EP4136449A1 - Dosage à écoulement latéral - Google Patents

Dosage à écoulement latéral

Info

Publication number
EP4136449A1
EP4136449A1 EP21717922.5A EP21717922A EP4136449A1 EP 4136449 A1 EP4136449 A1 EP 4136449A1 EP 21717922 A EP21717922 A EP 21717922A EP 4136449 A1 EP4136449 A1 EP 4136449A1
Authority
EP
European Patent Office
Prior art keywords
lateral flow
flow assay
assay according
zone
marking
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.)
Pending
Application number
EP21717922.5A
Other languages
German (de)
English (en)
Inventor
Friedrich Scholz
Theresa Heckmann
Lukas RÜTTINGER
Anne-Marie GAD
Hans-Hermann SÖFFING
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.)
Senova Gesellschaft fuer Biowissenschaft und Technik mbH
Original Assignee
Senova Gesellschaft fuer Biowissenschaft und Technik mbH
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 Senova Gesellschaft fuer Biowissenschaft und Technik mbH filed Critical Senova Gesellschaft fuer Biowissenschaft und Technik mbH
Publication of EP4136449A1 publication Critical patent/EP4136449A1/fr
Pending legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—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
    • G01N33/543—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
    • 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

Definitions

  • the present invention relates to a lateral flow assay, a method for quantifying an analyte and a device for reading out a lateral flow assay.
  • LFA Lateral Flow Assay
  • POCT point-of-care test
  • a liquid sample e.g. from a patient
  • the sample interacts with the individual components of the LFA and causes optical changes in the LFA.
  • optical indicators can be observed and evaluated by the user or by optical reading devices.
  • a read-out device typically has an optical sensor that detects the variation of the optical signal very accurately.
  • the LFAs are based on the principles of immunoassays, in which the presence of a substance, called an analyte, is detected in liquid samples such as blood, urine, saliva or environmental samples.
  • a liquid sample, which contains the analyte, is applied to the sample application field of the LFA.
  • an LFA consists of a sample zone, a reaction zone and a detection zone.
  • the sample zone is also known as the sample application field or sample pad.
  • the reaction zone comprises affinity molecules, which are also referred to as receptor elements.
  • the detection zone also includes affinity molecules.
  • a receptor element is inserted directly after the sample application area and a second receptor element is immo bilized in the so-called test zone of the LFA.
  • the two receptor elements are positioned in such a way that the first receptor molecule, after contact with the sample, is transported together with the analyte to the second receptor element by capillary flow forces.
  • the second receptor element which is fixed on the solid phase, binds the analyte or the first receptor element from the sample.
  • the analyte from the sample interacts with the receptor element directly after the sample application field.
  • This receptor element is usually conjugated to optical signal-emitting particles or molecules (marking).
  • the sample, with the analyte and the labeled receptor molecule (“labeling complex") then flows on to the test zone on which a second receptor element is immobilized.
  • the analyte and the two receptor elements interact, whereby an optical signal is formed on the test zone, depending on the marking used. This signal can be quantified by a readout device.
  • the first receptor element binds the analyte, which in turn binds to the second receptor element, one speaks of a sandwich assay.
  • the signal intensity on the test line increases with increasing analyte concentration.
  • the label can be bound to the analyte (or a structurally similar molecule).
  • This reagent marking complex
  • This reagent is then located in a defined amount directly after the sample application field and, after the sample application, migrates with the analyte from the sample to the test zone to which the receptor element is fixed.
  • the receptor element is bound to the marker and placed directly after the sample application area.
  • the analyte or a structure-like molecule is immobilized in the test zone. The analyte also migrates from the sample with the conjugate to the test zone.
  • the analyte from the sample, receptor molecule and the analyte introduced into the assay then interact with one another at the test zone and cause a signal in the test zone.
  • the signal intensity decreases with increasing analyte concentration.
  • the solid phase of the LFA which contains the test zone, provides a zone in which a receptor element or the analyte (or structure-like molecule), referred to as a ligand, is immobilized.
  • Porous materials such as nitrocellulose, nylon, cellulose acetate and other porous materials are used as the solid phase.
  • the sample task area mostly consists of fiberglass or cellulose membranes.
