EP1971863A1 - Utilisation de polymeres pour augmenter l'intensite du signal lors de l'execution de reactions de detection - Google Patents

Utilisation de polymeres pour augmenter l'intensite du signal lors de l'execution de reactions de detection

Info

Publication number
EP1971863A1
EP1971863A1 EP07703606A EP07703606A EP1971863A1 EP 1971863 A1 EP1971863 A1 EP 1971863A1 EP 07703606 A EP07703606 A EP 07703606A EP 07703606 A EP07703606 A EP 07703606A EP 1971863 A1 EP1971863 A1 EP 1971863A1
Authority
EP
European Patent Office
Prior art keywords
use according
interval
polyvinyl
molecular weight
polyvinylpyrrolidone
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Withdrawn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
EP07703606A
Other languages
German (de)
English (en)
Inventor
Peter Porschewski
Kerstin Steinert
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.)
Qiagen GmbH
Original Assignee
Qiagen GmbH
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 Qiagen GmbH filed Critical Qiagen GmbH
Publication of EP1971863A1 publication Critical patent/EP1971863A1/fr
Withdrawn legal-status Critical Current

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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
    • G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
    • 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
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding

Definitions

  • the present invention relates to the increase of the signal intensity with simultaneous reduction of non-specific background binding when carrying out detection reactions - in particular of immunoassays.
  • Immunoassays find i.a. in the analytics of medical diagnostics and human therapy a wide application.
  • ELISA and antibodies are routinely used in medical analytics labs to screen blood for relevant pathogens.
  • Polyclonal antibodies - a mixture of antibodies with different binding epitopes - are the most commonly used.
  • attempts have been made in recent years to establish new diagnostic methods that should be distinguished by greater sensitivity, lower costs and shorter processing times.
  • developments have become known from the prior art, based on the so-called.
  • Luminex xMap technology to develop detection techniques that meet the above requirements as much as possible.
  • microspheres or beads The basis of the Luminex xMAP technology are microscopically small spherical polymer particles, in particular polystyrene particles, commonly referred to in the art as microspheres or beads. These have in their interior at least two different fluorescent dyes. The variation of different proportions of the two dyes defines spectrally clearly distinguishable populations of beads. Thus, for example, one hundred different types of microspheres are available. Each species of the different beads can be coated with a specific detection reagent or antigen by simple coupling chemistry. In this way, it becomes theoretically possible to perform up to one hundred different detection reactions simultaneously in one sample. By the year 2004, about 30 of these detection reactions had already been established.
  • each MirosphDCpopulation binds with the appropriately coated detection reagent their specific target molecule.
  • the target molecules bound on the surface of the microspheres are recognized by a specific detection molecule, which - in the described example - carries a green fluorescent marker, also called a reporter.
  • Luminex 100 analyzer thousands of microspheres can be evaluated individually within seconds.
  • a first laser in the device is used to excite the red fluorescent dyes within the beads, which are classified based on their fluorescence emission.
  • a second laser is - in the present example - e.g. the green fluorescent dye of the reporter is excited and the intensity of the emitted light is recorded.
  • the identity of the antigen is specified, while qualitatively and quantitatively the antibodies specifically bound to the respective antigen can be detected by the second laser.
  • the principle of a LiquiChip immunoassay is based on the use of an (ELISA) antibody sandwich pair on LiquiChip beads.
  • an antibody of the sandwich pairs is immobilized covalently as a capture molecule (capture antibody) on the surface of the polystyrene beads.
  • the second antibody is used as the antigen-specific primary antibody.
  • This primary antibody can e.g. be biotinylated and detected using a streptavidin-phycoerythrin conjugate or by a corresponding (fluorophore) -marker-conjugated species-specific detection antibody.
  • the area in which the biomarker is detectable and quantifiable is called the dynamic range.
