Immunochromatographic analysis method, storage medium and system

Abstract

The invention provides an immunochromatographic analysis method, a storage medium and an immunochromatographic analysis system. The immunochromatographic analysis method comprises the following steps: providing an immunochromatographic analysis reagent card, and arranging a detection line on an NC membrane of a test strip of the reagent card; the detection line is at least coated with a capture object; a first reagent is provided, the first reagent comprises a first aptamer coupled with a tracer, and the tracer is at least used for indicating the existence and / or content of an object to be detected and generating an optical signal T; the optical signal T of the tracer is at least used for providing a detection result; and correcting the detection result by using a correction parameter to obtain a corrected detection result. The correction parameter is at least related to the coating amount of the captured substance on the detection line, and / or the correction parameter is at least related to the coating uniformity of the captured substance on the detection line, and / or the correction parameter is at least related to the speed of the chromatographic reaction.

Description

Technical Field

[0001] This application relates to the field of medical testing, specifically to an immunochromatographic analysis method, storage medium, and system. Background Technology

[0002] Immunochromatography belongs to the field of POCT (point-of-care testing) and has advantages such as convenient and fast testing, low overall cost, and the ability to be stored and transported at room temperature.

[0003] In optically-based quantitative immunochromatography, light emitted from a light source illuminates the immunoassay strip. A detector then collects the reflected light, transmitted light, fluorescence, or phosphorescence signals generated by the T line and / or control line (C line) of the immunoassay strip after illumination. The collected light signals are then converted into electrical signals, the magnitude of which is related to the concentration of the analyte, thereby quantitatively detecting the concentration of the analyte.

[0004] Current immunochromatographic analysis equipment generally detects the endpoint signal after chromatography and derives the corresponding test result based on this signal. However, differences in the raw materials used in chromatographic test strips can lead to variations in physical characteristics such as pore size and porosity between different chromatographic reagent cards, with particularly significant batch-to-batch differences. This affects the chromatography speed and the final test results, resulting in inaccurate results. Furthermore, differences in the manufacturing process of chromatographic test strips can cause variations in the amount of reagent embedded in the detection line, the fixation strength, and the uniformity of distribution, affecting the test results and leading to inaccurate results.

[0005] Therefore, existing immunochromatographic analysis methods cannot overcome the methodological defects of immunochromatography, resulting in poor accuracy and repeatability of test results. Summary of the Invention

[0006] To address the methodological shortcomings of immunochromatography, this application provides an immunochromatographic analysis method, storage medium, and system.

[0007] A first aspect of this application provides an immunochromatographic analysis method, comprising:

[0008] An immunochromatographic analysis reagent card is provided, the immunochromatographic analysis reagent card including a test strip, the test strip including at least a sample pad, an NC membrane and an absorbent pad, the sample pad being located upstream of the NC membrane, the NC membrane having a detection line; the detection line being at least coated with a trap.

[0009] A first reagent is provided, the first reagent comprising a first aptamer of a conjugated tracer, the tracer being used at least to indicate the presence and / or content of an analyte and to generate a light signal T; the light signal T of the tracer being used at least to provide a detection result;

[0010] The detection results are corrected using correction parameters to obtain corrected detection results.

[0011] The correction parameter is at least related to the amount of capture on the detection line, and / or the correction parameter is at least related to the uniformity of capture on the detection line, and / or the correction parameter is at least related to the rate of the chromatographic reaction.

[0012] Furthermore, the correction parameter is also related at least to the background signal of the reagent card and / or test strip, and / or, the correction parameter is also related at least to the HCT value of the sample, and / or, the correction parameter is also related at least to the dilution ratio of the sample.

[0013] The inventors discovered that the amount of precipitate coated on the detection line (including the initial coating amount before the chromatographic reaction and the effective coating amount after the chromatographic reaction), the uniformity of the precipitate coating on the detection line, and the speed of the chromatographic reaction are key factors affecting the accuracy of the test results. In addition, the background signal of the reagent card and / or test strip, the HCT value of the sample, and the sample dilution ratio are also factors that can affect the accuracy of the test results.

[0014] Immunochromatographic analysis involves capturing tracers directly and / or indirectly through traps on a detection line. These tracers generate optical signals, which at least indicate the presence and / or quantity of the analyte. However, the amount of traps on the detection line affects the amount of tracers directly and / or indirectly captured, thus affecting the magnitude of the optical signal generated by the tracers and consequently the detection results. Therefore, accurate determination of the amount of traps on the detection line is necessary. To address this, this application establishes a correction parameter related to the coating amount of traps on the detection line. By obtaining the actual coating amount of traps on the detection line, the corresponding correction parameter is derived, and the detection results are corrected to obtain the corrected results, thereby improving the accuracy of the detection results.

[0015] Similarly, the uniformity of the coating of the captured object on the detection line also affects the uniformity of the distribution of tracers directly and / or indirectly captured by the captured object, thereby affecting the uniformity of the optical signal generated by the tracers and consequently the detection results. Therefore, in order to obtain more accurate detection results, this application sets a correction parameter related to the coating uniformity of the captured object on the detection line. By obtaining the actual coating uniformity of the captured object on the detection line, the corresponding correction parameter is obtained, and the detection results are corrected to obtain the corrected detection results, which can improve the accuracy of the detection results.

[0016] A significant influencing factor in immunochromatographic analysis is the rate of the chromatographic reaction. Differences in the raw materials of the test strips can lead to varying reaction rates, resulting in different concentrations and / or proportions of the analyte reaching the detection line. Consequently, this affects the final test results, leading to inaccurate readings. Therefore, to obtain more accurate results, this application incorporates correction parameters related to the rate of the chromatographic reaction. By obtaining the actual rate of the chromatographic reaction, the corresponding correction parameters are derived, and the test results are corrected accordingly, resulting in improved accuracy. The chromatographic reaction in this application includes physical and / or chemical and / or biological reactions of the liquid on the test strip.

[0017] In immunochromatographic analysis, the background signal of the reagent card and / or test strip can interfere with the test results and affect their accuracy. Therefore, to obtain more accurate test results, this application sets correction parameters related to the background signal of the reagent card and / or test strip. By obtaining the background signal of the reagent card and / or test strip, the corresponding correction parameters are obtained, and the test results are corrected to obtain the corrected test results, which can improve the accuracy of the test results.

[0018] The HCT value and dilution ratio of a sample can affect the accuracy of the test results. Therefore, this application sets correction parameters related to the HCT value and dilution ratio of the sample to correct the test results and obtain corrected test results, which can improve the accuracy of the test results.

[0019] Furthermore, the correction parameters include at least one of the following: a first capture coating amount correction parameter a, which is related to the amount of capture on the detection line before the chromatographic reaction proceeds to the detection line; a second capture coating amount correction parameter b, which is related to the amount of capture on the detection line after the chromatographic reaction is completed; a first capture coating uniformity correction parameter c, which is related to the amount of capture on the detection line before the chromatographic reaction proceeds to the detection line; a second capture coating uniformity correction parameter d, which is related to the amount of capture on the detection line after the chromatographic reaction is completed; and an actual chromatography rate correction parameter e, which is related to the actual chromatographic reaction rate. This can also include any combination of two, three, four, or five of these parameters.

[0020] Preferably, the correction parameters include at least the following combinations of any two, any three, any four, or any five of the following: a first capture coating amount correction parameter a, which is related to the amount of capture on the detection line before the chromatographic reaction proceeds to the detection line; a second capture coating amount correction parameter b, which is related to the amount of capture on the detection line after the chromatographic reaction is completed; a first capture coating uniformity correction parameter c, which is related to the amount of capture on the detection line before the chromatographic reaction proceeds to the detection line; a second capture coating uniformity correction parameter d, which is related to the amount of capture on the detection line after the chromatographic reaction is completed; and an actual chromatography rate correction parameter e, which is related to the actual chromatographic reaction rate.

[0021] In this context, "completion of chromatographic reaction" generally refers to a specific time point after sample addition. For example, "completion of chromatographic reaction" can be any time point between 30 seconds and 15 minutes after sample addition.

[0022] For example, the correction parameter can be any one of a, b, c, d, and e, specifically a, b, c, d, or e. Taking the correction parameter as a as an example, let the detection result be X, and the corrected detection result be Y, then Y = f(a, X). Here, "f()" represents the correction function.

[0023] For example, the correction parameter can be any combination of two of a, b, c, d, and e, such as (a, b), (a, c), (a, d), (a, e), (b, c), (b, d), (b, e), (c, d), (c, e), or (d, e). Taking the correction parameter as (a, b) as an example, then Y = f(a, b, X).

