an assay strip.
  • FIG. 7 is a graph illustrating how assay results for an extended antibody of this invention vary as a function of temperature.
  • the assay used for preparing this figure employed an assay strip.
  • FIG. 8 is a graph illustrating how assay results for an extended antibody of 0 this invention vary as a function of temperature.
  • the assay used for preparing this figure employed an assay strip.
  • Analyte means the substance to be detected in a test sample.
  • An analyte can be any substances for which there exists a naturally occurring specific binding member (e.g., an antibody) or for which a specific binding 0 member can be prepared.
  • the analyte can bind to one or more specific binding members in an assay.
  • Analyte also includes any antigenic substances, haptens, antibodies, and combinations thereof.
  • the analyte can include a protein, a peptide, an amino acid, a hormone, a steroid, a vitamin, a drug including those administered for therapeutic purposes as well as those administered for illicit purposes, a 5 bacterium, a virus, metabolites of or antibodies to any of the above substances, nucleic acids, and ribonucleic acids.
  • Specific binding member means a member of a specific binding pair.
  • a specific binding pair comprises two different molecules, wherein one of the molecules through chemical or physical means specifically binds to the 0 other molecule.
  • An example of a specific binding pair is the antigen/antibody specific binding pair.
  • other specific binding pairs include, but are not limited to, biotin and avidin, carbohydrates and lectins, complementary nucleotide sequences (including probe and capture nucleic acid sequences used in DNA hybridization assays to detect a 5 target nucleic acid sequence), complementary peptide sequences, effector and receptor molecules, enzyme cofactors and enzymes, enzyme inhibitors and enzymes, and the like.
  • specific binding pairs can include members that are analogues of specific binding members originally contemplated.
  • a derivative or fragment of the analyte i.e., an analyte-analogue
  • Immunoreactive specific binding members include antigens, haptens, antibodies, and complexes thereof, including those formed by recombinant DNA methods or peptide synthesis.
  • “Indicator reagent,” as used herein, means a detectable material, in particulate form, directly or indirectly attached to a specific binding member.
  • “Capture reagent,” as used herein, means a specific binding member capable of binding the analyte or indicator reagent.
  • the capture reagent can be directly or indirectly attached to a substantially solid material to form a solid phase capture reagent complex.
  • the solid phase capture reagent complex can be used to separate the bound and unbound components of the assay.
  • the label of this invention comprises a colloidal particle having a spacer arm attached thereto and a specific binding member attached to the spacer arm. In effect, the specific binding member is extended from the surface of the colloidal particle by means of the spacer arm.
  • Colloidal particles suitable for use in preparing the label of this invention can be made of polymeric material, metallic material, non-metallic material, aggregated dye material, and other materials conventionally used to prepare colloidal particles for use in immunoassays.
  • Materials that are preferred for the colloidal particles include polymers having pyrrole units, e.g., polypyrrole, polymers having styrene units, polymers having acrylate units, and colloidal metals, e.g., colloidal gold, colloidal selenium.
  • the size of the colloidal particles can range from about 10 nm to about 5,000 nm in average diameter.
  • Nonchromophoric monomer refers to an organic monomer that is neither a pigment nor a dye and that has color or absorbance characteristics that make the unpolymerized substance unsuitable for use as a detectable label.
  • the polymeric particles absorb light in the visible, ultraviolet, or infrared regions at one or more wavelengths at which the nonchromophoric monomer does not.
  • the absorbance characteristics of the polymeric particle result from the extensively conjugated structure arising from the polymerization process.
  • the polymeric particle derives its characteristic color or absorbance from its polymeric structure rather than from the color or absorbance of the monomer from which it is made or from the addition of a dye or a pigment, i.e., a chromogen.
  • a dye or a pigment i.e., a chromogen.
  • pure monomeric pyrrole is a substantially nonchromophoric substance, but the poly(pyrrole) latex particle is black, due to its extensively conjugated structure and unpaired delocalized electrons (stable radicals).
  • substantially nonchromophoric means that impurities that impart a color to the monomer may be present, although typically, the pure form of the monomer is transparent or colorless.
  • the polymeric latex particles of this invention can be divided into at least six classes.
  • the first class comprises polymeric particles made from a single nonchromophoric monomeric material.
  • the second class of polymeric particles comprises a composite of both nonchromophoric monomers, which, upon polymerization, form a colored polymer, and nonchromophoric monomers which, upon polymerization, generally do not form a colored polymer.
