A semi-quantitative immunochromatographic detection method and an immunochromatographic semi-quantitative reagent card applicable thereto.

Abstract

This invention provides a semi-quantitative immunochromatographic detection method and an applicable semi-quantitative immunochromatographic reagent card. In this method, multiple detection lines are set, and the concentration of the target analyte in the sample is determined by combining the number of color-developed detection lines and their positions. This eliminates subjective judgments based on the human eye's interpretation of color intensity, thereby improving the accuracy of semi-quantitative detection using the immunochromatographic reagent card. Furthermore, the concentration exhibiting a hook-like effect on the detection lines, along with the colorimetric concentration value of the detection lines, is introduced to jointly divide the reading range of the immunochromatographic reagent card. This allows the number of concentration reading ranges on the immunochromatographic reagent card to exceed the number of detection lines. Simultaneously, since the concentration exhibiting a hook-like effect is often much greater than the colorimetric concentration, the usable reading range of the immunochromatographic reagent card is extended, improving its flexibility and applicability.

Description

Technical Field

[0001] This invention belongs to the field of immunochromatographic analysis technology, specifically, it relates to an immunochromatographic semi-quantitative detection method and an immunochromatographic semi-quantitative reagent card applicable thereto. Background Technology

[0002] Immunochromatographic reagent cards mainly include colloidal gold immunochromatographic reagent cards and fluorescent immunochromatographic reagent cards. Quantitative readings on most commercially available immunochromatographic reagent cards require the use of quantitative analysis instruments. The reliance on these instruments limits the widespread adoption of immunochromatographic reagent cards in impoverished and underdeveloped areas, as well as their application in large-scale on-site testing. Semi-quantitative immunochromatographic reagent cards have appeared on the market, claiming to be available from manufacturers. These cards typically utilize a color-changing tracer reaction of a detection line, using the human eye to determine the intensity of the colorimetric reaction and thus the concentration of the analyte in the sample. This semi-quantitative method has limited concentration range definition, restricting the applicable concentration range of the immunochromatographic reagent card. Furthermore, human colorimetric analysis is highly subjective and susceptible to errors, and different colorimetric reactions may interfere with each other, making it difficult to accurately detect different analytes on the same reagent card. For these reasons, currently available immunochromatographic reagent cards still fall short of market demand. Summary of the Invention

[0003] The purpose of this invention is to provide an immunochromatographic semi-quantitative detection method and an immunochromatographic semi-quantitative reagent card applicable thereto, so as to improve the accuracy and flexibility of on-site semi-quantitative detection using immunochromatographic reagent cards.

[0004] According to one aspect of the present invention, a semi-quantitative immunochromatographic detection method is provided, comprising the following steps: S1. Binding a target analyte in the test solution to a labeled antibody to form an antigen-labeled antibody pair; S2. Allowing the antigen-labeled antibody pair to undergo lateral chromatography on a chromatography membrane, wherein the chromatography membrane has n detection lines, n>1, the surface of the detection lines is coated with a specific antibody for binding to the target analyte, and the colorimetric concentration of the detection lines increases along the chromatography direction; S3. With m detection lines developing color, the concentration c of the target analyte is determined based on the colorimetric situation of the detection lines: if m=0, the colorimetric concentration value of the first detection line is D1, and a judgment is made that c<D1; if m=n, the colorimetric concentration value of the nth detection line is D... n , make D n The judgment of ≤c: If m < n, when the color concentrations corresponding to the undeveloped detection lines are all greater than the color concentrations corresponding to the developed detection lines, the color concentration D corresponding to the x-th detection line among the developed detection lines is... x Maximum, make D x ≤c<D x+1The determination is as follows: when there are y undeveloped detection lines with a lower colorimetric concentration than the developed detection lines, a hook effect is determined to have occurred, and the concentration at which the hook effect occurs at the y-th detection line is taken as D. y The concentration at which the hook effect appears on the (y+1)th detection line is D. y+1 , make D y ≤c<D y+1 The present invention improves the accuracy of semi-quantitative detection using the immunochromatographic reagent card by setting multiple detection lines and comprehensively judging the concentration of the target analyte in the sample based on the number of detection lines and their positions. This eliminates subjective judgments caused by the human eye interpreting the color intensity, thus improving the accuracy of semi-quantitative detection using the immunochromatographic reagent card provided by the present invention. Furthermore, when dividing the reading intervals, the concentration at which the detection line exhibits a hook-like effect is introduced, along with the colorimetric concentration value of the detection line, to participate in the division of the immunochromatographic reagent card's reading intervals. This allows the number of concentration reading intervals on the immunochromatographic reagent card to exceed the number of detection lines. Simultaneously, since the concentration exhibiting a hook-like effect is often much greater than the colorimetric concentration, the usable reading range of the immunochromatographic reagent card is extended, improving its flexibility and applicability. In summary, the immunochromatographic semi-quantitative detection method provided by the present invention effectively improves the detection efficiency and reliability of immunochromatographic detection.