[0024] For example, the correction parameter can be any combination of three of a, b, c, d, and e, such as (a, b, c), (a, b, d), (a, b, e), (a, c, d), (a, c, e), (a, d, e), (b, c, d), (b, c, e), (b, d, e), or (c, d, e). Taking the correction parameter as (a, b, c) as an example, then Y = f(a, b, c, X).

[0025] For example, the correction parameter can be any combination of four of a, b, c, d, and e, such as (a, b, c, d), (a, b, c, e), (a, b, d, e), (a, c, d, e), and (b, c, d, e). Taking the correction parameter as (a, b, c, d) as an example, then Y = f(a, b, c, d, X).

[0026] For example, the correction parameter can be a combination of five elements: a, b, c, d, and e, which is the combination (a, b, c, d, e). Then Y = f(a, b, c, d, e, X).

[0027] Among them, the first capture coating amount correction parameter a, which is related to the amount of capture on the detection line before the chromatography reaction proceeds, is related to the initial coating amount before the chromatography reaction. The second capture coating amount correction parameter b, which is related to the amount of capture on the detection line after the chromatography reaction is completed, is related to the effective coating amount after the chromatography reaction. The combination of a and b (e.g., ab, (ab) / b) can reflect the amount / rate of capture loss during the chromatography reaction.

[0028] Specifically, the first capture coating uniformity correction parameter c, which is related to the amount of capture on the detection line before the chromatographic reaction proceeds to the detection line, is related to the initial coating uniformity before the chromatographic reaction. The second capture coating uniformity correction parameter d, which is related to the amount of capture on the detection line after the chromatographic reaction, is related to the effective coating uniformity after the chromatographic reaction. The combination of c and d can reflect the proportion of capture loss in different regions of the detection line during the chromatographic reaction. Further combining a and / or b can reflect the amount of capture loss in different regions of the detection line during the chromatographic reaction.

[0029] The rate of the chromatographic reaction mainly refers to the speed of the physical chromatography process of the liquid on the test strip. Differences in the raw materials of the test strips can significantly affect the speed of this physical chromatography process, consequently impacting the chromatographic processes of various substances in the liquid. This results in variations in the content and / or proportion of the analyte reaching the detection line, and may also affect the amount and uniformity of the analyte coated on the detection line, ultimately leading to inaccurate test results. Therefore, combining e with a and / or b and / or c and / or d can better reflect the amount of analyte loss during the chromatographic reaction, and the amount of analyte loss in different regions of the detection line.

[0030] Furthermore, the correction parameters also include any one, any combination of two, any combination of three, any combination of four, or a combination of five of the following: a first background correction parameter f related to the background signal of the reagent card and / or test strip before the chromatographic reaction; a second background correction parameter g related to the background signal of the reagent card and / or test strip during the chromatographic reaction; a third background correction parameter h related to the background signal of the reagent card and / or test strip after the chromatographic reaction; an HCT correction parameter i related to the HCT value of the sample; and a dilution ratio correction parameter j related to the dilution ratio of the sample. Optionally, the correction parameters are any one, any combination of two, any combination of three, any combination of four, or a combination of five of the following: a first background correction parameter f related to the background signal of the reagent card and / or test strip before the chromatographic reaction; a second background correction parameter g related to the background signal of the reagent card and / or test strip during the chromatographic reaction; a third background correction parameter h related to the background signal of the reagent card and / or test strip after the chromatographic reaction; an HCT correction parameter i related to the HCT value of the sample; and a dilution ratio correction parameter j related to the dilution ratio of the sample.

[0031] Furthermore, the detection line is also coated with a first intrinsic parameter, which is used at least to indicate the amount and / or uniformity of the coating of the tracer captured by the detection line and to generate an optical signal. Preferably, the optical signal of the tracer and the optical signal of the first intrinsic parameter are distinguishable from each other.

[0032] Further, before the detection line, the first optical signal T1 of the first internal reference is obtained, and based on the first optical signal T1, the first capture amount correction parameter a and / or the first capture coating uniformity correction parameter c are obtained.

[0033] Further, after the chromatography reaction is completed, the second optical signal T2 of the first internal reference is obtained, and based on the second optical signal T2, the second trap coating amount correction parameter is obtained, and / or, the second trap coating uniformity correction parameter is obtained.

[0034] The completion of the chromatography reaction generally refers to a specific time point after sample addition. For example, the completion of the chromatography reaction can be any time point between the 30th second and the 15th minute after sample addition. In a preferred embodiment, the time interval between acquiring the tracer's optical signal T and acquiring the first internal reference's second optical signal T2 is less than or equal to 5 minutes. For example, it can be 4 minutes, 3 minutes, 2 minutes, 1 minute, 30 seconds, 20 seconds, 10 seconds, 5 seconds, or 0 seconds. The shorter the time interval between acquiring the tracer's optical signal T and acquiring the first intrinsic parameter's second optical signal T2, the better. If the time interval is too long, the coating amount and / or coating uniformity of the captured material on the detection line indicated by the second optical signal T2 may differ significantly from the coating amount and / or coating uniformity of the captured material on the detection line when the tracer's optical signal T is acquired. Therefore, the correction parameter b for the second captured material coating amount obtained based on the second optical signal T2, and the correction parameter d for the second captured material coating uniformity, cannot effectively correct the detection results, thus failing to guarantee the accuracy of the detection results. More preferably, the time interval between acquiring the tracer's optical signal T and acquiring the first intrinsic parameter's second optical signal T2 is less than or equal to 1 minute. Most preferably, the time interval between acquiring the tracer's optical signal T and acquiring the first intrinsic parameter's second optical signal T2 is less than or equal to 10 seconds.

[0035] Further, a second internal control is provided, which is at least used to indicate the rate of the chromatographic reaction and generate a light signal. The second internal control may be present in a reagent different from the first reagent. Preferably, the second internal control is present in the first reagent or on the test strip. As some examples, the second internal control may be a substance independent of the first aptamer of the coupled tracer, or it may be the first aptamer of the coupled tracer. As other examples, the second internal control may be present on the sample pad of the test strip; or, the test strip may further include a binding pad located between the sample pad and the NC membrane, on which the second internal control is present. Optionally, the second internal control is selected from at least one of a fluorescent signaling substance, an enzyme-catalyzing substance, an electromagnetic signaling substance, or a colored substance.

[0036] Further, the third optical signal T3 of the second internal reference during the chromatography reaction is acquired, and the chromatography speed correction parameter is obtained based on the third optical signal T3. The third optical signal T3 of the second internal reference can be the optical signal of the second internal reference at a certain moment during the chromatography reaction, the optical signal of the second internal reference during a certain time period during the chromatography reaction, or the optical signal of the second internal reference at multiple moments during the chromatography reaction.

[0037] Further, the captured object and the first internal reference are independently coated on the detection line; or, the first internal reference is directly and / or indirectly combined with the captured object. Preferably, the first internal reference is directly and / or indirectly combined with the captured object. When the first internal reference is directly and / or indirectly combined with the captured object, the coating amount of the first internal reference has better consistency with the coating amount of the captured object; the coating uniformity of the first internal reference also has better consistency with the coating uniformity of the captured object. That is, it is reliable to use the first internal reference to indicate the coating amount and / or coating uniformity of the captured object on the detection line; correspondingly, it is reliable to obtain a, b, c, and d by detecting the light signal generated by the first internal reference; correspondingly, the accuracy of the detection results can be improved by correcting the detection results with a and / or b and / or c and / or d.

[0038] Optionally, the test strip further includes a conjugate pad located between the sample pad and the NC membrane. Alternatively, the test strip may not have a conjugate pad, and the sample pad may be directly connected to the NC membrane.

[0039] Further, the trap is selected from at least one of avidin, streptavidin, anti-biotin antibody, anti-FITC antibody, or anti-DNP antibody. Alternatively, the trap is selected from at least one of antibody, antigen-binding fragment, aptamer, modified aptamer, aptamer, avidin, antigen, protein, polypeptide, multi-protein complex, exosome, microbial particle / fragment / disc, oligonucleotide, or low molecular weight compound.

[0040] Further, the first aptamer is selected from at least one of antibodies, antigen-binding fragments, aptamers, modified aptamers, aptamers, affinity molecules, antigens, proteins, peptides, multi-protein complexes, exosomes, microbial particles / fragments / debris, oligonucleotides, or low molecular weight compounds. Preferably, the antibody is selected from at least one of monoclonal antibodies, polyclonal antibodies, recombinant antibodies, chimeric antibodies, humanized antibodies, or camel antibodies. Preferably, the antigen-binding fragment is selected from at least one of Fab', Fab, F(ab')2, Fv, and scFV fragments.

