Microsphere coating method, immunochromatography detection kit, and preparation method and application of kit

Abstract

The invention discloses a microsphere coating method, an immunochromatography detection kit and a preparation method and application of the kit, and the microsphere coating method comprises the following steps: S1, preparing microspheres for marking antigens and antibodies to be detected and microspheres for marking quality control antibodies; and S2, coating the microspheres marked with the to-be-detected antigens and the to-be-detected antibodies and the microspheres marked with the quality control antibodies in the step S1 in a customized gun head or a reaction cup. Compared with the prior art, the antibody-labeled microsphere compound and the to-be-detected sample antigen are subjected to a more efficient reaction in advance, the problem of sufficiency of the reaction between the antibody-labeled microsphere and the to-be-detected antigen is thoroughly solved, the competition efficiency is improved, and the detection result is more accurate; moreover, the labeled antibody microspheres are solid reagents, can be directly used, and are high in sensitivity, high in accuracy, good in stability, wide in linear range and long in storage time, and the storage condition does not need refrigeration.

Description

Technical Field

[0001] The present invention belongs to the technical field of in vitro reagent instant detection, and in particular relates to a microsphere coating method, an immunochromatographic detection kit, a kit preparation method and an application. Background Art

[0002] Fluorescent immunochromatography is a detection and analysis method that combines immunoassay and chromatography. Fluorescent nanomaterials have great application value in labeling detection due to their unique structure and optical, electrical and magnetic properties. They can obtain the ability to analyze signals through markers. According to the markers, they can be divided into quantum dot chromatography, color fluorescent microsphere chromatography, upconversion luminescence chromatography and time-resolved immunochromatography. Fluorescent immunochromatography is widely used in the IVD industry, but it will have problems such as uneven antibody-gold label antibody binding and uneven layer development, and there is a lot of room for improvement.

[0003] At present, the components of the immunochromatographic reagent strip include a sample pad, a conjugate pad, a nitrocellulose membrane, and a water-absorbing pad, and the antibodies labeled with tracer particles are often coated on the conjugate pad. In Chinese patent document CN108181465A, a fluorescent chromatography kit for detecting 25-hydroxyvitamin D and a quantitative detection method thereof are disclosed, and the antibodies labeled with tracer particles in the reagent strip are still coated on the conjugate pad. In Chinese patent document CN202583195U, the detection reagent strip includes a water-absorbing pad, upper and lower sample pads, a conjugate pad, a nitrocellulose membrane, and the colloidal gold-labeled antibody is coated on the conjugate pad. Although this method innovatively uses upper and lower sample pads to adjust the position of the colloidal gold-labeled antibody, its position is still on the conjugate pad, and it cannot completely solve the problem of the sufficiency of the reaction between the colloidal gold-labeled antibody and the antigen. That is, the immunochromatographic reagent strips of the prior art have the problem of insufficient reaction between the labeled antibody microspheres and the antigen to be detected during detection. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a microsphere coating method that can enable the labeled antibody microspheres to fully react with the antigen to be detected.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is: a microsphere coating method, comprising the following steps:

[0006] Step S1: preparing microspheres labeled with the antigen and antibody to be tested and microspheres labeled with the quality control antibody;

[0007] Step S2: coating the microspheres labeled with the antigen and antibody to be tested and the microspheres labeled with the quality control antibody in step S1 in a customized gun tip or reaction cup.

[0008] The present invention pre-coats microspheres labeled with antigen antibodies to be tested and microspheres labeled with quality control antibodies in a gun tip or a reaction cup, so that the labeled antibody microsphere complex can react with the sample antigen to be tested more efficiently in advance, completely solving the problem of the sufficiency of the reaction between the labeled antibody microspheres and the antigen, improving the efficiency of competition, and making the detection results more accurate; moreover, the labeled antibody microspheres of the present invention are solid reagents, can be used directly, have high sensitivity, high accuracy, good stability, wide linear range, long storage time, and storage conditions do not require refrigeration.

[0009] Furthermore, in step S2, the microspheres labeled with the antigen and antibody to be tested and the microspheres labeled with the quality control antibody are coated in the customized gun tip or reaction cup with the spotting liquid as the coating liquid; the components of the spotting liquid include buffer, stabilizer, antifreeze, surfactant, protein stabilizer, macromolecular substance and preservative.

