WO2000042434A1 - Procede et dispositif pour determiner un analyte - Google Patents

Procede et dispositif pour determiner un analyte Download PDF

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Publication number
WO2000042434A1
WO2000042434A1 PCT/EP2000/000076 EP0000076W WO0042434A1 WO 2000042434 A1 WO2000042434 A1 WO 2000042434A1 EP 0000076 W EP0000076 W EP 0000076W WO 0042434 A1 WO0042434 A1 WO 0042434A1
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WO
WIPO (PCT)
Prior art keywords
analyte
zones
test strip
capacity
zone
Prior art date
Application number
PCT/EP2000/000076
Other languages
German (de)
English (en)
Inventor
Bernd Pevec
Original Assignee
Bernd Pevec
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 Bernd Pevec filed Critical Bernd Pevec
Priority to AU22882/00A priority Critical patent/AU2288200A/en
Priority to CA002358506A priority patent/CA2358506A1/fr
Priority to EP00901513A priority patent/EP1141713A1/fr
Publication of WO2000042434A1 publication Critical patent/WO2000042434A1/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
    • G01N33/54386—Analytical elements
    • 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
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A90/00—Technologies having an indirect contribution to adaptation to climate change
    • Y02A90/10—Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Definitions

  • the present invention relates to a method for the differential determination of an amount of at least one analyte in a sample using a solid phase and an apparatus for carrying out the method.
  • Test strips are often used in today's analytics. Test strips that meet the requirements of a rapid test are essentially based on the principle that a reagent is fixed on a solid matrix that reacts with the analyte to be detected. Under certain circumstances, a color change, a color change or another detectable property can be measured in a subsequent reaction. For example, staining serves as evidence of the presence of an agent to be identified.
  • WO-A-98/22824 relates to a so-called inhibitor assay.
  • This assay configuration is based on labeled compounds that correspond to or are identical to the analyte.
  • the marked compound is placed on the test strip and competes with the analyte present in a sample.
  • the marked connection leaves the original zone and can be verified.
  • a linear procedure is fundamentally not possible.
  • US-A-5,527,509 relates to an assay which determines the analyte by means of enzyme conversion of a low molecular weight analyte and subsequent color reaction. So-called capture assays, which are the subject of the present invention, are not mentioned there. A linear design of this assay is not possible.
  • US-A-5,252,496 relates to a color formation process. Basically, only qualitative assays are disclosed there. If the assay is carried out with two zones, one zone serves only as a control.
  • US-A-5,229,073 relates to a non-linear competitive immunoassay.
  • WO-A-98/36278 relates to a capture immunoassay which has different detection zones with capture components (capture reagents), each of which differ in the capture concentration, so that a linear execution of the assay described there is not possible.
  • test strips or similar systems What is common to many test strips or similar systems is that often only one type of staining takes place, so that such a test cannot be quantified. It only serves to determine one Yes / No statement. Either the analyte is present in a detectable amount, which is reflected in the formation of a color, or there is no coloring, so that the analyte does not exceed a previously determined limit value, that is to say it is below the detection limit of the respective test strip or test system. In order to be able to switch off systematic errors, a function check must also be provided by introducing a further reaction-independent parameter. This structure of test strips is sufficient for many applications. However, questions such as the severity of a stress or illness or change in the amount of an analyte during a rehabilitation process or a therapy attempt cannot be answered satisfactorily.
  • the technical problem underlying the invention therefore consists in avoiding the above-mentioned disadvantages of the systems already present in the prior art and in providing quantifying rapid tests.
  • the problem on which the invention is based is solved by a method for the differential determination of an amount of at least one analyte in a liquid sample using a test strip.
  • Differential determination is understood to mean an at least semi-quantitative determination, that is to say an analysis going beyond a purely qualitative determination.
  • This test strip must have at least two zones and ensure a lateral flow of the liquid sample or a portion of the sample through the test strip and the at least two zones.
