High specificity, high sensitivity of immunochromatography test paper and preparation method thereof, test kit and detection method thereof

Abstract

This invention discloses a highly specific and sensitive immunochromatographic test strip, comprising a first conjugation pad, a second conjugation pad, a nitrocellulose membrane coated with C and T lines, an absorption pad, and a base plate. The first conjugation pad is immobilized with antibody A conjugated to oxidized dextran, representing the antigen to be detected. The second conjugation pad is immobilized with ConA-conjugated colloidal gold and anti-DNP antibody conjugated to colloidal gold. The T line is coated with antibody B, representing the antigen to be detected. The C line is coated with DNP. The invention also discloses the preparation method of the test strip, the detection kit, and the detection method. This invention introduces the dextran and ConA system. ConA is a tetrameric globulin, with each subunit containing one sugar-binding site, and each ConA having four sugar-binding sites, thereby enhancing the detection signal, amplifying the signal, and improving detection sensitivity. It is suitable for the detection of various product types and has broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of immunochromatographic technology, and more particularly to a highly specific and highly sensitive immunochromatographic test strip, a method for preparing the test strip, a test kit, and a corresponding detection method. Background Technology

[0002] Currently, commonly used antigen detection techniques include: colloidal gold method, enzyme-linked immunosorbent assay (ELISA), fluorescence immunochromatography, and chemiluminescence method. The colloidal gold method is a novel immunolabeling technique that uses colloidal gold as a tracer marker for antigen-antibody reactions. Besides binding to proteins, colloidal gold can also bind to many other biomolecules, such as SPA, PNA, and ConA. ELISA has become a cutting-edge topic in analytical chemistry; it is a special reagent analysis method developed based on immunoenzyme technology. Fluorescence immunochromatography is a novel membrane detection technique based on antigen-antibody specific immune reactions. It uses a strip-shaped chromatographic material with a detection line (coated antibody or coated antigen) and a control line (anti-antibody) as the stationary phase, and the test solution as the mobile phase. Fluorescently labeled antibodies or antigens are immobilized on the connecting pads, and the analyte moves on the chromatographic strip through capillary action. Chemiluminescence method is a method that uses chemiluminescence to determine chemiluminescent reactants, catalysts, sensitizers, inhibitors, and reactants, catalysts, and sensitizers in coupling reactions. Summary of the Invention

[0003] The present invention mainly addresses the technical problems existing in the prior art, thereby providing a highly specific and sensitive immunochromatographic test kit detection device that can improve the specificity and sensitivity of colloidal gold immunochromatographic platform detection.

[0004] The above-mentioned technical problems of the present invention are mainly solved by the following technical solution: a highly specific and highly sensitive immunochromatographic test strip, characterized in that: it includes a first conjugate pad, a second conjugate pad, a nitrocellulose membrane coated with C-line and T-line, a sample absorption pad and a base plate, wherein the first conjugate pad, the second conjugate pad, the nitrocellulose membrane coated with C-line and T-line and the sample absorption pad are placed sequentially on the base 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 absorption pad overlaps with the tail of the nitrocellulose membrane;

[0005] Among them, antibody A conjugated with oxidized dextran is immobilized on the binding pad with the antigen to be detected;

[0006] The conjugate pad 2 is immobilized with ConA-conjugated colloidal gold and anti-DNP antibody-conjugated colloidal gold;

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

[0008] The C-line is enveloped by DNP.

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

[0010] Preferably, the first and second bonding pads are made of glass fiber, non-woven fabric or filter paper.

[0011] Preferably, the sampling pad is filter paper.

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

[0013] Preferably, the antibody A-conjugated oxidized dextran is prepared by mixing oxidized dextran with antibody A, reacting with NaCNBH3, reacting with ethylenediamine, and then reducing with NaBH4.

[0014] The present invention also discloses an immunochromatographic detection kit, comprising the above-mentioned immunochromatographic test strip and a cartridge, wherein the test strip is placed in the cartridge, the cartridge has a sample application well exposed at the corresponding part of the conjugate pad one of the test strip, a diluent addition well exposed at the corresponding part of the conjugate pad two of the test strip, and an observation window exposed at the corresponding parts of the C line and T line coated by the nitrocellulose membrane of the test strip.