  • the biomarker concentration increases, the binding saturates. However, the measurement at high concentrations above saturation is no longer possible. By contrast, low concentrations can not be detected due to the low sensitivity or because of the unspecific background signal.
  • the present invention thus has the object to enable an improved signal-to-background ratio (S / B).
  • the assay sensitivity can thus be improved, resulting in a larger dynamic range and an improved signal-to-background ratio (S / B).
  • a further advantage is that by achieving higher sensitivities, it is also possible to carry out assays with reduced sample quantities, especially if only a small initial amount of sample material is available.
  • proteinogenic Substances - such as BSA, casein or gelatin - or non-proteinogenic substances - especially detergents - can be used. These substances are used in the corresponding reaction buffers.
  • the detection antibodies are able to bind nonspecifically to the capture antibodies.
  • the buffers used in the prior art are usually standard buffers such as PBS or Tris buffer (pH 7 - 7.8).
  • detergents used in the corresponding buffers to reduce the non-specific binding that results from protein-protein or protein-matrix interactions have the serious disadvantage that they are too high in the specific antigen antibodies - Dissolve interactions or even denaturing effect on antigen and antibodies and thus ultimately have an inactivating effect.
  • the object underlying the present invention in view of the prior art described above, is now to overcome the described disadvantages of the prior art and not to optimize immunoassays - LiquiChip immunoassays - with respect to the increase of the signal intensity enzymatically mediated signal amplification - and to allow simultaneous reduction of non-specific background binding.
  • Another object of the present invention is to increase the signal intensity while reducing unspecific binding to so-called planar To enable protein arrays in which the detection of bound analytes on a substantially planar array.
  • a further object of the present invention is to provide assay conditions in a detection method which also make it possible to generate the highest possible specific signal with small sample quantities.
  • Polyvinyl derivatives in the sense of the invention are all derivatives of a polymer or oligomer obtained by polymerizing a monomer having a vinyl structure, in particular polyvinyl alcohol (PVA) or polyvinylpyrrolidone (PVP).
  • polyvinyl derivatives are understood as meaning oligomers or polymers with ester partial structures, such as, for example, Polyvinyl acetate, optionally partially saponified and copolymers, such.
  • soluble polyvinyl derivatives By using soluble polyvinyl derivatives, the disadvantages of the prior art can thus be overcome.
  • soluble polyvinyl derivatives can be used in a larger concentration range than detergents and cause an increase in signal intensity and sensitivity and at the same time contribute to the reduction of non-specific bonds. - This results in a improved signal-to-background ratio over a wider dynamic range.
  • soluble polyvinyl derivatives e.g. Polyvinyl alcohol (PVA) or polyvinylpyrrolidone (PVP) in the reaction buffers in all immunoassays or ELISAs, which are performed on hydrophobic surfaces, the signal intensities with simultaneous reduction of non-specific bonds.
  • PVA Polyvinyl alcohol
  • PVP polyvinylpyrrolidone
  • the use of polyvinyl derivatives can also improve the results of detection reactions which are carried out in a higher degree of reaction (multiplex assay).
  • the use of the polyvinyl derivatives opens up the possibility that, by increasing the sensitivity, it is also possible to analyze those parameters in parallel in the setup whose signal intensities prove to be too low under the previous conditions.
  • buffers comprising the mentioned polyvinyl derivatives improve protein-protein and protein-DNA interaction assays as well as DNA applications in LiquiChip assays on polystyrene surfaces as mentioned above.
  • the polyvinyl derivatives especially polyvinyl alcohol (PVA) or polyvinylpyrrolidone PVP contribute to prevent the unwanted aggregation of the beads.
  • the present invention allows an increase in the signal intensity - also with simultaneous reduction of non-specific binding in the detection of bound analytes by means of so-called planar protein arrays.
  • planar protein arrays contain the
  • Catcher molecules on a carrier e.g. Glass or glass slides, nitrocellulose, PVDF or polystyrene surfaces, and embodies, for example, an antibody, a fragment of an antibody, an aptamer, a peptide or an anticalin.
  • LiquiChip immunoassays involves the use of an (ELISA) antibody sandwich on LiquiChip beads.
  • an antibody of the sandwich pairs is covalent as
  • Capture molecule immobilized on the surface of polystyrene beads. These beads are incubated with a sample (e.g., serum, cell culture or tissue lysate, cell culture supernatant) containing the appropriate antigen.