  • Composite particles can be formed by combining one of the above-described polymers, or oligomers thereof, with moldable polymeric materials, such as poly(vinyl chloride), polystyrene, and poly(vinyl toluene), as well as hydrophilic polymeric materials, such as poly(acrylamide) and poly(N-vinyl pyrrolidone), and derivatives thereof.
  • the composite particles of blended materials may facilitate processing of the particle labels.
  • the third class of polymeric particles includes polymeric particles having repeating units of at least two different nonchromophoric monomers.
  • copolymers can be formed and used as labels according to the present invention.
  • examples of copolymers include, but are not limited to, poly(pyrrole-co-thiophene) and poly(pyrrole-co-phenylene).
  • the organic monomers include: pyrrole, benzene, toluene, aniline, thiophene, naphthalene, thiophenol and equivalent aromatic monomers, acetylene and equivalent nonaromatic monomers, and derivatives thereof.
  • the fourth class of polymeric particles comprises an organic or inorganic nucleus that can be coated with a polymer made by the polymerization of a plurality of nonchromophoric monomers.
  • a metallic colloidal particle can be coated with the colored organic polymeric latex.
  • the fifth class of polymeric particles comprises a polymeric particle made of a plurality of nonchromophoric monomers polymerized to form a substantially colorless polymeric particle which, upon further reaction with a substance that is not a dye or pigment, will become a colored polymeric particle.
  • An example of such a polymeric particle is poly(phenylene sulfide), a normally transparent and substantially colorless polymeric material, which becomes blue-green and finally blue-black upon further reaction with arsenic pentafluoride.
  • the sixth class of polymeric particle comprises a polymeric particle made of a plurality of nonchromophoric monomers which, upon polymerization, form a particle having detectable free radicals, and the resulting particle can be colored or substantially colorless. While such colorless particles may not be visually detectable, the stable free radical nature of these particles enables their detection and measurement with an electron paramagnetic resonance spectrometer.
  • Spacer arms suitable for use in preparing the label of this invention can be made of biological linking groups or synthetic polymeric linking groups.
  • Materials that are preferred for the spacer arm include antibodies, protein A, protein G, avidin, biotin, hydrophilic polymers, such as, for example, polymers having vinyl alcohol units, e.g., polyvinyl alcohol, polymers having acrylate units, e.g., hydroxy alkyl acrylate polymers, polymers having alkylene glycol units, e.g., polyethylene glycol, polymers having amide units, e.g., polyamides, polymers having peptide units, e.
  • the spacer arm must be sufficiently long to minimize steric hindrance resulting from the surface of the colloidal particle; however, the spacer arm must not be so long that non- specific entangling will result.
  • the length of the spacer arm ranges from about 5 nm to about 100 nm.
  • the spacer arm preferably has a high molecular weight, e.g., greater than about 1 ,000.
  • the spacer arm can be a single entity or a combination of two or more entities linked together, preferably by covalent bonds. However, it is much simpler to use a single entity as a spacer arm.
  • Specific binding members suitable for use in preparing the label of this invention can be made of specific binding proteins.
  • Materials that are preferred for the specific binding member include antibodies, enzymes, receptors, antigens, haptens, and epitopes. It is relatively easy to attach specific binding members to spacer arms attached to colloidal particles by covalent bonds to form indicator reagents useful in the present invention.
  • the specific binding member can be attached to the spacer arms attached to the colloidal particles either by adsorption, a linking group, or a surface-functional group.
  • Covalent attachment of colloidal particles to spacer arms can be accomplished by using carboxylic, amino, aldehyde, bromoacetyl, iodoacetyl, thiol, epoxy, and other reactive or linking groups, as well as by using residual free radicals and radical cations, through which a protein coupling reaction can be accomplished.
  • the label of this invention can be prepared by applying a layer of material suitable for forming spacer arms to the surface of the colloidal particle.
  • the material for preparing the spacer arms can be adsorbed onto the surface of the colloidal particle. Adsorption can be enhanced by chemical treatment. For example, the addition of periodate to the colloidal particle enhances adsorption of spacer arms to the colloidal particle.
  • spacer arms become attached to the colloidal particle by means of crosslinking of aldehyde groups generated from the oxidation resulting from addition of periodate to the colloidal particle.
  • the colloidal particle to which is attached the spacer arms can be treated with the specific binding member.
  • the specific binding member can be attached to a spacer arm by immunocomplex formation or covalent linkage.
  • the specific binding member becomes attached to the spacer arm by means of immunocomplex formation.
  • FIG. 1 illustrates in schematic form how the label of the present invention can be prepared.