[0010] Further, the buffer is one of phosphate buffer, Tris buffer, HEPES buffer or any combination thereof, and the concentration is 10-100 mM;

[0011] The stabilizer is bovine serum albumin, and the content of the bovine serum albumin is 2-20 g / L;

[0012] The antifreeze agent is glycerol, and the content of the glycerol is 1-10mL / L;

[0013] The preservative is ProClin 300, and the content of ProClin 300 is 0.2-2 mL / L;

[0014] The surfactant is one of Tween, Triton, EMULGEN series surfactants or any combination thereof, and the content is 0.1-10 mL / L;

[0015] The protein stabilizer is one of sucrose, trehalose, glucose, and mannitol or any combination thereof, and the content is 50-500 g / L;

[0016] The macromolecular substance is one of PVP, PVA, PEG or any combination thereof, and the content is 0.2-2 g / L.

[0017] Furthermore, the total content of the microspheres labeled with the antigen and antibody to be tested and the microspheres labeled with the quality control antibody in the customized gun tip or reaction cup is 5-50 μg, and the spotting volume is 3-200 μL.

[0018] Furthermore, the customized gun tip is a 200 μL gun tip with a scale, and the spotting position of the microspheres labeled with the antigen and antibody to be tested and the microspheres labeled with the quality control antibody is the 50 μL scale line of the customized gun tip.

[0019] Furthermore, the microspheres are selected from the group consisting of colored microspheres, time-resolved fluorescent microspheres, quantum dot microspheres, and colloidal gold microspheres.

[0020] Furthermore, the step S1 includes the following steps:

[0021] Step S11: preparing microspheres labeled with the antigen and antibody to be tested: activating the microspheres with 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysulfosuccinimide, adding the antigen and antibody to be tested, wherein the mass ratio of the antigen and antibody to be tested to the microspheres is 1:2-1:20, reacting at 37° C. for 1-4 h, adding a blocking agent for blocking, centrifuging, and storing, thereby obtaining the microspheres labeled with the antigen and antibody to be tested, which are ready for use;

[0022] Step S12: preparing microspheres labeled with quality control antibodies: activating the microspheres with 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysulfosuccinimide, adding quality control antibodies, wherein the mass ratio of the quality control antibodies to the microspheres is 1:2-1:20, reacting at 37° C. for 1-4 h, adding a blocking agent for blocking, centrifuging, and storing to obtain the microspheres labeled with the quality control antibodies for later use;

[0023] The step S2 comprises the following steps:

[0024] Step S21: preparing a customized gun tip or reaction cup: preparing the microspheres labeled with the antigen and antibody to be tested prepared in step S11 and the microspheres labeled with the quality control antibody prepared in step S12 with a spotting solution to prepare a microsphere mixture, adding the mixture to the gun tip or reaction cup, and drying the gun tip or reaction cup with the added microsphere mixture to obtain the customized gun tip or reaction cup.

[0025] Furthermore, in step S21, in the microsphere mixture, the content of the microspheres labeled with the antigen and antibody to be tested is 2-30 μg, and the content of the microspheres labeled with the quality control antibody is 2-30 μg; when the microsphere mixture is added to the gun tip or the reaction cup, the microsphere mixture added to the gun tip is 3-8 μL, and the microsphere mixture added to the reaction cup is 50-200 μL; when the gun tip or the reaction cup to which the microsphere mixture is added is dried, the gun tip or the reaction cup is placed in a vacuum dryer at a pressure of 100 Pa and dried for 8-12 hours.

[0026] Another problem to be solved by the present invention is to provide a method for preparing an immunochromatographic detection kit.

[0027] In order to solve the above technical problems, the technical solution adopted is: step S1 is carried out by the aforementioned microsphere coating method; and further comprising the following steps:

[0028] Step S2: preparing a nitrocellulose membrane: diluting the analog antigen of the antigen to be tested at a concentration of 0.1-2 mg / mL and the quality control secondary antibody at a concentration of 0.1-2 mg / mL with a coating solution, and then streaking onto the nitrocellulose membrane using a streaking instrument, with a coating volume of 0.5-1 μL / cm;

[0029] Step S3: preparing a conjugate pad: mixing 5-50 mM Tris-HCl, 0.2-1 mg / mL trehalose, 0.5-2 mL / L Tween-20 and 0.2-2 mL / L ProClin300, adjusting the pH to 7.4±1, spraying the mixture onto a blank glass fiber, and drying the mixture for later use;

[0030] Step S4: preparing a sample pad: mixing 5-50 mM Tris-HCl, 0.2-1 mg / mL trehalose, 0.5-2 mL / L Tween-20, 0.1-0.8 g / L erythrocyte monoclonal antibody, 0.2-1 g / L blocking agent and 0.2-2 mL / L ProClin 300, adjusting the pH to 7.4±1, spraying on a blank glass fiber, and drying for later use;