  • a sample e.g., serum, cell culture or tissue lysate, cell culture supernatant
  • the beads can optionally be washed.
  • Antigen should remain bound to the beads via the capture antibody.
  • the specifically bound antigen can be labeled, for example, with a second antigen-specific antibody and subsequently detected. This can be done, for example, with a fluorophore-labeled antibody directed against this second antibody.
  • non-specific binding of other assay components eg, other proteins from the sample
  • the incubation of the capture antibody beads with the sample (antigen) is carried out in a buffer containing a soluble polyvinyl derivative.
  • Suitable polyvinyl derivatives in the solution of the object according to the invention are all polyvinyl derivatives which are soluble or water-soluble under the given reaction conditions.
  • Preferred polyvinyl derivatives are such as e.g. Polyvinylpyrrolidone and polyvinyl alcohol in the form of their pure polymers, as copolymers with each other and / or with other suitable comonomers and as mixtures.
  • suitable copolymers With a view to the use of suitable copolymers is the use of any copolymer of vinyl alcohol or vinyl acetate and vinylpyrrolidone as well as copolymers of both - poly [vinylpyrrolidone-co-vinyl acetate] or poly [vinylpyrrolidone-co-vinyl alcohol] in addition to the above-mentioned copolymers, possible, provided that they have a suitable solubility behavior in an essentially aqueous environment.
  • the respective molecular weights can be varied within wide limits for the successful realization of the method according to the invention. Conveniently, they are in a range of 5,000 to 500,000 Da, preferably in an interval of 24,000 to
  • Polymers with mixtures of polyvinyl alcohol and polyvinylpyrrolidone being preferred.
  • polyvinyl alcohol alone or in mixtures with polyvinylpyrrolidone is conveniently a molecular weight range of 9,000 to 500,000 Da, preferably 31,000 to 186,000 Da and more preferably between 86,000 and 126,000 Da and between 146,000 and 186,000 Da.
  • a molecular weight range of 5,000 to 500,000 Da, preferably 10,000 to 450,000 Da, and more preferably 24,000 to 360,000 Da is practically suitable.
  • the concentration - individually or in combination - of the polyvinyl derivatives is for example in the LiquiChip-Imunoassays in an interval of 0.025 to 5% (w / v), preferably in an interval of 0.025 to 2% (w / v), and more preferably in one Interval from 0.1 to 1% (w / v).
  • the beads are preincubated with the buffer containing the polyvinyl derivative, followed by the addition of the sample and a further incubation.
  • the soluble polyvinyl derivatives e.g. preferably polyvinyl alcohol or polyvinylpyrrolidone - but may also be included in the following steps in the respective buffers, e.g. upon addition and incubation of the second antigen-specific antibody.
  • the present invention further relates to a method for increasing the signal intensity while reducing unspecific binding, which is characterized in that the detection of bound analytes on beads or on planar protein arrays in the presence of a polyvinyl derivative.
  • the present invention relates to a method in which the capture molecules are applied to glass (glasslides), nitrocellulose, PVDF (polyvinylidene fluoride) or on polystyrene surfaces and the detection reaction is carried out in the presence of a polyvinyl derivative.
  • the capture molecule may be, for example, an antibody, the fragment of an antibody, an aptamer, a peptide or an anticalin.
  • the present invention relates to a kit for carrying out an immunoassay containing at least one buffer with a polyvinyl derivative as component.
  • the present invention also relates to a kit in which the capture molecules are bound to the beads or to planar arrays.
  • the beads or planar arrays may have a carrier material made of glass, nitrocellulose, PVDF (polyvinylidene fluoride) or polystyrene.
  • Assay buffer II (contains no PVA or PVP):
  • Sodium chloride, NaH 2 PO 4 -H 2 O and the polyvinyl derivative were autoclaved in 900 ml of distilled water and dissolved. Thereafter, the pH is adjusted to 7.4 with sodium hydroxide solution (NaOH). Then BSA and Tween 20 are added and the Total volume filled up to 1 liter. Before use, the solution is filtered through a 0.45 ⁇ m filter.
  • the polymeric substances were also included in the first step in the binding of the antigens to the capture antibody beads in the incubation buffer.
  • assay buffer I 50 .mu.l of assay buffer I were initially charged and mixed with 25 .mu.l of sample dissolved in assay buffer II. This is followed by the addition of 25 .mu.l of assay buffer II containing each
  • SAPE Phycoerythrin