[0041] It should be noted that the immunochromatographic analysis method described in this application is applicable to traditional dry immunochromatographic analysis, semi-liquid immunochromatographic analysis, and whole-liquid immunochromatographic analysis.

[0042] In one embodiment, corresponding to dry immunochromatographic analysis, the first reagent is located on the test strip, which includes a sample pad, a conjugate pad, an NC membrane, and an absorbent pad arranged sequentially. The NC membrane has a detection line, and the first reagent is located on the sample pad and / or the conjugate pad of the test strip. The detection line is coated with a trap, which is at least one of antibody, antigen-binding fragment, aptamer, modified aptamer, aptamer, affinity, antigen, protein, polypeptide, multi-protein complex, exosome, microbial particle / fragment / disc, oligonucleotide, or low molecular weight compound.

[0043] In another embodiment, corresponding to semi-liquid phase immunochromatographic analysis, the test strip includes a sample pad, an NC membrane, and an absorbent pad arranged sequentially. The NC membrane has a detection line. The first reagent exists independently of the test strip. The detection line is coated with a trap, which is at least one of antibody, antigen-binding fragment, aptamer, modified aptamer, aptamer, affinity, antigen, protein, polypeptide, multi-protein complex, exosome, microbial particle / fragment / disc, oligonucleotide, or low molecular weight compound.

[0044] In another embodiment, corresponding to total liquid chromatography immunochromatographic analysis, the test strip includes a sample pad, an NC membrane, and an absorbent pad arranged sequentially; the detection line is coated with a trap; the first reagent exists independently of the test strip; the test strip further includes a second reagent, which is a second aptamer of a conjugate; the second reagent exists independently of the test strip; the trap is at least one of avidin, streptavidin, anti-biotin antibody, anti-FITC antibody, or anti-DNP antibody.

[0045] Optionally, the first aptamer and the second aptamer specifically recognize and / or bind to the same analyte; or, one of the first aptamer or the second aptamer competes with the analyte to recognize and / or bind to the other aptamer.

[0046] Further, the second aptamer is selected from at least one of antibodies, antigen-binding fragments, aptamers, modified aptamers, aptamers, affinity molecules, antigens, proteins, peptides, multi-protein complexes, exosomes, microbial particles / fragments / debris, oligonucleotides, or low molecular weight compounds. Preferably, the antibody is selected from at least one of monoclonal antibodies, polyclonal antibodies, recombinant antibodies, chimeric antibodies, humanized antibodies, or camel antibodies. Preferably, the antigen-binding fragment is selected from at least one of Fab', Fab, F(ab')2, Fv, and scFV fragments.

[0047] More specifically, when the first aptamer and the second aptamer specifically recognize and / or bind to the same analyte, the first aptamer and the second aptamer can be any molecule capable of specifically recognizing and binding to the analyte.

[0048] It should be understood that the first and second aptamers can be the same or different, as long as they both bind to the analyte, forming a sandwich-like multiplex complex of "tracer-first aptamer-analyte-second aptamer-connector". This complex is captured by the detection zone, and the content or presence of the analyte is calculated from the signal change generated by the tracer. For example, both the first and second aptamers are antibodies; they can be the same antibody or different antibodies. They can be different antibodies binding to the same epitope on the analyte, or they can be different antibodies binding to different epitopes on the analyte. When the first and second aptamers bind to the same epitope of the analyte, the analyte should have two or more repetitions of the same epitope. The first and second aptamers generally bind to different repetitions of the same epitope on the analyte. Furthermore, the signal generated by the tracer in the multiplex complex is proportional to the concentration of the analyte to a certain extent. By measuring the signal generated by the tracer in the detection zone, the presence of the analyte in the sample can be determined, or the concentration of the analyte can be calculated.

[0049] When one of the first aptamers or the second aptamer competes with the analyte to recognize and / or bind to the other aptamer, one of the first aptamers or the second aptamer is any molecule that can compete with the analyte to recognize and / or bind to the other aptamer.

[0050] It should be understood that the first aptamer can compete with the analyte to recognize and / or bind to the second aptamer, or the second aptamer can compete with the analyte to recognize and / or bind to the first aptamer, forming a multiple complex of "tracer-first aptamer-second aptamer-connector" during the competitive reaction with the analyte. The first aptamer and the analyte can be the same or different substances, as long as they can both specifically recognize and / or bind to the second aptamer. By competing with the first aptamer to bind to the second aptamer, and given the known amount of the coupled tracer-first aptamer in the system, the content or presence of the analyte can be calculated from the signal in the detection zone and changes in a preset signal value. Alternatively, the second aptamer and the analyte can be the same or different substances, as long as they can both specifically recognize and / or bind to the first aptamer. By competing with the second aptamer to bind to the first aptamer, and given the known amount of the coupled tracer-first aptamer in the system, the content or presence of the analyte can be calculated from the signal in the detection zone and changes in a preset signal value. Furthermore, by generating signals through tracers in multiple complexes, the signal intensity is inversely proportional to the concentration of the analyte to a certain extent. By measuring the signal generated by the tracers in the detection area, the presence of the analyte in the sample can be determined, or the concentration of the analyte can be calculated.

[0051] The capture and the linker are bound together with certain specificity and affinity. For example, you can choose "biotin-avidin, primary antibody-secondary antibody, antibody-protein, DNP (2,4-dinitrophenyl)-anti-DNP antibody, FITC (fluorescein isothiocyanate)-anti-FITC antibody", etc. Specifically, when the linker is biotin, the capture substance corresponds to avidin and / or streptavidin and / or anti-biotin antibodies; when the linker is avidin and / or streptavidin, the capture substance corresponds to biotin. When the linker is a primary antibody, the capture substance corresponds to a secondary antibody; when the linker is a secondary antibody, the capture substance corresponds to a primary antibody. When the linker is an antibody, the capture substance corresponds to a protein specifically recognized and / or bound by that antibody; when the linker is a protein, the capture substance corresponds to an antibody that can specifically recognize and / or bind to it. When the linker is DNP, the capture substance corresponds to anti-DNP antibodies; when the linker is anti-DNP, the capture substance corresponds to DNP. When the linker is FITC, the capture substance corresponds to anti-FITC antibodies; when the linker is anti-FITC antibodies, the capture substance corresponds to FITC.

[0052] In some specific implementations, the linker and trap connection system is a biotin-streptavidin linker system. It should be understood that one avidin molecule can bind four biotin molecules, and the binding is very stable. Furthermore, avidin and biotin can be conjugated to proteins, enzymes, luciferins, and other molecules without affecting their biological activity. One antibody molecule can conjugate multiple biotin molecules, and avidin can conjugate multiple biotin-antibody conjugates. This multi-stage amplification effect can further improve the sensitivity of the detection system.

[0053] In some specific implementation schemes, the test strip is also covered with a control line, which can be used to determine whether the test is valid or for positioning when the instrument reads the test results.

[0054] The tracer, first internal control, and / or second internal control can be any detectable labeled substance that can generate a signal detectable by visual or instrumental means. Exemplary examples include at least one of the following: fluorescent signaling substances, enzyme-catalyzed substances, chemiluminescent signaling substances, electromagnetic signaling substances, or colored substances. Depending on the reaction principle, as the analyte complex containing the tracer flows on the chromatography support, the tracer may be captured and aggregated or not captured on the detection line. The presence or concentration of the analyte is determined based on the tracer's signal, such as a color signal, fluorescence signal, or electromagnetic signal.

[0055] In some specific implementation schemes, the fluorescent signaling substance may be at least one of fluorescent microspheres, quantum dots, fluorescent proteins, or luciferin molecules; the enzyme catalytic substance may be at least one of horseradish peroxidase, alkaline phosphatase, glucose-6-phosphate dehydrogenase; the chemiluminescent signaling substance may be at least one of acridinium ester, thioester or sulfonamide, luminol, isoluminol or phenanthrene ester; the electromagnetic signaling substance may be at least one of magnetic particles or radioactive substances, more specifically, the radioactive substance may be at least one of 3H, 125I, 35S, 14C, 32P, or 33P; the colored substance may be at least one of metal colloidal particles or colored particles, more specifically, the metal colloidal particles may be selected from at least one of colloidal gold or colloidal platinum.

[0056] In some specific implementation schemes, the first internal control, the second internal control, and / or the tracer can be the same substance or different substances, as long as the signals they produce can be distinguished from each other. It should be understood that they can be distinguished by using different signaling substances to produce different signals, or by different detection methods, such as detecting and obtaining signals at different reaction stages or time points.