  • a component of a spacer arm e.g., anti-biotin
  • a specific binding member e.g., an antibody, such as, for example, anti-E1G antibody
  • an entity capable of specifically binding to the component of the spacer arm e.g., biotin
  • a spacer arm e.g., a polymeric material
  • a specific binding member e.g., an antibody, such as, for example, anti-E1 G antibody
  • the size of the colloidal particles can be varied in order to provide better assay results.
  • the colloidal particles preferably range in size from about 10 to about 5,000 nm, more preferably from about 50 nm to about 1 ,000 nm. Size of the colloidal particles can be controlled by using appropriate conditions for polymerization in the formation of the raw colloidal particles or by using an excess of specific binding member to prevent aggregation of the colloidal particles or by employing both conditions.
  • the colloidal particles can be washed by diafiltration, rather than by centrifugation. Further reduction of size of the colloidal particles can be achieved by sonication.
  • the label of this invention is preferably stored in a medium to protect specific reactivity of the specific binding member and to prevent nonspecific binding.
  • the medium may comprise proteins, carbohydrates, hydrophilic polymers, surfactants, salts, including buffer salts, and preservatives.
  • an assay device suitable for use with the present invention can have many configurations, several of which are dependent upon the material chosen for the solid phase.
  • the solid phase material can be any suitable chromatographic, bibulous, or porous material.
  • Solid-phase materials suitable for this invention include, but are not limited to, (1 ) a fiberglass, cellulose, or nylon pad for use in a flow-through assay device having one or more layers, containing one or more of the assay reagents, (2) a dipstick for a dip and read assay, (3) a test strip for chromatographic techniques (e.g., paper or glass fiber) or thin layer chromatographic techniques (e.g., nitrocellulose), in which the reagents are contained in separate zones of a single strip of solid phase material, or (4) an absorbent material well known to those skilled in the art.
  • the solid phase material can also include, but is not limited to, polyacrylamide beads, polystyrene beads or tubes, magnetic beads, a microtiter plate, and a glass or plastic
  • Natural, synthetic, or naturally-occurring materials that are synthetically modified can be used as a solid phase material.
  • the materials include polysaccharides, e.g., cellulose materials such as paper and cellulose derivatives such as cellulose acetate and nitrocellulose; silica; silicon particles; inorganic materials, such as deactivated alumina; or other inorganic particulate material uniformly dispersed in a porous polymeric matrix, with polymers such as, for example, vinyl chloride, copolymers of vinyl chloride and propylene, and a copolymer of vinyl chloride and vinyl acetate; cloth, both naturally occurring (e.g., cotton) and synthetic (e.g.
  • the solid phase material should have sufficient strength for immunoassay purposes or sufficient strength can be provided by means of a support.
  • the solid phase material should not interfere with the production of a detectable signal.
  • the labels of the present invention can be used in a variety of assay formats. In general, any assay configuration using specific binding members and a detectable label, such as, for example, organic polymeric latex particles, can be used. Specific binding assays are generally categorized into one of two major classes, homogeneous and heterogeneous assays.
  • reaction components or reagents are in the test solution and are not separated prior to the detection of the signal produced by the indicator reagent.
  • a solid phase material is used to allow the separation of bound from unbound reaction components, or a reagent of the initial solution is caused to precipitate and is subsequently removed from the test solution.
  • the indicator reagent of the present invention is detectable in both its bound and unbound forms and can be used in both homogeneous and heterogeneous assays. These assays may be further divided into sandwich and competitive assays, and variations thereof.
  • the label of this invention can be used as an indicator reagent in a competitive assay format in an immunochromatographic assay strip.
  • a typical assay strip can comprise a bibulous nitrocellulose membrane through which colloidal particles of sub-micron size can flow by capillary action.
  • the colloidal label will be captured by capture reagent immobilized on the nitrocellulose membrane.
  • the binding reaction between the analyte and the immobilized capture reagent will compete with the binding reaction between the colloidal label and the immobilized capture reagent on the membrane.
  • the zone will appear to be colored.
  • the optical density of the detection zone can be measured with a transmittance or reflectance light scanner that compares the optical density of the detection zone with background and a white reference standard. In some situations, results can be determined visually.
  • Computer driven micro-jetting process can be used to achieve penetration and homogeneous distribution of the capture reagent.
  • a typical assay strip will be described in greater detail in the working examples.
  • the label can be used for a self-performing assay with assay strips designed for immunoassays.
  • the label can be used in two modes — the wet mode and the dry mode. In the wet mode the label is mixed with the sample, the mixture is transferred to one end of the membrane strip directly or through a porous pad, and the label will flow spontaneously through the test strip. Typically, the test will be completed within 10 minutes, and often within five minutes. The results can be read either visually for qualitative results or instrumentally for quantitative results. However, it is preferable to use the label in the dry mode.
