RU2712249C1 - Quantitative immunochromatographic analysis method - Google Patents

Abstract

FIELD: biochemistry.SUBSTANCE: invention refers to biochemical diagnostics, particularly to methods for assessing antigen concentration when analysing blood serum, urine, food products, environmental samples and other fluids by immunochromatography. Described is a method of determining concentration of an analyte in an analysed blood sample using immunochromatographic test strips (IC-strips) containing at least one capture zone with at least with one affine reagent, comprising: application of the analysed sample on an IC-strip; recording and processing reaction results during passage of analyte through gripping areas with their subsequent interpretation, wherein: a) using an IM strip with a grip zone formed as an extended object, providing sample depletion with affinity reagents along the path of its movement under action of capillary forces; b) recording and processing of reaction results is carried out by measuring integral intensity of colouring of all areas of the capture area after passing the sample through said zone, wherein the obtained intensity of colouring is a measure of concentration of the analysed analyte in the sample, as well as by measuring the intensity of colouring the areas of the gripping zone, which is carried out from the beginning of the reaction with a certain periodicity, wherein the rate of growth of the colour of the region is a measure of the concentration of the analysed analyte in the sample, and the time for movement of the colour along the portions of the capture zone is a measure of viscosity of the solution and temperature, which is taken into account when determining concentration of the analyte, wherein the final concentration of the analyte in the sample is determined from the results of the comparison with the calibration data obtained for the analysed analyte.EFFECT: technical result is method enables high-accuracy determination of concentration of labelled analyte in sample using IC analysis.5 cl, 6 dwg, 1 tbl

Description

Technical field

The invention relates to the field of biochemical diagnostics, in particular, to methods for assessing the concentration of antigens in the analysis of blood serum, urine, food products, environmental samples and other fluids using immunochromatography.

State of the art

Currently, the method of immunochromatography (THEM), which is often called the Lateral Flow test in the English literature, is widely used in stationary medical and other laboratories, as well as in portable devices in off-laboratory conditions for quick and highly sensitive detection and determination of the content of a wide range of compounds in the studied samples. of practical interest, such as, for example, hormones, drugs and their metabolites, antibiotics, etc. (Lateral Flow Immunoassay. Eds. Wong RC T. HY - New York: Humana Press, 2009; M. Sajid, A.-N . Kawde, M. Da ud. Designs, formats and applications of lateral flow assay: A literature review. Journal of Saudi Chemical Society 2015, v. 19, pp. 689-7056 https://www.creative-diagnostics.com/Immunochromatography-guide.htm. )

The principle of IC is based on the ability of the analyzed compound (analyte) to move together with the fluid flow under the action of capillary forces along the immunochromatographic membrane, usually made in the form of a narrow strip (IC strip). The IH strip has one or more capture zones of immune complexes and a control capture zone, which are strips located across the longitudinal axis of the strip. The bands have a width of fractions of a millimeter to a millimeter and contain affinity substances (antibodies, antigens, or conjugates of low molecular weight haptens) capable of specifically binding to the analyte. Near the place of application of the analyzed sample, a reagent pad (usually made of fiberglass) containing an conjugate of antibodies with some marker is attached to the IH-strip, which allows the analyte to be further determined visually or using an instrument. The scheme of the IC strip for detecting antibodies by the sandwich method is shown in FIG. 1.

It is known that their tests are most widely used to visually assess the intensity of staining of the capture band after passing through the IH-strip of the test sample (http://faktor.ru/o_produktsii/iha). Many domestic and foreign companies offer THEM strips for conducting dozens of different clinical diagnostic tests in humans, animals, and in the environment (https://en.wikipedia.org/wiki/Lateral_low_test). It is also known that the intensity of staining of the capture band can be measured using instruments (Quantitative detection of analytes on immunochtomatographic strips, 1996, US patent 5569608, AE Urusov, A.V. Zherdev, T.A. Starovoitova, Yu.Yu. Vengerov, B .B. Dzantiev, Instrument registration of immunochromatographic tests. Clinical Laboratory Diagnostics, 2016, Volume 51, No. 3. P. 173-179). Some companies produce specialized instruments for recording the results of their analysis (www.synteco.ru/produktsiya/narkologiya, http://www.dirui-rus.com/katalog produktsii / analizatoryi mochi / h_100). When instrumental registration of ICs can be used for semi-quantitative determination of analyte content with the construction of calibration curves that allow you to calculate the analyte concentration in the sample from the color intensity of the capture zone of the IC strip.

