WO2021209576A1

WO2021209576A1 - Lateral flow assay

Info

Publication number: WO2021209576A1
Application number: PCT/EP2021/059846
Authority: WO
Inventors: Friedrich Scholz; Theresa Heckmann; Lukas RÜTTINGER; Anne-Marie GAD; Hans-Hermann SÖFFING
Original assignee: Senova Gesellschaft für Biowissenschaft und Technik mbH
Priority date: 2020-04-16
Filing date: 2021-04-16
Publication date: 2021-10-21
Also published as: EP4136449A1

Abstract

Disclosed is a lateral flow assay comprising - a sample zone for applying a sample, - a reaction zone with first affinity molecules, - a detection zone with second affinity molecules, characterized in that the first affinity molecules have an optically measurable marking that includes at least two different absorption maximums which have different peaks and are used for quantification purposes.

Description

Lateral Flow Assav

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.

Lateral Flow Assay (LFA) are widely used for the detection of a wide variety of analytes in different samples. The LFA are used in well-equipped laboratories or outside the laboratories by untrained staff as a point-of-care test (POCT). In a typical LFA, a liquid sample (e.g. from a patient) is applied to the sample application field of the LFA. The sample interacts with the individual components of the LFA and causes optical changes in the LFA. These 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.

Typically, 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.

If the sample is placed on the application field, 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. At the test zone, 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.

If 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.

If there is only one receptor element and an additional molecule that has structurally identical or structurally similar sections of the analyte, this is referred to as a competitive assay. For example, the label can be bound to the analyte (or a structurally similar molecule). This reagent (marking complex) 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. In another assay format, 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. In both competitive assay formats, 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.

Some 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

For the detection of larger analytes (1,000-1,000,000 Da), 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.

As already mentioned, 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. There are a variety of protocols that describe the attachment of the label to ligands. Traditionally, LFA are evaluated through the eye of the user. However, there are a large number of applications in which a quantification of the analyte concentration is necessary. For this reason, a wide variety of 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. In most cases, the light from the light source (for example an LED), which is either reflected or absorbed by the test zone, 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. For example, such readout units are described in EP 1484601, KR102005597B1, US 20150094227, US 7,297,529. A typical problem with the described lateral flow assays and the quantification of the signal is the limited dynamic range. 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.

Some measures have already been described in order to achieve better measuring ranges, for example by using fluorescence-based markings and evaluating the fluorescence intensity of the test bands (US20100135857A1; US10048259B2). Other inventions describe signal amplification reactions (US20160169887A1) or use photothermal sensors as a detection system and metal nanoparticles as marker molecules in order to increase the sensitivity and the dynamic range (US10094823B2; US9625385B2; US10094823B2). Improved measuring ranges can be achieved with the methods mentioned. However, this increases the metrological effort and thus the costs of the readout device.

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

- a sample zone for applying a sample

- a reaction zone with first affinity molecules

- a detection zone with second affinity molecules characterized in that 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.

As is customary in the prior art, 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.

It is crucial that 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.

It is important that the two absorption maxima have a different height so that they give different absorption values depending on the concentration of the analyte.

According to the invention, measurements are made at both absorption maxima. Depending on the concentration of the analyte, either 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. For example, the marking could be a blue and a red dye, which have absorption maxima of different heights.

However, preference is given to using a marker compound which has at least two different 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

Application of a sample to the lateral flow assay according to the invention

- Measure the signal at at least two wavelengths

- Calculate the concentration of the analyte.

The invention also relates to a device for reading out the lateral flow assay according to the invention

- At least one lighting device with at least two wavelengths

- a holding device for a lateral flow assay

- At least one detection device for measuring the at least two wavelengths of the lighting device

- an evaluation unit for calculating the concentration of the analyte.

Usually, 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.