[0031] Step S5: preparing a sample diluent: adding a buffer solution with a final concentration of 5-100 mM, a NaCl solution with a final concentration of 0.5-1 mg / mL, and a ProClin 300 solution with a volume content of 0.05-0.5% into a container, mixing them evenly, fixing the volume, and adjusting the pH to 6-8 after mixing to obtain a sample diluent; the buffer solution is one of a phosphate buffer solution, a Tris buffer solution, and a citrate buffer solution, or any combination thereof;

[0032] Step S6: preparing a reagent strip: pasting the nitrocellulose membrane prepared in step S2, the conjugate pad prepared in step S3, the sample pad and the absorbent pad prepared in step S4 to form a large plate, and cutting and assembling the large plate into a reagent strip;

[0033] Step S7: preparing a test kit: packaging the sample diluent prepared in step S5, the customized gun tip or reaction cup prepared in step S1, and the reagent strip prepared in step S6 to prepare a test kit for the antigen to be tested.

[0034] The present invention pre-coats microspheres labeled with antigen antibodies to be tested and microspheres labeled with quality control antibodies in a gun tip or a reaction cup, so that the labeled antibody microsphere complex can react with the sample antigen to be tested more efficiently in advance, completely solving the problem of the sufficiency of the reaction between the labeled antibody microspheres and the antigen, improving the efficiency of competition, and making the detection results more accurate; moreover, the labeled antibody microspheres of the present invention are solid reagents, can be used directly, have high sensitivity, high accuracy, good stability, wide linear range, long storage time, and storage conditions do not require refrigeration.

[0035] The third problem to be solved by the present invention is to provide an immunochromatographic detection kit prepared by the above-mentioned immunochromatographic detection kit preparation method.

[0036] In order to solve the above technical problems, the technical solution adopted is: the immunochromatographic detection kit includes a reagent strip and a sample diluent, the reagent strip includes a sample pad, a binding pad, a nitrocellulose membrane and a water absorbent pad stacked and connected in sequence from left to right, the kit also includes a customized gun tip or a reaction cup, and the customized gun tip or the reaction cup is coated with microspheres marked with antigens and antibodies to be tested and microspheres marked with quality control antibodies.

[0037] Compared with the prior art, the present invention pre-coats the microspheres labeled with the antigen and antibody to be tested and the microspheres labeled with the quality control antibody in the gun tip or reaction cup, so that the microsphere complex of the labeled antibody can react more efficiently with the antigen of the sample to be tested in advance, which completely solves the problem of the sufficiency of the reaction between the labeled antibody microsphere and the antigen, improves the efficiency of the competition, and makes the detection results more accurate; moreover, the labeled antibody microspheres of the present invention are solid reagents, which can be used directly, have high sensitivity, high accuracy, good stability, wide linear range, long storage time and storage conditions do not require refrigeration.

[0038] The fourth technical problem to be solved by the present invention is to provide the application of the aforementioned microsphere coating method in the detection of total triiodothyronine.

[0039] In order to solve the above technical problems, the technical scheme adopted is as follows: in the application of the microsphere coating method for total triiodothyronine detection, microspheres labeled with mouse T3 monoclonal antibody and microspheres labeled with chicken IgY antibody are coated in a customized gun tip or reaction cup; T3-BSA recombinant antigen and goat anti-chicken IgY antibody are coated on the nitrocellulose membrane, the concentration of the T3-BSA recombinant antigen is 1 mg / mL, the coating amount is 1 μL / cm, the concentration of the goat anti-chicken IgY antibody is 1 mg / mL, and the coating amount is 1 μL / cm.

[0040] Compared with the prior art, the present invention pre-coats microspheres labeled with mouse T3 monoclonal antibodies and microspheres labeled with chicken IgY antibodies in a gun tip or a reaction cup, that is, the microspheres labeled with the antigen and antibody to be tested are microspheres labeled with mouse T3 monoclonal antibodies, and the microspheres labeled with the quality control antibodies are microspheres labeled with chicken IgY antibodies; the microsphere complex of the labeled antibody is allowed to react more efficiently with the antigen of the sample to be tested in advance, which completely solves the problem of the sufficiency of the reaction between the labeled antibody microspheres and the antigen, improves the efficiency of the competition, and makes the detection results more accurate; and the labeled antibody microspheres of the present invention are solid reagents, can be used directly, have high sensitivity, high accuracy, good stability, wide linear range, long storage time, and storage conditions do not require refrigeration. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The following is further described in detail with reference to the accompanying drawings and embodiments of the present invention:

[0042] Figure 1 It is a schematic diagram of the structure of the reagent strip in the immunochromatographic detection kit of the present application;

[0043] In the figure: 1-sample pad; 2-binding pad; 3-nitrocellulose membrane; 4-absorbent pad; 31-test line; 32-quality control line. DETAILED DESCRIPTION

[0044] In order to deepen the understanding of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The embodiments are only used to explain the present invention and do not limit the protection scope of the present invention.

