WO1997023774A1 - Dosage immunologique faisant appel a des particules - Google Patents

Dosage immunologique faisant appel a des particules Download PDF

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Publication number
WO1997023774A1
WO1997023774A1 PCT/US1996/020139 US9620139W WO9723774A1 WO 1997023774 A1 WO1997023774 A1 WO 1997023774A1 US 9620139 W US9620139 W US 9620139W WO 9723774 A1 WO9723774 A1 WO 9723774A1
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WIPO (PCT)
Prior art keywords
particles
analyte
amount
particle
sample
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Application number
PCT/US1996/020139
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English (en)
Inventor
Mary Ann Childs
Anjana Prakash
David Bernstein
Original Assignee
Universal Healthwatch, Inc.
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 Universal Healthwatch, Inc. filed Critical Universal Healthwatch, Inc.
Priority to AU12943/97A priority Critical patent/AU1294397A/en
Publication of WO1997023774A1 publication Critical patent/WO1997023774A1/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
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • 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 generally to diagnostic assays and specifically to methods for improving the sensitivity of particle-based immunoassays.
  • Preferred embodiments of this invention provide improved gold particle-based immunoassays that are carried out in strip formats.
  • Diagnostic assays play an indispensable role in the treatment and management of disease. Many different types of diagnostic assays have been developed over the years in response to the need for detecting water borne substances in environmental samples, food and drink samples, and clinical samples such as blood, serum, plasma, urine, saliva, tissue biopsies, stool, sputum, and skin or throat swabs. Advantageously, such assays should give a quick result to allow timely treatment of a disease. In reality, however, many of these assays are limited by their speed. Another important parameter of assays is sensitivity. Developments in test technology have led to increasingly more sensitive tests that allow detection of newer analytes in trace quantities as well as for detecting disease indicators at the earliest time possible. Still, increases in sensitivity are desired for some assays.
  • a third limitation is convenience. Many assays require training of the test operator and complicated instrumentation. These assays cannot be used easily for rapid testing in the field, where electricity or instrumentation are sometimes difficult to obtain. Yet another limitation is the size of test sample required to obtain a test result and the amount of diagnostic test material required to obtain a test result. This limitation is a natural consequence of assay sensitivity. A test method that is more sensitive usually will require less test reagent and less sample to reach a result.
  • the gold particle immunoassay technique does not require instrumentation and can be read by eye. This characteristic makes the gold particle technique particularly suitable for use in third world countries and other countries such as Russia where electricity-based instrumentation based devices are not always easily purchased or used.
  • Colloidal gold particles can be prepared by reduction of a gold chloride with sodium citrate in an aqueous solution as described by Frens, Nature, 241: 20 (1973) . Gold particle size can be varied by changing the concentration of sodium citrate used to make colloidal gold.
  • gold particle assays can be made very specific and sensitive by the appropriate choice of antibody, much more needs to be done to make these assays more sensitive.
  • the gold particles used in the assay exist in a colloidal dispersion, which exhibits complex behavior that is difficult to predict.
  • a colloidal dispersion is a two phase system wherein each particle is separated from its liquid medium by a separation space.
  • the particle interface has characteristic adsorption and electrical potential properties which influence the behavior of binding reactions at the particle surface. Per unit weight, smaller particles have a greater surface volume and, under some circumstances react faster in binding reactions.
  • a convenient strip technique could be combined with gold particle detection by visual means to expand the use of immunoassays to regions of the world where instrumentation and electricity are not available.
  • analyte detection could be quickened and analytes could be detected at lower concentrations. That is, an increase in sensitivity would directly lead to earlier clinical treatment and thereby potentially decrease human suffering.
  • the present inventors have provided a method for determining the presence or amount of an analyte in a sample.
  • the method comprises the first step of contacting the sample with a reagent.
  • the reagent comprises a first group of particles having bound thereto a binding component capable of specifically recognizing the analyte, and a second group of particles having bound thereto a binding component capable of specifically recognizing the analyte.
  • the average diameter of the particles in the second group is smaller than the particles in said first group.
  • the second step of the method is determining the presence or amount of analyte- particle complexes as a detection or measure of the amount of analyte in the sample.
  • the presence or amount of analyte-particle complexes is determined after filtration through a membrane. In another embodiment of the invention, the presence or amount of analyte-particle complexes is determined after chromatographic movement through a porous support.
  • a method for enhancing the sensitivity of a gold particle immunoassay by employing less than an equivalent mass of selenium particles of larger size in the assay.
  • the present inventors believe that manipulation of the particle interface of the colloidal dispersion containing the particles might lead to more sensitive immunoassays. Indeed, while studying gold particle immunoassays the present inventors surprisingly found that inclusion of a different particle type such as colloidal selenium particles imparted new, desirable properties to the immunoassay. In fact, the present inventors surprisingly found that an immunoassay which used a combination of colloidal gold particles and colloidal selenium particles performed much better than the same immunoassay using only selenium particles or only gold particles. Indeed, the new combination allowed more than a ten fold improvement in assay sensitivity.