[0005] Preferably, the labeled antibodies include multiple primary antibodies that target different analytes, and the tracking signals of the tracer particles labeled with different primary antibodies do not interfere with each other; the specific antibodies include multiple secondary antibodies that target different analytes. By introducing multiple primary antibodies and multiple secondary antibodies targeting different analytes, multiplex detection of multiple analytes can be achieved simultaneously, improving detection efficiency.

[0006] Preferably, the tracer signal includes a colorimetric signal and / or a visible light signal. Both colorimetric and visible light signals can be recognized by the naked eye. Based on this, quantitative detection using immunochromatographic reagent cards can be made independent of quantitative analysis instruments, making readings convenient and facilitating the widespread application of immunochromatographic reagent cards in real-time detection.

[0007] Preferably, the tracer particles include visible light emitting particles, and the tracer signal includes a visible light signal emitted by the visible light emitting particles, wherein the excitation spectra of the visible light emitting particles do not overlap. This ensures that under different excitation light irradiation, at most one type of emitting tracer particle will exhibit a tracer light signal, eliminating interference effects caused by different emitting tracer particles emitting light, and improving detection accuracy.

[0008] Preferably, the emission spectra of the visible light luminescent particles do not overlap. This ensures that under different excitation light irradiation, at most only one type of luminescent tracer particle will exhibit a tracer light signal, eliminating interference from different luminescent tracer particles and improving detection accuracy.

[0009] Preferably, the method further includes the step of allowing the test solution to flow through the labeled antigen; along the chromatography direction, a test line and a control line are sequentially arranged, the surface of the control line being coated with a polyclonal antibody for binding to the labeled antigen; the immunochromatographic semi-quantitative detection method further includes S4: if the control line does not develop color, a judgment is made that the detection is invalid; if the control line develops color, a judgment is made that the detection is valid.

[0010] According to another aspect of the present invention, an immunochromatographic semi-quantitative reagent card suitable for the above-described immunochromatographic semi-quantitative detection method is provided. The reagent card includes a test strip, which includes a solid-phase carrier and a sample pad, a labeled conjugate pad, a reaction pad, and an absorbent pad connected end-to-end on the surface of the solid-phase carrier along the chromatography direction. The reaction pad is provided with a detection area and a control area. The detection area includes multiple detection lines, and the detection concentration limits of the target analyte on different detection lines increase sequentially along the chromatography direction. The control area is provided with a control line coated with a polyclonal antibody. The labeled conjugate pad contains a labeled antibody.

[0011] Preferably, the labeled antibody includes multiple first antibodies that target different analytes, the first antibodies are labeled by tracer particles, and the tracer signals of tracer particles labeled with different first antibodies do not interfere with each other; the specific antibody coated on each detection line includes multiple second antibodies that target different analytes, and the detection concentration limits of various analytes on different detection lines increase sequentially along the chromatography direction.

[0012] This scheme uses tracer particles with non-interfering tracer signals to distinguish different analytes, ensuring that the detection results of different analytes do not interfere with, overlap, or intersect. Different analytes have a one-to-one corresponding tracer judgment criterion on the same detection line, enabling multi-analyte detection of multiple different analytes. Furthermore, by setting multiple detection lines with progressively increasing colorimetric concentrations corresponding to different analytes, quantitative detection of different analytes can be achieved. The immunochromatographic semi-quantitative reagent card provided by this invention can achieve quantitative detection of multiple different analytes, offering significant flexibility and applicability. It eliminates the need for preparing large quantities of samples to detect different analytes, simplifying sample processing procedures and greatly improving the efficiency of quantitative detection.