  • the label can be impregnated in a porous pad, dehydrated via lyophilization or dry air, attached to one end of a membrane strip, and covered with an overseal or case into an assembled test strip.
  • the assembled assay strip is a miniature test kit including all essential reagent components. The user needs only to add the test sample to the pad and wait a few minutes for the test result.
  • the present invention allows the manufacturer of self-performing assays and automated assays performable via instruments to provide a diagnostic assay that is capable of generating consistent results, regardless of variations in environmental conditions, such as temperature and humidity.
  • the label of this invention exhibits a dose-response curve that varies only slightly over a wide temperature range, e.g., 13 °C to 37 °C when used in immunochromatographic assay strips. Prior to this invention, dose-response curves varied as much as fifty- fold or greater over a wide temperature range, e.g., 13 °C to 37 °C when used in immunochromatographic assay strips. This invention reduces variation in dose- response curves over the aforementioned temperature range to no more than five-fold, and often no more than three-fold.
  • a solution containing monoclonal anti-E1 G antibody (1.5 mg antibody in 750 ⁇ L of 0.1 M sodium bicarbonate, pH 8.3) was magnetically stirred.
  • a solution containing biotin-amidocaproylamidocaproate N-hydroxysuccinimide ester (1.5 mg antibody in 750 ⁇ L of 0.1 M sodium bicarbonate, pH 8.3) was magnetically stirred.
  • the incorporated biotin was quantified by a fluorescence polarization immunoassay (FPIA) method on a TDx® instrument (Abbott Laboratories), and the biotin-to-lgG molar ratio was 2.0.
  • FPIA fluorescence polarization immunoassay
  • a preservative Proclin-300®, Rohm and Haas was added at a concentration of 0.1% to the final solution of biotinylated anti-E1 G antibody.
  • borate buffer (5 mL, 20 mM borate, pH 10, 0.2% Brij 35 solution) at a temperature of 45 °C (water-bath) for two hours.
  • BIS-TRIS/BSA buffer (1.25 mL, 0.25 M BIS-TRIS, pH 7.0, 4% BSA, 0.1% Brij 35 solution)
  • the reaction mixture was mixed for three hours at a temperature of 2 to 8 °C, then 0.3 M H 5 l ⁇ 6 (555 ⁇ L, Aldrich Chemical Company), freshly dissolved in 0.5 M triethanolamine was added to the reaction mixture, and the resulting mixture mixed overnight.
  • the colloidal particles were washed with a MOPS/BSA buffer (25 mM MOPS, pH 7.2, 25 mM ethanolamine, 2% BSA, 0.1 % Brij 35 solution, 0.05% Proclin-300® preservative) on a miniature diafiltration washing device (Microgon®), and finally dispersed in a MES/casein diluent (0.1 M MES, pH 6.1 , 5% hydrolyzed casein, 0.024% Triton® surfactant, 0.04% bovine IgG, 0.1 % Proclin-300® preservative).
  • the concentration of colloid was adjusted by light scattering to an optical density of 4 (wavelength at 560 nm).
  • Monoclonal anti-biotin IgG antibody (0.225 mg) was mixed with polypyrrole -5 colloid (0.75% solids) in borate buffer (1 mL, 20 mM borate, pH 10, 0.2% Brij 35 solution) at a temperature of 45 °C (water-bath) for two hours.
  • borate buffer (1 mL, 20 mM borate, pH 10, 0.2% Brij 35 solution
  • BIS-TRIS/BSA buffer 250 ⁇ L, 0.25 M BIS-TRIS, pH 7.0, 4% BSA, 0.1 % Brij 35 solution
  • the reaction mixture was mixed for three hours at a temperature of 2 to 8 C C, then 0.3 0 M H 5 l ⁇ 6 (111 ⁇ L, Aldrich Chemical Company), freshly dissolved in 0.5 M triethanolamine was added to the reaction mixture, and the resulting mixture mixed overnight.
  • the colloidal particles were spun down, washed with a MOPS/BSA buffer (25 mM MOPS, pH 7.2, 25 mM ethanolamine, 2% BSA, 0.1 % Brij 35 solution, 0.05% Proclin-300® preservative), exchanged with wash buffer 5 through two centrifugations and sonications, and finally dispersed in MOPS/BSA buffer.