  • a particle useful for the present invention can be comprised of any material as long as it can be coated with a binding member and can stay in suspension. Examples of suitable materials are metal colloidal particles such as gold, silver, platinum, iridium, ruthenium and the like as well as non-metals such as selenium, sulfur and tellurium.
  • particles made from plastic and glass can be employed by one skilled in the art, particles comprised of a metal or other naturally colored substance are preferred because their aggregation can be more readily seen by the naked eye.
  • particles of colored latex, methacrylate, silica, and metal colloids such as colloidal gold, silver, or nonmetal colloids such as selenium, sulfur, tellurium.
  • Particles of gold and selenium are especially preferred because colloidal suspensions of these two substances can be readily made from their salts and form strong colors upon aggregation.
  • the particles of the invention participate in one or more binding reactions between a binding component and an analyte, in which the analyte is a corresponding bindable substance in an aqueous test sample.
  • the binding component can be, for example, one or more of the following: antibody, protein A, protein G, avidin, lectin, nucleic acid, a naturally occurring binder, and a synthetic binder.
  • the analyte can be any one of a large number of antigens, antibodies, or ligands of interest.
  • the binding component is preferably a protein and most preferably an antigen or antibody capable of specifically recognizing the corresponding analyte. Skilled artisans will recognize many possible binding components that can be used. Formation of analyte-particle complexes causes a color or color intensity change whereby the presence of an analyte can be visually determined. A wide variety of binding assay formats can be used to link one or more binding reactions to this color or color intensity change.
  • One format is a sandwich assay in which, for example, analyte binds two different particles or analyte binds an immobilized binding member (such as antibody bound to a membrane support) and a particle.
  • Another format is a competitive inhibition assay in which analyte ligand competes with another substance for binding. In this format the presence of analyte modulates particle to particle binding or particle to immobilized binding member binding.
  • Yet a third basic format is a displacement assay in which analyte in a test sample is detected by its ability to compete and displace a binding member. Such competition can, for example, be used to modulate the movement of particles within a moving stream of liquid such as sample fluid that has been taken up by an absorbent solid.
  • Other assay formats are known to those skilled in the art and can be used.
  • Both larger and smaller particles are coated with the binding component.
  • Conjugation of the binding component to the particles can be carried out using a number of methods known in the art including physical immobilization, covalent bonding, hydrophilic bonding, hydrophobic bonding, and ionic interaction.
  • an antibody capable of specifically recognizing the analyte of interest is adsorbed onto the particle surface, followed by washing and blocking with another protein such as bovine serum albumin.
  • both larger and smaller particles are small enough to stay in suspension long enough to participate in a binding reaction and are smaller than l um in their longest axis.
  • the larger particles will have an average diameter that is larger than the average diameter of the smaller particles.
  • the larger particles will have an average diameter that is greater in diameter by at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% 100% or more, than the average diameter of the smaller particles.
  • the larger particle is bigger than 50 nm, on average, in its largest dimension
  • the smaller particle is smaller than 50 nm, on average, in its largest dimension
  • the larger particle is most preferably a selenium colloid particle prepared by boiling a solution of selenium dioxide in the presence of ascorbate.
  • the smaller particle is most preferably a gold colloid particle prepared by boiling a solution of gold chloride in the presence of sodium citrate.
  • the larger and smaller particles are used together as one suspension.
  • the larger particle can be present in a greater, equal or lesser amount (expressed as gm/liter) than the smaller particle.
  • the larger particle is present in an equal amount or in lesser amount than the smaller particle.
  • the larger particle comprises between 10% to 50% of the total particle mixture and the smaller particle comprises between 50% and 90% of the total particle mixture.
  • the optimum ratio of larger to smaller particle for the particular assay employed is easily determined by one skilled in the art and can depart from these stated percentages, based on the mean particle size, degree of derivitization with binding component, total concentration of particles, pH, ionic strength, temperature, and other factors. In practice, one can determine the optimum use ratio by varying the ratio of large to small particles over the range 0.05 to 20 within a given set of reaction parameters.
  • the method of the present invention involves contacting a liquid sample having or suspected of having an analyte of interest, with the mixture of two particle types that are coated with at least one binding component.
  • the particle mixture may be present as a dried material that is re-wetted by the sample. More preferably the particle mixture is dried within an absorbent such as porous plastic, porous glass, glass fiber, paper, and the like.
  • the particle mixture is reconstituted by application of an aqueous sample suspected of containing the analyte to the absorbent. After an appropriate incubation time, (i.e.
  • the presence or amount of analyte/particle labelled constituent complexes is determined as a measure of the amount of analyte in the sample.
  • the relative amount of particle aggregation, or dispersal is determined by a color or color intensity change.
  • gold particles and selenium particles are coated with a first antibody for one epitope of an analyte molecule.
  • the particles are deposited onto one end of an absorbent material.
  • a second antibody that binds a separate epitope of the analyte is immobilized in a line on the absorbent material at some distance from the deposited particles.