  • the marking complexes are introduced into the pad adjoining the sample application area, with this membrane also mostly consisting of glass fiber and cellulose membranes.
  • LFA formats are also used as dipstick formats, in which a receptor molecule is also bound to the solid phase.
  • This test strip is then dipped into the sample containing the analyte.
  • This sample can then either release the marking complex or the marking complex can be added to the sample beforehand.
  • the marker complexes then interact with the analytes from the sample and / or the receptor molecule on the solid phase and either form a colored field or line in the test zone or not.
  • One LFA can also be used to detect multiple analytes. For example, when drugs are detected from biological samples, multiple drugs are detected. Such LFA are mostly offered with several test zones, whereby exactly one drug can be detected in one test zone. For the detection of small analytes (100-1000 Da), mostly competitive assay formats are used in which the signal in the test zone decreases with increasing analyte concentration, as shown in the following patents: US Pat. No. 4,703,017; U.S. 5,451,504; and US 5,798,273
  • sandwich assay formats are mostly used, in which the signal increases with increasing analyte concentration in the test zone.
  • Sandwich assay formats based on LFA are described, inter alia, in the following patents: US 20150094227.
  • the receptor molecules are marked with colored molecules or particles in order to make the complex of analyte and antibody or antibodies visible.
  • Typical markings for LFA are colored nanoparticles or dyes.
  • protocols that describe the attachment of the label to ligands.
  • LFA are evaluated through the eye of the user.
  • readout devices are used for quantification, which can deduce the analyte concentration in the sample from the intensity of the color of the test zone.
  • Such reading devices usually have a light source with which the test zone is irradiated and a detection unit which quantifies the light intensity of the light modified by the test zone.
  • the light from the light source for example an LED
  • the light from the light source is detected by one or more photodiodes or a camera.
  • Read-out units typically have several LEDs in order to quantify the various test and control zones. The measured signal is then converted into an analyte concentration by the device and shown to the user on a display.
  • such readout units are described in EP 1484601, KR102005597B1, US 20150094227, US 7,297,529.
  • LFA only have a measuring range of approx. 1.5-2 log levels. This is not sufficient for the quantification of some analytes. For example, it is necessary to precisely quantify the CRP in the blood in the concentration range of 0.5 - 150 pg / ml and thus over a measuring range of 2.5 log levels. The LFA procedures described for this are mostly inadequate.
  • the object of the present invention was to provide a lateral flow assay which allows an analyte to be quantified, in particular over a larger measuring range than in the prior art.
  • the task is solved by a lateral flow assay with
  • the first affinity molecules have an optically measurable marking which has at least 2 different absorption maxima of different heights, which are used for quantification.
  • the lateral flow assay according to the invention has a sample zone, a reaction zone and at least one detection zone.
  • the sample is applied in the sample zone in order to bind the first affinity molecules in the subsequent reaction zone.
  • Second affinity molecules are immobilized in the detection zone, so that complexes of first affinity molecules, analyte and second affinity molecules accumulate there in the sense of a sandwich assay.
  • the first affinity molecules raise an optically measurable mark that has two different absorption maxima of different heights, which can thus be used for quantification.
  • measurements are made at both absorption maxima.
  • the first measurements are in the area of the first absorption maximum or those in the second absorption maximum are in a range that allows good and reliable quantification.
  • the first and second affinity molecules can be antibodies or antibody fragments, for example. Protein A, protein G, streptavidin, neutravidin or derivatives thereof are also suitable. Further suitable affinity molecules are known to the person skilled in the art.
  • the optically measurable marking is preferably achieved by marking compounds which are selected from dyes, nanomaterials or combinations thereof.
  • Metal nanomaterials, latex nanomaterials, cellulose-based nanomaterials, silica nanomaterials or combinations thereof are particularly suitable as nanomaterials.
  • the optically measurable marking with two different absorption maxima can be achieved on the one hand by two different marking compounds.
  • the marking could be a blue and a red dye, which have absorption maxima of different heights.