  • MOPS/BSA buffer 25 mM MOPS, pH 7.2, 25 mM ethanolamine, 2% BSA, 0.1 % Brij 35 solution, 0.05% Proclin-300® preservative
  • Biotinylated anti-E1 G IgG antibody from Example I (800 ⁇ L, 0.464 mg) was introduced into 200 ⁇ L of the solution of anti-biotin colloid from Example III with 1 5 mL of the MOPS/BSA buffer, and the resulting mixture stirred at ambient temperature for 2 1/2 hours.
  • the colloid particles were spun down, washed with MOPS/BSA buffer, exchanged with wash buffer through two centrifugations and sonications, and finally dispersed in a MES/casein diluent (0.1 M MES, pH 6.1 , 5% hydrolyzed casein, 0.024% Triton® surfactant, 0.04% bovine IgG, 0.1 % Proclin-300® preservative).
  • the concentration of colloid was adjusted by light scattering to an optical density of 4 (wavelength at 560 nm).
  • Goat anti-mouse IgG antibody (0.53 mg) was mixed with polypyrrole (1 % solids) in borate buffer (4 mL, 20 mM borate, pH 10, 0.2% Brij 35 solution) at a temperature of 45 °C (water-bath) for two hours.
  • borate buffer (4 mL, 20 mM borate, pH 10, 0.2% Brij 35 solution) at a temperature of 45 °C (water-bath) for two hours.
  • BIS-TRIS/BSA buffer (1 mL, 0.25 M BIS-TRIS, pH 7.0, 4% BSA, 0.1% Brij 35 solution
  • the reaction mixture was mixed for three hours at a temperature of 2 to 8 °C, then 0.3 M H 5 I0 6 (555 ⁇ L, Aldrich Chemical Company), freshly dissolved in 0.5 M triethanolamine was added to the reaction mixture, and the resulting mixture mixed for 18 hours.
  • the colloidal particles were washed on a diafiltration washing device (Microgon®), washed with a MOPS/BSA buffer (25 mM MOPS, pH 7.2, 25 mM ethanolamine, 2% BSA, 0.1 % Brij 35 solution, 0.05% Proclin-300® preservative), with 10 volume exchanges with buffer.
  • MOPS/BSA buffer 25 mM MOPS, pH 7.2, 25 mM ethanolamine, 2% BSA, 0.1 % Brij 35 solution, 0.05% Proclin-300® preservative
  • Example VII Colloid coated with goat anti-mouse antibody from Example V (1 mL) was added to monoclonal anti-E1 G antibody in MOPS/BSA buffer (400 ⁇ g antibody/mL of buffer ) as above. The colloid was incubated overnight, and, without further wash, was diluted with the MES/casein diluent as Example IV to give a concentration corresponding to an optical density of 4 (wavelength of 560 nm).
  • an assay strip 10 includes a porous strip material 12 on which are located a sample mixing zone 14, a first capture zone 16, and a second capture zone 18.
  • the sample mixing zone 14 contains dried indicator reagent particles.
  • the indicator reagent particles contain first binding members that specifically bind to the analyte of interest.
  • the first capture zone 16, termed the "patient bar,” is a region of the porous strip material 12 in which the extent of reaction is a function of the concentration of the analyte of interest. More specifically, the first capture zone 16 contains second binding members attached to the strip material 12 that specifically bind to the first binding members. The second binding members compete with the analyte of interest for binding sites on the first binding member.
  • the extent of binding may be indicated by the level of optical reflectance at the first capture zone 16.
  • a second capture zone 18, termed the "control bar,” is also present.
  • the extent of binding is relatively insensitive to the concentration of the analyte of interest.
  • the second capture zone 18 contains third binding members attached to the porous strip material 12 that bind to the first binding members on the indicator reagent particles at sites unrelated to the analyte of interest.
  • the extent of binding in the patient bar 16 and the control bar 18 may be sensitive to factors such as the temperature at which the assay is performed and the age of the reagents within the porous strip material 12.
  • the function of the control bar is to indicate whether the reagents have deteriorated during storage.
  • the extent of binding in the control bar is inversely proportional to the level of deterioration.
  • the method of indicating the extent of binding in the second capture zone 18 will normally be the same as that for the first capture zone 16, although this is not necessary.
  • the assay utilizing the assay strip 10 shown in FIG. 2 is initiated when a known quantity of a liquid sample of a body fluid, such as a urine sample, suspected of containing the analyte of interest, is deposited on the sample mixing zone 14 of the porous strip material 12.
  • the first binding members on the indicator reagent particles tend to bind to the analyte of interest to an extent that is approximately proportional to the concentration of that analyte in the liquid sample.
  • the expression “flows through” means flows laterally through the strip material via the capillaries in the porous strip material.