  • a test fluid suspected of containing the analyte is contacted with an end of the absorbent material near the deposited particles. The aqueous sample fluid wicks along the absorbent material and, while doing so, re-wets and suspends the particles.
  • the absorbent is imbibing sample fluid
  • the first antibody on particle surfaces binds its epitope of the analyte.
  • the second antibody which has been immobilized to a separate region more distal to the sample application end encounters the particles. Particles that have bound analyte on their surfaces adhere to the immobilized antibody and form a color in a line in response to the presence of analyte.
  • particles are coated with antibodies that are targeted for various epitopes of the sample analyte.
  • test fluid is applied to a porous material in which particles have been deposited.
  • the particles are re-wetted and aggregate in response to analyte in the test sample.
  • the wetted particles contact one side of a membrane that has on its other side an absorbent which pulls fluid across the membrane.
  • a suitable membrane will have a porosity that is at least five times the mean diameter of the larger particles.
  • a preferred membrane is 5 um porosity nitrocellulose.
  • Analyte is detected as the formation of color on the membrane.
  • Most preferred in the context of this second embodiment is a membrane that has a second binding component which binds to ligand and which can capture particles.
  • Another embodiment of this invention provides particle assays in which the particles employed are dissimilar in composition an /or charge. That is, particles which are made from dissimilar metals (e.g. gold and platinum) , or from metals and non-metals (e.g. platinum and selenium) , or particles of different electrostatic charge (e.g., selenium and plastic), are used in the assay. As above, both particle types are coated with reagent and participate in the same binding reaction. The particles and assay formats which may be used to prepare such assays are described above.
  • V. cholera antibody Purified V. cholera antibody was commercially obtained from Global Diagnostics, Gainesville, Fl. The antibody was dialyzed against 0.002 M borax buffer pH 8.2, filtered through a 0.2 micron cellulose acetate filter, and diluted in the same buffer to a final concentration of 100 ug per milliliter.
  • a 4% solution of gold chloride was prepared by dissolving 360 mg of gold chloride (tetrachloroauric acid trihydrate) into 9 milliliters of deionized water.
  • a 1% solution of sodium citrate was prepared by dissolving 1.0 gram of sodium citrate into 100 milliliters of deionized water. Three liters of deionized water were placed into a 4 liter beaker and brought to a boil on a hot plate. Then 7.5 milliliters of the 4% gold chloride solution were added to the boiling water. Seventy two milliliters of the 1% sodium citrate solution were added to the beaker and the solution was boiled until its volume was reduced to 2.2 liters.
  • a colloidal gold solution was removed from the hot plate and allowed to cool to room temperature.
  • the cooled colloidal gold solution was filtered through a 0.2 micron cellulose acetate filter unit into a clean amber bottle.
  • Optical densities at 520 nm and 580 nm of the resultant filtrate were 1.54 and 0.455 respectively.
  • a one milliliter aliquot of colloidal selenium was mixed with an equal volume of colloidal gold.
  • the pH of this mixture was 5.0.
  • the combined selenium-gold mixture was centrifuged at 10000 x g for 1 minute to remove any aggregated material.
  • the pH of the supernatant was adjusted to 8.0 by the careful addition of 0.2 M potassium carbonate.
  • Protein protection studies were performed on each combination as follows: 1.0 ml colloid + 100 ul varying concentrations of anti-ViJrio cholera monoclonal antibody (80 ug/ml, 100 ug/ml, 120 ug/ml, 160 ug/ml, and 200 ug/ml) at various pH's (6.0, 7.0, 8.0, and 9.0) prepared by the addition of 0.2 M potassium carbonate. Minimum protein protection occurred at an antibody concentration of 10 ug per milliliter of selenium-gold mixture at pH 8.0 and pH 9.0.
  • nitrocellulose strips Mylar backed five micron nitrocellulose was cut into 22 mm x 4 mm strips (Grade 8980, Gelman Sciences, Ann Arbor, Michigan) . Rabbit anti-vibrio cholera affinity purified antibody (Louisiana State University, Baton Rouge) was diluted into 0.05 M sodium borate buffer Ph 8.2 to a concentration of 1.6 mg/ml. Then 1.5 microliters of antibody were spotted near the center of each nitrocellulose strip. The strips were dried in a vacuum desiccator.
  • a vinyl strip backed with acrylic adhesive was cut into 4 mm x 70 mm size portions. Each mylar backed nitrocellulose strip was affixed to a vinyl strip. An absorbent paper (type III 4 mm x 30 mm from Gelman Sciences) was affixed to each nitrocellulose/vinyl strip so as to overlap the top of the nitrocellulose strip. A glass fiber pad (Gelman Sciences, grade 8980) was affixed to overlap the bottom of each nitrocellulose strip. Assay for V. cholera
  • V. cholera 01 (ATCC strain #11628) and V. cholera non 01 (ATCC strain #14547) were grown in alkaline peptone water media overnight at 35 degrees C. The organisms were centrifuged at 5,000 x g for 5 minutes and each pellet was resuspended in phosphate buffered saline pH 7.2 to an optical density at 650nm of 0.6 (approximately 10* organisms per ml) . Log, 0 dilutions of each organism were made into an extraction buffer consisting of 1% Triton X-100 in 0.02 M Tris with 1% bovine serum albumin and O.l M NaCl at pH 8.0. One hundred microliters of each diluted organism suspension was placed into a separate 10 mm x 75 mm test tube. To each pair of tubes was added 50 ul of the following mixtures of colloidal gold/selenium antibody conjugates:
  • test strip was placed in each tube and the fluid in each tube was allowed to diffuse up and through the nitrocellulose strip. After 10 minutes each strip was examined for the presence of an orange-purple color in its middle zone where antibody had been mobilized. The results were as follows:
  • V. cholera assay test was at least 10 times as sensitive when a mixture of selenium particles and gold particles were used compared to when only selenium particles were used or when only gold particles were used.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Cell Biology (AREA)
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Abstract