  • the two different absorption maxima used according to the invention are preferably at least 50 nm apart, preferably at least 70 nm apart and even more preferably at least 100 nm apart.
  • the one absorption maximum is preferably at least 1.5 times higher than the other absorption maximum.
  • the ratio is preferably at least 2 or at least 3. It does not matter whether the absorption maximum, which is higher than the other absorption maximum, is at a higher or lower wavelength.
  • the absorbance or transmission of the marker molecules is measured at both absorption maxima. Due to the different height of the two absorption maxima, different measuring ranges are obtained.
  • the lateral flow assay of the present invention can also contain other common features, such as a control zone in which the correct The functionality of the test is checked and a suction zone to promote the migration of the samples.
  • the invention also relates to a method for quantifying an analyte comprising the steps
  • the invention also relates to a device for reading out the lateral flow assay according to the invention
  • At least one detection device for measuring the at least two wavelengths of the lighting device
  • detection conjugates are used for labeling with LFAs, which have a defined absorption maximum, such as gold nanoparticles with an absorption maximum between 520-540 nm.
  • the dynamic range of the method is determined by the fact that the color in the test zone is saturated with a few nanoparticles and can no longer be resolved by the reading devices. This problem also occurs when using other colored nanomaterials such as latex nanoparticles, cellulose-based nanoparticles or other metal-based metal nanoparticles. When using less colored nanomaterials, the dynamic range can be shifted to higher concentrations, in which case the The sensitivity of the method is lower and the overall width of the dynamic range does not change.
  • marking complexes are used which have at least two different absorption maxima at clearly un different wavelengths.
  • the absorption maxima of the marking complex differ in height.
  • the marking complexes can be detected with different sensitivities using optical measuring methods.
  • small amounts of the marking complex for example if the amount of analyte corresponds to the detection limit of the measurement method, only the stronger absorption maximum of the marking complex is recognized, with the second, weaker absorption maximum not being able to be detected.
  • the quantifiable amount of the marking complex is limited accordingly by the measuring system. From a corresponding amount of marking complex, the measuring system can no longer resolve the optical signal of different amounts of the marking complex. If there are two absorption maxima of the same marking complex available, the amount of marking complex can still be quantified when using the weaker absorption maxima if the measurement method has already reached saturation when measuring the stronger absorption maxima.
  • the markings used have two or more absorption maxima in the range between 200-2000 nm.
  • gold nanorods, silver nanoplates or silica nanoshells are used.
  • latex or silica nanoparticles containing various dyes can also be used.
  • the different absorption maxima are typically at least 100 nm apart.
  • spectra of gold nanorods with different absorption spectra are shown by way of example. These nanorods have an absorption maxima in the range between 520-540 nm and a second absorption maxima in the range between 600-800 nm.
  • the shape and size of the marking can be used for the Different application, but it is preferred that the materials are homogeneous in size and shape within a process.
  • the gold nanorods in the spectra shown by way of example have a length between 50-70 nm and a diameter of 10-20 nm.
  • nanomaterials are also bound to one or more affinity molecules on the surface.
  • the ligands were bound to the nanomaterials either by passive adsorption or covalently by chemical reactions.
  • Antibodies are used, for example, as ligands.
  • a further aspect of the method is a reading device which can quantify the amount of the marking complex in the test zone.
  • the amount of the marking complex accumulated in the test zone is quantified at two different wavelengths.
  • a preferred device be seated one or more light sources, which emits light according to the different wavelengths of the absorption maxima.
  • the device has one or more detectors that detect the light that is reflected or absorbed by the test zone.
  • the detector can be a camera, which images the test zone and the discoloration of the test band is then quantified by the device.
  • Another possibility for a detector are photodiodes, which detect the light that is reflected or absorbed on the membrane.
  • the reading device quantifies the amount of the marking complex using light of the two different wavelengths.
  • the wavelengths of the light correspond to the absorption maxima of the marking complex.