  • At least two zones are designed in such a way that they are able to make a statement about the amount of the at least one analyte, in that a first zone has a predefined capacity for binding the at least one analyte and if this capacity is exceeded, by the amount of the at least one analyte which is too large for this capacity, the at least one analyte is bound in at least one second zone, the capacity of the at least two zones being essentially the same in each case and the binding of the at least one analyte in the at least first and at least one second zone is measured by a detectable property.
  • the amount of the at least one analyte is preferably determined by an activity of the at least one analyte.
  • the analyte can be bound specifically and / or non-specifically on the test strip.
  • the specific binding of the at least one analyte preferably takes place via an affinity for another ligand or affinity for the material used for the test strip.
  • binding via immunoaffinity by binding partners bound specifically or unspecifically to the surface of the test strip is possible.
  • An example of the non-specific binding of the at least one analyte on the Test strip is the precipitation of the at least one analyte on the test strip used.
  • the specific binding between the at least one analyte and an affinity binding partner comes, for example, through receptor / ligand interaction, enzyme / substrate interaction, antigen / antibody interaction and other affinity binding, such as avidin, streptavidin, protein A, IgG and others known to the person skilled in the art Systems, established.
  • the defined capacity for binding the at least one analyte on the test strip is determined as a function of the amount of the at least one analyte to be expected in a sample to be analyzed, in order to at least exceed the capacity of the at least first zone to be able to detect an analyte in the second zone.
  • the difference in capacity of the capture component applied in the capture and assay according to the invention in the first and second zones is preferably not more than + 10%, in particular + 5%. It should generally be noted that the more precisely the capacity of the catcher can be set in the at least two zones, the more precisely the linearity of the assay according to the invention is. If more than two zones are used, it can be advantageous to provide the zone beyond that with a higher amount of catcher, that is to say to increase the capacity of this zone in order to achieve better detection. When using several zones, the last zone is preferably provided with a higher capacity of the catcher.
  • the advantage of the present invention is to be seen in the fact that essentially equal amounts of binding agent (scavenger) are applied.
  • FIG. 1 explains the method of operation of the method according to the invention by way of example.
  • FIG. 2 shows a result of the method according to the invention if three zones, each with a defined capacity, are provided.
  • FIG. 3 relates to a device with the test strip according to FIG. 4.
  • FIG. 4 relates to the test strip according to the invention.
  • FIG. 1 shows schematically the fixation of an antibody on a solid matrix, for example nitrocellulose.
  • the phase coated in lb) is then treated with the analyte.
  • the analyte binds to the corresponding antibody via immunoaffinity.
  • This is then added to the detection in 1 c, for example with a further antibody which binds to the antigen and is in turn labeled, in particular by a gold colloid. This indicates the presence of the analyte.
  • Enough capacity of the first If the zone is not sufficient to bind all analytes in the sample, these diffuse further with other sample components on the solid porous support in the direction of the at least second zone, in which the analytes are then bound by the antibodies immobilized there.
  • an analyte it is therefore possible to differentiate whether amounts of an analyte are present in a sample below the detection limit, above the detection limit or significantly above the detection limit.
  • the maximum bindable amount of analyte is thus bound in the first zone and if the amount of analyte present is exceeded, further binding will take place in the at least second zone or further zones.
  • an analysis target can be processed in a variety of ways.
  • the number of zones By increasing the number of zones, a fine division of the measuring range to be recorded can be achieved. For practical reasons, however, the number of zones will regularly be between three and ten. The upper limit will be determined regularly by the readability or interpretability of the results.
  • Figure 2 shows the example of a medical immunoassay.
  • the amount of C-reactive proteins bound in plasma is approximately between 0.2 and 200 mg / 1, values up to approximately 10 mg / 1 being considered normal. Concentrations above this value indicate different forms of bacterial infections.
  • the plasma concentration of C-reactive proteins reacts very quickly to positive treatment, in practice all concentrations actually occur in the range mentioned.
  • the situation shown schematically in FIG. 2 now shows three different zones which can interact immunologically with C-reactive proteins.