[0057] Further, the captured substance and the first internal reference are substances with similar properties; more preferably, the captured substance and the first internal reference have similar molecular weights and / or isoelectric points, and the first internal reference can generate a detectable signal; most preferably, the captured substance and the first internal reference are the same substance.

[0058] In some specific implementations, the second internal reference may be coupled to the connector together with the tracer, or the second internal reference may exist as an integral part of the tracer, with the tracer being the second internal reference.

[0059] The term "specific recognition" or "specific binding" as used in this article can refer to the interaction between an antibody, protein, or peptide and a second chemical substance, wherein the interaction depends on the presence of a specific structure (e.g., an antigenic structure) or a determinant / epitope on the chemical substance, such as an antibody recognizing and binding to a specific antigenic structure, the mutual recognition and binding of a receptor and ligand, or the mutual recognition and binding of biotin and avidin.

[0060] The term "coupled" as used herein broadly refers to a stable bond between two substances resulting from any chemical, physical, or physicochemical interaction (e.g., covalent bond, hydrogen bond, electrostatic interaction, polar attraction, van der Waals attraction, hydrophobic interaction, or adsorption). Unless otherwise stated, this term is intended to encompass both: direct binding / conjugation between two substances, such as the direct binding / conjugation of an antibody to an antigen on a protein, and indirect binding / conjugation between two substances through one or more intermediate means, such as the association between an antibody and a polynucleotide by means of one or more oligonucleotides and / or labels. Therefore, when used with respect to two substances, "coupled" refers to two substances bound / conjugated by any such direct or indirect means.

[0061] The analyte should not be particularly limited and can be any biomolecule or biological cell that needs to be detected and / or quantified in a sample. Non-limiting examples of analytes include antigens, antibodies, antigen-binding fragments, proteins, peptides, multi-protein complexes, hormones, exosomes, oligonucleotides, or low-molecular-weight compounds. The analyte can be detected in any sample of interest and is not particularly limited to biological samples, such as, but not limited to, bodily fluids (e.g., urine, saliva, blood, serum, plasma, sweat), extracts (e.g., cell extracts), and solutions containing proteins and / or DNA (e.g., reaction mixtures).

[0062] A second aspect of this application provides a computer-readable storage medium including a program that can be executed by a processor to implement any of the aforementioned immunochromatographic analysis methods.

[0063] A third aspect of this application provides an immunochromatographic analysis system, including a signal detection module and a processor, wherein the signal detection module is used to detect optical signals, and the processor is used to execute a program in a computer-readable storage medium as described above.

[0064] The beneficial effects of this application are as follows:

[0065] The inventors of this application discovered that the coating amount of the captured material on the detection line (including the initial coating amount before the chromatographic reaction and the effective coating amount after the chromatographic reaction), the coating uniformity of the captured material on the detection line, and the speed of the chromatographic reaction are key factors affecting the accuracy of the detection results. Therefore, correction parameters related to the coating amount of the captured material on the detection line, correction parameters related to the coating uniformity of the captured material on the detection line, and correction parameters related to the speed of the chromatographic reaction are set. By using correction parameters to correct the detection results, the accuracy of the detection results can be effectively guaranteed.

[0066] Meanwhile, by analyzing various factors and their interrelationships, a combination of correction parameters was established. This involved combining at least two of the following parameters: a first capture coating amount correction parameter related to the amount of capture on the detection line before the chromatographic reaction proceeds to the detection line; a second capture coating amount correction parameter related to the amount of capture on the detection line after the chromatographic reaction; a first capture coating uniformity correction parameter related to the amount of capture on the detection line before the chromatographic reaction proceeds to the detection line; a second capture coating uniformity correction parameter related to the amount of capture on the detection line after the chromatographic reaction; and an actual chromatography rate correction parameter related to the actual chromatographic reaction rate. This combination better corrects the detection results and improves their accuracy.

[0067] In the immunochromatographic analysis process, by introducing a first internal control and / or a second internal control, the chromatography process is dynamically monitored, achieving high-precision detection of analytes; reducing the difference between raw material rolls of NC membrane, the complexity of production is reduced; and the requirements for the detection environment, such as temperature and humidity, are reduced.

[0068] Furthermore, the immunochromatographic analysis method of this application is applicable to traditional dry immunochromatographic analysis, semi-liquid immunochromatographic analysis, and whole-liquid immunochromatographic analysis, and has a wide range of applicable scenarios, which can effectively improve the detection accuracy of various immunochromatographic modes. Detailed Implementation

[0069] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0070] In this document, "and / or" includes any and all combinations of one or more of the listed related items.

[0071] In this article, "multiple" means two or more, that is, it includes two, three, four, five, etc.

[0072] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0073] In this specification, certain embodiments may be disclosed in a range-bound format. It should be understood that this "range-bound" description is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered as having specifically disclosed all possible subranges and independent numerical values ​​within those ranges. For example, range The description should be considered as having specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the range.

[0074] In the following embodiments, the tracer, first internal control, and second internal control are all illustrated by way of fluorescent signaling substances. Accordingly, the various light signals obtained in each embodiment are all fluorescent signals. In other embodiments, the tracer, first internal control, and second internal control are each independently selected from at least one of fluorescent signaling substances, enzyme catalytic substances, electromagnetic signaling substances, or colored substances; the light signals of the tracer, the first internal control, and the second internal control are independently selected from at least one of reflected light signals, transmitted light signals, fluorescent light signals, or phosphorescent light signals.

[0075] In the first type of implementation, the immunochromatographic analysis method corresponds to dry immunochromatographic analysis, including: providing an immunochromatographic analysis reagent card, the immunochromatographic analysis reagent card including a test strip, the test strip including a sample pad, a conjugation pad, an NC membrane and an absorbent pad arranged sequentially, the NC membrane having a detection line; the detection line being coated with a capture substance and a first internal control;

[0076] A first reagent is provided and disposed on the conjugate pad. The first reagent is a first aptamer of a conjugate tracer. The tracer is used to indicate the content of the analyte and generate a light signal T. A detection result is obtained based on the light signal T. The detection result is corrected using correction parameters to obtain a corrected detection result.

[0077] In one implementation, the correction parameter is related to the amount of trapping material on the detection line. In other implementations, the correction parameter is related to the uniformity of trapping material coating on the detection line. Still other implementations, the correction parameter is related to the rate of the chromatographic reaction.

[0078] In one embodiment, the correction parameter is a combination of a first capture coating amount correction parameter a, which is related to the amount of capture on the detection line before the chromatography reaction proceeds to the detection line, and a second capture coating amount correction parameter b, which is related to the amount of capture on the detection line after the chromatography reaction is completed, i.e., the (a, b) combination.

[0079] In other implementations, the correction parameter can be any one of a, b, c, d, e, or a combination of (a, c), (a, d), (a, e), (b, c), (b, d), (b, e), (c, d), (c, e), or (d, e), (a, b, c), (a, b, d), (a, b, e), (a, c, d), (a, c, e), (a, d, e), (b, c, d), (b, c, e), (b, d, e), or (c, d, e), (a, b, c, d), (a, b, c, e), (a, b, d, e), (a, c, d, e), (b, c, d, e), (a, b, c, e).

[0080] The following is an example, using the combination of modified parameters (a, b).

[0081] After the chromatography reaction is completed (in this example, the time point after sample addition is taken as the time point after the chromatography reaction is completed), the light signal T of the tracer is obtained, and the detection result X of the analyte is obtained based on the light signal T.

[0082] Obtain the first optical signal T1 of the first internal control before sample addition, and the second optical signal T2 of the first internal control after the chromatography reaction is completed (in this example, the time point after the chromatography reaction is completed is 5 minutes after sample addition). Using the fluorescence intensity of the first optical signal T1 as a and the fluorescence intensity of the second optical signal T2 as b, the effective coating rate of the first internal control before and after the chromatography reaction is n = b / a.

[0083] As an example, the method of correcting the detection result using correction parameters is as follows: the corrected detection result is Y = f(a, b, X) = X * a / b.

[0084] The following is an example, taking the correction parameter as either a or b.

[0085] After the chromatography reaction is completed (in this example, the time point after sample addition is taken as the time point after the chromatography reaction is completed), the light signal T of the tracer is obtained, and the detection result X of the analyte is obtained based on the light signal T.

