An immunochromatographic test strip for signal amplification, its preparation method, a detection kit thereof and a detection method thereof

Abstract

The present invention discloses an immunochromatographic test strip for signal amplification, which comprises a first conjugate pad, a second conjugate pad, a nitrocellulose membrane coated with a C line and a T line, a sample absorbing pad and a bottom plate; wherein the first conjugate pad is fixed with antibody A against the antigen to be detected conjugated with oxidized dextran; the second conjugate pad is fixed with ConA-conjugated quantum dot fluorescent microspheres and anti-DNP antibody-conjugated quantum dot fluorescent microspheres; the T line is coated with antibody B against the antigen to be detected; and the C line is coated with DNP. The preparation method of the test strip, a detection kit and a detection method are also disclosed. The present invention introduces a dextran and ConA system. ConA is a tetrameric globulin, and each subunit contains 1 sugar-binding site, and each ConA has 4 sugar-binding sites, thereby enhancing the detection signal, realizing signal amplification and improving the detection sensitivity. It is suitable for the detection of various product types and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of immunochromatography, and in particular to an immunochromatographic test strip with signal amplification, a preparation method of the test strip, a detection kit and a corresponding detection method. Background Art

[0002] Currently, the commonly used detection technologies for antigens are: colloidal gold method, enzyme-linked immunosorbent assay (ELISA), fluorescence immunochromatography, chemiluminescence method, etc. The colloidal gold method is a new type of immunolabeling technology that uses colloidal gold as a tracer marker in antigen-antibody reactions. In addition to binding to proteins, colloidal gold can also bind to many other biological macromolecules, such as SPA, PNA, ConA, etc.; ELISA has become a frontier topic in the field of analytical chemistry. It is a special reagent analysis method and a new type of immunoassay technology developed on the basis of immunoenzyme technology; Fluorescence immunochromatography technology is a new type of membrane detection technology based on specific antigen-antibody immune reactions. A strip-shaped fiber chromatography material with a detection line (coated antibody or coated antigen) and a quality control line (anti-antibody) fixed thereon is used as the stationary phase, and the test solution is used as the mobile phase. The fluorescence-labeled antibody or antigen is fixed on the conjugate pad, and the analyte moves on the chromatography strip through capillary action; The chemiluminescence method is a method for measuring chemiluminescent reactants, catalysts, sensitizers, inhibitors, and reactants, catalysts, and sensitizers in coupling reactions using chemiluminescence. Summary of the Invention

[0003] The present invention mainly solves the technical problems existing in the prior art, and thus provides an immunochromatographic test strip cassette detection device with signal amplification, which can improve the specificity and sensitivity of the immunochromatographic platform detection.

[0004] The above technical problems of the present invention are mainly solved by the following technical solutions: An immunochromatographic test strip with signal amplification includes a first conjugate pad, a second conjugate pad, a nitrocellulose membrane coated with a C line and a T line, a sample pad, and a bottom plate. The first conjugate pad, the second conjugate pad, the nitrocellulose membrane coated with the C line and the T line, and the sample pad are sequentially placed on the bottom plate. The head of the second conjugate pad overlaps with the tail of the first conjugate pad, the head of the nitrocellulose membrane overlaps with the tail of the second conjugate pad, and the head of the sample pad overlaps with the tail of the nitrocellulose membrane;

[0005] Among them, the first conjugate pad is fixed with antibody A conjugated with oxidized dextran for the antigen to be detected;

[0006] The second conjugate pad is fixed with ConA-conjugated quantum dot fluorescent microspheres and anti-DNP antibody-conjugated quantum dot fluorescent microspheres;

[0007] The T line is coated with antibody B for the antigen to be detected;

[0008] The C line is coated with DNP.

[0009] Preferably, the bottom plate is made of PVC.

[0010] Preferably, the binding pads I and II are made of glass fiber, non-woven fabric or filter paper.

[0011] Preferably, the sample absorption pad is made of filter paper.

[0012] Preferably, the oxidized dextran is obtained by oxidizing dextran with sodium periodate.