[0013] Preferably, the tracer particles include colorimetric particles and / or visible light emitting particles.

[0014] Preferably, a detection zone and a quality control zone are sequentially arranged on the reaction pad along the chromatography direction. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the structure of the test strip contained in the immunochromatographic semi-quantitative reagent card provided by the present invention;

[0016] The attached diagram is labeled as follows: 1 solid support, 2 sample pad, 3 marker binding pad, 4 reaction pad, 5 absorbent pad, 6 detection area, 7 quality control area. Detailed Implementation

[0017] The technical features of the technical solution provided by the present invention will be further clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] Example 1

[0019] This embodiment provides an immunochromatographic semi-quantitative reagent card, which includes a test strip, the structure of which is as follows: Figure 1 As shown, the test strip includes a solid support 1 and a sample pad 2, a marker binding pad 3, a reaction pad 4, and an absorbent pad 5, which are connected end-to-end along the chromatography direction on the surface of the solid support 1. The reaction pad has a detection area 6 and a control area 7. The detection area 6 has multiple detection lines (T1, T2...T...). n-1 T n ), and there is one quality control line in quality control area 7.

[0020] In this embodiment, each detection line is coated with a second antibody targeting the analyte. The detection concentration limits of various analytes on different detection lines increase sequentially along the chromatography direction. Detection lines T1, T2...T n-1 T n The corresponding colorimetric concentration values ​​of the target analytes are C1, C2...C n-1 C n And C1 <C2<……C n-1 <C n Detection lines T1, T2...T n-1 T n The concentration values ​​at which the hook-like effect of the target analyte were observed were D1, D2, ..., D. n-1 D n And D1 <D2<……D n-1 <D nThe control line C is designed to monitor whether the reagent card is invalid. Control area 7 contains control line C, which is coated with a polyclonal antibody. The labeling pad 3 is coated with multiple primary antibodies targeting different analytes. Different primary antibodies are labeled with different tracer particles, and the tracer signals from the different tracer particles do not interfere with each other.

[0021] In this embodiment, the solid support 1 is a PVC base plate, the sample pad 2 is a glass cellulose membrane, the marker binding pad 3 is a glass cellulose membrane, the reaction pad 4 is a nitrocellulose membrane (NC membrane), and the absorbent pad 5 is a glass cellulose membrane.

[0022] Among them, multiple detection lines (T1, T2...T) n-1 T n The detection areas 6 are evenly spaced, and the distance between two adjacent detection lines can be 0.5 to 2 cm. When using the immunochromatographic semi-quantitative reagent card provided in this embodiment, the target sample is added to the sample pad 2, and the target sample is subjected to chromatography towards the absorbent pad 5, passing through the sample pad 2 and the marker binding pad 3 in sequence, and then entering the detection area 6 on the reaction pad 4 for detection. The detection principle of the immunochromatographic semi-quantitative reagent card provided in this embodiment is as follows: When the target sample (containing antigen) is dropped onto the sample pad 2, the target sample will be chromatographically separated along the direction of the absorbent pad 5. When the target sample is chromatographically separated onto the labeled binding pad 3, the target will bind to the first antibody on the labeled binding pad 3 that is labeled with tracer particles and can target the target, forming an antigen-first antibody complex. The antigen-first antibody complex will continue to be chromatographically separated towards the absorbent pad. When the antigen-first antibody complex flows through the T1 detection line, the second antibody coated on the detection line T1 that can target the target will capture the antigen-first antibody complex. The excess antigen-first antibody complex will continue to flow to the detection line T2, and the second antibody coated on the detection line T2 that can target the target will capture the antigen-first antibody complex, and so on, until the last detection line T1. n After capturing the antigen-first antibody complex, the reagent continues to flow to control line C. Only when control line C develops color can the test result of the reagent card be considered a valid result; otherwise, the reagent card is invalid and the test result cannot be considered a valid result.

[0023] It should be noted that both the first and second antibodies are monoclonal antibodies targeting the analyte. The analyte in the sample binds specifically to the first and second antibodies, forming a double-antibody sandwich structure. Because the labeling pad 3 of the multi-detection immunochromatographic semi-quantitative reagent card provided in this embodiment is coated with multiple first antibodies targeting different analytes, and different first antibodies are labeled with different tracer particles, the tracer signals of the different tracer particles do not interfere with each other.

