WO1993005395A1 - Methode de repartition a particules colorees - Google Patents

Methode de repartition a particules colorees Download PDF

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Publication number
WO1993005395A1
WO1993005395A1 PCT/DK1992/000277 DK9200277W WO9305395A1 WO 1993005395 A1 WO1993005395 A1 WO 1993005395A1 DK 9200277 W DK9200277 W DK 9200277W WO 9305395 A1 WO9305395 A1 WO 9305395A1
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WO
WIPO (PCT)
Prior art keywords
affinity
substance
aqueous medium
phases
particles
Prior art date
Application number
PCT/DK1992/000277
Other languages
English (en)
Inventor
Jørgen FOLKERSEN
Søren LEMONIUS
Original Assignee
Folkersen Joergen
Lemonius Soeren
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 Folkersen Joergen, Lemonius Soeren filed Critical Folkersen Joergen
Publication of WO1993005395A1 publication Critical patent/WO1993005395A1/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
    • G01N33/58—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
    • G01N33/587—Nanoparticles
    • 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
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
    • G01N33/5375—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody by changing the physical or chemical properties of the medium or immunochemicals, e.g. temperature, density, pH, partitioning

Definitions

  • the invention relates to a method for visually detecting a substance contained in a biological fluid involving a biospecific affinity reaction.
  • the substance to be detected can be a chemical group, molecule, such as a hapten or a bio olecule having protein, carbohydrate or lipid structure, a virus, and a cell or fragments thereof.
  • Biospecific affinity reactions such as the binding of protein-A to immunoglobulin, biotin binding to avidin, lectin binding to carbohydrates, and immuno-chemical reactions have been used in methods of detecting substances in biological fluids, such as blood, urine, saliva, milk, or any liquid medium containing substances of biological origin, such as extracts of animal or plant tissue.
  • Heterogenous assays involve one or more separation steps to enable the partial or complete separation or removal of selected assay components, which interfere with the analytical signal.
  • Conventional enzyme immunoassays and radioimmunoassays belong to this group of assay constructions.
  • the separation is most frequently mediated by simple washing procedure, as e.g. washing of solid phase bound reactants or precipitation of aggregates by e.g. polyethylene glycol.
  • homogenous assays such as agglutination assays, wherein an agglutination reaction between an antigen or antibody and a particle carried ligand takes place on a glass plate or the like, do no involve such separation steps, since the analytical signal can be generated in the unlimited presence of all the assay components.
  • the assays can further be divided according to the way the analy ⁇ tical signal is generated.
  • Most high sensitive assays require an amplification of the analytical signal, mediated by accumulation of indieatable chemical products such as enzyme generated colours or accumulation of radioactive decay of an isotope.
  • Accumulation of light from flourescent markers also belongs to this group, which may be designated as Accumulating amplification assays, which are also characterised by the chemical alteration and comsumption of one of the assay components during signal generation. This kind of signal generation is required when fully quantitative and high sensitive assays are constructed. Such assays can not be performed by unskilled persons.
  • US patent No. 4,312,944 discloses a method of improving the separation of the assay components of a heterogeneous bioassay, more specifically the separation of conjugate formed in an affinity reaction from the reaction medium by adding a system of two immiscible aqueous liquid phases, wherein the conjugate and the unreacted labelled reactant are assymmetrically partitioned before quantification of the reactants can take place.
  • the methodology disclosed also belongs to the group of heterogeneous and accumu ⁇ lating amplification assays.
  • the object of the invention is to provide a method of detecting a substance in a biological fluid, whereby the unskilled person in a simple procedure can obtain a direct visual signal without the need for separation steps or analytical equipment.
  • the present method does not apply the aqueous two phase technology for the separation of bound and free assay components before signal generation can be analysed, but applies the aqueous two-phase technology to generate the signal .
  • the result of the present invention is therefore a novel assay-construction resulting in a homogenous and nonamplified assay method.
  • a further object of the invention is to provide a simple diagnostic kit for visual detection of substances in biological fluids.
  • the aqueous medium comprising at least two phases comprises prefer ⁇ ably an aqueous solution of at least one polymer and at least one salt or at least two polymers and optionally at least one salt, in which a separation of phases will take place when the solution is left in the gravitational field.