  • the expression “flow- through” refers to an assay device wherein a flowable material flows through one or more layers of material. As used herein, the expressions "flow through”, “flows through”, and the like is intended to encompass all embodiments, whether they involve lateral flow or flow from one layer through another layer.
  • the extent of binding between the indicator reagent particles and the assay strip at the first capture zone is a function of the concentration of the analyte of interest in the sample of body fluid; the higher the concentration of the analyte of interest, the lower the extent of binding of the indicator reagent particles at the first capture zone.
  • the body fluid sample continues to flow through the porous strip material 12, carrying with it the remainder of the indicator reagent particles, to the second capture zone 18, i.e., the control bar.
  • control bar 18 At the control bar 18 are the third binding members fixed to the porous strip material 12 and that tend to bind to the first binding members on the indicator reagent particles.
  • the amount of binding at the control bar 18 is indicative of the condition of the assay and reagents, and may be used to adjust the quantification of binding at the patient bar 16 to correct for assay temperature and reagent aging.
  • Preformed immune complex of anti-E1 G antibody/anti-biotin polypyrrole colloid as described in Example IV was diluted in a MES/casein/sugar diluent (0.1 M MES, pH 6.1 , 2.5% hydrolyzed milk, 2% Trehalose, 0.012% Triton® surfactant, 0.02% bovine IgG, 0.05% Proclin-300® preservative).
  • concentration of colloid was adjusted to correspond to an optical density of 2 (wavelength of 560 nm).
  • the colloid was introduced into a long strip (13 mm in width) of fiberglass pad (Lydall®), 2 mL of solution per foot.
  • the colloid impregnated strip was dried by being passed through a tunnel with flow of warm air.
  • the assay strip was assembled with ribbons of nitrocellulose membrane, onto which were jetted a patient bar containing E1 G-bovine IgG conjugate and a control bar containing goat anti-mouse antibody.
  • the assembled assay strip was cut into narrower strips having a width of 4 mm. Each assay strip 10, which is illustrated in FIG.
  • An E1G assay strip was assembled in a manner similar to that used in Example VII except that a blank Lydall® pad was used in place of the Lydall® pad impregnated with dried colloid.
  • the strip assay was run on a strip reader, which was a reflectance scanner. The strip reader was able to handle six strips in parallel and was controlled through a personal computer with data collection capability.
  • the protocol for running the assay was as follows: (1 ) 25 ⁇ L of a E1 G urine calibrator was transferred to a disposable 1 mL vial, (2) 25 ⁇ L of colloid containing anti-El G antibody was transferred to a vial, (3) the contents of the vials were mixed, and immediately, 40 ⁇ L of the mixture were transferred to the Lydall® pad of a strip that was set on the reader.
  • the running time of each individual assay was about five minutes, and the complete cycle of testing and data processing on the reader took about 10 minutes for each run.
  • FIG. 3 indicates that the dose-response of anti-E1G antibody immobilized on polypyrrole colloid varied significantly with respect to temperature.
  • FIG. 4 indicates that the dose-response of anti-E1G antibody immobilized on polypyrrole colloid via the anti-biotin spacer arm varied little with respect to temperature.
  • FIG. 5 indicates that the dose-response of anti-E1 G antibody immobilized on polypyrrole colloid via the goat anti-mouse antibody spacer arm varied little with respect to temperature.
  • the E1G assay strip used in this example was assembled in the manner as was used in Example VII.
  • the strip assay was run on the same strip reader as described in Example VII. There was no need for sample mixing.
  • a sample (40 ⁇ L) was simply added to the exposed end of Lydall pad of each strip set on the reader.
  • FIG. 6 shows test results of E1G calibration curves generated at three different temperatures — 13 °C, 25 °C, and 37 °C.
  • FIG. 6 indicates that the dose- response of anti-E1 G antibody immobilized on polypyrrole colloid via the anti- biotin spacer arm varied little with respect to temperature when used in the dry mode type of assay.
  • Anti-E1 G Antibody Linked Bv Bis[polvoxvethvlene bis(amine And Glutaraldehvde Spacer Arm To Polvpvrrole Colloid
  • the colloid was washed with phosphate buffer (20 mM sodium phosphate, pH 8.4, 0.1 % Brij 35 solution) on a miniature diafiltration washing device (Microgon®), and colloid containing bis(polyoxyethylene bis[amine]) (10 mL) was recovered. The recovered colloid (2.5 mL) was slowly added to a phosphate buffer (20 mM sodium phosphate, pH 8.4, 0.1 % Brij 35 solution) on a miniature diafiltration washing device (Microgon®), and colloid containing bis(polyoxyethylene bis[amine]) (10 mL) was recovered. The recovered colloid (2.5 mL) was slowly added to a
  • MOPS/BSA buffer 25 mM MOPS, pH 7.2, 25 mM ethanolamine, 2% BSA, 0.1 % Brij 35 solution, 0.05% Proclin-300® preservative.