One of the directions in the development of IC methods is the use of test strips to simultaneously determine the concentration of several analytes in the test sample. For this purpose, an IH strip is made in such a way that it contains several capture zones with affinity reagents specific for different analytes (Single or multiple analyte semi quantitative / quantitative rapid diagnostic flow test system for large molecules. 2001, R. Buck, US patent 6,258,548. Multi-analyte assay. 2007, Japan patent JP 5214723 B2). Another direction in the development of IC methods is to increase sensitivity. To this end, to detect the binding of analytes to affinity reagents, markers with fluorescence when exposed to visible light are used (Method and test strip for determining an analyte. 2002, Jorg Bausback US patent 6,335,205 B1, Lateral flow quantitative assay method and strip and laser-induced fluorescence detection device therefor, Kie-Bong Nahm, et. al. 2002, US patent 7371582 B2).

The prior art also known to conduct quantitative studies using their strips by measuring the rate of accumulation of color during the passage of the analyte with the conjugate along the strip (company Scienion). However, the reliability and accuracy of the results obtained using this method is doubtful, since it is known that the viscosity and temperature, which affect the speed of dyeing of the test strip, are not taken into account in this method.

However, the most important indicator of their tests is their accuracy. When passing through the IC strip of the test sample, only part of the analyte interacts with the affinity reagents of the capture zone. The other part of the sample with analyte moves further along the strip, reaching the control strip, or generally leaving into the suction pad; the amount of analyte bound to the capture zone depends on its concentration in the test sample, as well as on the viscosity of the sample and the temperature at which the reaction takes place (https://dspace.lboro.ac.uk/dspace-jspui/bitstream /2134/12905/1/Thesis-2004-Li.pdf).

It is known that one of the main directions in the development of modern IC methods is the development of methods for increasing the accuracy of determining bound analyte (Truus Posthuma-Trumpie, Jakob Korf Lateral Flow Assay: Its Strengths, Weaknesses, Opportunities and Threats. A Literature Survey, Analytical and Bioanalytical Chemistry, 2008, v. 393 (2), pp. 569-582. U. Babu, F. Aberl, M. Scheibenzuber, R. Sambursky, R. VanDine, J. Sambursky. Method to increase specificity and / or accuracy of lateral flow immunoassays. 2016, US patent 9.250,236 B2, Dongfang Li, 2004, Development of immunoassay screening methods using long wavelength fluorescence Doctoral Thesis Loughborough University). To increase the accuracy of the analysis, some companies offer special techniques and devices that can reduce the influence of the viscosity of the test sample (Viscosity independent paper microfluidic inhibition. W. Guo, J. Hansson, and W. van der Wijngaart. Proceedings of The 20th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2016 (pp. 13-14) .and also conduct them at a constant temperature (https://atl-ltd.ru/wp-content/uploads/2015/06/heatsensor-duo.png However, these methods complicate and increase the cost of analysis, which limits the scope of the method.

To increase the accuracy of the method of IC, it is necessary to identify the entire analyte, which is applied to the strip from the test sample. In this regard, the closest solutions to the proposed invention are methods that describe the use of IH strips containing several capture zones with the same affinity reagents. This method of conducting their analysis is described in several patents (Method for the determination of analyte concentration if a lateral flow sandwich immunoassay exhibiting high-dose Hai-Hang Kuo, 2001, US patent 6,183,972 B1. Device and method for obtaining clinically significant analyte ratios Hai- Hang Kuo 2002, US patent 6,436,721 B1). The authors of the patented solutions suggest using such strips to expand the dynamic range of the measured analyte concentrations. The part of the analyte that did not contact the first capture band moves further along the IH-strip and reacts with the next capture zone. The authors of the proposed patents propose using each capture zone to determine the analyte concentration in one range, but do not propose using this approach to increase the accuracy of measurements. In addition, two or three additional bands do not allow constructing the analyte depletion curve when passing through the IH-strip, do not give full confidence that the entire analyte was associated with the capture zones. In addition, the staining intensity of each capture zone will continue to depend on the speed of movement of the sample along the IH strip.

SUMMARY OF THE INVENTION

The technical problem is the lack of solutions that can accurately determine the concentration of the analyte labeled with a marker in the sample using their analysis.

The technical result is achieved by the fact that for the analysis they use an IH strip with a capture zone formed in the form of an extended continuous or discontinuous strip containing affinity molecules for the analyte, which ensure its depletion along the strip under the action of capillary forces. The results of the analysis are recorded by measuring the concentration of the marker by the integrated intensity of the staining of the capture zone during or after the sample passes through this zone, while the obtained value of the staining intensity, as well as the nature of the change in the intensity of staining inside the capture zone, characterize the concentration and integral content of the analyte under study in the sample .