  • the polymers are preferably dextran having a molecular weight of from about 50,000"to about 150,000, preferably from about 60,000 to about 100,000, more preferably from 70,000 to 80,000, polyethylene glycol having a molecular weight of from about 400 to about 10000 preferably about 1000, or other polymers, such as polyvinylpyrro- lidon or modified polymers, which will separate into different phases in an aqueous medium (P.A. Albertsson, Partition of Cell Particles and Macromolecules, 3rd (1986) Wiley-Interscience Public- cations).
  • the salts are preferably NaCl, C1, or NH.SO,.
  • the overall water content of the phase-system can be up to about 95%, which makes most biomolecules soluble in the system.
  • biomolecules The distribution of biomolecules depends on the properties of the biomolecules and on the phase system constituents. Proteins distri ⁇ bute according to their surface properties. Factors such as charge, hydrophobic/hydrophilic patches and size have been shown to play an important role.
  • the separation properties of two- or multiple-phase systems can be manipulated by choice of polymers, molecular weight of polymers, concentration of polymers, choise of salt, salt concentration and pH.
  • Distribution of cells or particles in two-phase systems usually happens between one of the interphases and one of the phases. Both in theori and practise particle size shows correlation to adsorption on the interphase (P.A. Albertsson, 1986, above).
  • the first step in the separation of the phases is the forma ⁇ tion of icells consisting of the individual phase components in the other phase components. These micells will move up or down depending on the type of phase component.
  • the indicator particles used in the method of the invention can be any type of visible or coloured particles having an average diameter less than 100 n such as coloured, cellulose latex, or polystyren/ vinyl particles, or metal particles, such as gold particles that can be dispersed in an aqueous medium and onto which the ligand can be noncovalently or covalently bound.
  • the indicator particles comprise preferably a maximum density of ligand. The detection limit of the assay is dependent on both the ligand density on the indicator particles and the concentration of the particles.
  • the change of affinity of the coloured particle mediated by the reaction with the analyte is caused by at least 2 mechanisms of great practical importance to the construction of the assay.
  • the first mechanism is related to the change of surface property of the coloured particle resulting from the coupling of the analyte alone, because the particle acquire some of the surface properties of the analyte instead of its own surface property.
  • the second mechanism is related alone to the possible aggregation of coloured particles caused by a crosbinding between analyte and ligand of several particles. Aggregation of small particles to a larger average size results in a relative increase in an affinity to an interphase.
  • the first mechanism dominates if the analyte and ligand have one or just a few available binding sites, whereas the second mechanism is favoured if the analyte and ligand has several or many binding sites.
  • Use of e.g. monoclonal antibodies will tend to favour the first mechanism, whereas the use of polyclonal antibodies will favour the second mechanism.
  • Polyclonal antibodies with a very narrow specificity towards an epitope on the analyte that are exposed only in one part of the molecule will favour the first mechanism.
  • the range of the assay is defined as the minimal dose-change of analyte, resulting in a total phase-shift of all coloured particles, it can be concluded that the first mechanism gives a comparatively broder assay range than the second mechanism.
  • This basic property to the present invention therefore favours the use of polyclonal antibodies, because a narrow range is desired in most qualitative tests. A narrow range results visually in a complete shift of coloured particles from one phase to another by the addition of very small amounts of analyte. Mixture of monoclonal and polyclonal antibodies or mixture of several types of monoclonal antibodies reacting with different epitopes of the analyte constitute a tool for the adjustment of the assay range.
  • the sensitivity of the assay might be defined as the minimal dose change of analyte that results in the smallest visible redistribution of a fraction of coloured particles.
  • the sensitivity are primarily dependent of the ligand density of the coloured particles.
  • a low ligand density results i a very sensitive assay, which are only limited by the smallest concentration of coloured particles visible to the naked eye, and the length of the incubation time.
  • a high concentration of coloured particles bearing a high ligand density will tend to lower the sensitivity of an assay.
  • Different analytical purposes require different sensitivity and range of the assay. The above mentioned parameters make it possible to construct qualitative assays with a wide variety of properties.