[0024] The interpretation method of the multiplex immunoassay chromatography semi - quantitative reagent card provided by the present invention is as follows:

[0025] 1. If the control line C does not show color, it indicates that the reagent card is invalid, and the test result of this time is invalid, and a re - test is required.

[0026] 2. If the control line shows color, it indicates that the test result is valid, and further analysis is carried out based on the color development result of the test line:

[0027] (1) If no test line shows color, it is judged that the concentration c of the target to be detected < C1;

[0028] (2) If there is a colored test line:

[0029] a. The color development concentration value corresponding to the uncolored test line is higher than the color development concentration value corresponding to the colored test line. If only the test line T1 shows color, it is judged that C1≤c < C2. If the test lines T1 and T2 show color, it is judged that C2≤c < C3, and so on, and the concentration range of the target to be detected can be obtained;

[0030] b. If all n test lines in the test area are colored, it is judged that C n ≤c;

[0031] c. The color development concentration value corresponding to the uncolored test line is lower than the color development concentration value corresponding to the colored test line. If only the test line T1 is uncolored, it indicates that the hook - effect appears in the test line T1 (at this time C n <<c), and it is judged that D1≤c < D2. If the test lines T1 and T2 are uncolored, it indicates that the hook - effect appears in the test line T2, and it is judged that D2≤c < D3, and so on, and the concentration range of the target to be detected can be obtained.

[0032] Example 2

[0033] Based on the immunoassay chromatography semi - quantitative reagent card provided in Example 1, this example realizes the semi - quantitative detection of human cardiac troponin I (CTnI). Refer to Figure 1In this embodiment, the labeling pad 3 of the immunochromatographic semi-quantitative reagent card is coated with colloidal gold-labeled mouse anti-human CTnI antibody I and colloidal gold-labeled chicken IgY. The colloidal gold-labeled mouse anti-human CTnI antibody I corresponds to the first antibody coated on the binding pad 3 in the immunochromatographic semi-quantitative reagent card provided in Example 1 (the colloidal gold-labeled mouse anti-human CTnI antibody I is a monoclonal antibody specifically targeting CTnI). Along the chromatography direction, four detection lines (T1, T2, T3, T4) and one control line C are sequentially drawn on the detection area 6. The distance between two adjacent detection lines is 1 cm, and the distance between the control line C and the detection line T4 is also 1 cm. The detection lines are coated with mouse anti-human CTnI antibody II, which corresponds to the second antibody coated on the detection lines in the immunochromatographic semi-quantitative reagent card provided in Example 1 (the mouse anti-human CTnI antibody II is a monoclonal antibody specifically targeting CTnI). The four detection lines have different CTnI colorimetric concentrations based on the varying concentrations of the coated mouse anti-human CTnI antibody II. The CTnI colorimetric concentration for detection line T1 is set to C1, and the concentration at which a hook effect occurs is set to D1. For detection line T2, the CTnI colorimetric concentration is set to C2, and the concentration at which a hook effect occurs is set to D2. For detection line T3, the CTnI colorimetric concentration is set to C3, and the concentration at which a hook effect occurs is set to D3. For detection line T4, the CTnI colorimetric concentration is set to C4, and the concentration at which a hook effect occurs is set to D4. The order of C4 > C3 > C2 > C1, D4 > D3 > D2 > D1, and D1 >> C4. The concentration of CTnI in the sample is c. The sample is flowed laterally through the NC membrane via detection lines T1, T2, T3, T4, and the control line C. If the control line C develops color normally, the test result is valid. If the control line C does not develop color, the test result is invalid and needs to be repeated.

[0034] When the quality control line C is colored:

[0035] 1. If none of the test lines show color, then c < C1, and the test result is negative;

[0036] 2. If only the detection line T1 shows color, the test result is positive, and C1≤c<C2;

[0037] 3. Detection lines T1 and T2 show color development, while detection lines T3 and T4 do not show color development. The test result is positive, and C2≤c<C3.

[0038] 4. If test lines T1, T2, and T3 all show color, but test line T4 does not show color, the test result is positive, and C3≤c<C4;

[0039] 5. If all four test lines (T1, T2, T3, and T4) show color, the test result is positive, and C4 ≤ c.

