EP0324008A1 - Procede d'analyse de phase solide codee par coloration - Google Patents

Procede d'analyse de phase solide codee par coloration

Info

Publication number
EP0324008A1
EP0324008A1 EP88906515A EP88906515A EP0324008A1 EP 0324008 A1 EP0324008 A1 EP 0324008A1 EP 88906515 A EP88906515 A EP 88906515A EP 88906515 A EP88906515 A EP 88906515A EP 0324008 A1 EP0324008 A1 EP 0324008A1
Authority
EP
European Patent Office
Prior art keywords
solid phase
antigen
dye
antibody
solution
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP88906515A
Other languages
German (de)
English (en)
Other versions
EP0324008A4 (en
Inventor
Mats Gustaf Ranby
Nils Arvid Bergsdorf
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
CYTRX BIOPOOL Ltd
Original Assignee
CYTRX BIOPOOL Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by CYTRX BIOPOOL Ltd filed Critical CYTRX BIOPOOL Ltd
Publication of EP0324008A1 publication Critical patent/EP0324008A1/fr
Publication of EP0324008A4 publication Critical patent/EP0324008A4/en
Withdrawn legal-status Critical Current

Links

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
    • G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
    • 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
    • G01N33/531—Production of immunochemical test materials
    • 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
    • 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
    • G01N33/54306—Solid-phase reaction mechanisms
    • 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
    • G01N33/583—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with non-fluorescent dye label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/972—Plasminogen activators
    • G01N2333/9726—Tissue plasminogen activator