[0015] This invention also discloses a method for preparing the above-mentioned highly specific and highly sensitive immunochromatographic test strip, characterized by comprising the following steps:

[0016] 1. Preparation of binding pad

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

[0018] 1.2 Oxidized dextran-conjugated antibody: Oxidized dextran was mixed with antibody A against the target antigen and reacted, then reduced to a binary conjugate with NaCNBH3, then aminated with ethylenediamine, then reduced with NaBH4, and finally dialyzed to remove ethylenediamine and NaBH4.

[0019] 1.3 Preparation of Binding Pad 1: The oxidized dextran-conjugated antibody obtained in the previous step was immobilized onto a glass fiber pad to prepare Binding Pad 1;

[0020] 2. Preparation of binding pad II

[0021] 2.1 ConA / anti-DNP antibody conjugated colloidal gold: K2CO3 was added to the colloidal gold solution, followed by ConA or anti-DNP antibody for conjugation. After blocking with blocking buffer, the colloidal gold particles were centrifuged and stored in a preservation solution for later use.

[0022] 2.2 Preparation of Binding Pad II: ConA-conjugated colloidal gold and anti-DNP antibody-conjugated colloidal gold were immobilized onto a glass fiber pad to prepare Binding Pad II;

[0023] 3. Preparation of nitrocellulose membranes coated with C-lines and T-lines

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

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

[0026] 3.3 The C-line antibody and T-line antibody were coated onto a nitrocellulose membrane, respectively, and dried to obtain a nitrocellulose membrane coated with C-line and T-line.

[0027] 4. Assembly

[0028] The conjugate pad 1, conjugate pad 2, nitrocellulose membrane coated with C and T lines, and sample absorbent pad are placed sequentially on the base plate. The head of conjugate pad 2 overlaps with the tail of conjugate pad 1, the head of nitrocellulose membrane overlaps with the tail of conjugate pad 2, and the head of sample absorbent pad overlaps with the tail of nitrocellulose membrane to obtain colloidal gold immunochromatographic test strip.

[0029] Preferably, step 1.1, dextran oxidation, specifically involves dissolving a certain amount of dextran in a 20 mmol / L phosphate buffer solution with pH 7.0, and preparing a 100 mg / mL sodium periodate aqueous solution. The sodium periodate aqueous solution is then added to the dextran solution to achieve a molar ratio of NaIO4 to monosaccharide units in the dextran of 1:3, resulting in a final dextran concentration of 40 mg / mL. After reacting with gentle stirring at 30°C in the dark for 3 hours, excess ethylene glycol is added to terminate the oxidation reaction. The reaction is then continued with stirring at 30°C in the dark for another 15 minutes before being removed and dialyzed with deionized water or a 20 mmol / L phosphate buffer solution with pH 7.0.

[0030] Preferably, in step 1.2, the oxidized dextran-conjugated antibody is prepared by mixing oxidized dextran with antibody A against the target antigen and reacting for 2 hours, then reducing it to a binary conjugate with NaCNBH3, then adding ethylenediamine, ammonifying for 2 hours, reducing with NaBH4 for 2 hours, and finally dialysis to remove ethylenediamine and NaBH4.

[0031] Preferably, step 2.1, the conjugation of ConA / anti-DNP antibody with colloidal gold, is as follows: take colloidal gold solution, add 0.1 mol / L K2CO3 to adjust to a suitable pH, add ConA / anti-DNP antibody, mix well, let stand to react, after the reaction is complete, slowly add blocking buffer, mix well, let stand to block, centrifuge to remove supernatant, add washing buffer to wash, centrifuge to remove supernatant, add storage solution and mix well for later use.

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

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

[0034] This invention also discloses a highly specific and sensitive immunochromatographic detection method, based on the above-mentioned immunochromatographic detection kit, characterized by the following steps:

[0035] (1) Turn on the colloidal gold immunoassay analyzer;

[0036] (2) Take 50 μL of the sample to be tested and add it to the sample well of the immunochromatographic test strip. After 5 to 10 minutes, take 100 μL of diluent and add it to the diluent addition well.