[0086] Obtain the first optical signal T1 of the first internal control before sample addition, and the second optical signal T2 of the first internal control after the chromatography reaction is completed (in this example, the time point after sample addition is taken as the completion time of the chromatography reaction). Use the fluorescence intensity of the first optical signal T1 as a, and the fluorescence intensity of the second optical signal T2 as b.

[0087] Obtain the factory-set fluorescence intensity of the captured material on the detection line of this batch of reagent cards, denoted as a0.

[0088] As an example, the method of correcting the detection result using the correction parameter is as follows: the corrected detection result is Y = f(a, X) = X * a0 / a, or Y = f(b, X) = X * a0 / b.

[0089] The following is an example, using the combination of modified parameters (a, b, d).

[0090] After the chromatography reaction is completed (in this example, the time point after sample addition is taken as the time point after the chromatography reaction is completed), the light signal T of the tracer is obtained, and the detection result X of the analyte is obtained based on the light signal T.

[0091] Obtain the first optical signal T1 of the first internal control before sample addition, and the second optical signal T2 of the first internal control after the chromatography reaction is completed (in this example, the time point after the chromatography reaction is completed is 5 minutes after sample addition). Let the fluorescence intensity of the first optical signal T1 be a, the fluorescence intensity of the second optical signal T2 be b, and the uniformity of the second optical signal T2 be d (for example, the fluorescence signal region can be divided into 100 small rectangular regions of the same size, the fluorescence signal intensity values ​​in each small rectangular region can be obtained, and then the uniform distribution standard deviation of these fluorescence signal intensity values ​​can be calculated, with the uniform distribution standard deviation being used as the uniformity d).

[0092] As an example, the method of correcting the detection result with correction parameters is: the corrected detection result is Y = f(a, b, d, X) = X*a(bd) / (b*b).

[0093] The following is an example, using the combination of modified parameters (a, b, c, d).

[0094] After the chromatography reaction is completed (in this example, the time point after sample addition is taken as the time point after the chromatography reaction is completed), the light signal T of the tracer is obtained, and the detection result X of the analyte is obtained based on the light signal T.

[0095] Obtain the first optical signal T1 of the first internal control before sample addition, and the second optical signal T2 of the first internal control after the chromatography reaction is completed (in this example, the time point after sample addition is taken as the completion time of the chromatography reaction). Let the fluorescence intensity of the first optical signal T1 be a, the fluorescence intensity of the second optical signal T2 be b, the uniformity of the first optical signal T1 be c (for example, the fluorescence signal region can be divided into 100 small rectangular regions of the same size, the fluorescence signal intensity value in each small rectangular region can be obtained, and then the uniform distribution standard deviation of these fluorescence signal intensity values ​​can be calculated, and the uniform distribution standard deviation can be used as the uniformity c), and the uniformity of the second optical signal T2 be d (for example, the fluorescence signal region can be divided into 100 small rectangular regions of the same size, the fluorescence signal intensity value in each small rectangular region can be obtained, and then the uniform distribution standard deviation of these fluorescence signal intensity values ​​can be calculated, and the uniform distribution standard deviation can be used as the uniformity d).

[0096] As an example, the method of correcting the detection result with correction parameters is: the corrected detection result is Y = f(a, b, c, d, X) = X * a * a(bd) / [b * b * (ac)].

[0097] In a preferred embodiment, the method further includes providing a second intrinsic parameter, which is used to indicate the rate of the chromatography reaction and generate an optical signal; acquiring a third optical signal T3 of the second intrinsic parameter during the chromatography reaction; and obtaining a chromatography rate correction parameter e based on the third optical signal T3.

[0098] The following is an example, using the combination of modified parameters (a, b, c, d, e).

[0099] After the chromatography reaction is completed (in this example, the time point after sample addition is taken as the time point after the chromatography reaction is completed), the light signal T of the tracer is obtained, and the detection result X of the analyte is obtained based on the light signal T.

[0100] Obtain the first optical signal T1 of the first internal control before sample addition, and the second optical signal T2 of the first internal control after the chromatography reaction is completed (in this example, the time point after sample addition is taken as the completion time of the chromatography reaction). Let the fluorescence intensity of the first optical signal T1 be a, the fluorescence intensity of the second optical signal T2 be b, the uniformity of the first optical signal T1 be c (for example, the fluorescence signal region can be divided into 100 small rectangular regions of the same size, the fluorescence signal intensity value in each small rectangular region can be obtained, and then the uniform distribution standard deviation of these fluorescence signal intensity values ​​can be calculated, and the uniform distribution standard deviation can be used as the uniformity c), and the uniformity of the second optical signal T2 be d (for example, the fluorescence signal region can be divided into 100 small rectangular regions of the same size, the fluorescence signal intensity value in each small rectangular region can be obtained, and then the uniform distribution standard deviation of these fluorescence signal intensity values ​​can be calculated, and the uniform distribution standard deviation can be used as the uniformity d).

[0101] In this embodiment, the dynamic mobility of the second internal reference is used as the chromatography rate correction parameter e. The dynamic mobility of the second internal reference refers to its ability to migrate between the stationary phase and the mobile phase. In chromatography, dynamic mobility is an important parameter describing the migration rate of the second internal reference in the chromatography column.

[0102] Obtain the baseline fluorescence signal T3 of the second internal control during the chromatography reaction at 1 min incubation. 1min and baseline fluorescence signal T3 at 3 minutes of incubation 3min The baseline fluorescence signal is the average fluorescence signal from 50-100 points out of 500 signal acquisition points in the chromatographic detection. In this embodiment, as an example, e = T3. 1min / T3 3min .

[0103] As an example, the method of correcting the detection result with correction parameters is as follows: The corrected detection result is Y = f(a, b, c, d, e, X) = X * a * a(bd) / [b * b * (ac) * e].

[0104] In a preferred embodiment, the first internal participant is a different fluorescent signaling substance.

[0105] In some alternative embodiments, the capture material is at least one of an antibody, an antigen-binding fragment, an aptamer, a modified aptamer, an aptamer, an affinity compound, an antigen, a protein, a polypeptide, a multi-protein complex, an exosome, a microbial particle / fragment / disc, an oligonucleotide, or a low molecular weight compound.

[0106] In some preferred embodiments, the trap and the first internal reference are substances with similar properties; more preferably, the trap and the first internal reference have similar molecular weights and / or isoelectric points, and the first internal reference can generate a detectable signal; most preferably, the trap and the first internal reference are the same substance.

[0107] In some optional embodiments, the completion of the chromatographic reaction generally refers to a specific time point after sample addition. For example, the completion of the chromatographic reaction can be any time point between the 30th second and the 15th minute after sample addition. In a preferred embodiment, the time interval between acquiring the light signal T of the tracer and acquiring the second light signal T2 of the first internal reference is less than or equal to 5 minutes. For example, it can be 4 minutes, 3 minutes, 2 minutes, 1 minute, 30 seconds, 20 seconds, 10 seconds, 5 seconds, or 0 seconds.

[0108] In the second type of implementation, the immunochromatographic analysis method corresponds to semi-liquid phase immunochromatographic analysis, including: providing an immunochromatographic analysis reagent card, the immunochromatographic analysis reagent card including a test strip, the test strip including a sample pad, an NC membrane and an absorbent pad arranged in sequence, the NC membrane having a detection line; the detection line being coated with a capture substance and a first internal control;

[0109] A first reagent is provided, which is located in the premixed cell at the end of the reagent card and exists independently of the test strip. The first reagent is the first aptamer of the coupled tracer, which is used to indicate the content of the analyte and generate a light signal T. A detection result is obtained based on the light signal T. The detection result is corrected with correction parameters to obtain a corrected detection result.

[0110] In one implementation, the correction parameter is related to the amount of trapping material on the detection line. In other implementations, the correction parameter is related to the uniformity of trapping material coating on the detection line. Still other implementations, the correction parameter is related to the rate of the chromatographic reaction.

[0111] In one implementation, the correction parameter can be any one of a, b, c, d, e, or a combination of (a, b), (a, c), (a, d), (a, e), (b, c), (b, d), (b, e), (c, d), (c, e), or a combination of (d, e), (a, b, c), (a, b, d), (a, b, e). Combinations: (a, c, d), (a, c, e), (a, d, e), (b, c, d), (b, c, e), (b, d, e), or (c, d, e), (a, b, c, d), (a, b, c, e), (a, b, d, e), (a, c, d, e), (b, c, d, e), (a, b, c, d, e).

[0112] The following is an example, using the combination of modified parameters (a, b).

[0113] After the chromatography reaction is completed (in this example, the time point after sample addition is taken as the time point after the chromatography reaction is completed), the light signal T of the tracer is obtained, and the detection result X of the analyte is obtained based on the light signal T.