Definitions

  • the present invention relates to an improved solid phase analysis method, and more particularly, relates to a method of color-coding assays for the detection of antigens or antibodies by immunological solid phase methods.
  • t-PA tissue plasminogen activator
  • Clinical technicians frequently perform chemical assays in which a number of reagents are added sequentially to a container followed by the addition of a sample to be tested.
  • the technician may utilize a rack of test tubes, a micro test plate or some other container as the solid phase. Aliquots of reagent or sample are normally added one at a time to each tube or microtest plate well from a larger vessel. Normally, the reagents are first added to all of the wells and then an aliquot of the sample solution is added individually to each well. If the technician is interrupted or otherwise distracted during the process of adding sample, a tube or well can be neglected resulting in an erroneous test result. The technician is normally unable to determine whether any particular well contains sample because the reagent solutions are nearly identical to the sample solutions due to the fact that all are normally colorless or lightly colored liquids.
  • the improved solid phase analysis method described above utilizes two microtest plate wells for each sample. Both wells are coated with an antigen-specific antibody, and the second well is exposed to the same antigen- specific antibody solution while the first well is exposed to a non ⁇ specific antibody. The sample containing antigen is then added and quantitative antigen values obtained. The value obtained for the sample in the second well is subtracted from the value obtained for the same sample in the first well so that the portion resulting from non-specific adsorption (or bonding) cancels out.
  • an improved method of detecting an antigen in a sample comprising the steps of coating a first solid phase and a second solid phase with an antigen-specific antibody, adding a first dye to the first solid phase, exposing the first solid phase to a standard solution with a known amount of the antigen, exposing the second solid phase to a solution with an unknown amount of antigen, and determining the amount of antigen bound to the first solid phase and to the second solid phase.
  • a second dye with a color different from the first dye can be added to the second solid phase.
  • Either the first dye or the second dye or both dyes can have the capability of changing colors when the sample to be tested is added. For example, if bromphenol blue is added to a solid phase to which a sample containing blood or plasma is to be tested, the bromphenol blue will change from purple to blue when the plasma sample is added. This aids the technician who is running the test to monitor the addition of the samples.
  • the present invention is especially useful in assays utilizing microtest plates wherein large numbers of samples are added to many wells during the typical assay. Because the wells in the microtest plates are color-coded, it is much easier for the technician to tell which wells have sample added. Accordingly, it is an object of the present invention to provide a method of reducing sampling error in immunological assays.
  • Another object of the present invention is to provide a method for distinguishing a solution to which a sample has been added from a solution devoid of sample.
  • Another object of the present invention is to provide a method for distinguishing a solution to which a sample has been added from a solution devoid of sample by a color change.
  • Another object of the present invention is to provide a method for distinguishing lyophilized reagents from one another after reconstitution.
  • Another object of the present invention is to provide a method for distinguishing one solid phase from another by using various biochemical dyes. Another object of the present invention is to provide a color- coded immunoassay kit.
  • Fig. 1 is a top plan view of a microtest plate embodying the present invention.
  • an improved method of detecting a substance in a sample comprising the steps of immobilizing an antigen-specific antibody to the surface of a solid phase, adding a dye to the solid phase, adding sample containing unknown amounts of the antigen and then determining the amount of antigen bound to the surface of the solid phase. Determining the amount of antigen bound to the surface of the solid phase is well known to those of ordinary skill in the art.
  • the present invention also includes an improved method of detecting an antigen in a sample, comprising the steps of coating a first solid phase and a second solid phase with an antigen-specific antibody, adding a first dye to the first solid phase, exposing the first solid phase to a standard solution with a known amount of the antigen, exposing the second solid phase to a solution with an unknown amount of antigen, and determining the amount of antigen bound to the first solid phase and to the second solid phase.
  • a second dye with a color different from the first dye can be added to the second solid phase. Either the first dye or the second dye or both dyes can have the capability of changing colors when the sample to be tested is added.
  • bromphenol blue can change from purple to blue when the plasma sample is added. This aids the technician who is running the test to monitor of addition of the samples.
  • the sample may induce a pH change when it is added.
  • many of the pH indicator dyes may be suited for use in the present invention.
  • the color change may be governed by a pH change or it may be governed by other factors such as ionic charge, concentration of a component, etc.
  • the same concept can be applied to immunological assays for measuring antigen-specific antibody.
  • antibody-specific antigen is coated on the solid phase.
  • the sample solutions have known or unknown amounts of antibody in the solution.