[0013] Preferably, the antibody A conjugated oxidized dextran is obtained by mixing oxidized dextran with antibody A, adding NaCNBH3 for reaction, then adding ethylenediamine for reaction, and then adding NaBH4 for reduction.

[0014] The present invention also discloses an immunochromatographic detection kit for signal amplification, which includes the above-mentioned immunochromatographic test strip and a cassette. The test strip is placed in the cassette. The cassette exposes a sample adding hole at a corresponding position of the binding pad I of the test strip, exposes a diluent adding hole at a corresponding position of the binding pad II of the test strip, and exposes an observation window at a corresponding position of the C line and T line coated on the nitrocellulose membrane of the test strip.

[0015] The present invention also discloses a preparation method of the above-mentioned immunochromatographic test strip for signal amplification, which is characterized by including the following steps:

[0016] 1. Preparation of binding pad I

[0017] 1.2 Oxidation of dextran: Dextran is oxidized with sodium periodate. After the reaction, excessive ethylene glycol is added to terminate the oxidation reaction, and oxidized dextran is obtained by dialysis.

[0018] 1.2 Conjugation of oxidized dextran with antibody: After oxidized dextran is mixed and reacted with antibody A against the target antigen, it is reduced to a binary conjugate with NaCNBH3, then ethylenediamine is added for ammoniation, and then it is reduced with NaBH4. After that, ethylenediamine and NaBH4 are removed by dialysis.

[0019] 1.3 Preparation of binding pad I: The oxidized dextran conjugated antibody obtained in the previous step is fixed on a glass fiber pad to obtain binding pad I.

[0020] 2. Preparation of binding pad II

[0021] 2.1 Conjugation of ConA / anti-DNP antibody with quantum dot fluorescent microspheres: After the quantum dot fluorescent microspheres are activated, ConA or anti-DNP antibody is added for conjugation reaction, and then it is blocked for standby.

[0022] 2.2 Preparation of binding pad II: The ConA conjugated quantum dot fluorescent microspheres and the anti-DNP antibody conjugated quantum dot fluorescent microspheres are fixed on a glass fiber pad to obtain binding pad II.

[0023] 3. Preparation of the nitrocellulose membrane coated with the C line and the T line

[0024] 3.1 Dilute antibody B against the target antigen with the coating buffer to obtain the T line antibody;

[0025] 3.2 Dilute DNP-BSA with the coating buffer to obtain the C line antibody;

[0026] 3.3 Coat the C line antibody and the T line antibody on the nitrocellulose membrane respectively, and dry to obtain the nitrocellulose membrane coated with the C line and the T line;

[0027] 4. Assembly

[0028] Place the first conjugate pad, the second conjugate pad, the nitrocellulose membrane coated with the C line and the T line, and the sample absorption pad on the bottom plate in sequence. The head of the second conjugate pad overlaps with the tail of the first conjugate pad, the head of the nitrocellulose membrane overlaps with the tail of the second conjugate pad, and the head of the sample absorption pad overlaps with the tail of the nitrocellulose membrane to obtain the quantum dot fluorescence immunochromatographic test strip.

[0029] Preferably, the dextran oxidation in step 1.1 is specifically as follows: Dissolve a certain amount of dextran with 20 mmol / L phosphate buffer at pH 7.0, and prepare a 100 mg / mL sodium periodate aqueous solution. Add the sodium periodate aqueous solution to the above dextran solution so that the molar ratio of NaIO4 to the monosaccharide unit in dextran is 1:3, and the final concentration of dextran is 40 mg / mL. Stir the reaction weakly at 30 °C in the dark for 3 hours, then add an excessive amount of ethylene glycol to terminate the oxidation reaction. Continue to stir the reaction at 30 °C in the dark for 15 minutes and then take it out, and perform dialysis with deionized water or 20 mmol / L phosphate buffer at pH 7.0.

[0030] Preferably, the conjugation of oxidized dextran with antibody in step 1.2 is specifically as follows: After the oxidized dextran and antibody A against the target antigen are mixed and reacted for 2 hours, they are reduced to a binary conjugate with NaCNBH3, then ethylenediamine is added, ammoniated for 2 hours, and then reduced with NaBH4 for 2 hours. After that, dialysis is performed to remove ethylenediamine and NaBH4.