  • concentration of colloid coated with anti-E1 G antibody was adjusted to an optical density of 25 (wavelength at 560 nm) in MOPS/BSA for storage.
  • Colloid containing bis(polyoxyethylene bis[amine]) (2.5 mL) prepared as described in Example X was slowly added to a vigorously mixed solution of polyoxyethyiene bis(p-nitrophenyl carbonate) (7.5 mg in H2O, 2.5 ml); the pH of 35 the mixture was adjusted to 8.4, and the mixture stirred at a temperature of 2 to 8 °C overnight.
  • the resulting colloid was washed and exchanged with phosphate buffer (20 mM sodium phosphate, pH 7.1 , 0.1 % Brij 35 solution) on a miniature diafiltration washing device (Microgon®); 3.5 mL of colloid was recovered; the recovered colloid was introduced into 1 mL of anti-E1 G antibody (3 mg of antibody in 0.1 M phosphate, pH 10.0, 0.1 % Brij 35 solution), the pH was adjusted to 9.5, and the resulting mixture mixed gently at a temperature of 2 to 8 °C overnight.
  • phosphate buffer 20 mM sodium phosphate, pH 7.1 , 0.1 % Brij 35 solution
  • Microgon® miniature diafiltration washing device
  • the resulting colloid was then mixed with BIS-TRIS/BSA buffer (1 mL, 0.25M BIS-TRIS, pH 7.0, 4% BSA, 0.1 % Brij 35 solution) for 1 hour and then washed and exchanged with MOPS/BSA buffer (25 mM MOPS, pH 7.2, 25 mM ethanolamine, 2% BSA, 0.1 % Brij 35 solution, 0.05% Proclin-300® preservative).
  • MOPS/BSA buffer 25 mM MOPS, pH 7.2, 25 mM ethanolamine, 2% BSA, 0.1 % Brij 35 solution, 0.05% Proclin-300® preservative.
  • the concentration of colloid containing anti-E1 G antibody was adjusted to an optical density of 46 (wavelength at 560 nm) in the MOPS/BSA buffer for storage.
  • Example VIII The E1G assay strips and assay protocol were the same as described in Example VIII.
  • Example XI was diluted to provide an optical density of 4 (wavelength at 560 nm) in MES/casein diluent (0.1 M MES, pH 6.1 , 5% hydrolyzed casein, 0.024% Triton® surfactant, 0.04% bovine IgG, 0.1% Proclin-300® preservative) for wet mode testing.
  • FIG. 7 shows two dose response curves for two kinds of colloids containing anti-E1G antibody linked via polymeric spacer arms at ambient temperature (22 °C).
  • FIG. 8 shows three dose-response curves generated at three temperatures, 13 °C, 22 °C, and 37 °C, with the colloid of Example X.
  • FIG. 8 indicates that the dose-response of anti-E1 G antibody immobilized on polypyrrole colloid via the polymeric spacer arm varied little with respect to temperature when used in the wet mode type of assay.

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Abstract

La présente invention concerne une étiquette constituée d'une particule colloïdale sur laquelle tient un bras et d'un élément primaire de liaison spécifique qui tient sur le bras. La particule colloïdale est réalisée de préférence à partir d'une matière polymère et d'une solution colloïdale de métal. Le bras est de préférence un groupe de liaison de nature biologique ou synthétique. L'élément primaire de liaison spécifique est de préférence un anticorps ou un antigène. En utilisation sur des bandelettes de dosage immunitaire par chromatographie, l'étiquette de cette invention présente une courbe dose-réaction ne variant que légèrement dans une large plage de températures, par exemple de 13 °C à 37 °C.