[0037] (3) Continue incubation for 10-15 minutes, then place the kit into the colloidal gold immunoassay analyzer to read the test results.

[0038] The high-specificity and high-sensitivity fluorescence immunoassay method of this invention follows this technical route: First, an antibody A specific to a particular antigen is coupled with oxidized dextran and immobilized on conjugation pad 1. Concanavalin A (ConA) and anti-DNP antibody are then coupled with colloidal gold, respectively. The two colloidal gold labels are mixed and immobilized on conjugation pad 2. Next, another antibody B targeting the same antigen is coated onto a specific zone of a nitrocellulose membrane to form a detection line (T line), and DNP-BSA is coated onto a specific zone of the nitrocellulose membrane to form a control line (C line), parallel to the T line. This process assembles and prepares the colloidal gold immunochromatographic test strip. After the sample is added to conjugation pad 1, the oxidized dextran-coupled antibody A reacts with the antigen in the sample to generate an antigen-antibody A-oxidized dextran complex. Continuing to move forward, upon passing conjugation pad 2, the oxidized dextran on this complex binds to the ConA-colloidal gold complex, forming an antigen-antibody A-oxidized dextran-ConA-colloidal gold complex. At this point, diluent is added to conjugation pad 2. Under the action of the diluent, the sample continues to move forward. When it reaches the T-line region where another antibody B is immobilized, the antigen in the sample reacts with this antibody B, ultimately forming an antibody B-antigen-antibody A-oxidized dextran-ConA-colloidal gold complex. If the sample does not contain the target antigen, no fluorescent complex will form at the T-line. As the sample continues to move forward, regardless of whether the sample contains the target antigen, the anti-DNP antibody labeled with colloidal gold will bind to the DNP-BSA coated at line C. The signal intensity of lines C and T can be read using a matching colloidal gold immunoassay analyzer, and the sample concentration can be calculated based on the pre-set standard curve in the SD card to provide quantitative detection results. This technology, by introducing the dextran and ConA system (ConA is a tetrameric globulin, each subunit containing one sugar-binding site, and each ConA having four sugar-binding sites), enhances the detection signal, amplifies the signal, and improves detection sensitivity. This invention is suitable for the detection of various product types and has broad application prospects. Attached Figure Description

[0039] Figure 1 This is a schematic diagram of the structure of the highly specific and highly sensitive immunochromatographic test strip of the present invention.

[0040] Figure 2 This is a structural diagram of the immunochromatographic detection kit of the present invention.

[0041] Figure 3 The regression equation is for the detection results of the S100-β kit. Detailed Implementation

[0042] The preferred embodiments of the present invention will now be described in detail with reference to 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 providing a clearer and more explicit definition of the scope of protection of the present invention.

[0043] Unless otherwise specified, all reagents or instruments used are commercially available products.

[0044] Example 1: Dextran oxidation

[0045] A certain amount of dextran was dissolved in 20 mmol / L phosphate buffer (pH 7.0), and a 100 mg / mL sodium periodate aqueous solution was prepared (protected from light). A certain amount of sodium periodate aqueous solution was added to the dextran solution to make the molar ratio of NaIO4 / dextran monosaccharide units 1:3, resulting in a final dextran concentration of 40 mg / mL. After reacting with gentle stirring at 30°C in the dark for 3 hours, excess ethylene glycol was added to terminate the oxidation reaction. The reaction was continued at 30°C in the dark with stirring for another 15 minutes. The mixture was then removed and dialyzed with deionized water or 20 mmol / L phosphate buffer (pH 7.0). (Dextran with molecular weights of 500 kDa and 70 kDa was dialyzed using a dialysis bag with a molecular weight cutoff of 12000; dextran with a molecular weight cutoff of 10 kDa was dialyzed using a dialysis bag with a molecular weight cutoff of 3500.)