[0031] Preferably, the conjugation of ConA / anti-DNP antibody with quantum dot fluorescent microspheres in step 2.1 is specifically as follows: Take the quantum dot fluorescent microspheres, add MES, centrifuge, then add a small amount of MES and ultrasonically resuspend. Then first add NHS and mix well, then add EDC. The activation reaction time is not less than half an hour. Add MES, centrifuge, remove the supernatant, then ultrasonically clean with LMES and buffer solution in sequence, resuspend with buffer solution, then add Con-A or anti-DNP antibody to start the conjugation reaction, then add glycine and ethanolamine, add BSA for blocking, wash with PBST, wash with the preservation solution, and resuspend and preserve with the preservation solution.

[0032] Preferably, in step 3.1, the antibody B against the target antigen is diluted to 1.0 - 2.0 mg / mL with the coating buffer solution.

[0033] Preferably, in step 3.2, the DNP-BSA is diluted to 1.0 - 2.0 mg / mL with the coating buffer solution.

[0034] The present invention also discloses a quantum dot fluorescence immunochromatographic detection method with signal amplification, which is carried out based on the above immunochromatographic detection kit, and is characterized in that its steps include:

[0035] (1) Turn on the quantum dot fluorescence immunoassay analyzer;

[0036] (2) Take 50 μL of the sample to be tested and drop it into the sample addition hole of the immunochromatographic test strip. After 5 - 10 min, take 100 μL of the diluent and drop it into the diluent addition hole.

[0037] (3) Continue to incubate for 10 - 15 min, and put the kit into the quantum dot fluorescence immunoassay analyzer to read the test result.

[0038] The technical route of the fluorescence immunoassay method for the reaction signal amplification of concanavalin and glucose in the present invention is as follows: First, couple the antibody A specific to a certain specific antigen with oxidized dextran and fix it on the binding pad 1; couple concanavalin (ConA) with quantum dot fluorescent microspheres and anti-DNP antibody with quantum dot fluorescent microspheres respectively, mix the two quantum dot fluorescent markers and fix them on the binding pad 2; then coat another antibody B against this antigen on a specific zone of the nitrocellulose membrane to form a test line (T line), and coat DNP-BSA on a specific zone of the nitrocellulose membrane to form a quality control line (C line), parallel to the T line, and assemble and prepare a quantum dot fluorescence immunochromatographic test strip. After adding the sample at the binding pad 1, the antibody A coupled with oxidized dextran will react with the antigen in the sample to generate an antigen-antibody A-oxidized dextran complex, and continue to move forward. When passing through the binding pad 2, the oxidized dextran on this complex will bind to the ConA-quantum dot fluorescence complex to form an antigen-antibody A-oxidized dextran-ConA-quantum dot fluorescence complex. At this time, add a diluent at the binding pad 2. Under the action of the diluent, the sample continues to move forward. When chromatographing to the T line area where another antibody B is fixed, the antigen in the sample will react with this antibody B, and finally generate an antibody B-antigen-antibody A-oxidized dextran-ConA-quantum dot fluorescence complex. If there is no antigen to be detected in the sample, no fluorescence complex will be formed at the T line; the sample continues to move forward. Whether there is the antigen to be detected in the sample or not, the anti-DNP antibody labeled with quantum dot fluorescence will bind to the DNP-BSA coated on the C line. The signal intensities of the C and T lines can be read by a supporting quantum dot fluorescence immunoassay analyzer, and the sample concentration can be calculated according to the standard curve preset in the SD card to give a quantitative detection result. Due to the introduction of the dextran and ConA system in this technology, ConA is a tetrameric globulin, each subunit contains 1 sugar-binding site, and each ConA has 4 sugar-binding sites, thereby enhancing the detection signal, realizing signal amplification, and improving the detection sensitivity. It is suitable for the detection of various product types and has broad application prospects. Brief Description of the Drawings

[0039] Figure 1 It is a schematic structural diagram of the signal-amplified immunochromatographic test strip of the present invention.

[0040] Figure 2 It is the immunochromatographic detection kit of the present invention.