[0114] Obtain the first optical signal T1 of the first internal control before sample addition, and the second optical signal T2 of the first internal control after the chromatography reaction is completed (in this example, the time point after the 4-minute mark after sample addition is taken as the completion time of the chromatography reaction). Using the fluorescence intensity of the first optical signal T1 as a and the fluorescence intensity of the second optical signal T2 as b, the effective coating rate of the first internal control before and after the chromatography reaction is n = b / a.

[0115] As an example, the method for correcting the detection results using correction parameters is as follows: the corrected detection result is Y = f(a, b, X) = K * X * a / b. Where K is the temperature compensation factor.

[0116] The following is an example, taking the correction parameter as either a or b.

[0117] After the chromatography reaction is completed (in this example, the time point after sample addition is taken as the time point after the chromatography reaction is completed), the light signal T of the tracer is obtained, and the detection result X of the analyte is obtained based on the light signal T.

[0118] Obtain the first optical signal T1 of the first internal control before sample addition, and the second optical signal T2 of the first internal control after the chromatography reaction is completed (in this example, the time point after sample addition is taken as the completion time of the chromatography reaction). Use the fluorescence intensity of the first optical signal T1 as a, and the fluorescence intensity of the second optical signal T2 as b.

[0119] Obtain the factory-set fluorescence intensity of the captured material on the detection line of this batch of reagent cards, denoted as a0.

[0120] As an example, the method of correcting the detection result using the correction parameter is as follows: the corrected detection result is Y = f(a, X) = X * a0 / a, or Y = f(b, X) = X * a0 / b.

[0121] The following is an example, taking the combination of correction parameters (a, b, d) as an example, and also including HCT correction parameters.

[0122] After the chromatography reaction is completed (in this example, the time point after sample addition is taken as the time point after the chromatography reaction is completed), the light signal T of the tracer is obtained, and the detection result X of the analyte is obtained based on the light signal T.

[0123] Obtain the first optical signal T1 of the first internal control before sample addition, and the second optical signal T2 of the first internal control after the chromatography reaction is completed (in this example, the time point after sample addition is taken as the completion time of the chromatography reaction). Let the fluorescence intensity of the first optical signal T1 be a, the fluorescence intensity of the second optical signal T2 be b, and the uniformity of the second optical signal T2 be d (for example, the fluorescence signal region can be divided into 100 small rectangular regions of the same size, the fluorescence signal intensity values ​​in each small rectangular region can be obtained, and then the uniform distribution standard deviation of these fluorescence signal intensity values ​​can be calculated, with the uniform distribution standard deviation being used as the uniformity d).

[0124] As an example, the method for correcting the detection results using correction parameters is as follows: The corrected detection result is Y = f(a, b, d, X) = i * X * a(bd) / (b * b). i is the HCT correction parameter for the sample.

[0125] The following is an example, using the combination of modified parameters (a, b, c, d).

[0126] After the chromatography reaction is completed (in this example, the time point after sample addition is taken as the time point after the chromatography reaction is completed), the light signal T of the tracer is obtained, and the detection result X of the analyte is obtained based on the light signal T.

[0127] Obtain the first optical signal T1 of the first internal control before sample addition, and the second optical signal T2 of the first internal control after the chromatography reaction is completed (in this example, the time point after sample addition is taken as the time point after the chromatography reaction is completed). Let the fluorescence intensity of the first optical signal T1 be a, the fluorescence intensity of the second optical signal T2 be b, the uniformity of the first optical signal T1 be c (for example, the fluorescence signal region can be divided into 100 small rectangular regions of the same size, the fluorescence signal intensity value in each small rectangular region can be obtained, and then the uniform distribution standard deviation of these fluorescence signal intensity values ​​can be calculated, and the uniform distribution standard deviation can be used as the uniformity c), and the uniformity of the second optical signal T2 be d (for example, the fluorescence signal region can be divided into 100 small rectangular regions of the same size, the fluorescence signal intensity value in each small rectangular region can be obtained, and then the uniform distribution standard deviation of these fluorescence signal intensity values ​​can be calculated, and the uniform distribution standard deviation can be used as the uniformity d).

[0128] As an example, the method of correcting the detection result with correction parameters is: the corrected detection result is Y = f(a, b, c, d, X) = X * a * a(bd) / [b * b * (ac)].

[0129] In a preferred embodiment, the method further includes providing a second intrinsic parameter, which is used to indicate the rate of the chromatography reaction and generate an optical signal; acquiring a third optical signal T3 of the second intrinsic parameter during the chromatography reaction; and obtaining a chromatography rate correction parameter e based on the third optical signal T3.

[0130] The following is an example, using the combination of modified parameters (a, b, c, d, e).

[0131] After the chromatography reaction is completed (in this example, the time point after sample addition is taken as the time point after the chromatography reaction is completed), the light signal T of the tracer is obtained, and the detection result X of the analyte is obtained based on the light signal T.

[0132] Obtain the first optical signal T1 of the first internal control before sample addition, and the second optical signal T2 of the first internal control after the chromatography reaction is completed (in this example, the time point after sample addition is taken as the time point after the chromatography reaction is completed). Let the fluorescence intensity of the first optical signal T1 be a, the fluorescence intensity of the second optical signal T2 be b, the uniformity of the first optical signal T1 be c (for example, the fluorescence signal region can be divided into 100 small rectangular regions of the same size, the fluorescence signal intensity value in each small rectangular region can be obtained, and then the uniform distribution standard deviation of these fluorescence signal intensity values ​​can be calculated, and the uniform distribution standard deviation can be used as the uniformity c), and the uniformity of the second optical signal T2 be d (for example, the fluorescence signal region can be divided into 100 small rectangular regions of the same size, the fluorescence signal intensity value in each small rectangular region can be obtained, and then the uniform distribution standard deviation of these fluorescence signal intensity values ​​can be calculated, and the uniform distribution standard deviation can be used as the uniformity d).

[0133] In this embodiment, the dynamic mobility of the second internal reference is used as the chromatography rate correction parameter e. The dynamic mobility of the second internal reference refers to its ability to migrate between the stationary phase and the mobile phase. In chromatography, dynamic mobility is an important parameter describing the migration rate of the second internal reference in the chromatography column.

[0134] Obtain the baseline fluorescence signal T3 of the second internal control during the chromatography reaction at 1 min incubation. 1min and baseline fluorescence signal T3 at 3 minutes of incubation 3min The baseline fluorescence signal is the average fluorescence signal from 50-100 points out of 500 signal acquisition points in the chromatographic detection. In this embodiment, as an example, e = T3. 1min / T3 3min .

[0135] As an example, the method of correcting the detection result with correction parameters is as follows: The corrected detection result is Y = f(a, b, c, d, e, X) = X * a * a(bd) / [b * b * (ac) * e].

[0136] In a preferred embodiment, the first internal participant is a different fluorescent signaling substance.

[0137] In some alternative embodiments, the capture material is at least one of an antibody, an antigen-binding fragment, an aptamer, a modified aptamer, an aptamer, an affinity compound, an antigen, a protein, a polypeptide, a multi-protein complex, an exosome, a microbial particle / fragment / disc, an oligonucleotide, or a low molecular weight compound.

[0138] In some preferred embodiments, the trap and the first internal reference are substances with similar properties; more preferably, the trap and the first internal reference have similar molecular weights and / or isoelectric points, and the first internal reference can generate a detectable signal; most preferably, the trap and the first internal reference are the same substance.

[0139] In some optional embodiments, the completion of the chromatographic reaction generally refers to a specific time point after sample addition. For example, the completion of the chromatographic reaction can be any time point between the 30th second and the 15th minute after sample addition. In a preferred embodiment, the time interval between acquiring the light signal T of the tracer and acquiring the second light signal T2 of the first internal reference is less than or equal to 5 minutes. For example, it can be 4 minutes, 3 minutes, 2 minutes, 1 minute, 30 seconds, 20 seconds, 10 seconds, 5 seconds, or 0 seconds.

[0140] In the third type of implementation, the immunochromatographic analysis method corresponds to total liquid phase immunochromatographic analysis, including: providing an immunochromatographic analysis reagent card, the immunochromatographic analysis reagent card including a test strip, the test strip including a sample pad, an NC membrane and an absorbent pad arranged in sequence, the NC membrane having a detection line; the detection line being coated with a capture substance and a first internal control;

[0141] A first reagent is provided, which is a first aptamer of the conjugated tracer; the first reagent exists independently of the reagent card; a second reagent is provided, which is a second aptamer of the conjugated linker; the second reagent exists independently of the reagent card; the first aptamer and the second aptamer specifically bind to the same analyte.