[0045] In this embodiment, a microsphere coating method comprises the following steps:

[0046] Step S1: preparing microspheres labeled with the antigen and antibody to be tested and microspheres labeled with the quality control antibody;

[0047] Step S2: coating the microspheres labeled with the antigen and antibody to be tested and the microspheres labeled with the quality control antibody in step S1 in a customized gun tip or reaction cup.

[0048] In step S2, the microspheres labeled with the antigen and antibody to be tested and the microspheres labeled with the quality control antibody are coated in the customized gun tip or reaction cup with the spotting liquid as the coating liquid; the components of the spotting liquid include buffer, stabilizer, antifreeze, surfactant, protein stabilizer, macromolecular substance and preservative.

[0049] The buffer is a Tris buffer, and the concentration of the Tris buffer is 50 mM;

[0050] The stabilizer is bovine serum albumin, and the content of the bovine serum albumin is 15g / L;

[0051] The antifreeze agent is glycerol, and the content of the glycerol is 8 mL / L;

[0052] The preservative is ProClin 300, and the content of ProClin 300 is 1 mL / L;

[0053] The surfactant is Tween20, and its content is 5mL / L;

[0054] The protein stabilizer is sucrose, and its content is 100g / L;

[0055] The macromolecular substance is PVP-10, and its content is 0.5 g / L.

[0056] In other embodiments, the buffer may also be a phosphate buffer or a HEPES buffer, or any combination of a phosphate buffer, a Tris buffer, and a HEPES buffer, and the concentration of the buffer may be any value between 10 and 100 mM; the components of the phosphate buffer include disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate dihydrate, the components of the Tris buffer include tris(hydroxymethyl)aminomethane hydrochloride, and the components of the HEPES buffer include 4-hydroxyethylpiperazineethanesulfonic acid;

[0057] The content of bovine serum albumin can be any value between 2-20 g / L;

[0058] The content of glycerol can be any value between 1-10mL / L;

[0059] The content of ProClin 300 can be any value between 0.2-2 mL / L;

[0060] The surfactant may also be a Triton or EMULGEN series surfactant, or any combination of Tween, Triton, and EMULGEN series surfactants, and its content may be any value in the range of 0.1-10 mL / L;

[0061] The protein stabilizer may also be trehalose, glucose or mannitol, or any combination of sucrose, trehalose, glucose and mannitol, and its content may be any value in the range of 50-500 g / L;

[0062] The macromolecular substance may also be PVA or PEG, or any combination of PVP, PVA, and PEG, and its content may be any value in the range of 0.2-2 g / L.

[0063] The total content of the microspheres labeled with the antigen and antibody to be tested and the microspheres labeled with the quality control antibody in the customized gun tip is 11 μg, and the spotting volume is 5 μL.

[0064] In other embodiments, the total content of the microspheres labeled with the antigen and antibody to be tested and the microspheres labeled with the quality control antibody in the customized gun tip or reaction cup can also be any value between 5 and 50 μg, and the spotting volume can be any value between 3 and 200 μL.

[0065] The custom-made gun tip is a 200 μL gun tip with a scale, and the spotting position of the microspheres labeled with the antigen and antibody to be tested and the microspheres labeled with the quality control antibody is the 50 μL scale line of the custom-made gun tip.

[0066] The microsphere is one of colored microspheres, time-resolved fluorescent microspheres, quantum dot microspheres or colloidal gold microspheres.

[0067] Furthermore, the step S1 includes the following steps:

[0068] Step S11: preparing microspheres labeled with the antigen and antibody to be tested: activating the microspheres with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysulfosuccinimide, adding the antigen and antibody to be tested, wherein the mass ratio of the antigen and antibody to be tested to the microspheres is 1:5, reacting at 37° C. for 3 h (or any time from 1 to 4 h), adding a blocking agent for blocking, centrifuging, and storing to obtain the microspheres labeled with the antigen and antibody to be tested, and storing for later use; in other embodiments, the mass ratio of the microspheres to the antigen and antibody to be tested can also be any value in the range of 1:2-1:20;

[0069] Step S12: preparing microspheres labeled with quality control antibodies: activating the microspheres with 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysulfosuccinimide, adding quality control antibodies, the mass ratio of the quality control antibodies to the microspheres is 1:5, reacting at 37° C. for 3 h (it can also be 1-4 h, any time can be selected), adding a blocking agent for blocking, centrifuging, and storing to obtain the microspheres labeled with the quality control antibodies for later use; in other embodiments, the mass ratio of the microspheres to the antigen-antibody to be tested can also be any value in the range of 1:2-1:20;

[0070] The step S2 comprises the following steps:

[0071] Step S21: preparing a customized gun tip or reaction cup: preparing the microspheres labeled with the antigen and antibody to be tested prepared in step S11 and the microspheres labeled with the quality control antibody prepared in step S12 with a spotting solution to prepare a microsphere mixture, adding the mixture to the gun tip or reaction cup, and drying the gun tip or reaction cup with the added microsphere mixture to obtain the customized gun tip or reaction cup.