Cette invention permet d'améliorer la sensibilité de dosages immunologiques faisant appel à des particules en ajoutant aux premières particules des particules d'un second type et de plus grande taille. Cette technique est particulièrement utile dans l'élargissement de la gamme de sensibilité de dosages immunologiques colloïdaux à base d'or, et peut être utilisée sous un format pratique tel qu'une languette. Dans un mode de réalisation, des particules de sélénium d'une plus grande taille sont ajoutées à un système de dosage de type sandwich utilisant des particules d'or d'une moindre taille, lesdites particules de sélénium permettant d'améliorer le développement couleur des particules d'or. Cette technique peut être appliquée à d'autres systèmes de dosage à base de particules, dans lesquels on souhaite améliorer la sensibilité de dosage.
PCT/US1996/020139 1995-12-22 1996-12-23 Dosage immunologique faisant appel a des particules WO1997023774A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU12943/97A AU1294397A (en) 1995-12-22 1996-12-23 Particle assisted immunoassay

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US57710895A 1995-12-22 1995-12-22
US08/577,108 1995-12-22

Publications (1)

Publication Number Publication Date
WO1997023774A1 true WO1997023774A1 (fr) 1997-07-03

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WO (1) WO1997023774A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7470379B2 (en) 2000-03-20 2008-12-30 Massachusetts Institute Of Technology Inorganic particle conjugates

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4859612A (en) * 1987-10-07 1989-08-22 Hygeia Sciences, Inc. Metal sol capture immunoassay procedure, kit for use therewith and captured metal containing composite
US5141850A (en) * 1990-02-07 1992-08-25 Hygeia Sciences, Inc. Porous strip form assay device method
US5518887A (en) * 1992-03-30 1996-05-21 Abbott Laboratories Immunoassays empolying generic anti-hapten antibodies and materials for use therein

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4859612A (en) * 1987-10-07 1989-08-22 Hygeia Sciences, Inc. Metal sol capture immunoassay procedure, kit for use therewith and captured metal containing composite
US5141850A (en) * 1990-02-07 1992-08-25 Hygeia Sciences, Inc. Porous strip form assay device method
US5518887A (en) * 1992-03-30 1996-05-21 Abbott Laboratories Immunoassays empolying generic anti-hapten antibodies and materials for use therein

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7470379B2 (en) 2000-03-20 2008-12-30 Massachusetts Institute Of Technology Inorganic particle conjugates
US8034259B2 (en) 2000-03-20 2011-10-11 Massachusetts Institute Of Technology Inorganic particle conjugates
US8192646B2 (en) 2000-03-20 2012-06-05 Massachusetts Institute Of Technology Inorganic particle conjugates

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Publication number Publication date
AU1294397A (en) 1997-07-17

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