[0046] Example 2: Oxidized dextran conjugated antibody A

[0047] Oxidized dextran and antibody were mixed at a 1:1 molar ratio and reacted at 4°C for 2 hours. Then, 10 times the molar amount of protein NaCNBH3 was added and reacted at 4°C for 2 hours. Next, ethylenediamine was added and reacted at 4°C for 2 hours. Finally, the mixture was reduced with 4 times the molar amount of ethylenediamine NaBH4 for 2 hours. Then, ethylenediamine and NaBH4 were removed by dialysis.

[0048] Example 3: Preparation of binding pad 1

[0049] The conjugate from Example 2 was diluted to 0.02–0.04 mg / mL with conjugation pad fixative and fixed onto a glass fiber pad, then dried in a 37°C oven for 24 hours. The conjugation pad fixative was a 50 mM PB buffer solution with pH 7.2–7.4, containing 0.5% Tween-20, 0.5% PVPK-30, 5% sucrose, 1% casein-Na, 2% protein protectant, and 0.02% PC-300.

[0050] Example 4: Conjugandarin (ConA) coupled with colloidal gold

[0051] Colloidal gold was prepared by the trisodium citrate reduction method, and the steps are as follows: 0.01% HauCl4 aqueous solution was prepared, and 100 ml of the solution was heated to boiling; 6.5 ml of 1% trisodium citrate aqueous solution was added while stirring; the solution was heated to boiling for 15 min; after cooling to room temperature, ddH2O was added to make up to 500 ml to obtain a colloidal gold solution. The particle diameter of the colloidal gold was approximately 30-40 nm.

[0052] Colloidal gold can also be prepared using other conventional methods or by purchasing existing colloidal gold solution products.

[0053] Take 1 mL of colloidal gold solution, add 6–8 μL of 0.1 mol / L K₂CO₃, add 0.02–0.03 mg of ConA, mix well, and let stand for one hour. After the reaction is complete, slowly add 0.1 mL of 10% sodium casein, mix well, and let stand for one hour to block the reaction. Centrifuge at 6000 g, 2–8 °C, for 40 min, remove the supernatant, add washing buffer, mix well, and repeat once. After centrifugation, remove the supernatant, add preservation solution, and mix well.

[0054] Example 5: Anti-DNP antibody conjugated with colloidal gold

[0055] Take 1 mL of colloidal gold solution, add 6–8 μL of 0.1 mol / L K₂CO₃, add 0.02–0.03 mg of DNP antibody, mix well, and let stand for one hour. After the reaction is complete, slowly add 0.1 mL of 10% casein sodium, mix well, and let stand for one hour to block. Centrifuge at 6000 g, 2–8 °C, for 40 min, remove the supernatant, add washing buffer, mix well, and repeat once. After centrifugation, remove the supernatant, add preservation solution, and mix well.

[0056] Example 6: Preparation of binding pad 2

[0057] The conjugates from Examples 4 and 5 were diluted with conjugate pad fixative to a final concentration of 0.02–0.04 mg / mL, fixed onto glass fiber pads, and dried in a 37°C oven for 24 hours. The conjugate pad fixative was a 50 mMMPB buffer solution with pH 7.2–7.4, containing 0.5% Tween-20, 0.5% PVPK-30, 5% sucrose, 1% casein-Na, 2% protein protectant, and 0.02% PC-300.

[0058] Example 7: Preparation of nitrocellulose membrane coated with C-line and T-line

[0059] Antibody B was diluted to 1.0–2.0 mg / mL with coating buffer; C-line streaking buffer was prepared by diluting DNP-BSA to 1.0–2.0 mg / mL with coating buffer; the coating buffer was 10 mM PB containing 1% sucrose. Using a gold-sprayed streaking apparatus, C-line and T-line antibodies were coated onto nitrocellulose membranes fixed to a substrate; the streaked plates were then dried in a forced-air drying oven at 37°C for 24 hours and cut to the required size.

[0060] Example 8: Assembly

[0061] like Figure 1 As shown, conjugate pad 1, conjugate pad 2, nitrocellulose membrane 5 coated with C-line 3 and T-line 4, and sample absorbent pad 6 are sequentially placed on the base 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 absorbent pad overlaps with the tail of the nitrocellulose membrane. The base plate is made of PVC, and the sample absorbent pad is made of highly absorbent filter paper, thus completing the preparation of the colloidal gold immunochromatographic test strip.