[0072] Further, in step S21, in the microsphere mixture, the content of the microspheres labeled with the antigen and antibody to be tested is 8 μg, and the content of the microspheres labeled with the quality control antibody is 8 μg; when the microsphere mixture is added to the gun tip or the reaction cup, the microsphere mixture added to the gun tip is 5 μL (it can also be arbitrarily selected within the range of 3-8 μL), and the microsphere mixture added to the reaction cup is 100 μL (it can also be arbitrarily selected within the range of 50-200 μL); when the gun tip or the reaction cup to which the microsphere mixture is added is dried, the gun tip or the reaction cup is placed in a vacuum dryer with a pressure of 100 Pa and dried for 10 hours. In other embodiments, in the microsphere mixture, the content of the microspheres labeled with the antigen and antibody to be tested can also be any value between 2-30 μg, and the content of the microspheres labeled with the quality control antibody can be any value between 2-30 μg; the drying time can also be any value between 8-12 hours.

[0073] In this embodiment, the preparation method of the immunochromatographic detection kit comprises the following steps:

[0074] Step S1 is performed using the aforementioned microsphere coating method; and further comprises the following steps:

[0075] Step S2: preparing a nitrocellulose membrane: diluting the analog antigen of the antigen to be tested at a concentration of 1 mg / mL and the quality control secondary antibody at a concentration of 1 mg / mL with a coating solution, and then streaking onto the nitrocellulose membrane using a streaking instrument, with a coating amount of 1 μL / cm; in other embodiments, the concentration of the analog antigen of the antigen to be tested can also be any value in the range of 0.1-2 mg / mL, the concentration of the quality control secondary antibody can be any value in the range of 0.1-2 mg / mL, and the streaking coating amount can be any value in the range of 0.5-1 μL / cm;

[0076] Step S3: preparing a conjugate pad: mixing 30 mM Tris-HCl, 1 mg / mL trehalose, 0.5 mL / L Tween-20 and 1 mL / L ProClin 300, adjusting the pH to 7.4±1, spraying the mixture on a blank glass fiber, and drying the mixture for later use; in other embodiments, the concentration of Tris-HCl may be any value in the range of 5-50 mM, the concentration of trehalose may be any value in the range of 0.2-1 mg / mL, the volume content of Tween-20 may be any value in the range of 0.5-2 mL / L, and the volume content of ProClin 300 may be any value in the range of 0.2-2 mL / L;

[0077] Step S4: preparing a sample pad: after mixing Tris-HCl with a concentration of 20 mM, trehalose with a concentration of 0.5 mg / mL, Tween-20 with a volume content of 1 mL / L, erythrocyte monoclonal antibody with a concentration of 0.5 g / L, a blocking agent with a concentration of 0.5 g / L, and ProClin 300 with a volume content of 1 mL / L, the pH is adjusted to 7.4±1, and the mixture is sprayed on a blank glass fiber, dried, and set aside; in other embodiments, the concentration of Tris-HCl can also be any value in the range of 5-50 mM, the concentration of trehalose can be any value in the range of 0.2-1 mg / mL, the volume content of Tween-20 can be any value in the range of 0.5-2 mL / L, the concentration of erythrocyte monoclonal antibody can be any value in the range of 0.1-0.8 g / L, the concentration of the blocking agent can be any value in the range of 0.2-1 g / L, and the volume content of ProClin 300 can be any value in the range of 0.2-2 mL / L;

[0078] Step S5: preparing a sample diluent: adding a Tris buffer with a final concentration of 10 mM, a NaCl with a final concentration of 0.85 mg / mL, and a ProClin 300 with a volume content of 0.1% into a container, mixing evenly, fixing the volume, and adjusting the pH to 6-8 after mixing to obtain a sample diluent; in other embodiments, the buffer may also be a phosphate buffer or a citrate buffer, or any combination of a phosphate buffer, a citrate buffer, and a Tris buffer, and the final concentration may be any value in the range of 5-100 mM, the components of the phosphate buffer include disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate dihydrate, the components of the Tris buffer include tris(hydroxymethyl)aminomethane hydrochloride, and the components of the citrate buffer include citric acid and sodium citrate; the final concentration of NaCl may be any value in the range of 0.5-1 mg / mL; the volume content of ProClin 300 may be any value in the range of 0.05-0.5%; if it is a special formula, the sample diluent may also be purchased externally;

[0079] Step S6: preparing a reagent strip: pasting the nitrocellulose membrane prepared in step S2, the conjugate pad prepared in step S3, the sample pad and the absorbent pad prepared in step S4 to form a large plate, and cutting and assembling the large plate into a reagent strip;

[0080] Step S7: preparing a test kit: packaging the sample diluent prepared in step S5, the customized gun tip or reaction cup prepared in step S1, and the reagent strip prepared in step S6 to prepare a test kit for the antigen to be tested.