The technical result of this invention is to increase the accuracy of determining the concentration of analyte in the test sample for the time required for the passage of the sample along the capture zone of the IC strip. As the sample moves along the capture zone of the IQ strip, the analyte is depleted with affinity reagents and the staining intensity of the capture zone (capture zones) made in the form of an extended object (a longitudinal strip or a set of individual spots along the longitudinal axis of the IX strip) decreases. Determination of the integrated intensity of the coloration of the capture zone (capture zones), as well as the registration of changes in the intensity of the coloration as the sample passes along the strip, allows to significantly reduce the effect on the results of quantitative analysis of the analyte (s) in the sample viscosity of the sample and the ambient temperature and increase the accuracy analysis. Additionally, using the same IH strip, it is possible to measure the color intensity of the capture zones for a control sample containing a known amount of reference analyte.

Determination of the analyte concentration in the sample is achieved by measuring the intensity of the coloration of the capture zones of the IH strip and subsequent analysis of the data obtained, in the case where the capture zones are presented as a continuous strip of a certain width or as several spots of a certain diameter containing affinity reagents and located along the longitudinal axis THEM strips. In this case, using the instrument, (A) the intensity of coloring of each capture zone after passing the sample along the IH strip is recorded, and (or) (B) the rate of change in the color intensity of the capture zones as the sample passes along the IH strip.

Brief Description of the Drawings

In FIG. 1. presents a diagram of a standard IC strip when conducting analysis by the sandwich method, where:

1. Sample application area (pillow)

2. The area containing the conjugate (pillow)

3. Test area

4. The control zone

5. Suction cushion

6. Start of analysis (application of the sample)

7. Advance of conjugate complexes with antigen in a strip

8. The final stage of analysis. Unbound reagents passed into the suction pad.

9. Antigen

10. Conjugate (antibodies to the antigen containing the label)

11. Antigen-conjugate complexes

12. Specific antibodies

13. Secondary antibodies

14. Conjugate complexes with secondary antibodies

15. Secondary antibody-conjugate-antigen complexes

In FIG. Figure 2 presents images of the capture zones of ten IH strips made in the form of spots with the same concentration of affinity reagents (antibodies to total IgE) and located along the longitudinal axis of the IH strip. The numbers below the images (from 2 to 1000 units / ml) indicate the concentration of the analyte in the test sample. Marker - Su5 fluorochrome, images are inverted. Move the sample from left to right.

In FIG. 3 shows the fluorescence intensity of the individual spots of the IC strip after analyzing the images shown in FIG. 2. Each curve corresponds to one analyte concentration (IgE). Analyte concentrations are indicated to the right of the graph. The seventh spot differs from the background only at the highest concentration of IgE (1000 units)

In FIG. 4 schematically shows the options for the location of zones (zones) capture on the IH strip. 16 - the capture zone is a continuous strip containing antibodies to the analyte; 17 - capture zones are individual spots containing the same concentration of affinity reagents; 18 - capture zones are individual spots containing an increasing concentration of affinity reagents.

In FIG. Figure 5 shows the images of the IR strips in the manufacture of the capture zone as a continuous strip in the light of fluorescence of the marker after immunochromatography with samples (representing blood serum) containing different amounts of analyte (analyte - total IgE). As a probe, anti-IgE was selected at a concentration of 1 mg / ml, deposited in the form of a strip running along the EM strip. Marker - dye Su5. A line is plotted along each image, and a change in the fluorescence intensity of Cy5 dye along this line is shown below the images.

A - IgE concentration - 50 mg / ml.

B - IgE concentration - 25 mg / ml.

B - The concentration of IgE - 12.5 mg / ml.

In FIG. Figure 6 shows the image of the capture zones of the IC strips after analysis of samples with different viscosities. A line is plotted along each image, and a change in the fluorescence intensity of Cy5 dye along the line is shown below the images.

A. 10% bovine serum albumin (BSA) by volume (low viscosity sample) was added to the sample.

B. 10% by volume of glycerol (sample with increased viscosity) was added to the sample.

The implementation of the invention

Below is a more detailed description of the claimed invention. The present invention may be subjected to various changes and modifications understood by one skilled in the art upon reading this description. Such changes do not limit the scope of claims. For example, markers for visualization can be changed, and a device for recording analysis results, an image analysis program, probes deposited on an EM strip, etc.