[0062] like Figure 2 As shown, the assembled test strip is placed into the cartridge 8, and the sample application well 9 and diluent addition well 10 are exposed at the corresponding part of the conjugation pad of the test strip. The observation window 11 is exposed at the corresponding part of the C line and T line coated by the nitrocellulose membrane of the test strip, thus completing the preparation of the colloidal gold immunochromatographic detection device.

[0063] Example 9: Detection results of the S100-β kit

[0064] The S100-β detection kit was prepared using the methods described in Examples 1-8 above. 50 μL of standard sample was added to well 9 of the immunochromatographic test strip. After 5-10 minutes, 100 μL of diluent was added to well 10. Incubation continued for 10-15 minutes. The kit was then placed in a colloidal gold immunoassay analyzer to read the results. The test results are shown in Table 1. A four-parameter Logistic curve was fitted with concentration on the x-axis and signal value (T / C) on the y-axis. The R² value was 0.999. The regression curve is shown in Table 1. Figure 3 The regression equation is:

[0065] Four-parameter Logistic curve fitting

[0066] Equation: y = (AD) / [1 + (x / C)^B] + D

[0067] A = 78.85141

[0068] B = -0.87812

[0069] C = 49.79200

[0070] D = -0.09688

[0071] r^2 = 0.99964.

[0072] Table 1. Test results of S100-β reagent kit samples

[0073] Concentration ng / mL Signal value (T / C) 0 0.01 0.051 0.11 0.13 0.26 0.24 0.54 0.67 1.49 1.16 2.88 2.38 5.12 5.66 9.97 7.89 12.94 8.18 13.29 9.44 14.98 10.12 15.43 .

[0074] The diluent, in 1000 parts by weight, includes 1-5 parts Tween-20, 1-5 parts casein sodium salt, 1-5 parts bovine serum albumin, 1-5 parts polyethylene glycol 200, 10-50 parts sucrose, 0.1-0.5 parts PC-300, and the remainder phosphate buffer.

[0075] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions conceived without inventive effort should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. A highly specific and highly sensitive immunochromatographic test strip, characterized in that: Includes conjugation pad one, conjugation pad two, nitrocellulose membrane covering C-line and T-line, sample suction pad and base plate; Among them, antibody A conjugated with oxidized dextran is immobilized on the binding pad with the antigen to be detected; The conjugate pad 2 is immobilized with ConA-conjugated colloidal gold and anti-DNP antibody-conjugated colloidal gold; T-line is coated with antibody B containing the antigen to be detected; C-line is enclosed by DNP; The oxidized dextran is formed by oxidizing dextran with sodium periodate; The antibody A-coupled oxidized dextran is prepared by mixing oxidized dextran with antibody A, reacting with NaCNBH3, reacting with ethylenediamine, and then reducing with NaBH4.

2. The immunochromatographic test strip according to claim 1, characterized in that: The conjugation pad one, conjugation pad two, nitrocellulose membrane covering C-line and T-line, and sampling pad are placed sequentially on the base plate. The head of conjugation pad two overlaps with the tail of conjugation pad one, the head of nitrocellulose membrane overlaps with the tail of conjugation pad two, and the head of sampling pad overlaps with the tail of nitrocellulose membrane.

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

4. The immunochromatographic test strip according to claim 1, characterized in that: The first and second bonding pads are made of glass fiber, non-woven fabric or filter paper.

5. The immunochromatographic test strip according to claim 1, characterized in that: The sampling pad is filter paper.

6. A highly specific and highly sensitive immunochromatographic assay kit, characterized in that: The test strip includes any one of claims 1-5, and a cartridge, wherein the test strip is placed inside the cartridge, and the cartridge exposes a sample application well and a diluent addition well at a corresponding location on the conjugate pad of the test strip, and an observation window is exposed at a corresponding location on the nitrocellulose membrane-coated C-line and T-line of the test strip.