  • the present invention can be utilized to analyze either monoclonal antibodies or polyclonal antibodies which are antigen-specific.
  • the present invention is especially useful in assays utilizing microtest plates wherein large numbers of samples are added to many wells during the typical assay. Because the wells in the microtest plates are color-coded, it is much easier for the technician to tell which wells have sample added.
  • a first dye can be added to the first row of wells which are coated with either antigen or antibody, depending upon whether antibodies or antigens are being measured.
  • a first dye is added to the first row of wells and a second dye with a color different from the first dye can be added to the second row of wells in the plate.
  • the first dye is then added to the third row and the process is repeated until all of the wells are colored.
  • the improved solid phase analysis method utilizes two microtest plate wells for each sample to account- for the amount of physical adsorption or coating of antibodies on the inner surface of the wells. Both wells are coated with an antigen-specific antibody, and the second well is exposed to the same antigen-specific antibody solution while the first well is exposed to a non-specific antibody. The sample containing antigen is then added and quantitative antigen values obtained. The value obtained for the sample in the second well is subtracted from the value obtained for the same sample in the first well so that the amount of non-specific adsorption (or bonding) is removed.
  • the improved solid phase analysis method utilizes a second vessel or well in a microtest plate for each sample, it is even more likely that a mistake could be made in adding reagents or sample to the wells of the microtest plate.
  • the improved solid phase analysis method of the present invention includes any addition of a dye in any order to an immunological assay so that the addition of reagents and samples to be tested is made easier to monitor. It is within the contemplation of the present invention that dyes could be added individually to the various wells utilized in a typical immunoassay system.
  • solid phases such as a series of test tubes or, more preferably, wells in a microtest plate, such as that used in an ELISA type assay, are coated with an antigen-specific antibody.
  • Example I The microtest plates are coated with antibody by incubating in the wells of the plates between approximately 30-300 ⁇ l of a solution containing between approximately 1-100 mg/1 antibody and between approximately 0.1-1.0 M/l NaHC ⁇ 3 while shaking carefully for about two to four hours at 20-30 ⁇ C. The wells are then emptied and the contents washed with PBS Tween.
  • Dyes that can be used in the present invention include, but are not limited to, phenol red, bromphenol blue and, bromcresol green, alizarin red S, bromcresol purple, bromthymol blue, neutral red, brilliant yellow and methyl orange.
  • phenol red bromphenol blue and, bromcresol green
  • alizarin red S bromcresol purple
  • bromthymol blue neutral red
  • brilliant yellow and methyl orange phenol red
  • Fig. 1 the following description is one embodiment of the present invention.
  • the typical microtest plate 10 is made up of approximately twelve vertical columns 12, each including approximately eight wells 14. Horizontal rows are often designated with letters 13 from A to H and vertical columns are often designated with numbers 15 from 1 to 12 as indicated in
  • the left-most row 16 is prefilled with between approximately 100-200 ⁇ l of normal, or non-specific antibody, 5-15 mg/ml, dissolved in PBS
  • the column 18, designated column 2, immediately adjacent to the left-most column 16 is prefilled with 100-200 ⁇ l of a specific antibody, 5-15 mg/ml, also dissolved in PBS Tween.
  • the specific antibody is preferably the same antigen-specific antibody as used for the coating.
  • next adjacent column 20, designated column 3 is prefilled with normal antibody and the next adjacent column 22, designated column 4, is prefilled with antigen-specific antibody. Thereafter the columns 12 are alternatively prefilled with normal antibody or antigen-specific antibody.
  • a dye having a distinctive color, such as phenol red, is added to each well in the two left-most columns 16 and 18.
  • a dye having a different color is added to the next column 20.
  • This dye can be selected from the group of dyes that change color when exposed to the fluid to be tested. For example, bromphenol blue changes color from purple to blue when a solution containing albumin is added. Thus, if plasma is being tested, bromphenol blue is an excellent dye to use.
  • a dye having a color that can be distinguished from the dyes in the wells of columns 16, 18 and 20 is added to the wells in column 22.
  • this dye is methyl orange.
  • wells are alternatingly filled with the latter two biochemical dyes so that all wells which contain the non-specific antibody also contain the biochemical dye which changes color when exposed to a biological fluid.
  • the left-most columns 16 and 18 are then filled with equal amounts standard plasmas containing increasingly larger concentrations of antigen in concentrations from 0 ng/ml to approximately 30 ng/ml.
  • a biochemical filler solution such as mannitol, can be added to all the wells.
  • the filler serves the purpose of physically stabilizing the contents of the wells so that the contents will remain in the wells after the microtest plate is lyophilized.
  • the mannitol filler also protects the protein components from denaturing during the lyophilization process.
  • Other substances can be used as fillers including, but not limited to, albumin, glycine, polyethylene glycol and Pluronic® F-68 (BASF Corporation, Parsippanny, NJ).
  • Blood samples are collected in the conventional manner and are centrifuged within several minutes at 1000-3000 x g for several minutes.