A membrane in which there are capture zones with specific affinity reagents to the analyte under study, and along which the sample under investigation can move due to capillary forces, can act as IH strips. Nitrocellulose membranes are most widely used for the manufacture of IC strips. The membrane can also be polytetrafluoroethylene, nylon, etc. The pore size determines the rate of diffusion of the sample across the membrane, which, in turn, determines the time of the analysis, since at larger pores the sample moves faster along the IH-strip.

The capture zones of the IC strips may contain the same or different concentration of affinity reagents, and, depending on the concentration of affinity reagents, the amount of analyte bound to them changes. As affinity reagents can be antigens or antibodies to the studied analytes.

When carrying out the analysis by the sandwich method, the test sample is applied to the beginning of the IH strip (similar to the study using the standard strip shown in Fig. 1), then the sample moves along the IH strip, and the analyte deposited on the strip binds to the conjugate containing any marker and located zone 2, forming an analyte conjugate complex. The marker may be colloidal gold, dye, fluorochrome or other substance, the concentration of which can be determined using an appropriate analyzer. This complex moves along the IH strip and interacts with affinity reagents immobilized in the capture zone or zones (located as individual spots or a continuous strip), as a result of which, depending on the marker used, these zones become colored or begin to fluoresce.

The studied samples can be any biological fluids containing substances (analytes) to which there are specific affinity substances immobilized in the capture zones. Registration of the intensity of coloring of the capture zones is carried out using the device, depending on the type of marker used. For example, when using a marker in the form of colloidal gold, a reflectometer recording the intensity of reflection is used; when using fluorescent markers, a fluorimeter is used to measure the fluorescence intensity of the sections of the IH strip.

Below is a description of examples of implementation of the invention, showing the results of the analysis of 10 samples with capture zones made in the form of individual spots with the same affinity reagent content on a nitrocellulose IC strip. Two rows of 7 spots each containing the same amount of affinity reagent (anti-IgE at a concentration of 1 mg / ml) are applied to the strip. Samples contain various analyte concentrations (see FIG. 2). When a sample passes through the first capture zone (first test zone), a part of the analyte containing the fluorescent dye Cu5 as a marker binds to reagents immobilized in this zone, while the amount of bound analyte depends on its concentration in the sample, as well as on the speed of the sample on their strip. The analyte, which did not contact the first spot, continues to move along the IH-strip and interacts with affinity reagents located in the second spot, etc. It is seen that the spots located closer to the beginning of the strip are colored more strongly; as the sample moves along the strip, the sample is depleted, as a result of which the following spots are less colored. In FIG. Figure 3 shows the distribution of spot fluorescence intensity. As can be seen from the data presented in figure 3, even at a high analyte concentration in the test sample, the last spot gives a signal that almost matches the background, which indicates a high degree of analyte depletion as it moves along the IH strip. The number of spots and the content of affinity substance in them are selected in such a way that the color intensity of the last spot does not differ from the background color, that is, so that after the sample passes through all the capture zones in the sample applied to the strip, there is no analyte left, since almost the entire analyte contacted capture zone reagents. Usually, when 7 spots are located along the strip of 7, the last spot is no longer stained, which indicates that as the sample moves along the strip of stripes, it is completely bound to the capture zone reagents. The intensity of the coloration of the capture zones is measured as the sample passes through the IH strip. In our case, the measurement was carried out using a device similar to that described previously (Yu. Lysov, V. Barsky, D. Urasov, R. Urasov, A. Cherepanov, D. Mamaev, Y. Yegorov, A. Chudinov, S. Surzhikov, A Rubina, O. Smokdovskaya, A. Zasedatelev. Biomedical Optics Express, Vol. 8, No. 11, Nov 2017, 4799) and programs, e.g. Imageware (D. Gryadunov, E. Dementieva, V. Mikhailovich, T. Nasedkina , A. Rubina, E. Savvateeva, E. Fesenko, A. Chudinov, D. Zimenkov, A. Kolchinsky, and A. Zasedatelev, "Gel-based microarrays in clinical diagnostics in Russia", Expert Rev. Mol. Diagn. 11 (8), 839-853 (2011).

In FIG. Figure 3 shows the magnitude of the signals of individual spots of the IC strips after analysis of samples with different analyte concentrations. Even at a high analyte concentration in the test sample, the last spot gives a signal that practically coincides with the background, which indicates a high degree of analyte depletion as it moves along the IH strip.

