WO1990008322A1 - Procede immunologique de test de concentration - Google Patents

Procede immunologique de test de concentration Download PDF

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Publication number
WO1990008322A1
WO1990008322A1 PCT/AU1990/000008 AU9000008W WO9008322A1 WO 1990008322 A1 WO1990008322 A1 WO 1990008322A1 AU 9000008 W AU9000008 W AU 9000008W WO 9008322 A1 WO9008322 A1 WO 9008322A1
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WO
WIPO (PCT)
Prior art keywords
test
species
test strip
antigen
antibody
Prior art date
Application number
PCT/AU1990/000008
Other languages
English (en)
Inventor
John Douglas Wilson
Richard John Thorogood
Original Assignee
Nathan, Pamela, Anne
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 Nathan, Pamela, Anne filed Critical Nathan, Pamela, Anne
Publication of WO1990008322A1 publication Critical patent/WO1990008322A1/fr

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Classifications

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

Definitions

  • This invention relates to the quantification of molecules in solution by immunological techniques, in particular by the use of test strips.
  • concentration of a species of test antigen or antibody in a test solution can be determined by allowing the test solution to migrate along the length of a test strip with primary antibody or antigen able to bind the test molecule immobilised on it.
  • the antigen or antibody to be tested is found on a part of a strip proximal to the point at which the solution containing the antigen or antibody is loaded, and a sharp cut-off point is found beyond which no antigen or antibody is detectable.
  • the distance between the cut-off point and the point at which the antigen or antibody-containing solution is applied is directly proportional to the concentration of the antigen or antibody in the solution.
  • this can be used as a method of determining the concentration of any antigen or antibody in a solution.
  • the present method differs in some respects from conventional immunodiffusion techniques; the most obvious being that the present method relies on capillary action to bring the antigen or antibody to be tested in juxtaposition with primary antibody or antigen rather than relying on diffusion. Another major difference is that the primary antibody or antigen is immobilised on the porous material, rather than being relatively mobile within a gel.
  • the invention could be said to reside in a method of quantifying the concentration of one species of test immu no reactive molecules in a test solution, including the steps of:- applying the test solution to one end of a test strip, said test strip including a sheet of porous material and a second species of immunoreactive molecules immobilised on said sheet of porous material, the said second species of immunoreactive molecules being selected so as to be able to bind to the said one species of test immunoreactive molecules, allowing the test solution to migrate toward an opposite end of the test strip, substantially washing out any of the one species of test immunoreactive molecules not bound to the said second species of immunoreactive molecules, and applying an indicator means for the visualisation of any of the said one species of test immunoreactive molecules retained on the test strip
  • the porous material of the test-strip may be material such as various grades of paper, nitrocellulose, or nylon membrane.
  • the visualisation means includes the steps of washing said strip to a pre-determined degree of stringency, adding a secondary antibody, with a specificity that is reactive with the first immunoreactive molecule, to said strip, said secondary antibody being bound by a detection stimulator, and applying detection means.
  • the detection stimulator is radiolabelling of the secondary antibody.
  • the detection stimulator is an enzyme capable of producing colour change to said strip on the addition of substrate.
  • the enzymes could be enzymes such as Horse Radish Peroxidase or Alkaline Phosphatase.
  • the detection stimulator is the biotinylation of the secondary antibody.
  • the porous material is nylon membrane and the method of coating includes the steps of agitating a sheet of nylon membrane in a solution of primary antibody, blocking any remaining unreacted sites by the addition of a concentrated solution of protein, said protein being unreactive with the visualising means.
  • This protein could, for example, be gelatine.
  • the invention could be said to reside in a method of quantifying the concentration of a species test antigen in a test solution comprising the steps of:- applying the said test solution to one end of a test strip, the said test strip including a sheet of porous material that has a species of primary antibody immobilized on the said sheet of porous material, the said species of primary antibody being selected so as to be able to bind to the said species test antigen, allowing the test solution to migrate toward an opposite end of the test strip, substantially washing out any of the said species of antigen not bound to the species of primary antibody, and applying an indicator means for the visualisation of the said species of test antigen retained on the test strip.