  • a 10 to 100 ⁇ l aliquot from each plasma sample to be tested is placed in each of two adjacent wells, starting at column 20, containing one of the biochemical dyes and pre-filled with normal or specific antibody, respectively, in a concentration of 5-15 mg/1 dissolved in PBS Tween. Therefore, an uppermost well 30 in the first adjacent row 20 will contain the same sample as an uppermost well 32 in the second adjacent row 22 of wells.
  • pairs 40 of adjacent wells, containing solutions of distinguishable colors will contain an aliquot of the same sample.
  • the kit When plasma is added to the wells containing normal antibody and bromphenol blue, the color of the solution changes from purple to blue enabling the technician to distinguish between wells which already contain a plasma sample and wells to which a plasma sample has not yet been added.
  • the kit has the wells with the standard plasmas colored red, the wells with the non-specific antibody are colored purple, and the wells with the antigen-specific antibody are colored yellow.
  • Such a kit is much easier to use and the likelihood of sampling error is greatly reduced because the technician conducting the tests can easily monitor addition of samples.
  • the standards and samples are incubated for 3 hours at 20-30°C while shaking carefully.
  • the substrate reaction is interrupted by adding 50 ⁇ l of 4-5 M/l of H 2 S ⁇ 4.
  • the absorbance at 492 nm is recorded.
  • the difference in absorbance between the wells filled with normal antibody and the wells filled with specific antibody is calculated for the respective plasma samples. This represents the antigen-specific part of the response; this is compared with the standard curve wherein antigen is diluted in antigen depleted blood plasma.
  • the following is a (2-step) method for determination of human tissue plasminogen activator antigen in plasma utilizing polyclonal and monoclonal antibodies against t-PA.
  • t-PA Human single-chain tissue plasminogen activator
  • PAM 1 Sepharose monoclonal antibody
  • Sephacryl SA-200 gel filtration on Sephacryl SA-200 superfine.
  • the final product is concentrated to about 1 mg/ml and stored in liquid nitrogen until used.
  • the final product gives a single band on sodium dodecylsulphate polyacrylamide gel electrophoresis under reducing conditions. Protein content is determined by amino acid analysis after acid hydrolysis.
  • Polyclonal antiserum against t-PA is raised by immunization of 8 goats with highly purified human single chain t-PA.
  • the IgG fraction is isolated by ammonium sulfate precipitation and Protein A affinity chromatography.
  • the IgG is dialyzed against 20 mmol/L phosphate buffer pH 7.2 containing 150 mmol/1 NaCI and concentrated to 10 mg/ml and stored at -20°C or colder until used. Protein content is determined using an absorptivity of 1.6 mg-1 ml-1 cm-1.
  • Non-immune goat IgG is isolated by ammonium sulfate precipitation and Protein A affinity chromatography.
  • the IgG is dialyzed against 20 mmol/L phosphate buffer pH 7.2 containing 150 mmol/1 NaCl and concentrated to 10 mg/ml and stored at -20°C or colder until used. Protein content is determined using an absorptivity of 1.6 mg-1 ml-1 cm- ⁇ .
  • An IgGlyk monoclonal antibody with high affinity against single and two-chain t-PA (PAM 3) is purified from mouse ascites by ammonium sulfate precipitation and Protein-A affinity chromatography.
  • the IgG is dialyzed against 100 mmol 1 NaHC03 pH 9.2 and concentrated to 20 mg/ml and stored at -20°C or colder until used. Protein content is determined using an abso ⁇ tivity of
  • PAM 3/HRP conjugate for 1000 micro-test plates is performed as follows: 100 mg Horseradish peroxidase (Sigma type VI (St. Louis, MO., USA) is dissolved with 2 ml 1.25% glutaraldehyde (dissolved in 50 mmol/1 sodium phosphate buffer pH 7.2 containing 150 mmol/L NaCl). The solution is incubated 18 hours at 25°C in dark. After incubation, the solution is diluted to 9 ml with 100 mm NaHC ⁇ 3 and dialyzed 8 hours against the same buffer. 0.3 ml PAM 3 antibody (20 mg/ml) is added and the mixture is incubated 18 hours at 25°C in dark.
  • the absorbances at 403 nm and 280 nm is measured, and the A403/A280 ratio is calculated and found to be between 0.2 and 0.3.
  • the conjugate is concentrated to a protein concentration of approximately 1 mg/ml and then stored at -20°C or colder until used.
  • t-PA depleted human plasma Human plasma found free of hepatitis and AIDS antibodies is t-PA depleted by immunoadso ⁇ tion on sepharose bound polyclonal goat antibodies against human t-PA.
  • Bovine serum albumin (No. A-7638) is purchased from Sigma, St. Louis, MO., USA. Other Reagents Mannitol, Phenol red, Methyl orange a n d Polyoxiethylenesorbitanmonolaurate (Tween 20) are purchased from Kebo AB, Sweden.
  • Hydrogen peroxide solution (30%) is purchased from Labassco, Sweden,
  • EDTA Sodium Chloride, Sodium hydrogen carbonate, and o-phenylenedamine are purchased from Merck, Darmstadt, FRG
  • Micro-test plates Micro-test plates (Immuno plate II) is purchasedjfrom Nune, Denmark. PBS-Tween buffer, 10 mmol/1 sodium phosphate buffer containing pH 7.4 containing 140 mmol/1 NaCl, and 0.05% Tween 20.
  • PET-buffer 30 mmol/1 phosphate buffer pH 7.25 containing 5.5 mmol/l EDTA, 140 mmol/1 NaCl, and 0.05% Tween 20. 4.5 mol/1 H2SO4.
  • citric acid 38.4 g citric acid is diluted to 625 ml with distilled water. When all is in solution 2.4 g 1, 2-phenylenedamine (O.P.D.) is added. When all is in solution 2.4 g 1, 2-phenylenedamine (O.P.D.) is added. When all is in solution 2.4 g 1, 2-phenylenedamine (O.P.D.) is added. When all is in solution 2.4 g 1, 2-phenylenedamine (O.P.D.) is added. When
  • O.P.D. O.P.D. is dissolved, the solution is sterile filtered and lyophilized in 2.5 ml portions.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
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  • Investigating Or Analysing Biological Materials (AREA)