[0081] In this embodiment, an immunochromatographic detection kit prepared by the aforementioned immunochromatographic detection kit preparation method includes a reagent strip and a sample diluent, wherein the reagent strip includes a sample pad 1, a conjugate pad 2, a nitrocellulose membrane 3 and a water-absorbing pad 4 stacked and connected in sequence from left to right, and the kit also includes a customized gun tip or a reaction cup, wherein the customized gun tip or the reaction cup is coated with microspheres labeled with the antigen and antibody to be tested and microspheres labeled with the quality control antibody.

[0082] In this embodiment, the aforementioned microsphere coating method is applied to the detection of total triiodothyronine, wherein the customized gun tip or reaction cup is coated with microspheres labeled with mouse T3 monoclonal antibody and microspheres labeled with chicken IgY antibody; the nitrocellulose membrane is coated with T3-BSA recombinant antigen and goat anti-chicken IgY antibody, wherein the concentration of the T3-BSA recombinant antigen is 1 mg / mL and the coating amount is 1 μL / cm, and the concentration of the goat anti-chicken IgY antibody is 1 mg / mL and the coating amount is 1 μL / cm.

[0083] Specifically, the total triiodothyronine detection kit comprises a reagent strip, a sample diluent and a customized gun tip, wherein the reagent strip comprises a sample pad 1, a binding pad 2, a nitrocellulose membrane 3 and a water absorbent pad 4, and the nitrocellulose membrane 3 is coated with a detection line 31 and a quality control line 32.

[0084] The preparation method of the conjugate pad 2 is as follows: first, a conjugate pad treatment solution is prepared, 10 mM Tris-HCl, 0.5 mg / mL trehalose, 1 mL / L Tween-20 and 1 mL / L ProClin 300 are mixed, and the pH is adjusted to 7.4 to obtain the conjugate pad treatment solution, the conjugate pad treatment solution is treated on a blank glass fiber, and dried at 45° C. for 6-8 hours for standby use;

[0085] The preparation method of the sample pad 1 is as follows: first, a sample pad treatment solution is prepared, 10 mM Tris-HCl, 1 mg / mL trehalose, 1 mL / L Tween-20, 0.5 g / L erythrocyte monoclonal antibody, 0.5 g / L blocking agent and 1 mL / L ProClin 300 are mixed, and the pH is adjusted to 7.4 to obtain the sample pad treatment solution, the sample pad treatment solution is treated on a blank glass fiber, and dried at 45° C. for 6-8 hours for standby use;

[0086] The specific coating method of the nitrocellulose membrane 3 is as follows: 1 mg / mL of T3-BSA recombinant antigen and 1 mg / mL of goat anti-chicken IgY antibody are diluted with coating solution, and then respectively drawn on the nitrocellulose membrane as the detection line 31 and the quality control line 32 with a film drawing instrument, and the coating volume is 1 μL / cm, and then dried for later use;

[0087] The sample diluent is T3 / T4 dissociation agent (manufacturer: Jiangsu Huakong Biotechnology Co., Ltd.; item number: 5034H);

[0088] Preparation of fluorescent microspheres labeled with mouse T3 monoclonal antibody: After activating fluorescent microspheres (manufacturer: Bangs Laboratories) with 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (manufacturer: Sigma-Aldrich) and N-hydroxysulfosuccinimide (manufacturer: Sigma-Aldrich), mouse T3 monoclonal antibody (manufacturer: Jiangsu Huakong Biotechnology Co., Ltd.) was added, the mass ratio of mouse T3 monoclonal antibody to fluorescent microspheres was 1:5, and the reaction was carried out at 37° C. for 3 h, and then a blocking agent was added for blocking, centrifugation, and storage to obtain fluorescent microspheres labeled with mouse T3 monoclonal antibody for standby use;