Under reproducible conditions for setting the analysis for each sample, the staining intensity of the capture zone is proportional to the analyte concentration in the test sample. In the case of the same concentration of developing substances in spots with affinity reagents, the intensity of their staining will naturally decrease with distance from the start, and the analyte content in the test sample can be determined in different ways.

1. As in the standard analysis of the IC strips, the staining intensity of any spot after passing through the sample reflects the initial analyte concentration in the sample for a given viscosity of the sample and a given temperature. It is this method of recording the results of immunochromatographic analysis that is used when using standard IR strips. Registration of the staining intensity of all spots gives more accurate information about the concentration of the analyte in the test sample.

2. The rate of increase in the intensity of staining of the spot over time also reflects the concentration of analyte in the sample. In this case, the signal rise rate, in addition to the initial analyte concentration, also depends on the viscosity of the sample and temperature.

3. The time difference between the appearance of a reliable signal between adjacent spots characterizes the viscosity of the sample and the temperature of the analysis.

4. The sum of the staining intensities of all spots (all capture zones) is a measure of the analyte concentration in the test sample, regardless of the viscosity of the sample and temperature.

The capture zones of the EM strip can be made in different ways. In FIG. 4 shows an image of an IK strip in which the capture zone is a strip of a certain width containing the same concentration of affinity reagents along the strip. Capture zones can be represented as individual spots in which the concentration of affinity reagents remains constant or gradually increases.

In FIG. Figure 5 shows examples of determining the concentration of IgE in serum containing various concentrations of IgE. For determination, a strip of nitrocellulose NC-150 from Sartorius, 5 × 50 mm in size, was made. On this strip, the capture zone was made in the form of a single longitudinal strip containing a total Anti-IgE allergen at a concentration of 1 mg / ml. The strip length was 3 mm; the width was about 0.8 mm. On one side of the strip, several layers of filter paper were attached to absorb the solution applied to the strip.

Sera containing IgE were kept in a solution of the conjugate containing Cy5 as a marker for 2 min, then the resulting mixture (various serum proteins bound to the colored IgE conjugate and pure conjugate) was applied to the edge of the STRIP, opposite to to which filter paper was attached, and allowed to diffuse freely. After 30 minutes An image of the strip was obtained in the light of Su5 fluorescence using a microfluorimeter (Yu. Lysov, V. Barsky, D. Urasov, R. Urasov, A. Cherepanov, D. Mamaev, Y. Yegorov, A. Chudinov, S. Surzhikov, A. Rubina , O. Smokdovskaya, A. Zasedatelev. Biomedical Optics Express, Vol. 8, No. 11, Nov 2017, 4799) and the images were digitized.

The image of the nitrocellulose strip shows that as the analyzed sample advances, the content of the marker (Cy5) in the capture zone containing anti-IgE decreases. At the same time, the marker content in the center of the band drops faster than at the edges, the anti-IgE band after the reaction has two “tails”. This phenomenon is reproduced in all experimental settings with different concentrations of IgE in serum and is explained by the fact that the sample moves along one front along the entire width of the strip, and in addition, the molecules in the moving complex move not only along the nitrocellulose membrane, but also constantly diffuse across membranes. Therefore, in the center of the band with anti-IgE, a regular depletion of the sample with colored IgE occurs; and due to the transverse diffusion of fresh portions of IgE, additional coloring of the edges of the strip is observed, and IgE depletion at the edges of the strip is not observed. In addition, there is an increase in the fluorescence intensity of the entire strip caused by nonspecific adsorption of the components of the diffusing mixture of molecules, including those containing Cy5, with nitrocellulose.

The same figure (Fig. 5) shows how the fluorescence intensity of the marker changes along a line drawn along the axis of the capture zone. In FIG. 5A, 5B, 5B show how the marker content changes along the axis of the capture zone during immunochromatography of samples with different IgE contents. It is seen that, as IgE decreases in the sample, the peak height and the rate of decrease in staining intensity along the axis decrease. This is the result of the fact that, for a given anti-IgE concentration, a decrease in the IgE concentration in the sample leads to its faster depletion as it moves along the capture zone.

When the IC is repeated, the IgE analyte concentration is determined in the same serum, the area under the curve reflecting the change in marker fluorescence along the capture zone varies within ± 5%. The values of the signal maximum and the areas under the curves for different dilutions of this serum are shown in the Table, which presents the values of the areas under the curves characterizing the change in marker concentration (Cy5) when passing samples containing a decreasing analyte concentration (total IgE) along the band containing the affinity reagent .