  • the reader can then calculate the concentration of the analyte.
  • a schematic representation is shown in FIG.
  • the invention also relates to a method for quantifying an analyte comprising the steps:
  • gold nanorods have plasmon resonance, which can be influenced by the size of the rods, the absorption properties can be controlled by the synthesis conditions.
  • the absorption spectrum of a 10 nm diameter and 40 nm long gold nanorod has two absorption maxima.
  • An absorption maximum, caused by the transversely directed surface plasmon resonance of the gold nanorod, is at 525 nm.
  • the second absorption maximum, caused by the longitudinally directed surface plasmon resonance, is at 800 nm.
  • the absorption maximum at 800 nm has an absorption 4.1 times as high like the maximum at 525 nm. Marking complex
  • gold nanorods are used to produce a detection molecule.
  • anti-CRP antibodies were passively bound to the citrate-stabilized gold nanorods by adding the antibody directly to a solution of the nanorods. After a 30-minute incubation, the remaining binding sites on the nanorods were blocked with BSA and the marking complex was washed by centrifugation at 8000 g and resuspension in water. The solution was adjusted to an OD 10 at 525 nm.
  • the marking complex was introduced into a glass fiber membrane with an Airjet Quanti module from the Biodot XYZ Series dosing platform. With the spray function, the marking complex was sprayed onto the pad at a pressure of approx. 4 psi and a metering rate of 10 pl cm -1.
  • the glass fiber membrane with the introduced marking complex was then dried for at least two hours at 37 ° C. in a drying cabinet.
  • the Cavro unit of the Biodot dosage platform was used to apply 1 pg of anti-CRP Ab / ml in PB buffer at a dosage rate of 1 ml / cm. After the coating, the membrane was dried at 37 ° C. for 1 h.
  • a sample zone, a reaction zone, a detection zone and a suction zone were glued to a backing card and then cut into 4 mm wide strips.
  • the reading device consists of an illumination source which has two LEDs with wavelengths of 525 nm and 800 nm. These LEDs were placed in such a way that they emit the light evenly onto the detection zone of the lateral flow assay.
  • a camera is located above the detection zone, which takes a picture of the detection zone. The device takes both an image with the camera under illumination of 525 nm and an image with illumination at 800 nm. An algorithm can then be used to calculate a concentration of the CRP in the sample from the images.
  • FIG. 3 shows the measuring range of the method when using 525 nm as the detection wavelength and when using 800 nm as the wavelength, as well as the resulting entire measuring range.
  • Silver nanomaterials were produced which have two different absorption maxima due to plasmon resonance.
  • One absorption maximum was 400 nm and the second at 600 nm.
  • the absorption maximum at 600 nm has an absorption that is 5 times as high as the maximum at 400 nm.
  • Anti-TSH antibodies were conjugated to these silver nanomaterials and this conjugate was then used as a labeling complex.
  • a nitrocellulose membrane was also coated with anti-TSH antibodies.
  • the marking complex was added to the sample and the coated nit rocellulose was immersed in this sample, incubated for 5 minutes and then evaluated with a reading device which can quantify the marking complex accumulated in the detection zone at two different wavelengths. Over a measuring range of 0.05-100 mIII / ml TSH with a coefficient of variation of ⁇ 15% TSH could be detected in the TSH-containing samples.
  • silica nanoparticles Two different dyes with different absorption maxima and extinction coefficients were included in silica nanoparticles. One absorption maximum was 450 nm and the second was 620 nm. The absorption maximum of the silica nanoparticle at 620 nm has an absorption 2.5 times as high as the maximum at 450 nm.
  • Anti-IgE antibodies were covalently immobilized on these silicon nanoparticles. This marking complex was placed in a glass fiber membrane and dried in vacuo. To coat a nitrocellulose membrane, 1 pg / cm of a mixture of four known egg allergens (ovomucoid, ovalbumin, ovotransferrin and lysozymes) in PBS buffer was added to a nitrocellulose membrane. For the production of ready-to-use test strips, a sample zone, a detection zone, a reaction zone and a suction zone were glued to a backing card and then cut into 4 mm wide strips. These test strips were placed in a case.
  • the readout unit After applying 100 ⁇ l of a sample to the sample zone, the test was placed in the readout unit.
  • the readout unit has two LEDs with wavelengths of 450 nm and 620 nm and can detect the reflected light at 450 nm as well as at 620 nm in the test zone through a line of photodiodes. It was possible to detect IgE over a measuring range of 2-5000 kU / l with a coefficient of variation of ⁇ 15% in the egg allergen-specific IgE-containing samples.