  • the ligand can be any type of reactant, such as an antibody, an antigen or a lectin, which can participate in a biological affinity reaction.
  • a sample of from about 10 ⁇ to about 1000 _!, preferably from about 25 ⁇ l to 100 ⁇ l, more preferably about 50 ⁇ l , of a biological fluid is mixed with from about 1 ml to about 10 ml, preferably from about 1,5 ml to about 5 ml, more preferably about 2 ml of an aqueous medium comprising two phases, consisting of a solution of a relatively hydrophilic polymer, such ' as dextran, and a solution of a relatively hydrophobic polymer, such as polyethylene glycol, and containing coloured indicator particles having a ligand bound to the surfaces.
  • a relatively hydrophilic polymer such ' as dextran
  • a relatively hydrophobic polymer such as polyethylene glycol
  • the mixture is agitated at short intervals for from 3 to 60 minutes preferably for about 10 to 30 minutes and is left in the gravita ⁇ tional field.
  • a positive reaction between a substance of the biological fluid and the ligand has taken place on the surface of the indicator particles the surface properties of the indicator particles have changed and accordingly the particles' affinity for the phases have changed resulting in a different distribution in the aqueous medium, which can be visualized directly after 5 to 20 minutes notwithstanding that the time for equilibration of the phase system is much longer.
  • the time for a visual response can be shor- tened when the aqueous medium is centrifuged after reaction or by the addition of a magnetic compound having affinity for one of the phases and then applying a magnetic field.
  • a sample of from about 10 ⁇ l to about 1000 ⁇ l preferably from about 25 ⁇ l to about 100 ⁇ l, more preferably about 50 ⁇ l
  • the mixture is introduced in an aqueous medium like the one described above, the mixture is agitated, and a positive reaction is visualized directly as described above.
  • a sample of from about 10 ⁇ l to about 1000 ⁇ l , preferably from 25 ⁇ l to 100 ⁇ l , more preferably about 50 ⁇ l is mixed with an aqueous medium like the ones described above and containing indicator particles of one colour having a non-sense ligand bound to the surfaces and indicator particles of a colour distinct from the first and having a ligand bound to the surfaces.
  • the mixture is agitated as previously described and is left in the gravitational field.
  • the invention further relates to a diagnostic kit for visual detec ⁇ tion of a substance in biological fluids
  • a diagnostic kit for visual detec ⁇ tion of a substance in biological fluids comprising a transparent test tube containing an aqueous medium comprising at least two phases and containing at least one type of dispersed, coloured indicator particles comprising a ligand having affinity to the substance to be detected, which particles change affinity for at least one of the phases of the aqueous medium when a binding result- ing from an affinity reaction between the ligand and the substance to be detected has taken place.
  • the desired detection limit of albumin in human urine is 20 ⁇ g albumin/ml urine.
  • Affinity purified monospecific polyclonal anti-human serum albumin is bound to particles of carboxylated polystyren/vinyl having a particle size of 44 nm and having an intensely blue colour using carbodiimid according to the procedure described by Bauminger and Wilchek in "Methods of Enzymology, Vol. 70, p. 151-160, 1980.
  • the anti-albumin is preferably bound to the particles in a density corresponding to 88 ⁇ g antibody/mg particles and the indicator particles are preferably adjusted to 0.125% of particle mass.
  • a sample of human urine (50 ⁇ l) is mixed with 1,95 ml of the aqueous medium containing coloured indicator particles comprising anti-human serum albumin prepared as described above.
  • the mixture is agitated at 5 minutes' intervals for 30 minutes, left and observed after 10-20 minutes.
  • the indicator par ⁇ ticles will initiate a distribution in the phase system according to the presence of albumin above the detection limit in the sample.
  • the unreacted indicator particles have affinity for the top-phase comprising the polyethylene glycol. If the sample contains more than 20 ⁇ g albumin/ml urine the indicator particles carrying a conjugate between the anti-human serum albumin and albumin will distribute to the inter-phase.
  • a negative control test can be performed for com ⁇ parison.
  • Indicator particles are prepared as in Example 1.
  • a sample of human urine (50 ⁇ l) is mixed with 80 ⁇ l of the indicator particles and left for 10 minutes. Then the mixture is introduced in 1,90 ml of the aqueous medium described above, the mixture is agitated, left and observed after 10 to 20 minutes. Immediately following agitation the indicator particles will initiate a distri ⁇ bution in the aqueous medium according to the presence of albumin above the detection limit in the sample.
  • the distribution of the indicator particles is the same as described in Example 1.