PCT/US1997/003886 1996-03-14 1997-03-12 Elements de liaison prenant naissance sur des particules et destines a des dosages immunologiques WO1997034150A1 (fr)

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WO2000000826A1 (fr) * 1998-06-30 2000-01-06 Cambridge Sensors Limited Analyse immunometrique
WO2001071344A2 (fr) * 2000-03-17 2001-09-27 Quantum Design, Inc. Procede et dispositif d'essai immuno-chromatographique
EP1761774A1 (fr) * 2004-07-01 2007-03-14 The Central Science Laboratory, "CSL", Representing The Secretary of State for Environment, Food and Rural Affairs Systeme de detection d'analytes
WO2007049157A2 (fr) * 2005-10-24 2007-05-03 Manawatu Biotech Investments Ltd. (Mbil) Surveillance et gestion d'un cycle d'ovulation
US20080139399A1 (en) * 2006-06-29 2008-06-12 Invitrogen Dynal As Particles containing multi-block polymers
US7919333B2 (en) 2003-11-25 2011-04-05 Magnamedics Gmbh Spherical and magnetical silicagel carriers having an increase surface for purifying nucleic acids
WO2012162346A1 (fr) 2011-05-23 2012-11-29 Charm Sciences, Inc. Kit d'analyse à flux latéral et procédé de détection d'une substance de poids moléculaire élevé
EP1147416B1 (fr) * 1999-01-23 2013-05-29 Minerva Biotechnologies Corporation Interaction entre des especes immobilisees sur des colloides et des especes existant sur des structures colloidales
JP2014209117A (ja) * 2013-03-29 2014-11-06 積水メディカル株式会社 イムノクロマト用着色粒子及びこれを用いた診断用イムノクロマト試薬
WO2016207842A1 (fr) * 2015-06-25 2016-12-29 Commissariat A L'energie Atomique Et Aux Energies Alternatives Dispositif a flux lateral avec prelevement

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EP0428412A2 (fr) * 1989-11-16 1991-05-22 Ortho Diagnostic Systems Inc. Procédé pour la production d'un réactif à sol métallique contenant des particules de métaux colloidaux d'une taille présélectionnées

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Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000000826A1 (fr) * 1998-06-30 2000-01-06 Cambridge Sensors Limited Analyse immunometrique
EP1147416B1 (fr) * 1999-01-23 2013-05-29 Minerva Biotechnologies Corporation Interaction entre des especes immobilisees sur des colloides et des especes existant sur des structures colloidales
WO2001071344A2 (fr) * 2000-03-17 2001-09-27 Quantum Design, Inc. Procede et dispositif d'essai immuno-chromatographique
WO2001071344A3 (fr) * 2000-03-17 2002-01-10 Quantum Design Inc Procede et dispositif d'essai immuno-chromatographique
US6607922B2 (en) 2000-03-17 2003-08-19 Quantum Design, Inc. Immunochromatographic assay method and apparatus
US7919333B2 (en) 2003-11-25 2011-04-05 Magnamedics Gmbh Spherical and magnetical silicagel carriers having an increase surface for purifying nucleic acids
EP1761774A1 (fr) * 2004-07-01 2007-03-14 The Central Science Laboratory, "CSL", Representing The Secretary of State for Environment, Food and Rural Affairs Systeme de detection d'analytes
WO2007049157A3 (fr) * 2005-10-24 2007-11-15 Manawatu Biotech Invest Ltd Mb Surveillance et gestion d'un cycle d'ovulation
WO2007049157A2 (fr) * 2005-10-24 2007-05-03 Manawatu Biotech Investments Ltd. (Mbil) Surveillance et gestion d'un cycle d'ovulation
US20080139399A1 (en) * 2006-06-29 2008-06-12 Invitrogen Dynal As Particles containing multi-block polymers
US9738745B2 (en) * 2006-06-29 2017-08-22 Life Technologies As Particles containing multi-block polymers
WO2012162346A1 (fr) 2011-05-23 2012-11-29 Charm Sciences, Inc. Kit d'analyse à flux latéral et procédé de détection d'une substance de poids moléculaire élevé
EP2715358A1 (fr) * 2011-05-23 2014-04-09 Charm Sciences, Inc. Kit d'analyse à flux latéral et procédé de détection d'une substance de poids moléculaire élevé
EP2715358A4 (fr) * 2011-05-23 2014-12-17 Charm Sciences Inc Kit d'analyse à flux latéral et procédé de détection d'une substance de poids moléculaire élevé
JP2014209117A (ja) * 2013-03-29 2014-11-06 積水メディカル株式会社 イムノクロマト用着色粒子及びこれを用いた診断用イムノクロマト試薬
WO2016207842A1 (fr) * 2015-06-25 2016-12-29 Commissariat A L'energie Atomique Et Aux Energies Alternatives Dispositif a flux lateral avec prelevement
FR3038056A1 (fr) * 2015-06-25 2016-12-30 Commissariat Energie Atomique Dispositif a flux lateral avec prelevement
US11002733B2 (en) 2015-06-25 2021-05-11 Commissariat A L'energie Atomique Et Aux Energies Alternatives Lateral flow device comprising sampling

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