  • the following three are regular Read conditions visually: - no coloring, (+) weak and + intensive coloring. If the situation shown in FIG. 2 as situation 1, namely weak coloring in the first zone and no coloring in the second and third zones, is shown in a test strip, the amount of C-reactive protein is in the range from 0.2 to 5 mg /1. The capacity of the respective zones was matched to these quantities.
  • Situation 2 now shows an increased plasma concentration of C-reactive protein in that the staining is correspondingly more intense.
  • This situation corresponds to a content of 5 to 10 mg / 1
  • situation 3 the maximum possible coloration is formed in the first zone and a weaker coloration in the second zone, whereas zone 3 is not colored at all, which indicates an amount of 11 to 15 mg / 1 indicates
  • situation 4 shows strong coloring in the first and second zones
  • the third zone is not colored, which corresponds to a plasma level of 16 to 25 mg / 1
  • situation 5 shows maximum coloring in and in the first two zones
  • Zone 3 weak staining corresponding to a plasma level of 25 to 50 mg / 1
  • situation 6 shows strong staining in all three areas, from which it can then be concluded that> 50 mg / 1 is present.
  • This three-zone model given by way of example therefore allows a six-stage differentiation by simply combining the number of colored zones and the intensity of dyeing.
  • the first zone With a capacity between 0.2 and 5 mg / 1, a functional control is also inherently available, since the first zone must be weakly positive in practically every patient (basal level).
  • analyte C-reactive protein CRP
  • rheumatoid factors CRP
  • troponin I troponin I
  • creatinine kinase CK-MB
  • myoglobin antigens of the organisms Samonella, Legionella, E. coli (EHEC), Aspergillus flavus and fumigatus, glucose, Dioxin, PCB, cocaine (ecgonine), heroin, amphetamine and other pathologically indicative or environmentally relevant substances
  • ECP C-reactive protein
  • CK-MB creatinine kinase
  • myoglobin antigens of the organisms Samonella, Legionella, E. coli (EHEC), Aspergillus flavus and fumigatus
  • glucose, Dioxin PCB
  • cocaine ecgonine
  • heroin amphetamine and other pathologically indicative or environmentally relevant substances
  • two or more analytes can advantageously also be determined in parallel.
  • the following pairs come into consideration as analytes that can be determined in pairs with clinical or environmental analytical relevance: CRP and antistreptolysin, troponin I and CK-MB, myoglobin and CK-MB, CK-MB, myoglobin and troponin I, cocaine, heroin and amphetamine , Samonella and E. coli.
  • a material that has a certain porosity is in principle possible, such as porous supports comprising nitrocellulose, nylon, PTFE, polystyrene, latex, glass fibers, silica gel, aluminum oxide, cellulose, dextran, agarose , Polyvinyl alcohol, also as microspheres or "magnetobeads", optionally preactivated with BrCN, NPCF, NHS chloroformate, tresyl chloride, squaric acid esters and / or corresponding chlorosilanes.
  • porous supports comprising nitrocellulose, nylon, PTFE, polystyrene, latex, glass fibers, silica gel, aluminum oxide, cellulose, dextran, agarose , Polyvinyl alcohol, also as microspheres or "magnetobeads", optionally preactivated with BrCN, NPCF, NHS chloroformate, tresyl chloride, squaric acid esters and / or corresponding chlorosilanes.
  • the method according to the invention is therefore particularly user-friendly since individual analytical problems can be solved by selecting the concentrations which are to be detected in the individual zones. For example, the user himself, e.g. B. by covering the zones with one with the analyte in Interacting agent to tackle individual analysis problems.
  • the test strip according to the invention which is suitable for carrying out the method according to the invention has an essentially porous carrier 2 which can be penetrated by liquids and at least two zones 3, 4 with at least one catcher for at least one analyte, the at least one analyte directly or indirectly to the Captor is bound and the zones 3, 4 each have a previously defined binding capacity for the at least one analyte, which is essentially the same and the catchers are the same for the respective analytes.
  • a typical test strip is shown in Figure 4.
  • the device according to the invention for carrying out the method according to the invention receives the test strip according to the invention.
  • the device has a housing in which the test strip is arranged.
  • the housing consists of an upper side which has at least one opening at the locations of the at least two zones of the test strip, which extends over the region of the at least two zones of the test strip and at least one further opening for application of at least one sample and / or auxiliary substances to be examined for performing an assay that allows contact of a liquid with the test strip.
  • the device preferably has a further opening for receiving auxiliary reagents.
  • the device instead of the one opening 12 which extends over the entire area of zones 3, 4 of the test strip, it has as many openings as corresponds to the number of zones of the test strip. To read the result in the zones of the test strip, it is necessary that the openings in the housing are essentially congruent with the zones of the test strip.