7. A method for preparing a highly specific and highly sensitive immunochromatographic test strip, characterized in that... Includes the following steps: Preparation of binding pad 1 1.1 Dextran oxidation: Dextran was oxidized with sodium periodate. After the reaction, excess ethylene glycol was added to terminate the oxidation reaction, and oxidized dextran was obtained by dialysis. 1.2 Oxidized dextran-conjugated antibody: Oxidized dextran was mixed with antibody A against the target antigen and reacted, then reduced to a binary conjugate with NaCNBH3, then aminated with ethylenediamine, then reduced with NaBH4, and finally dialyzed to remove ethylenediamine and NaBH4. 1.3 Preparation of Binding Pad 1: The oxidized dextran-conjugated antibody obtained in the previous step was immobilized onto a glass fiber pad to prepare Binding Pad 1; Preparation of binding pad II 2.1 ConA / anti-DNP antibody conjugated colloidal gold: K2CO3 was added to the colloidal gold solution, followed by ConA or anti-DNP antibody for conjugation. After blocking with blocking buffer, the colloidal gold particles were centrifuged and stored in a preservation solution for later use. 2.2 Preparation of Binding Pad II: ConA-conjugated colloidal gold and anti-DNP antibody-conjugated colloidal gold were immobilized onto a glass fiber pad to prepare Binding Pad II; Preparation of nitrocellulose membranes coated with C- and T-lines 3.1 Dilute antibody B against the target antigen with coating buffer to obtain T-line antibody; 3.2 Dilute DNP-BSA with coating buffer to obtain C-line antibody; 3.3 The C-line antibody and T-line antibody were coated onto a nitrocellulose membrane, respectively, and dried to obtain a nitrocellulose membrane coated with C-line and T-line. Assembly The conjugation pad 1, conjugation pad 2, nitrocellulose membrane coated with C and T lines, and sample pad are placed sequentially on the base plate. The head of conjugation pad 2 overlaps with the tail of conjugation pad 1, the head of nitrocellulose membrane overlaps with the tail of conjugation pad 2, and the head of sample pad overlaps with the tail of nitrocellulose membrane to obtain colloidal gold immunochromatographic test strip. Step 1.1 Dextran oxidation is as follows: Dissolve a certain amount of dextran in 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 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 excess ethylene glycol to terminate the oxidation reaction. Continue to react with stirring at 3°C ​​in the dark for 15 minutes, and then remove the solution and dialyze it with deionized water or 20 mmol / L phosphate buffer at pH 7.

0. Step 1.2 Oxidized dextran conjugated antibody: Oxidized dextran is mixed with antibody A against the target antigen and reacted for 2 hours. Then, it is reduced to a binary conjugate with NaCNBH3. Ethylenediamine is then added, and after amination for 2 hours, it is reduced with NaBH4 for 2 hours. Finally, dialysis is used to remove ethylenediamine and NaBH4. Step 2.1 ConA / anti-DNP antibody conjugated to colloidal gold is as follows: Take colloidal gold solution, add 0.1 mol / L K2CO3 to adjust to a suitable pH, add ConA / anti-DNP antibody, mix well, let stand to react, after the reaction is complete, slowly add blocking buffer, mix well, let stand to block, centrifuge to remove supernatant, add washing buffer to wash, centrifuge to remove supernatant, add storage solution and mix well for later use; In step 3.1, antibody B against the target antigen is diluted to 1.0–2.0 mg / mL using coating buffer; In step 3.2, DNP-BSA is diluted to 1.0–2.0 mg / mL with coating buffer.

8. A highly specific and sensitive immunochromatographic detection method for non-diagnostic purposes, based on the highly specific and sensitive immunochromatographic detection kit of claim 6, characterized in that... The steps include: (1) turning on the colloidal gold immunoassay analyzer; (2) taking 50 μL of the sample to be tested and adding it to the sample well of the immunochromatographic test strip, and after 5 to 10 minutes, taking 100 μL of diluent and adding it to the diluent well; (3) continuing to incubate for 10 to 15 minutes, and then placing the kit into the colloidal gold immunoassay analyzer to read the test results.

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