Abstract

Procédé amélioré de détection d'un antigène dans un échantillon comprenant les étapes d'enrobage d'une première phase solide (18) et d'une seconde phase solide (22) avec un anticorps spécifique d'antigène, d'adjonction d'un premier colorant à la première phase solide (18), d'exposition de la première phase solide (18) à une première solution de référence avec une quantité connue de l'antigène, d'exposition de la seconde phase solide (22) à une solution avec une quantité inconnue d'antigène, et de détermination de la quantité d'antigène lié à la première phase solide (18) et à la seconde phase solide (22). Dans un autre mode de réalisation de la présente invention, un second colorant ayant une couleur différente du premier colorant peut être ajouté à la seconde phase solide (22).
EP19880906515 1987-07-06 1988-07-01 A color-coded solid phase analysis method Withdrawn EP0324008A4 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US7003087A 1987-07-06 1987-07-06
US70030 1987-07-06

Publications (2)

Publication Number Publication Date
EP0324008A1 true EP0324008A1 (fr) 1989-07-19
EP0324008A4 EP0324008A4 (en) 1990-11-22

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EP19880906515 Withdrawn EP0324008A4 (en) 1987-07-06 1988-07-01 A color-coded solid phase analysis method

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EP (1) EP0324008A4 (fr)
JP (1) JPH02500861A (fr)
AU (1) AU2077588A (fr)
WO (1) WO1989000292A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992015883A1 (fr) * 1991-03-08 1992-09-17 Amoco Corporation Analyses ameliorees comprenant des composes organiques colores
GB2339018B (en) * 1998-06-22 2003-07-30 Ortho Clinical Diagnostics Specific binding assays using methyl orange
GB0405999D0 (en) * 2004-03-17 2004-04-21 Cozart Bioscience Ltd Procedure for manufacture of strips for lateral flow immunochromatographic devices
JP4616819B2 (ja) * 2006-11-20 2011-01-19 デンカ生研株式会社 着色担体を用いたアッセイ装置及びその作製方法

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Publication number Priority date Publication date Assignee Title
US3957436A (en) * 1974-04-29 1976-05-18 Kallestad Laboratories, Inc. Resultant color step indicator
US4461829A (en) * 1981-09-14 1984-07-24 Miles Laboratories, Inc. Homogeneous specific binding assay element and lyophilization production method

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
No further relevant documents have been disclosed. *
See also references of WO8900292A1 *

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AU2077588A (en) 1989-01-30
WO1989000292A1 (fr) 1989-01-12
JPH02500861A (ja) 1990-03-22
EP0324008A4 (en) 1990-11-22

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