[0040] 6. If test line T1 does not show color, but test lines T2, T3, and T4 all show color, the test result is positive. Test line T1 also shows a hook effect, and D1≤c<D2.

[0041] 7. If test lines T1 and T2 do not develop color, but test lines T3 and T4 develop color, the test result is positive. Furthermore, test lines T1 and T2 exhibit a hook-like effect, and D2≤c<D3.

[0042] 8. Detection lines T1, T2, and T3 do not show color, only detection line T4 shows color, the test result is positive, and detection lines T1, T2, and T3 all show a hook effect, and D3≤c<D4.

[0043] Example 3

[0044] This embodiment uses the immunochromatographic semi-quantitative reagent card provided in Example 1 as a basis to achieve semi-quantitative detection of human cardiac troponin I (CTnI) and human D-dimer in a multi-parameter configuration. (Refer to...) Figure 1 In this embodiment, the marker conjugate pad 3 of the semi-quantitative reagent card is coated with colloidal gold-labeled mouse anti-human CTnI monoclonal antibody I, europium latex microsphere-labeled mouse anti-human D-dimer antibody I, and colloidal gold-labeled chicken IgY. The colloidal gold-labeled mouse anti-human CTnI antibody I and europium latex microsphere-labeled mouse anti-human D-dimer antibody I correspond to the two different first antibodies coated on the conjugate pad 3 in the immunochromatographic semi-quantitative reagent card provided in Example 1 (colloidal gold-labeled mouse anti-human CTnI antibody I is a monoclonal antibody specifically targeting CTnI, and europium latex microsphere-labeled mouse anti-human D-dimer antibody I is a monoclonal antibody specifically targeting human D-dimer). Along the layer... In the analytical direction, four detection lines (T1, T2, T3, T4) and one control line C are sequentially marked on detection area 6. The distance between two adjacent detection lines is 1 cm, and the distance between control line C and detection line T4 is also 1 cm. Each detection line is coated with mouse anti-human CTnI antibody II and mouse anti-human D-dimer antibody II. Mouse anti-human CTnI antibody II and mouse anti-human D-dimer antibody II correspond to the two different second antibodies coated on the detection lines in the immunochromatographic semi-quantitative reagent card provided in Example 1 (mouse anti-human CTnI antibody II is a monoclonal antibody specifically targeting CTnI, and mouse anti-human D-dimer antibody II is a monoclonal antibody specifically targeting human D-dimer). The four detection lines have different CTnI colorimetric concentration values ​​and mouse anti-human D-dimer colorimetric concentration values ​​based on the concentration differences of the coated mouse anti-human CTnI antibody II and mouse anti-human D-dimer antibody II. The settings are as follows:

[0045] For detection line T1, the colorimetric concentration value for CTnI is C1#, and the concentration value at which the hook effect appears is D1#. For mouse anti-human D-dimer, the colorimetric concentration value is C1*, and the concentration value at which the hook effect appears is D1*.

[0046] For detection line T2, the colorimetric concentration value for CTnI is C2#, and the concentration value at which the hook effect appears is D2#. For mouse anti-human D-dimer, the colorimetric concentration value is C2*, and the concentration value at which the hook effect appears is D2*.

[0047] For detection line T3, the colorimetric concentration value for CTnI is C3#, and the concentration value at which the hook effect appears is D3#. For mouse anti-human D-dimer, the colorimetric concentration value is C3*, and the concentration value at which the hook effect appears is D3*.

[0048] For detection line T4, the colorimetric concentration value for CTnI is C4#, and the concentration value at which the hook effect appears is D4#. For mouse anti-human D-dimer, the colorimetric concentration value is C4*, and the concentration value at which the hook effect appears is D4*.

[0049] C4#>C3#>C2#>C1#, D4#>D3#>D2#>D1#, and D1#>>C4#;

[0050] C4*>C3*>C2*>C1*, D4*>D3*>D2*>D1*, and D1*>>C4*.