  • the invention could be said to reside in an arrangement for quantifying the concentration of one species of test immunoreactive molecules in a test solution
  • a test strip holder a test strip including a sheet of porous material that has a second species of immunoreactive molecules immobilised on said sheet of porous material, a vessel for holding the test solution of one species of test immunoreactive molecules up to a first level, said second species of immunoreactive molecules being selected as being able to bind the said one species of test immunoreactive molecules, said test strip holder being adapted to hold said test strip within the said vessel so that the first level defines a loading point on the test strip said arrangement further including indicator means for visualisation of the said one species of test immunoreactive molecules retained on the test strip.
  • the invention could be said to reside in an arrangement for quantifying the concentration of first immunoreactive molecules in a solution
  • a test strip holder a test strip including a sheet of porous material that has second immunoreactive molecules immobilised on it a vessel for holding the solution of the first immunoreactive molecules up to a first level, the said vessel being adapted for washing of uncomplexed first immunoreactive molecules from said test strip.
  • said first immunoreactive adapted to be bound by the said first immunoreactive molecules
  • said test strip holder being adapted to hold said test strip within the said vessel so that the first level defines a loading point on the test strip
  • said apparatus further including means for visualisation of the said first immunoreactive molecules complexed to second immunoreactive molecules and retained on the test strip.
  • test immunoreactive molecule will now be referred to as antigen and the second species of immunoreactive molecule will be referred to as antibody or primary antibody. It is to be understood however that the first species of test immunoreactive molecule can be either antibody or an antigen, and the "primary antibody” can be either antibody or antigen.
  • the dried, antibody-coated test strip is dipped to a specified depth and for a specified period of time into the test solution. During this time, the antigen-containing test solution is drawn up the strip by capillary diffusion and as the test antigen comes into contact with the immobilised primary antibody it is removed from the migrating solution and becomes bound, via the primary antibody, to the strip.
  • test antigen As more test antigen is drawn from the test solution reservoir, it must pass further up the test strip before coming into contact with uncomplexed antibody and thereby becoming bound to the strip.
  • the primary antibody on the lower part of the test strip will become saturated rapidly and antigen will reach progressively higher regions of the strip before being bound by the primary antibody on the strip.
  • test antigen is present in extremely high concentrations, the antibody on the test strip might become completely saturated and free antigens would travel with the solvent front. In this situation, after development of the assay, the entire strip would be coloured and obviously the result would not be considered quantitative.
  • test antigen is present in extremely low concentrations the antibody on the part of the strip which is dipped into the antigen- containing test solution might not become fully saturated during the time period of the assay. In this case, there would be no migration of the test antigen up the strip and following development of the assay, the coloured area which would correspond to the portion of the strip dipped in the antigen containing test solution would not be truly quantitative.
  • the height of bound antigen will be proportional to the concentration of antigen in the test solution.
  • the solvent for the antigen containing test solution is envisaged as being aqueous, although other solutions may be applicable provided that antibody binding is not inhibited.
  • the method includes the steps of comparing the distance from loading point to cut off point of an unknown immunoreactive molecule contain solution to that distance for solutions of known concentration of the immunoreactive molecule.
  • FIG.1 is a a typical example the relationship between distance between the loading point and the cut off point on a test strip and the concentration in the test solution,
  • FIG.2 is a perspective exploded view of a first embodiment of an arrangement according to the invention.
  • FIG.3 is a plan view of the washing vessel from above
  • FIG.4 is a cross-sectional view from A-A of figure 3 of the washing vessel.
  • FIG.5 is a more detailed view of a clip for holding the test strips.
  • test molecule is Human IgG.