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

Abstract

L'invention concerne l'utilisation de dérivés de polyvinyle pour augmenter l'intensité du signal tout en réduisant simultanément les liaisons d'arrière-plan non spécifiques lors de l'exécution de réactions de détection, notamment de dosages immunologiques.
EP07703606A 2006-01-03 2007-01-03 Utilisation de polymeres pour augmenter l'intensite du signal lors de l'execution de reactions de detection Withdrawn EP1971863A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102006000707A DE102006000707A1 (de) 2006-01-03 2006-01-03 Verwendung von Polymeren zur Erhöhung der Signalintensität bei der Durchführung von Nachweisreaktionen
PCT/EP2007/050033 WO2007077238A1 (fr) 2006-01-03 2007-01-03 Utilisation de polymeres pour augmenter l'intensite du signal lors de l'execution de reactions de detection

Publications (1)

Publication Number Publication Date
EP1971863A1 true EP1971863A1 (fr) 2008-09-24

Family

ID=38135831

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Application Number Title Priority Date Filing Date
EP07703606A Withdrawn EP1971863A1 (fr) 2006-01-03 2007-01-03 Utilisation de polymeres pour augmenter l'intensite du signal lors de l'execution de reactions de detection

Country Status (4)

Country Link
US (1) US20090131267A1 (fr)
EP (1) EP1971863A1 (fr)
DE (1) DE102006000707A1 (fr)
WO (1) WO2007077238A1 (fr)

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WO2013071055A1 (fr) * 2011-11-10 2013-05-16 Wellstat Diagnostics, Llc Dosage pour autoanticorps associés au diabète
US20130224770A1 (en) * 2012-02-29 2013-08-29 General Electric Company Antibody Diluent Buffer
ES2527887B1 (es) * 2013-08-02 2015-11-19 Linear Chemicals, S. L. Procedimiento para cuantificar la presencia de un antígeno de Helicobacter pylori en una muestra biológica de origen fecal y kit para cuantificar la presencia de dicho antígeno mediante dicho método
CN106596920B (zh) * 2016-11-24 2018-10-26 上海透景诊断科技有限公司 链霉亲和素藻红蛋白冷冻稳定剂及其制备方法和应用
CN110392831B (zh) 2017-01-27 2023-09-05 豪夫迈·罗氏有限公司 用于在相互作用测定中调节信号强度的方法
CN110297085A (zh) * 2019-06-13 2019-10-01 武汉博士德生物工程有限公司 一种用于增强免疫印迹检测信号的抗体稀释液
CN113933506A (zh) * 2021-10-29 2022-01-14 北京利德曼生化股份有限公司 测定人体壳多糖酶3样蛋白1(chi3l1)含量的磁微粒化学发光检测试剂盒
GR20220100698A (el) * 2022-08-18 2024-03-12 Πανεπιστημιο Πατρων, Μεθοδος βελτιωσης της ανιχνευσιμοτητας των δοκιμων που βασιζονται σε ταινιες ξηρων αντιδραστηριων

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Also Published As

Publication number Publication date
WO2007077238A1 (fr) 2007-07-12
DE102006000707A1 (de) 2007-07-05
US20090131267A1 (en) 2009-05-21

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