[0051] The concentration of CTnI in the sample is c a The concentration of mouse anti-human D-dimer was c. b The sample is flowed laterally through the NC membrane, passing sequentially through detection lines T1, T2, T3, T4, and control line C. During testing, under fluorescent light, the colloidal gold appears red, and its color signal can be read visually. At this time, the europium latex microspheres appear milky white and therefore do not affect the color signal of the colloidal gold. Under ultraviolet light, the europium latex microspheres emit bright red fluorescence, while the colloidal gold does not emit light and therefore does not affect the fluorescence signal of the europium latex microspheres. Under fluorescent light, normal color development at control line C indicates a valid test result; no color development at control line C indicates an invalid test result, requiring retesting.

[0052] If the control line C shows color under fluorescent light, continue to perform semi-quantitative detection of CTnI under fluorescent light as follows:

[0053] 1. If none of the test lines show color, then c a <C1#, the test result is negative;

[0054] 2. If only the T1 detection line shows color, the test result is positive, and C1#≤ca <C2#;

[0055] 3. Detection lines T1 and T2 show color development, while detection lines T3 and T4 do not. The test result is positive, and C2# ≤ c. a <C3#;

[0056] 4. If test lines T1, T2, and T3 all develop color, but test line T4 does not develop color, the test result is positive, and C3# ≤ c a <C4#;

[0057] 5. If all four test lines (T1, T2, T3, and T4) show color, the test result is positive, and C4# ≤ c a ;

[0058] 6. If test line T1 does not develop color, but test lines T2, T3, and T4 all develop color, the test result is positive. Furthermore, test line T1 exhibits a hook-like effect, and D1# ≤ c. a <D2#;

[0059] 7. Detection lines T1 and T2 show no color development, while detection lines T3 and T4 show color development. The test result is positive. Furthermore, detection lines T1 and T2 exhibit a hook-like effect, and D2# ≤ c. a <D3#;

[0060] 8. Detection lines T1, T2, and T3 show no color development, only detection line T4 shows color development, indicating a positive result. Furthermore, detection lines T1, T2, and T3 all exhibit a hook-like effect, and D3# ≤ c. a <D4#.

[0061] If the quality control line C shows color under fluorescent light, then the mouse anti-human D-dimer should be semi-quantitatively detected under ultraviolet light as follows:

[0062] 1. If none of the test lines show color, then c b <C1*, the test result is negative;

[0063] 2. If only the detection line T1 shows color, the test result is positive, and C1*≤c b <C2*;

[0064] 3. Detection lines T1 and T2 show color development, while detection lines T3 and T4 do not. The test result is positive, and C2*≤c b <C3*;

[0065] 4. If test lines T1, T2, and T3 all develop color, but test line T4 does not develop color, the test result is positive, and C3*≤c b <C4*;

[0066] 5. If all four test lines (T1, T2, T3, and T4) show color, the test result is positive, and C4*≤c b ;

[0067] 6. If test line T1 does not develop color, but test lines T2, T3, and T4 all develop color, the test result is positive. Furthermore, test line T1 exhibits a hook-like effect, and D1*≤c b <D2*;

[0068] 7. If test lines T1 and T2 do not develop color, but test lines T3 and T4 do, the test result is positive. Furthermore, test lines T1 and T2 exhibit a hook-like effect, and D2*≤c. b <D3*;

[0069] 8. Detection lines T1, T2, and T3 show no color development, only detection line T4 shows color development, indicating a positive result. Furthermore, detection lines T1, T2, and T3 all exhibit a hook-like effect, and D3*≤c b <D4*.

[0070] The immunochromatographic semi-quantitative reagent card provided in this embodiment utilizes tracer particles with non-overlapping and non-crossing tracer signals to label monoclonal antibodies that specifically target different analytes. A sandwich antibody conjugate is constructed using these monoclonal antibodies and the analytes, ensuring that different analytes have corresponding tracer criteria on the same detection line. This enables the multi-analyte detection of various analytes. Furthermore, by setting multiple detection lines with progressively increasing colorimetric concentrations corresponding to different analytes, multi-analyte semi-quantitative detection of different analytes can be achieved.

[0071] In other specific embodiments, if a third target object is present, semi-quantitative detection by the naked eye can be achieved by introducing a third type of tracer particle. For example, semi-quantitative detection of the third target object by the naked eye can also be achieved by introducing tracer particles that can emit other signals (such as green light) under other special conditions (such as blue light irradiation), and so on.