Abstract

L'invention concerne un dosage à écoulement latéral comportant - une zone d'échantillon servant à appliquer un échantillon, - une zone de réaction dotée de molécules de première affinité, - une zone de détection dotée de molécules de seconde affinité, caractérisé en ce que les molécules de première affinité présentent un marquage mesurable optiquement qui comprend au moins deux maxima d'absorption différents qui présentent des pics différents et sont utilisés à des fins de quantification.
EP21717922.5A 2020-04-16 2021-04-16 Dosage à écoulement latéral Pending EP4136449A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP20169903 2020-04-16
PCT/EP2021/059846 WO2021209576A1 (fr) 2020-04-16 2021-04-16 Dosage à écoulement latéral

Publications (1)

Publication Number Publication Date
EP4136449A1 true EP4136449A1 (fr) 2023-02-22

Family

ID=70292903

Family Applications (1)

Application Number Title Priority Date Filing Date
EP21717922.5A Pending EP4136449A1 (fr) 2020-04-16 2021-04-16 Dosage à écoulement latéral

Country Status (2)

Country Link
EP (1) EP4136449A1 (fr)
WO (1) WO2021209576A1 (fr)

Family Cites Families (16)

* Cited by examiner, † Cited by third party
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US4703017C1 (en) 1984-02-14 2001-12-04 Becton Dickinson Co Solid phase assay with visual readout
US5451504A (en) 1991-07-29 1995-09-19 Serex, Inc. Method and device for detecting the presence of analyte in a sample
US5798273A (en) 1996-09-25 1998-08-25 Becton Dickinson And Company Direct read lateral flow assay for small analytes
US6394952B1 (en) * 1998-02-03 2002-05-28 Adeza Biomedical Corporation Point of care diagnostic systems
US6136610A (en) 1998-11-23 2000-10-24 Praxsys Biosystems, Inc. Method and apparatus for performing a lateral flow assay
JP2002303629A (ja) * 2001-04-06 2002-10-18 Matsushita Electric Ind Co Ltd 免疫クロマトデバイス及びそれを用いた被検物質測定方法
ES2323311T3 (es) 2003-06-04 2009-07-13 Inverness Medical Switzerland Gmbh Disposicion optica para dispositivo de lectura de analisis.
US20100135857A1 (en) 2007-05-02 2010-06-03 Fluidyx Pt Ltd Portable device for reading a fluorescent-labelled, membrane based assay
JP2015509201A (ja) * 2012-01-31 2015-03-26 リージェンツ オブ ザ ユニバーシティ オブ ミネソタ 熱コントラスト検定およびリーダー
PL2812675T3 (pl) 2012-02-06 2021-12-13 The Regents Of The University Of California Przenośny czytnik szybkich testów diagnostycznych
GB201317458D0 (en) 2013-10-02 2013-11-13 Spd Swiss Prec Diagnostics Gmbh Improved pregnancy test device and method
US10048259B2 (en) 2013-11-19 2018-08-14 National Tsing Hua University Portable fluorescence detection system
US20160169887A1 (en) 2014-12-15 2016-06-16 Church & Dwight, Co., Inc. Systems, devices and methods for a lateral flow assay with solution enhancement
US10094823B2 (en) * 2015-01-23 2018-10-09 Tokitae Llc Photothermal spectroscopy assay readers, and related assay kits and methods
US9625385B2 (en) 2015-02-24 2017-04-18 Tokitae Llc Photothermal spectroscopy systems for offset synchronous testing of flow assays and methods of using same
CA3079759A1 (fr) * 2017-11-04 2019-05-09 Sona Nano Tech Limited Nanoparticules metalliques et leurs procedes de production

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