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  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
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  • Engineering & Computer Science (AREA)
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  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
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  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
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Abstract

Méthode de détection visuelle d'une substance présente dans un liquide biologique à l'aide d'une réaction d'affinité biospécifique, dans laquelle on mélange un échantillon d'un liquide biologique avec un milieu aqueux comportant au moins deux phases et contenant au moins un type de particules indicatrices colorées et dispersées comportant un ligand présentant une affinité avec la substance à détecter. Lesdites particules modifient leur affinité avec au moins une des phases du milieu aqueux lorsqu'il se produit une fixation due à une réaction d'affinité entre le ligand et la substance à détecter, et que l'on fait réagir le mélange ainsi formé. Par la suite, on constate la modification de la répartition des particules indicatrices dans le milieu aqueux.
PCT/DK1992/000277 1991-09-12 1992-09-14 Methode de repartition a particules colorees WO1993005395A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DK159291A DK159291D0 (da) 1991-09-12 1991-09-12 Fremgangsmaade til paavisning af et stof indeholdt i en biologisk vaeske
DK1592/91 1991-09-12

Publications (1)

Publication Number Publication Date
WO1993005395A1 true WO1993005395A1 (fr) 1993-03-18

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PCT/DK1992/000277 WO1993005395A1 (fr) 1991-09-12 1992-09-14 Methode de repartition a particules colorees

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DK (1) DK159291D0 (fr)
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016026916A3 (fr) * 2014-08-19 2016-05-19 Ecole Polytechnique Federale De Lausanne (Epfl) Base de transfert de phase de détection chimique

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0011837A1 (fr) * 1978-11-28 1980-06-11 MATTIASSON, Bo Gustav Méthode d'essai faisant intervenir des réactions d'affinité biospécifique
GB2078370A (en) * 1980-06-13 1982-01-06 Nat Res Dev Binding assays
US4579661A (en) * 1983-05-02 1986-04-01 Pharmacia Ab Process in the purification of biologically active substances

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0011837A1 (fr) * 1978-11-28 1980-06-11 MATTIASSON, Bo Gustav Méthode d'essai faisant intervenir des réactions d'affinité biospécifique
GB2078370A (en) * 1980-06-13 1982-01-06 Nat Res Dev Binding assays
US4579661A (en) * 1983-05-02 1986-04-01 Pharmacia Ab Process in the purification of biologically active substances

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DIALOG INFORMATION SERVICES, File 154, Medline, Accession No. 07589107, Medline Accession No. 91108107, LIM, P. et al.: "A Tube Latex Based on Colour Separation for the Detection of IqM Antibodies to Either One of Two Different Microorgamisms"; & J IMMUNOL METHODS, 31 Dec. 1990, 135 (1-2), p. 9-14. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016026916A3 (fr) * 2014-08-19 2016-05-19 Ecole Polytechnique Federale De Lausanne (Epfl) Base de transfert de phase de détection chimique

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DK159291D0 (da) 1991-09-12
AU2594992A (en) 1993-04-05

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