[0142] The tracer is used to indicate the content of the analyte and generate a light signal T; the detection result is obtained based on the light signal T; the detection result is corrected with correction parameters to obtain the corrected detection result.

[0143] In one implementation, the correction parameter is related to the amount of trapping material on the detection line. In other implementations, the correction parameter is related to the uniformity of trapping material coating on the detection line. Still other implementations, the correction parameter is related to the rate of the chromatographic reaction.

[0144] In one implementation, the correction parameter can be any one of a, b, c, d, e, or a combination of (a, b), (a, c), (a, d), (a, e), (b, c), (b, d), (b, e), (c, d), (c, e), or a combination of (d, e), (a, b, c), (a, b, d), (a, b, e). Combinations: (a, c, d), (a, c, e), (a, d, e), (b, c, d), (b, c, e), (b, d, e), or (c, d, e), (a, b, c, d), (a, b, c, e), (a, b, d, e), (a, c, d, e), (b, c, d, e), (a, b, c, d, e).

[0145] The following is an example, using the combination of modified parameters (a, b).

[0146] After the chromatography reaction is completed (in this example, the time point after sample addition is taken as the time point after the chromatography reaction is completed), the light signal T of the tracer is obtained, and the detection result X of the analyte is obtained based on the light signal T.

[0147] Obtain the first optical signal T1 of the first internal control before sample addition, and the second optical signal T2 of the first internal control after the chromatography reaction is completed (in this example, the time point after the chromatography reaction is completed is 6 minutes after sample addition). Using the fluorescence intensity of the first optical signal T1 as a and the fluorescence intensity of the second optical signal T2 as b, the effective coating rate of the first internal control before and after the chromatography reaction is n = b / a.

[0148] As an example, the method for correcting the detection results using correction parameters is as follows: the corrected detection result is Y = f(a, b, X) = K * X * a / b. Where K is the temperature compensation factor.

[0149] The following is an example, taking the correction parameter as either a or b.

[0150] After the chromatography reaction is completed (in this example, the time point after sample addition is taken as the time point after the chromatography reaction is completed), the light signal T of the tracer is obtained, and the detection result X of the analyte is obtained based on the light signal T.

[0151] Obtain the first optical signal T1 of the first internal control before sample addition, and the second optical signal T2 of the first internal control after the chromatography reaction is completed (in this example, the time point after sample addition is taken as the completion time of the chromatography reaction). Use the fluorescence intensity of the first optical signal T1 as a, and the fluorescence intensity of the second optical signal T2 as b.

[0152] Obtain the factory-set fluorescence intensity of the captured material on the detection line of this batch of reagent cards, denoted as a0.

[0153] As an example, the method of correcting the detection result using the correction parameter is as follows: the corrected detection result is Y = f(a, X) = X * a0 / a, or Y = f(b, X) = X * a0 / b.

[0154] The following is an example, taking the combination of correction parameters (a, b, d) as an example, and also including HCT correction parameters.

[0155] After the chromatography reaction is completed (in this example, the time point after sample addition is taken as the time point after the chromatography reaction is completed), the light signal T of the tracer is obtained, and the detection result X of the analyte is obtained based on the light signal T.

[0156] Obtain the first optical signal T1 of the first internal control before sample addition, and the second optical signal T2 of the first internal control after the chromatography reaction is completed (in this example, the time point after the chromatography reaction is completed is 6 minutes after sample addition). Let the fluorescence intensity of the first optical signal T1 be a, the fluorescence intensity of the second optical signal T2 be b, and the uniformity of the second optical signal T2 be d (for example, the fluorescence signal region can be divided into 100 small rectangular regions of the same size, the fluorescence signal intensity values ​​in each small rectangular region can be obtained, and then the uniform distribution standard deviation of these fluorescence signal intensity values ​​can be calculated, with the uniform distribution standard deviation as the uniformity d).

[0157] As an example, the method for correcting the detection results using correction parameters is as follows: The corrected detection result is Y = f(a, b, d, X) = i * X * a(bd) / (b * b). i is the HCT correction parameter for the sample.

[0158] The following is an example, using the combination of modified parameters (a, b, c, d).

[0159] After the chromatography reaction is completed (in this example, the time point after sample addition is taken as the time point after the chromatography reaction is completed), the light signal T of the tracer is obtained, and the detection result X of the analyte is obtained based on the light signal T.

[0160] Obtain the first optical signal T1 of the first internal control before sample addition, and the second optical signal T2 of the first internal control after the chromatography reaction is completed (in this example, the time point after the chromatography reaction is completed is 6 minutes after sample addition). Let the fluorescence intensity of the first optical signal T1 be a, the fluorescence intensity of the second optical signal T2 be b, the uniformity of the first optical signal T1 be c (for example, the fluorescence signal region can be divided into 100 small rectangular regions of the same size, the fluorescence signal intensity value in each small rectangular region can be obtained, and then the uniform distribution standard deviation of these fluorescence signal intensity values ​​can be calculated, and the uniform distribution standard deviation can be used as the uniformity c), and the uniformity of the second optical signal T2 be d (for example, the fluorescence signal region can be divided into 100 small rectangular regions of the same size, the fluorescence signal intensity value in each small rectangular region can be obtained, and then the uniform distribution standard deviation of these fluorescence signal intensity values ​​can be calculated, and the uniform distribution standard deviation can be used as the uniformity d).

[0161] As an example, a method for correcting the detection results using correction parameters is as follows: the corrected detection result is Y = f(a, b, c, d, X) = K*X*a*a(bd) / [b*b*(ac)]. Where K is the temperature compensation factor.

[0162] In a preferred embodiment, the method further includes providing a second intrinsic parameter, which is used to indicate the rate of the chromatography reaction and generate an optical signal; acquiring a third optical signal T3 of the second intrinsic parameter during the chromatography reaction; and obtaining a chromatography rate correction parameter e based on the third optical signal T3.

[0163] The following is an example, using the combination of modified parameters (a, b, c, d, e).

[0164] After the chromatography reaction is completed (in this example, the time point after sample addition is taken as the time point after the chromatography reaction is completed), the light signal T of the tracer is obtained, and the detection result X of the analyte is obtained based on the light signal T.

[0165] Obtain the first optical signal T1 of the first internal control before sample addition, and the second optical signal T2 of the first internal control after the chromatography reaction is completed (in this example, the time point after the chromatography reaction is completed is 6 minutes after sample addition). Let the fluorescence intensity of the first optical signal T1 be a, the fluorescence intensity of the second optical signal T2 be b, the uniformity of the first optical signal T1 be c (for example, the fluorescence signal region can be divided into 100 small rectangular regions of the same size, the fluorescence signal intensity value in each small rectangular region can be obtained, and then the uniform distribution standard deviation of these fluorescence signal intensity values ​​can be calculated, and the uniform distribution standard deviation can be used as the uniformity c), and the uniformity of the second optical signal T2 be d (for example, the fluorescence signal region can be divided into 100 small rectangular regions of the same size, the fluorescence signal intensity value in each small rectangular region can be obtained, and then the uniform distribution standard deviation of these fluorescence signal intensity values ​​can be calculated, and the uniform distribution standard deviation can be used as the uniformity d).

[0166] In this embodiment, the dynamic mobility of the second internal reference is used as the chromatography rate correction parameter e. The dynamic mobility of the second internal reference refers to its ability to migrate between the stationary phase and the mobile phase. In chromatography, dynamic mobility is an important parameter describing the migration rate of the second internal reference in the chromatography column.

[0167] Obtain the baseline fluorescence signal T3 of the second internal control during the chromatography reaction at 1 min incubation. 1min and baseline fluorescence signal T3 at 3 minutes of incubation 3min The baseline fluorescence signal is the average fluorescence signal from 50-100 points out of 500 signal acquisition points in the chromatographic detection. In this embodiment, as an example, e = T3. 1min / T3 3min .

[0168] As an example, the method of correcting the detection result with correction parameters is as follows: The corrected detection result is Y = f(a, b, c, d, e, X) = X * a * a(bd) / [b * b * (ac) * e].

[0169] In a preferred embodiment, the first internal participant is a different fluorescent signaling substance.

[0170] In some alternative embodiments, the trap is selected from at least one of avidin, streptavidin, anti-biotin antibody, anti-FITC antibody, or anti-DNP antibody.