[0041] Figure 3 It is the regression equation of the detection result of the interleukin-6 kit. Detailed Embodiments

[0042] The preferred embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention.

[0043] For the reagents or instruments used, unless otherwise specified by the manufacturer, they are all conventional products that can be obtained by purchasing in the market.

[0044] Example 1: Oxidation of dextran

[0045] Dissolve a certain amount of dextran in 20 mmol / L phosphate buffer at pH 7.0, and prepare an aqueous solution of sodium periodate at 100 mg / mL (protected from light). Add a certain amount of the aqueous sodium periodate solution to the above dextran solution so that the molar ratio of NaIO4 to the monosaccharide units in dextran is 1:3, and the final concentration of dextran is 40 mg / mL. After reacting with gentle stirring at 30 °C in the dark for 3 hours, add an excess of ethylene glycol to terminate the oxidation reaction, continue stirring at 30 °C in the dark for 15 minutes and then take out, and perform dialysis with deionized water or 20 mmol / L phosphate buffer at pH 7.0 (dextran with a molecular weight of 500 kd and 70 kd is dialyzed with a dialysis bag with a cut-off molecular weight of 12,000, and dextran with a molecular weight of 10 kd is dialyzed with a dialysis bag with a cut-off molecular weight of 3500).

[0046] Example 2: Conjugation of oxidized dextran with antibody A

[0047] Mix oxidized dextran and antibody in a molar ratio of 1:1, react at 4 °C for 2 hours, add NaCNBH3 with a molar amount 10 times that of the protein concentration, react at 4 °C for 2 hours, then add ethylenediamine, react at 4 °C for 2 hours, and then reduce with NaBH4 with a molar amount 4 times that of ethylenediamine for 2 hours. After that, dialyze to remove ethylenediamine and NaBH4.

[0048] Example 3: Preparation of conjugate pad 1

[0049] Dilute the conjugate in Example 2 with a conjugate pad fixing solution to 0.02 - 0.04 mg / mL and fix it on a glass fiber pad, and place it in an oven at 37 °C for drying for 24 h. The conjugate pad fixing solution is 50 mM PB buffer at pH = 7.2 - 7.4, containing 0.5% Tween-20, 0.5% PVPK-30, 5% sucrose, 1% casein-Na, 2% protein protectant, 0.02% PC-300.

[0050] Example 4: Conjugation of concanavalin A (ConA) with quantum dot fluorescent microspheres

[0051] ① Quantum dot fluorescent microsphere BUFF replacement: Take 10 mg of quantum dot fluorescent microspheres, add MES buffer solution with pH 5.5 - 6.0 until full and centrifuge (200 nm, 14000 rpm, 30 min; 120 nm, 20000 rpm, 30 min). Add 200 μL of MES and resuspend by ultrasonic treatment for later use.

[0052] ② Activation: Add 0.5 - 1 mg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and 0.6 mg of NHS (N-hydroxysuccinimide) to every 10 mg of quantum dot fluorescent microspheres (add NHS first, mix, and then add EDC. For large-scale activation, EDC must be added dropwise while stirring). For large-scale reactions, less EDC can be used. Taking 10 mg of quantum dot fluorescent microspheres as an example, supplement MES to make the volume up to 400 - 500 μL, and react by rotation at 37 °C for about 1 hour (rotation speed 40 - 60 rpm).

[0053] ③ Washing: After activation, add MES buffer solution until full, centrifuge (14000 rpm, 30 min), discard the supernatant, add 200 μL of MES and resuspend by ultrasonic treatment, repeat once, add 50 mM HEPES (all the following are 50 mM) with pH 8.0 and resuspend by ultrasonic treatment, supplement HEPES until full and centrifuge, and resuspend with HEPES for later use.

[0054] ④ Coupling: Add 0.1 - 0.5 mg of ConA, make the volume up to 400 - 500 μL, mix well and react at 37 °C for 3 hours.

[0055] ⑤ Blocking 1: Add 70 μL of 2 M glycine and 140 μL of 1 M ethanolamine and block at 37 °C for 1 hour.

[0056] ⑥ Blocking 2: Add 200 μL of 200 mg / mL BSA and block overnight at 4 °C.