According to the data presented in the Table, there is a correlation between the concentration of the analyte and the measured values.

It should be noted that in this experiment, their determination was carried out under identical conditions for serum with the same viscosity value. To test how the proposed method works when analyzing sera with different viscosities, we made two dilutions of the same serum. In the first case, 10% by volume of glycerol was added to the initial serum; in the second case, 10 by volume of BSA was added to the same serum. Thus, we obtained two samples with the same analyte concentration (in this case, the total IgE analyte was analyzed), but with different viscosities. In FIG. Figure 6 shows the change in the maximum intensity and the shape of the curve of the change in the fluorescence intensity of the marker for one and this serum, with the addition of glycerol or buffer. Images of the IR strips after interaction with affinity reagents in the capture zone in the light of fluorescence of the marker are shown in FIG. 6. Judging by the data presented in FIG. 6, both the maximum value and the shape of the curve of the marker content along the capture zone change with a change in serum viscosity. So, for a sample diluted with glycerol, the maximum value of the marker signal is 31000.e., while for a serum diluted with a buffer -28000. At the same time, the area under the curve reflecting the interaction of the marker with affinity reagents remains constant. These data once again indicate that only the measurement of the coloration of the capture zone in standard TH = strips is not enough for accurate measurements at that time.

When conducting competitive research, the IH-strip also contains a reagent pad and capture zones (http://www.ivdtechnology.com/article/concurrent-engineering-lateral-flow-diagnostics), however, in this case, the concentration of affinity reagents in the capture zones and the number of capture zones is selected so that in the first spot located along the direction of movement of the sample, there is no color.

In the case of applying several different extended capture zones containing affinity reagents to different analytes onto an IK strip, such a strip can be used for parallel multiparameter analysis of several analytes.

To increase the accuracy of measurements on an IH strip, several sets of strips or spots containing affinity reagents identical to the first set, parallel to the first set and located across the longitudinal axis of the IH strip, can be arranged.

An additional increase in the accuracy of determining the concentration of analyte can be achieved by adjusting for the viscosity of the sample and the temperature of the analysis. Such a correction can be made by adding to the test sample any other, control analyte in a known concentration. At the same time, capture bands with affinity reagents specific to the control reagent should be located on the IH strip. When calibrating the ИХ strips by the manufacturer, correction coefficients should be in the strip passport. These coefficients must be entered if the curve for changing the brightness of the control capture zone with affinity reagents in the control analyte differs from that for normal viscosities or temperatures.

Claims (10)

1. The method of determining the concentration of analyte in the test blood sample using immunochromatographic test strips (IH strips) containing at least one capture zone with at least one affinity reagent, including - applying to the IC strip of the test sample, - registration and processing of the reaction results during the passage of the analyte through the capture zone with their subsequent interpretation, characterized in that: a) use an IH-strip with a capture zone formed in the form of an extended object, which ensures depletion of the sample by affinity reagents along its movement under the action of capillary forces; b) registration and processing of the reaction results is carried out by measuring the integrated intensity of the staining of all portions of the capture zone after passing the sample through this zone, while the obtained value of the staining intensity is a measure of the concentration of the analyte under study in the sample, as well as by measuring the intensity of staining of the portions of the capture zone, which produce from the beginning of the reaction with a certain periodicity, while the growth rate of the staining of the plot is a measure of the concentration of the analyte in the sample, and the advance of coloration along portions of the grip region is a measure of the solution viscosity and temperature, which take into account in determining the analyte concentration, wherein the final concentration of the analyte in the sample is determined by comparison with calibration data obtained for the test analyte. 2. The method according to p. 1, characterized in that the extended object, providing depletion of the sample, is formed in the form of a line or a set of spots. 3. The method according to p. 1, characterized in that the concentration of the affinity reagent in the capture zone is constant or changes according to a predetermined law along the length of the capture zone. 4. The method according to p. 1, characterized in that a control analyte is introduced into the sample, the presence of which is potentially impossible in the test sample, in a known concentration, while the IH strip contains a capture zone or zones with affinity reagents specific to the control analyte. 5. The method according to p. 1, characterized in that the processing of the results is carried out by comparing the integrated intensity of the staining of the given capture zone, as well as the nature of the changes in the intensity of the staining inside the capture zone and / or the rate of change in the intensity of the coloring of this capture zone or its sections with previously obtained calibration data for the control analyte are determined by the correction factors used to determine the concentration of the analyte under study.

Images