[0171] In some preferred embodiments, the trap and the first internal reference are substances with similar properties; more preferably, the trap and the first internal reference have similar molecular weights and / or isoelectric points, and the first internal reference can generate a detectable signal; most preferably, the trap and the first internal reference are the same substance.

[0172] In some optional embodiments, the completion of the chromatographic reaction generally refers to a specific time point after sample addition. For example, the completion of the chromatographic reaction can be any time point between the 30th second and the 15th minute after sample addition. In a preferred embodiment, the time interval between acquiring the light signal T of the tracer and acquiring the second light signal T2 of the first internal reference is less than or equal to 5 minutes. For example, it can be 4 minutes, 3 minutes, 2 minutes, 1 minute, 30 seconds, 20 seconds, 10 seconds, 5 seconds, or 0 seconds.

[0173] The present invention also provides a computer-readable storage medium including a program that can be executed by a processor to implement an immunochromatographic analysis method in any of the foregoing embodiments.

[0174] The present invention also provides an immunochromatographic analysis system, comprising: a signal detection module for detecting optical signals; and a processor for executing the program in a computer-readable storage medium as described above.

[0175] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. An immunochromatographic assay method characterized by, The application relates to an immuno-chromatographic analysis method, comprising the following steps: providing an immuno-chromatographic analysis reagent card, which comprises a test strip, the test strip comprising at least a sample pad, a NC membrane and a water absorption pad, the sample pad being located upstream of the NC membrane, and a detection line being provided on the NC membrane; at least a capture agent being coated on the detection line; providing a first reagent, the first reagent comprising a first aptamer coupled with a tracer, the tracer being used at least for indicating the presence or absence and / or content of a target substance and generating a light signal T; the light signal T of the tracer being used at least for providing a detection result; correcting the detection result by using a correction parameter to obtain a corrected detection result; the correction parameter being related at least to the coating amount of the capture agent on the detection line, and / or, the correction parameter being related at least to the coating uniformity of the capture agent on the detection line, and / or, the correction parameter being related at least to the speed of the chromatographic reaction.

2. The immuno-chromatographic analysis method according to claim 1, wherein: the correction parameter comprises at least: a first capture agent coating amount correction parameter related to the coating amount of the capture agent on the detection line before the chromatographic reaction reaches the detection line, a second capture agent coating amount correction parameter related to the coating amount of the capture agent on the detection line after the chromatographic reaction is completed, a first capture agent coating uniformity correction parameter related to the coating amount of the capture agent on the detection line before the chromatographic reaction reaches the detection line, a second capture agent coating uniformity correction parameter related to the coating amount of the capture agent on the detection line after the chromatographic reaction is completed, a real chromatographic speed correction parameter related to the speed of the actual chromatographic reaction, a combination of any two, a combination of any three, a combination of any four, or a combination of five.

3. The immuno-chromatographic analysis method according to any one of claims 1-2, wherein: the detection line is further coated with a first internal reference, the first internal reference being used at least for indicating the coating amount and / or coating uniformity of the capture agent on the detection line and generating a light signal; the light signal of the tracer and the light signal of the first internal reference can be distinguished from each other; before the chromatographic reaction reaches the detection line, a first light signal T1 of the first internal reference is obtained, based on the first light signal T1, a first capture agent coating amount correction parameter is obtained, and / or a first capture agent coating uniformity correction parameter is obtained; and / or, after the chromatographic reaction is completed, a second light signal T2 of the first internal reference is obtained, based on the second light signal T2, a second capture agent coating amount correction parameter is obtained, and / or a second capture agent coating uniformity correction parameter is obtained.

4. The immuno-chromatographic analysis method according to any one of claims 1-3, wherein: a second internal reference is provided, the second internal reference being used at least for indicating the speed of the chromatographic reaction and generating a light signal; a third light signal T3 of the second internal reference during the chromatographic reaction is obtained, based on the third light signal T3, a chromatographic speed correction parameter is obtained.

5. The immuno-chromatographic analysis method according to claim 3, wherein: the capture agent and the first internal reference are independently coated on the detection line; or, the first internal reference is directly and / or indirectly combined with the capture agent.

6. An immunochromatographic assay method according to claim 3, characterized by: The first internal reference is selected from at least one of a fluorescent signaling substance, an enzyme catalytic substance, an electromagnetic signaling substance, or a colored substance; preferably, the first internal reference is different from the tracer.

7. An immunochromatographic assay method according to claim 4, wherein: The second internal reference is selected from at least one of a fluorescent signaling substance, an enzyme catalytic substance, an electromagnetic signaling substance, or a colored substance.

8. The immuno-chromatographic assay method of any one of claims 1-7, wherein: The test strip further comprises a conjugate pad, which is located between the sample pad and the NC membrane; or the sample pad is directly connected to the NC membrane.

9. The immuno-chromatographic assay method of any one of claims 1-7, wherein: The light signal of the tracer, the light signal of the first internal reference, and the light signal of the second internal reference are independently selected from at least one of a reflected light signal, a transmitted light signal, a fluorescent light signal, or a phosphorescent light signal.

10. The immuno-chromatographic assay method of any one of claims 1-7, wherein: The capture is selected from at least one of avidin, streptavidin, anti-biotin antibody, anti-FITC antibody, or anti-DNP antibody; Alternatively, the capture is selected from at least one of an antibody, an antigen-binding fragment, an aptamer, a modified aptamer, an aptamer, an affimer, an antigen, a protein, a polypeptide, a multi-protein complex, an exosome, a microbial particle / piece / fragment, an oligonucleotide, or a low-molecular-weight compound.

11. An immunochromatographic assay method according to any one of claims 1 to 7, characterised in that: The first aptamer is selected from at least one of an antibody, an antigen-binding fragment, an aptamer, a modified aptamer, an aptamer, an affimer, an antigen, a protein, a polypeptide, a multi-protein complex, an exosome, a microbial particle / piece / fragment, an oligonucleotide, or a low-molecular-weight compound. Preferably, the antibody is selected from at least one of a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a chimeric antibody, a humanized antibody, or a camelid antibody. Preferably, the antigen-binding fragment is selected from at least one of a Fab', a Fab, a F(ab')2, a Fv, and a scFV fragment.

12. An immunochromatographic assay method according to any one of claims 1 to 7, characterised in that: The tracer is selected from at least one of a fluorescent signaling substance, an enzyme catalytic substance, an electromagnetic signaling substance, or a colored substance.

13. The immuno-chromatographic assay method of any one of claims 1-12, wherein: The first reagent is located on the test strip, which comprises a sample pad, a conjugate pad, an NC membrane, and an absorbent pad arranged in sequence, and the first reagent is located on the sample pad or the conjugate pad of the test strip; or the test strip comprises a sample pad, an NC membrane, and an absorbent pad arranged in sequence, and the first reagent is independent of the test strip; The capture is at least one of an antibody, an antigen-binding fragment, an aptamer, a modified aptamer, an aptamer, an affimer, an antigen, a protein, a polypeptide, a multi-protein complex, an exosome, a microbial particle / piece / fragment, an oligonucleotide, or a low-molecular-weight compound.

14. The immuno-chromatographic assay method of any one of claims 1-12, wherein: A second reagent is provided, which is a second aptamer coupled to a linker; The test strip comprises a sample pad, an NC membrane, and an absorbent pad arranged in sequence; The first reagent is independent of the test strip; the second reagent is independent of the test strip. The capture is at least one of avidin, streptavidin, anti-biotin antibody, anti-FITC antibody, or anti-DNP antibody.

15. The immuno-chromatographic assay method of claim 14, wherein: The first aptamer and the second aptamer specifically recognize and / or bind to the same analyte; And / or, one of the first aptamer and the second aptamer competes with the analyte to recognize and / or bind to the other aptamer.

16. The immuno-chromatographic assay method of claim 14 or 15, wherein: The second aptamer is at least one of an antibody, an antigen-binding fragment, an aptamer, a modified aptamer, an aptibody, an affibody, an antigen, a protein, a polypeptide, a multi-protein complex, an exosome, a microbial particle / piece / fragment, an oligonucleotide, or a low-molecular-weight compound; Preferably, the antibody is at least one of a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a chimeric antibody, a humanized antibody, or a camelid antibody; Preferably, the antigen-binding fragment is at least one of a Fab', a Fab, a F(ab')2, a Fv, and a scFV fragment.

17. A computer-readable storage medium, characterized in that, A program executable by a processor to implement the immuno-chromatographic assay method of any one of claims 1-16.

18. An immunochromatographic assay system characterized by comprising: Comprising: A signal detection module for detecting an optical signal; A processor for executing the program in the computer-readable storage medium of claim 17.