[0089] Preparation of fluorescent microspheres labeled with chicken IgY antibody: After activating fluorescent microspheres (manufacturer: Bangs Laboratories) with 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (manufacturer: Sigma-Aldrich) and N-hydroxysulfosuccinimide (manufacturer: Sigma-Aldrich), chicken IgY antibody (manufacturer: Jiangsu Huakong Biotechnology Co., Ltd.) was added, the mass ratio of chicken IgY antibody to fluorescent microspheres was 1:5, and the reaction was carried out at 37° C. for 3 h, and then a blocking agent was added for blocking, centrifugation, and storage to obtain fluorescent microspheres labeled with chicken IgY antibody for later use;

[0090] Preparation of customized gun tips: The customized gun tips include spotting solution and fluorescent microspheres labeled with antibodies, wherein the spotting solution is a coating solution of the fluorescent microspheres labeled with antibodies; the customized gun tip is a gun tip with a scale, and the fluorescent microspheres labeled with antibodies are coated with the coating solution and fixed at a specific position of the gun tip to prevent clogging of the gun tip or inability to dissolve the coating during the test. The specific preparation method is as follows: 8 μg of fluorescent microspheres labeled with mouse T3 monoclonal antibody and 3 μg of fluorescent microspheres labeled with chicken IgY antibody are prepared into a fluorescent particle mixture with a spotting solution. If the fluorescent particle mixture is too little, the spotting solution is too little, and the spotting solution is prone to slipping. If the fluorescent particle mixture is too much, the fluorescent particle mixture is prone to form a closed loop and block the gun tip. Therefore, 5 μL of the fluorescent particle mixture is appropriate. Take 5 μL of the fluorescent particle mixture and spot it on the 50 μL scale line in the 200 μL gun tip with a scale. Put the spotted gun tip into a vacuum dryer, set the pressure to 100 Pa, and dry it for 10 hours to obtain the customized gun tip.

[0091] The specific formula of the above-mentioned spotting solution is: 50mM Tris buffer, 10g / L bovine serum albumin, 5mL / L propylene glycol, 1mL / L Tween20, 50g / L sucrose, 1g / L PVP-10 and 1mL / L preservative ProClin 300; in other embodiments, the content of the bovine serum albumin can also be any value between 2-20g / L.

[0092] Prepare the reagent strip: Place the nitrocellulose membrane 3, conjugate pad 2, sample pad 1 and absorbent pad 4 prepared above on a plate. Figure 1 As shown, the large plates are pasted and assembled, and then cut and assembled into reagent strips; wherein, the preparation method of the absorbent pad 4 is prior art and will not be described here.

[0093] Preparation of a kit: The sample diluent, customized pipette tip and reagent strip prepared above are packaged to prepare a total triiodothyronine detection kit.

[0094] After the above kit was prepared, the conventional gold-spraying kit and the process kit of the present invention were tested using the national standard for triiodothyronine immunoassay. The national standard is numbered 150550 and was dissolved in 10 mM phosphate buffer (pH 7.3±0.1) containing 0.8% bovine serum albumin and diluted to the following 6 concentration points. The test results are shown in Table 1 below.

[0095] Table 1 Comparison results of the conventional gold spraying kit and the process kit of the present invention in the test of national standard products

[0096]

[0097] As can be seen from Table 1, the test value of the process of the present invention is obviously better than that of the conventional process, and the accuracy is higher. The process adopted by the kit of the present invention combines the fluorescent microspheres labeled with the antibody with the antigen in advance, and the reaction is more sufficient, thus avoiding the problem of poor adequacy of gold spraying and re-dissolution in the conventional process.

[0098] The above-mentioned kit was placed at room temperature (10-30°C) for 0, 3, 6, 9, 12, 15, 18, 21, and 24 months, respectively. The total triiodothyronine detection kit was tested by diluting two levels with the national standard. The test results are shown in Table 2 below.

[0099] Table 2 Test results of two levels of dilution of national standard sample of the kit of the present invention

[0100]

[0101] As can be seen from Table 2, the test kit of the present invention is stable within 24 months when stored at room temperature (10-30° C.).

[0102] Compared with the prior art, the present invention pre-coats fluorescent microspheres labeling the antigen and antibody to be tested and fluorescent microspheres labeling the quality control antibody in the gun tip, so that the fluorescent microsphere complex of the labeled antibody and the antigen of the sample to be tested can produce a more efficient reaction in advance, which completely solves the problem of the sufficiency of the reaction between the labeled antibody fluorescent microsphere and the antigen, improves the efficiency of competition, and makes the detection result more accurate; moreover, the labeled antibody fluorescent microsphere of the present invention is a solid reagent, which can be used directly, has high sensitivity, high accuracy, good stability, wide linear range, long storage time and storage conditions do not require refrigeration.

[0103] It should be noted that the specific application of the above-mentioned microsphere coating method includes not only the application in the detection of total triiodothyronine, but also the application in the detection of other items.