  • test strip is based on nylon membrane, and the preparation of the test strip is as follows:- 1. A sheet of nylon membrane (Hybond-N, Amersham International pic) is immersed in coating buffer (0.1 M Sodium carbonate/ bicarbonate buffer pH 9.6) (1 ml/cm 2 of test strip) and gently agitated on a rocking platform for five minutes at room temperature to ensure complete wetting of the membrane.
  • coating buffer 0.1 M Sodium carbonate/ bicarbonate buffer pH 9.6
  • the coating buffer is then removed and replaced with Rabbit anti- Human IgG (IgG fraction) diluted in coating buffer to give 10 ⁇ g/cm 2 and 0.5ml/c ' m 2 .
  • the membrane is then gently agitated as before at room temperature for two hours.
  • the membrane is then washed three times in phosphate buffered saline pH7.2 with Tween 20 (0.1%) for five minutes each wash, followed by gentle agitation in blocking buffer (3% gelatin) for one hour at room temperature.
  • the method of determining the concentration of the test molecule according to this second example is as follows.
  • test strips are mounted into test strip holders, one end of which are immersed into a vessel containing the test solution, up to a line marked in the vessel.
  • the test solution can be made up with Phosphate Buffered Saline pH 7.2 (PBS).
  • PBS Phosphate Buffered Saline pH 7.2
  • the test strips are left immersed and undisturbed in the test solution containing vessel for 15 minutes to allow the test solution to migrate to the other end.
  • the test strips are washed after removing them from the test solution by gently shaking them in a washing buffer (such as PBS-Tween 20 (0.1%) (PBS-Tw)) to remove unbound test molecules.
  • a washing buffer such as PBS-Tween 20 (0.1%) (PBS-Tw)
  • the test strips are immersed in a conjugate solution, which in this example is Anti-human IgG conjugated to Horse Radish Peroxidase and diluted in PBS-Tw with gelatin (1%).
  • the strips are removed from the conjugate solution after 5 minutes and then washed, as before in PBS-Tw to remove unbound secondary antibody.
  • the test strips are then immersed in enzyme substrate solution which in this specific example is 100 ⁇ g/ml 3,3'- D ⁇ aminobenzidine tetrahydrochloride, 0.2 ⁇ l/ml 30%Hydrogen peroxide, in 100mM citrate buffer pH5.0. After 5 minutes the test strips are rinsed under tap water for 30 seconds and the length of the coloured zone in each strip is measured.
  • FIG. 1 A graph of the relationship between length of the coloured zone and concentration of IgG in the test solution is shown in FIG. 1. Shown are the lengths of the coloured zone of five samples (in centimetres) against the concentration of IgG in the test solution. The plotted points are shown as filled in square boxes
  • the assay in this example has been adjusted to detect material in the range of approximately 2 - 50 ⁇ g/ml whereas the sensitivity of the test can be increased so as to detect lower levels of test molecules.
  • a lower detection level of 100ng/ml is achievable by altering the parameters of the test strip.
  • the lower detection level can be adjusted up or down by alteration of the concentration of the coating antibody solution.
  • the range can also be adjusted by varying parameters such as the length of the test strip and incubation (dip) time.
  • Test strips 1 made of nylon - membrane or nitrocellulose paper are held by test strip holders which comprises plastic clips 2 that clip around the test strips and "push fit" into a holder base 3.
  • the holder base 3 has a tapered edge to ensure a leak free fit into the washing vessel 4.
  • the test strip clips 2 are made of plastics material and include two hinged flaps 5 at either end of the clips 2 with a tongue and groove locking mechanism to lock the flaps into a closed position on the ⁇ holder.
  • the test strips can be fitted into the clips 2 when the flaps are in the open position and are locked into position by snapping the hinged flaps 5 shut.
  • the washing vessel 4 is used for (i) holding the test solution (ii) as a washing vessel and (iii) for holding the enzyme substrate solution.
  • the washing vessel 4 comprises a base 6 with walls 7 of the vessel extending upwardly therefrom. Two internal shoulders 8 extend longitudinally along the base, leaving there between a narrow channel. The narrow channel serves to locate the strip holders centrally and also reduces the volume of sample solution required.
  • Conjugate tubes 9 are provided that can be supported by a stand 10.
  • the conjugate tubes contain freeze dried, enzyme conjugated antisera having specificity for the antigens detected by the antisera on the corresponding nylon strips.
  • the test strips supported by the clips 2 are adapted to fit into the conjugate tubes.
  • this assay can be used to measure any antigenic material to which an antibody can be raised.
  • the assay can be used for measuring antibody concentration by immobilising purified antigen on the strip, dipping the strip into an antibody-containing sample (e.g. blood) and developing the strip with an enzyme-labelled antigen.
  • An example of the usage of the assay in this situation might be for measuring the immunity of a population to a particular disease. The ease of use of this assay makes it particularly applicable for use "in the field”.