  • FIG. 3 shows a preferred embodiment of the device according to the invention.
  • an opening 12 is provided which extends over the area of the at least two zones 3, 4 of the test strip 1.
  • an opening 14 is provided, with the aid of which the sample to be examined is applied. If necessary, auxiliaries for carrying out the method according to the invention can also be added through the opening 14.
  • the opening 14 has a wall and is connected to the test strip 1. After application of the sample, this pulls into the test strip due to the porosity of the test strip and, due to capillary forces, migrates in a lateral direction through the test strip and therefore through zones 3, 4.
  • the size of the opening 14 determines the volume that the opening can accommodate. The sample volume is precisely determined by the exact diameter and the height of the opening.
  • the sample receiving volume is increased by means of a tube which can be inserted into the opening. It is in particular possible to provide a defined volume by inserting a tube with a defined volume (defined wall thickness and length), which is matched to the quantitative or semi-quantitative assay that can be carried out with the device according to the invention.
  • the further opening 15 is suitable for receiving auxiliary reagents.
  • the opening 14 can be adapted to the respective needs by further additions. Is z. B. directly under the opening 14 in contact with the test strip, a commercially available membrane that is able to separate blood cells from plasma through its defined pore opening, an analysis of plasma parameters can be carried out directly in the test strip.
  • This membrane can be, for example, a glass fiber filter with a suitable density or a plastic membrane with narrow channels, advantageously with asymmetrical channels that allow blood to penetrate. This fulfills the requirements for a quick test for on-site analysis.
  • interference parameters can be selectively filtered out using suitable media. If the opening 14 z. B. filled with aluminum oxide, a sample can be degreased directly, which is of great importance in food analysis. Similarly, particles can be removed by using a filter membrane or soil samples can be examined directly by using a filter together with an absorbent medium (e.g. activated carbon).
  • an absorbent medium e.g. activated carbon
  • test strip according to the invention can be produced in a manner known per se (WO-A-95/13542).
  • test strips can be produced which can be used by the customer himself.
  • the zones on the test strip that are to be used for the detection of the analyte are only preactivated, for example by chemical activation or coating using immunoreactive substances, for example protein A, protein G, streptavidin, etc.
  • the user then only needs to modify his analyte with complementary structures or to bind antibodies against the analyte in the zones in order to use the test strip for his purposes.
  • the quantitative determination of C-reactive protein is carried out in the following way: the reactive zones 3, 4 and a corresponding third zone are loaded with 2.3 ⁇ g each of an anti-human CRP antibody from rabbits on a 4 cm wide nitrocellu strip. The excess reactive groups of the nitrocellulose are then saturated with skimmed milk. Then a test strip consisting of the nitrocellulose, a porous glass fiber strip for receiving the anti-CRP-gold conjugate and a blood piasma-separating glass fiber membrane are mounted on a self-adhesive plastic film. After the application of thick paper strips to hold the necessary washing liquids, 5 mm wide strips are placed in a housing as shown in Figure 3.
  • the opening 14 is filled with 20 ⁇ l of blood (this corresponds reproducibly to an amount of plasma which is suitable for carrying out the test and which reaches the test strip). After 2 minutes, the opening 15 is filled with buffer.
  • the plasma is guided over the three reactive zones.
  • the buffer first passes through the glass fiber filter with the lyophilized gold-antibody conjugate and from there across the zones to which the CRP has already bound. At the points where CRP is present, the gold conjugate also binds and thus produces a color.
  • troponin I and CK-MB can also be measured in parallel in an analogous manner. Both parameters are important indicators of a myocardial infarction, but depending on the cause of the myocardial damage, the amount increases in time, so that their parallel determination provides important diagnostic information.
  • two zones are applied with a monoclonal mouse anti-human troponin I or CK-MB antibody.
  • the difference to the test according to Example 1 is that lyophilized antibody-enzyme conjugates are used instead of the antibody-gold conjugate. These antibodies are directed against a second domain of the analyte.
  • anti-troponin I is conjugated with peroxidase, anti-CK-MB with alkaline phosphatase.
  • BCIP / INT a phosphatase reagent which leaves an orange precipitate in the place of the phosphatase
  • TMB a peroxidase reagent, blue dye