[0057] ⑦ Washing: Add PBST to wash twice, add buffer solution to wash once, and then make the volume up to about 3 - 4 mL for storage and later use.

[0058] Example 5: Conjugation of anti-DNP antibody with quantum dot fluorescent microspheres

[0059] ① Quantum dot fluorescent microsphere BUFF replacement: Take 10 mg of quantum dot fluorescent microspheres, add MES with pH 5.5 - 6.0 until full and centrifuge (200 nm, 14000 rpm, 30 min; 120 nm, 20000 rpm, 30 min). Add 200 μL of MES and resuspend by ultrasonic treatment for later use.

[0060] ② Activation: Add 0.5 - 1 mg of EDC and 0.6 mg of NHS to every 10 mg of quantum dot fluorescent microspheres (add NHS first, mix, and then add EDC). For a large number of reactions, less EDC can be used. Taking 10 mg of quantum dot fluorescent microspheres as an example, make up with MES to a volume of 400 - 500 μL, and react by rotation at 37 °C for about 1 hour (rotation speed 40 - 60 rpm).

[0061] ③ Washing: After activation, fill it up with MES, centrifuge (14000 rpm for 30 min), discard the supernatant, add 200 μL of MES and resuspend by ultrasonic treatment, repeat once, add 50 mM HEPES (all the following are 50 mM) with pH 8.0 and resuspend by ultrasonic treatment, fill it up with HEPES and centrifuge, and resuspend with HEPES for later use.

[0062] ④ Coupling: Add 0.1 - 0.5 mg of antibody, make up the volume to 400 - 500 μL, mix well and react at 37 °C for 3 hours.

[0063] ⑤ Blocking 1: Add 70 μL of 2 M glycine and 140 μL of 1 M ethanolamine and block at 37 °C for 1 hour.

[0064] ⑥ Blocking 2: Add 200 μL of 200 mg / ml BSA and block overnight at 4 °C.

[0065] ⑦ Washing: Add PBST to wash twice, add buffer to wash once, and then make up the volume to about 3 - 4 mL for storage and later use.

[0066] Example 6: Preparation of Conjugate Pad 2

[0067] Mix the conjugates in Example 4 and Example 5, dilute with conjugate pad fixing solution, and the final concentration is 0.02 - 0.04 mg / mL. Fix them on the glass fiber pad and dry in an oven at 37 °C for 24 h. The conjugate pad fixing solution is 50 mM PB buffer with pH = 7.2 - 7.4, containing 0.5% Tween - 20, 0.5% PVPK - 30, 5% sucrose, 1% casein - Na, 2% protein protectant, 0.02% PC - 300.

[0068] Example 7: Preparation of Nitrocellulose Membrane Coated with C - line and T - line

[0069] Dilute antibody B to 1.0 - 2.0 mg / mL with coating buffer; Preparation of C - line scribing solution: Dilute DNP - BSA to 1.0 - 2.0 mg / mL with coating buffer; The coating buffer is 10 mM PB containing 1% sucrose. Using a gold - spraying scribing instrument, coat the C - line antibody and T - line antibody on the nitrocellulose membrane fixed on the bottom plate respectively; Place the scribed large plate in a forced - air drying oven and dry at 37 °C for 24 h, and cut it into the required size.

[0070] Example 8: Assembly

[0071] As shown Figure 1 in the figure, the conjugate pad 1, conjugate pad 2, nitrocellulose membrane 5 coated with C line 3 and T line 4, and sample absorption pad 6 are sequentially placed on the bottom plate 7. The head of conjugate pad 2 overlaps with the tail of conjugate pad 1, the head of the nitrocellulose membrane overlaps with the tail of conjugate pad 2, and the head of the sample absorption pad overlaps with the tail of the nitrocellulose membrane. The bottom plate is made of PVC, and the sample absorption pad is made of filter paper with good water absorption, thus completing the preparation of the quantum dot fluorescence immunochromatographic test strip.

[0072] As shown Figure 2 in the figure, the assembled test strip is placed into the cartridge 8. The sample adding hole 9 and the diluent adding hole 10 are exposed at the corresponding positions of conjugate pad 1 of the test strip, and the observation window 11 is exposed at the corresponding positions of the C line and T line coated on the nitrocellulose membrane of the test strip, thus completing the preparation of the quantum dot fluorescence immunochromatographic detection device.