[0072] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention, but such modifications or substitutions are all within the scope of protection of the present invention.

Claims

1. A semi-quantitative immunochromatographic detection method, characterized in that, Includes the following steps: S1. The target analyte in the test solution is bound to the labeled antibody to form an antigen-labeled antibody pair, wherein the labeled antibody includes multiple first antibodies that target different target analytes respectively, and the tracer signals of tracer particles labeled with different first antibodies do not interfere with each other; S2. The antigen-labeled antibody pair undergoes lateral chromatography on a chromatographic membrane, wherein the chromatographic membrane is provided with n detection lines, n>1, and the surface of the detection lines is coated with specific antibodies for binding to the target analyte. The colorimetric concentration of the detection lines increases along the chromatographic direction, wherein the specific antibodies include multiple secondary antibodies that target different target analytes respectively. S3. If m detection lines develop color, determine the concentration c of the target analyte based on the color development of the detection lines: If m=0, use the colorimetric concentration value of the detection line described in Article 1 as D1, and make a judgment that c < D1; If m=n, the colorimetric concentration value of the nth detection line is D. n , make D n The condition for ≤c; If m < n When the colorimetric concentrations corresponding to the undeveloped detection lines are all greater than the colorimetric concentrations corresponding to the developed detection lines, the colorimetric concentration D corresponding to the x-th detection line among the developed detection lines is... x Maximum, make D x ≤c<D x+1 The judgment; When there are y undeveloped detection lines with a lower colorimetric concentration than the developed detection lines, a hook effect is determined to have occurred, and the concentration at which the hook effect occurs at the y-th detection line is defined as D. y The concentration at which the hook-like effect occurs in the detection line described in the (y+1)th line is D. y+1 , make D y ≤c<D y+1 The judgment.

2. The immunochromatographic semi-quantitative detection method as described in claim 1, characterized in that: The tracer signal includes colorimetric signals and / or visible light signals.

3. The immunochromatographic semi-quantitative detection method as described in claim 2, characterized in that: The tracer particles include visible light emitting particles, and the tracer signal includes a visible light signal emitted by the visible light emitting particles, wherein the excitation spectra of the visible light emitting particles do not overlap.

4. The immunochromatographic semi-quantitative detection method as described in claim 3, characterized in that: The emission spectra of the visible light emitting particles do not overlap.

5. The immunochromatographic semi-quantitative detection method according to any one of claims 1 to 4, characterized in that: It also includes the step of passing the test solution through the labeled antigen; Along the chromatography direction, the detection line and the control line are sequentially arranged, and the surface of the control line is coated with a polyclonal antibody for binding to the labeled antigen; The immunochromatographic semi-quantitative detection method further includes S4: if the control line does not develop color, the detection is deemed invalid; if the control line develops color, the detection is deemed valid.

6. An immunochromatographic semi-quantitative reagent card suitable for the immunochromatographic semi-quantitative detection method as described in any one of claims 1 to 4, characterized in that: The reagent card includes a test strip, which includes a solid support and a sample pad, a marker binding pad, a reaction pad, and an absorbent pad that are connected end to end along the chromatography direction on the surface of the solid support. The reaction pad is provided with a detection area and a quality control area. The detection area includes multiple detection lines. The detection concentration limits of the target analyte on different detection lines increase sequentially along the chromatography direction. The quality control area is provided with quality control lines, which are coated with polyclonal antibodies. The labeled binding pad contains the labeled antibody.

7. The immunochromatographic semi-quantitative reagent card as described in claim 6, characterized in that: The labeled antibody includes multiple first antibodies that target different analytes respectively. The first antibodies are labeled by tracer particles, and the tracer signals of the tracer particles labeled with different first antibodies do not interfere with each other. Each detection line is coated with a specific antibody that includes multiple secondary antibodies that target different analytes, and the detection concentration limits of each analyte on different detection lines increase sequentially along the chromatography direction.

8. The immunochromatographic semi-quantitative reagent card as described in claim 7, characterized in that: The tracer particles include colorimetric particles and / or visible light emitting particles.

9. The immunochromatographic semi-quantitative reagent card as described in claim 6, characterized in that: The detection zone and the quality control zone are sequentially arranged on the reaction pad along the chromatography direction.

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