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Abstract

L'invention concerne un procédé et un dispositif permettant la détermination différentielle d'une quantité d'au moins un analyte contenu dans un échantillon liquide, cela au moyen d'une bande test qui comporte au moins deux zones dont la constitution leur permet de donner une information sur la quantité du ou des analytes, la bande test garantissant un écoulement latéral traversant de l'échantillon ou d'une fraction d'échantillon et garantissant la présence des zones qui sont au moins au nombre de deux. Selon l'invention, une première zone présente une capacité prédéfinie à fixer le ou les analytes et, en cas de dépassement de cette capacité par la quantité trop importante du ou des analytes pour cette capacité, il se produit une liaison du ou des analytes dans au moins une seconde zone, la capacité de la seconde zone étant sensiblement la même que celle de la première zone, et la fixation du ou des analytes dans la première et la seconde zone est mesurée en fonction d'une caractéristique détectable.
PCT/EP2000/000076 1999-01-09 2000-01-07 Procede et dispositif pour determiner un analyte WO2000042434A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU22882/00A AU2288200A (en) 1999-01-09 2000-01-07 Method and device for determining an analyte
CA002358506A CA2358506A1 (fr) 1999-01-09 2000-01-07 Procede et dispositif pour determiner un analyte
EP00901513A EP1141713A1 (fr) 1999-01-09 2000-01-07 Procede et dispositif pour determiner un analyte

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP99100410 1999-01-09
EP99100410.2 1999-01-09

Publications (1)

Publication Number Publication Date
WO2000042434A1 true WO2000042434A1 (fr) 2000-07-20

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PCT/EP2000/000076 WO2000042434A1 (fr) 1999-01-09 2000-01-07 Procede et dispositif pour determiner un analyte

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EP (1) EP1141713A1 (fr)
AU (1) AU2288200A (fr)
CA (1) CA2358506A1 (fr)
WO (1) WO2000042434A1 (fr)

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EP1546722A2 (fr) * 2002-06-07 2005-06-29 Cholestech Corporation Cassette de dosage immunologique automatise, appareil et procede associes
EP1664296A1 (fr) * 2003-08-28 2006-06-07 Canon Kabushiki Kaisha Support de sonde et procede de quantification de substances cibles au moyen du support de sonde
US7238519B2 (en) 2002-06-07 2007-07-03 Cholestech Corporation Automated immunoassay cassette, apparatus and method
US7393697B2 (en) 2003-06-06 2008-07-01 Advantage Diagnostics Corporation Diagnostic test for analytes in a sample
WO2010022543A1 (fr) * 2008-08-29 2010-03-04 红电医学科技股份有限公司 Bandelette de test pour liquide
US7763433B2 (en) 2004-07-01 2010-07-27 Forsite Diagnostics Limited Analyte detection system
US7795038B2 (en) 2002-04-09 2010-09-14 Cholestech Corporation High-density lipoprotein assay device and method
US7824879B2 (en) 2007-01-09 2010-11-02 Cholestech Corporation Device and method for measuring LDL-associated cholesterol
US7964370B2 (en) 2008-10-17 2011-06-21 Actherm Inc Analytical strip and detecting method using the same
WO2012020122A1 (fr) 2010-08-13 2012-02-16 Dentognostics Gmbh Procédé visant à éviter des résultats faussement positifs dans le cadre d'un processus de détection d'un indicateur de l'inflammation dans une solution de bain de bouche destinée à recueillir le liquide créviculaire gingival
EP2419724A2 (fr) * 2009-04-15 2012-02-22 Relia Diagnostic Systems, Inc. Extension de la plage dynamique d'une bandelette réactive
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
JP2014528067A (ja) * 2011-09-08 2014-10-23 ネクサス・ディーエックス・インコーポレイテッドＮｅｘｕｓ Ｄｘ， Ｉｎｃ． マルチレベル分析物アッセイ
CN106290881A (zh) * 2015-06-01 2017-01-04 上海凯创生物技术有限公司 沙门氏菌近红外荧光法检测试剂盒

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CN108254550B (zh) * 2016-12-28 2019-10-25 广州瑞博奥生物科技有限公司 检测ck-mb的时间分辨荧光免疫层析试纸条、试剂盒及其制备方法
CN108254563B (zh) * 2016-12-28 2019-10-29 广州瑞博奥生物科技有限公司 检测cTnI的时间分辨荧光免疫层析试纸条、试剂盒及其制备方法
EP4058810A4 (fr) * 2019-11-15 2023-12-20 President And Fellows Of Harvard College Dispositif et procédé de détection d'analytes

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