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  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
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  • Cell Biology (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)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne un procédé de quantification d'une espèce d'antigène de test, ou d'anticorps de test dans une solution de test à l'aide d'une bande de test. On plonge ladite bande de test en matière poreuse telle que du papier de nitrocellulose de membrane en nylon, sur laquelle une espèce d'antigène ou d'anticorps primaire est immobilisé, au niveau d'une extémité, dans une solution de test. On laisse la solution de test migrer vers l'autre extrémité de ladite bande de test. On lave les bandes de test puis on détecte l'antigène ou l'anticorps de test lié selon un procédé tel que le développement coloré par un anticorps secondaire à liaison enzymatique dirigé vers l'antigène ou l'anticorps de test. On compare la longueur de la surface colorée avec la longueur de la surface colorée par une concentration de la même espèce d'antigène ou d'anticorps afin de calculer la concentration.
PCT/AU1990/000008 1989-01-11 1990-01-11 Procede immunologique de test de concentration WO1990008322A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AUPJ222189 1989-01-11
AUPJ2221 1989-01-11

Publications (1)

Publication Number Publication Date
WO1990008322A1 true WO1990008322A1 (fr) 1990-07-26

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PCT/AU1990/000008 WO1990008322A1 (fr) 1989-01-11 1990-01-11 Procede immunologique de test de concentration

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WO (1) WO1990008322A1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0921397A1 (fr) * 1997-12-08 1999-06-09 Kreatech Biotechnology B.V. Procédé d'identification d'une espèce mycobactérienne
WO2000025135A1 (fr) * 1998-10-22 2000-05-04 Horticulture Research International Systeme de detection par bandelette reactive
WO2010043075A1 (fr) * 2008-10-17 2010-04-22 红电医学科技股份有限公司 Pièce de test pour tester un liquide et procédé de test associé
US8133718B2 (en) 2008-10-17 2012-03-13 Actherm Inc Analytical strip and detecting method using the same
US8367015B2 (en) 2009-03-23 2013-02-05 Actherm Inc Analytical strip and the manufacturing method thereof
US8372660B2 (en) 2008-10-09 2013-02-12 Actherm Inc Quantitative analyzing method
JP2020530556A (ja) * 2017-07-27 2020-10-22 ベラックス バイオメディカル インコーポレイテッド 逐次的ラテラルフローデバイス

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU1056376A (en) * 1975-01-27 1977-07-28 A.B. Kabi Diagnostic method involving immuno-chemical quantification
AU1684983A (en) * 1982-07-15 1984-01-19 Syva Co. Enzyme chromatograph immunoassay
AU5362486A (en) * 1985-02-14 1987-08-20 Abbott Laboratories Concentrating immunochemical test strip
EP0246643A2 (fr) * 1986-05-20 1987-11-25 DNA PLANT TECHNOLOGY OPERATING CO. (under the laws of the State of Delaware) Procédé pour préparer un bâtonnet d'échantillonnage afin d'accomplir une analyse diagnostique
AU1359588A (en) * 1987-03-27 1988-09-29 Becton Dickinson & Company Solid phase assay
AU1430588A (en) * 1987-04-07 1988-10-13 Syntex (U.S.A.) Inc. Immunoassay device

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU1056376A (en) * 1975-01-27 1977-07-28 A.B. Kabi Diagnostic method involving immuno-chemical quantification
AU1684983A (en) * 1982-07-15 1984-01-19 Syva Co. Enzyme chromatograph immunoassay
AU5362486A (en) * 1985-02-14 1987-08-20 Abbott Laboratories Concentrating immunochemical test strip
EP0246643A2 (fr) * 1986-05-20 1987-11-25 DNA PLANT TECHNOLOGY OPERATING CO. (under the laws of the State of Delaware) Procédé pour préparer un bâtonnet d'échantillonnage afin d'accomplir une analyse diagnostique
AU1359588A (en) * 1987-03-27 1988-09-29 Becton Dickinson & Company Solid phase assay
AU1430588A (en) * 1987-04-07 1988-10-13 Syntex (U.S.A.) Inc. Immunoassay device

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0921397A1 (fr) * 1997-12-08 1999-06-09 Kreatech Biotechnology B.V. Procédé d'identification d'une espèce mycobactérienne
WO1999030162A1 (fr) * 1997-12-08 1999-06-17 Kreatech Biotechnology B.V. METHODE D'IDENTIFICATION D'UNE ESPECE DE $i(MYCOBACTERIUM)
WO2000025135A1 (fr) * 1998-10-22 2000-05-04 Horticulture Research International Systeme de detection par bandelette reactive
US8372660B2 (en) 2008-10-09 2013-02-12 Actherm Inc Quantitative analyzing method
WO2010043075A1 (fr) * 2008-10-17 2010-04-22 红电医学科技股份有限公司 Pièce de test pour tester un liquide et procédé de test associé
US7964370B2 (en) 2008-10-17 2011-06-21 Actherm Inc Analytical strip and detecting method using the same
CN102171566A (zh) * 2008-10-17 2011-08-31 红电医学科技股份有限公司 流体检测试片及其测试方法
US8133718B2 (en) 2008-10-17 2012-03-13 Actherm Inc Analytical strip and detecting method using the same
US8367015B2 (en) 2009-03-23 2013-02-05 Actherm Inc Analytical strip and the manufacturing method thereof
JP2020530556A (ja) * 2017-07-27 2020-10-22 ベラックス バイオメディカル インコーポレイテッド 逐次的ラテラルフローデバイス

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