[0073] Example 9: Detection results of interleukin-6 kit

[0074] The interleukin-6 detection kit was prepared by the method of the above Examples 1-8. 50 μL of the standard sample was dropped into the sample adding hole 9 of the immunochromatographic test strip. After 5-10 minutes, 100 μL of the diluent was dropped into the diluent adding hole 10, and incubation was continued for 10-15 minutes. Then the kit was put into the quantum dot fluorescence immunoassay analyzer to read the detection results. The test results are shown in Table 1. With the concentration as the abscissa and the signal value (T / C) as the ordinate, a four-parameter Logistic curve fitting was performed, and the R2 value was 0.999. The regression curve is shown in Figure 3 , and the regression equation is:

[0075] Equation: y=(A-D) / [1+(x / C)^B]+D

[0076] Where:

[0077] A = 88.76990

[0078] B = -1.01141

[0079] C = 3719.90518

[0080] D = 0.35346

[0081] r^2 = 0.99908.

[0082] Table 1 Test results of standard samples of interleukin-6 kit

[0083]

Claims

1. An immunochromatographic test strip for signal amplification, characterized in that: It includes a conjugate pad 1, a conjugate pad 2, a nitrocellulose membrane coated with C line and T line, a sample absorption pad and a bottom plate; Among them, the conjugate pad 1 is fixed with antibody A conjugated with oxidized dextran for the antigen to be detected; the oxidized dextran is formed by oxidizing dextran with sodium periodate. The antibody A conjugated with oxidized dextran is obtained by mixing oxidized dextran and antibody A, adding NaCNBH3 for reaction, then adding ethylenediamine for reaction, and then adding NaBH4 for reduction; The conjugate pad 2 is fixed with ConA-conjugated quantum dot fluorescent microspheres and anti-DNP antibody-conjugated quantum dot fluorescent microspheres; The T line is coated with antibody B for the antigen to be detected; The C line is coated with DNP.

2. The immunochromatographic test strip according to claim 1, characterized in that: The conjugate pad 1, the conjugate pad 2, the nitrocellulose membrane coated with C line and T line, and the sample absorption pad are sequentially placed on the bottom plate. The head of the conjugate pad 2 overlaps with the tail of the conjugate pad 1, the head of the nitrocellulose membrane overlaps with the tail of the conjugate pad 2, and the head of the sample absorption pad overlaps with the tail of the nitrocellulose membrane.

3. The immunochromatographic test strip according to claim 1, characterized in that: The bottom plate is made of PVC.

4. The immunochromatographic test strip according to claim 1, characterized in that: The conjugate pad 1 and the conjugate pad 2 are made of glass fiber, non-woven fabric or filter paper.

5. The immunochromatographic test strip according to claim 1, characterized in that: The sample absorption pad is made of filter paper.

6. An immunochromatographic detection kit for signal amplification, characterized in that: It includes the immunochromatographic test strip according to any one of claims 1-5, and a cartridge. The test strip is placed in the cartridge. The cartridge exposes a sample addition hole and a diluent addition hole at the corresponding position of the conjugate pad 1 of the test strip, and exposes an observation window at the corresponding position of the nitrocellulose membrane coated with C line and T line of the test strip.

7. A preparation method of an immunochromatographic test strip for signal amplification, characterized in that It includes the following steps:

1. Prepare the conjugate pad 1 1.1 Dextran oxidation: Dextran is oxidized with sodium periodate. After the reaction, an excessive amount of ethylene glycol is added to terminate the oxidation reaction, and the oxidized dextran is obtained by dialysis; 1.2 Conjugation of oxidized dextran with antibody: After the oxidized dextran reacts with antibody A against the target antigen, it is reduced to a binary conjugate with NaCNBH3, then ethylenediamine is added for amination, and then it is reduced with NaBH4. After that, dialysis is carried out to remove ethylenediamine and NaBH4; 1.3 Preparation of conjugate pad 1: The oxidized dextran-conjugated antibody obtained in the previous step is immobilized on a glass fiber pad to obtain conjugate pad 1; 2. Preparation of conjugate pad 2 2.1 Conjugation of ConA / anti-DNP antibody with quantum dot fluorescent microspheres: After the quantum dot fluorescent microspheres are activated, ConA or anti-DNP antibody is added for conjugation reaction, and then it is blocked and reserved; 2.2 Preparation of conjugate pad 2: The ConA-conjugated quantum dot fluorescent microspheres and the anti-DNP antibody-conjugated quantum dot fluorescent microspheres are immobilized on a glass fiber pad to obtain conjugate pad 2; 3. Preparation of nitrocellulose membrane coated with C line and T line 3.1 Dilute antibody B against the target antigen with a coating buffer to obtain the T line antibody; 3.2 Dilute DNP-BSA with a coating buffer to obtain the C line antibody; 3.3 Coat the C-line antibody and the T-line antibody on the nitrocellulose membrane respectively, and dry it to obtain the nitrocellulose membrane coated with the C-line and the T-line.

4. Assembly Place the conjugate pad 1, the conjugate pad 2, the nitrocellulose membrane coated with the C-line and the T-line, and the sample absorption pad on the bottom plate in sequence. The head of the conjugate pad 2 overlaps with the tail of the conjugate pad 1, the head of the nitrocellulose membrane overlaps with the tail of the conjugate pad 2, and the head of the sample absorption pad overlaps with the tail of the nitrocellulose membrane to obtain the quantum dot fluorescence immunochromatographic test strip. Specifically, the oxidation of dextran in step 1.1 is as follows: dissolve a certain amount of dextran with 20 mmol / L phosphate buffer solution at pH 7.0, and prepare an aqueous solution of sodium periodate at 100 mg / mL. Add the aqueous solution of sodium periodate to the above dextran solution so that the molar ratio of NaIO4 to the monosaccharide unit of dextran is 1:3, and the final concentration of dextran is 40 mg / mL. After reacting with weak stirring at 30 °C in the dark for 3 hours, add an excessive amount of ethylene glycol to terminate the oxidation reaction, continue to stir and react at 30 °C in the dark for 15 minutes, then take it out and dialyze it with deionized water or 20 mmol / L phosphate buffer solution at pH 7.

0. Specifically, the conjugation of oxidized dextran and antibody in step 1.2 is as follows: after the oxidized dextran and antibody A against the target antigen are mixed and reacted for 2 hours, it is reduced to a binary conjugate with NaCNBH3, then ethylenediamine is added, ammoniated for 2 hours, and then reduced with NaBH4 for 2 hours, and then dialyzed to remove ethylenediamine and NaBH4. Specifically, the conjugation of ConA / anti-DNP antibody and quantum dot fluorescent microspheres in step 2.1 is as follows: take quantum dot fluorescent microspheres, add MES, centrifuge, add a small amount of MES and resuspend by ultrasonic wave, then add NHS and mix well, then add EDC, and the activation reaction time is not less than half an hour. Add MES, centrifuge, remove the supernatant, then wash with LMES and buffer solution by ultrasonic wave in sequence, resuspend with buffer solution, then add Con-A or anti-DNP antibody to start the conjugation reaction, then add glycine and ethanolamine, add BSA for blocking, wash with PBST, wash with the preservation solution, and resuspend and preserve with the preservation solution. In step 3.1, dilute the antibody B against the target antigen to 1.0 - 2.0 mg / mL with the coating buffer. In step 3.2, dilute DNP-BSA to 1.0 - 2.0 mg / mL with the coating buffer.

8. A signal-amplified immunochromatographic detection method, which is carried out based on the signal-amplified immunochromatographic detection kit according to claim 6, and is characterized in that The steps include: (1) Turn on the quantum dot fluorescence immunoassay analyzer; (2) Take 50 μL of the sample to be tested and drop it into the sample addition hole of the immunochromatographic test strip. After 5-10 minutes, take 100 μL of diluent and drop it into the diluent addition hole; (3) Continue to incubate for 10-15 minutes, and put the kit into the quantum dot fluorescence immunoassay analyzer to read the test result.

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