In the method described here, marking complexes are used which have at least two different absorption maxima at clearly un different wavelengths. In addition, the absorption maxima of the marking complex differ in height. In this way, the marking complexes can be detected with different sensitivities using optical measuring methods. In the case of 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. At higher concentrations of the marking complex, 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. Typically, gold nanorods, silver nanoplates or silica nanoshells are used. However, latex or silica nanoparticles containing various dyes can also be used. The different absorption maxima are typically at least 100 nm apart. In FIG. 2, 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.

These 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. In addition, 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. To emit light of different wavelengths, either different LEDs can be used or a light source is used and then the emitted light is modified by optical components such as optical filters or grids so that only light of defined wavelengths reaches the test zone. 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. Using suitable algorithms, 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:

Applying a sample to a lateral flow assay with a sample zone for applying a sample to a reaction zone with first affinity molecules of a detection zone with second affinity molecules, the first affinity molecules having an optically measurable marking which has at least 2 different absorption maxima of different heights, which are used for quantification will

Measuring the signal at at least two wavelengths, the at least two wavelengths corresponding to absorption maxima of the optical marking

Calculate the concentration of the analyte.

Examples

example 1

Synthesis and properties of gold nanorods

Since 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

These gold nanorods are used to produce a detection molecule. For this purpose, 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.

Nanorod-based LFA

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 (reaction zone) was then dried for at least two hours at 37 ° C. in a drying cabinet. To coat a nitrocellulose membrane, 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. For the production of ready-to-use test strips, 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.

Readout device

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.

procedure

100 ml of a sample are applied to the sample zone of the LFA and incubated for 10 minutes. After 10 minutes, the discoloration of the detection zone is measured with the reader and the CRP concentration is output. It was possible to detect CRP with a coefficient of variation of <15% in the CRP-containing samples over a measuring range of 0.5-200 pg. 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.

Example 2

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. For a late ral flow assay, 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. Example 3

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.

After applying 100 μl 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.

Claims

1. Lateral flow assay with a sample zone for applying a sample of a reaction zone with first affinity molecules of a detection zone with second affinity molecules, characterized in that 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.

2. Lateral flow assay according to claim 1, wherein the first and second affi nity molecules are independently antibodies, antibody fragments, antigens, antigen fragments, antigen-like structures, protein A, protein G, streptavidin or neutravidin.

3. Lateral flow assay according to one of claims 1 or 2, wherein the optically measurable marking is carried out by marking compounds selected from dyes, nanomaterials or combinations thereof.

4. Lateral flow assay according to one of the preceding claims, wherein the nanomaterials are metal nanomaterials, latex nanomaterials, cellulose-based nanomaterials, silica nanomaterials or combinations thereof.

5. Lateral flow assay according to one of the preceding claims, wherein the optically measurable marking comprises two different Markierungsverbindun gene.

6. Lateral flow assay according to any one of the preceding claims, wherein the optically measurable marker comprises a marker compound which has at least 2 different absorption maxima of different heights.

7. Lateral flow assay according to one of the preceding claims, wherein a control zone is also present.

8. Lateral flow assay according to one of the preceding claims, wherein a suction zone is also present.

9. Lateral flow assay according to one of the preceding claims, wherein two of the at least two absorption maxima are at a distance of at least 100 nm.

10. Lateral flow assay according to one of the preceding claims, wherein one of the at least two absorption maxima is at least 1.5 times higher than the absorption maximum of another absorption maximum.

11. A method for quantifying an analyte comprising the steps

Application of a sample to a lateral flow assay according to one of Claims 1 to 10

Calculate the concentration of the analyte.

12. Device for reading out the lateral flow assay according to one of claims 1 to 10 comprising at least one lighting device with at least two wavelengths and a holding device for a lateral flow assay at least one detection device for measuring the at least two wavelengths of the lighting device; an evaluation unit for calculating the concentration of the analyte.

13. Use of a lateral flow assay according to one of claims 1 to 10 for determining the concentration of an analyte by measuring the optical absorption at two different wavelengths which correspond to the absorption maxima of the optical marking.