[0104] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A microsphere coating method, characterized in that: The following steps are involved: Step S1: preparing microspheres labeled with the antigen and antibody to be tested and microspheres labeled with the quality control antibody; Step S2: coating the microspheres labeled with the antigen and antibody to be tested and the microspheres labeled with the quality control antibody in step S1 in a customized gun tip or reaction cup.

2. The microsphere coating method according to claim 1, characterized in that: In step S2, the microspheres labeled with the antigen and antibody to be tested and the microspheres labeled with the quality control antibody are coated in the customized gun tip or reaction cup with the spotting liquid as the coating liquid; the components of the spotting liquid include buffer, stabilizer, antifreeze, surfactant, protein stabilizer, macromolecular substance and preservative.

3. The microsphere coating method according to claim 2, characterized in that: The buffer is one of phosphate buffer, Tris buffer, HEPES buffer or any combination thereof, and the concentration is 10-100 mM; the stabilizer is bovine serum albumin, and the content of the bovine serum albumin is 2-20 g / L; The antifreeze agent is glycerol, and the content of the glycerol is 1-10mL / L; The preservative is ProClin 300, and the content of ProClin 300 is 0.2-2 mL / L; The surfactant is one of Tween, Triton, EMULGEN series surfactants or any combination thereof, and the content is 0.1-10 mL / L; The protein stabilizer is one of sucrose, trehalose, glucose, and mannitol or any combination thereof, and the content is 50-500 g / L; The macromolecular substance is one of PVP, PVA, PEG or any combination thereof, and the content is 0.2-2 g / L.

4. The microsphere coating method according to claim 1, characterized in that: The total content of the microspheres labeled with the antigen and antibody to be tested and the microspheres labeled with the quality control antibody in the customized gun tip or reaction cup is 5-50 μg, and the spotting volume is 3-200 μL.

5. The microsphere coating method according to claim 4, characterized in that: The custom-made gun tip is a 200 μL gun tip with a scale, and the spotting position of the microspheres labeled with the antigen and antibody to be tested and the microspheres labeled with the quality control antibody is the 50 μL scale line of the custom-made gun tip.

6. The microsphere coating method according to claim 1, characterized in that: The microsphere is one of colored microspheres, time-resolved fluorescent microspheres, quantum dot microspheres or colloidal gold microspheres.

7. The microsphere coating method according to claim 1, characterized in that: The step S1 comprises the following steps: Step S11: preparing microspheres labeled with the antigen and antibody to be tested: activating the microspheres with 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysulfosuccinimide, adding the antigen and antibody to be tested, wherein the mass ratio of the antigen and antibody to be tested to the microspheres is 1:2-1:20, reacting at 37° C. for 1-4 h, adding a blocking agent for blocking, centrifuging, and storing, thereby obtaining the microspheres labeled with the antigen and antibody to be tested, which are ready for use; Step S12: preparing microspheres labeled with quality control antibodies: activating the microspheres with 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysulfosuccinimide, adding quality control antibodies, wherein the mass ratio of the quality control antibodies to the microspheres is 1:2-1:20, reacting at 37° C. for 1-4 h, adding a blocking agent for blocking, centrifuging, and storing to obtain the microspheres labeled with the quality control antibodies for later use; The step S2 comprises the following steps: Step S21: preparing a customized gun tip or reaction cup: preparing the microspheres labeled with the antigen and antibody to be tested prepared in step S11 and the microspheres labeled with the quality control antibody prepared in step S12 with a spotting solution to prepare a microsphere mixture, adding the mixture to the gun tip or reaction cup, and drying the gun tip or reaction cup with the added microsphere mixture to obtain the customized gun tip or reaction cup.

8. The microsphere coating method according to claim 7, characterized in that: In the step S21, in the microsphere mixture, the content of the microspheres labeled with the antigen and antibody to be tested is 2-30 μg, and the content of the microspheres labeled with the quality control antibody is 2-30 μg; when the microsphere mixture is added to the gun tip or the reaction cup, the microsphere mixture added to the gun tip is 3-8 μL, and the microsphere mixture added to the reaction cup is 50-200 μL; when the gun tip or the reaction cup to which the microsphere mixture is added is dried, the gun tip or the reaction cup is placed in a vacuum dryer at a pressure of 100 Pa and dried for 8-12 hours.

9. A method for preparing an immunochromatographic detection kit, characterized in that: Step S1 is performed using the microsphere coating method according to any one of claims 1 to 8; and further comprises the following steps: Step S2: preparing a nitrocellulose membrane: diluting the analog antigen of the antigen to be tested at a concentration of 0.1-2 mg / mL and the quality control secondary antibody at a concentration of 0.1-2 mg / mL with a coating solution, and then streaking onto the nitrocellulose membrane using a streaking instrument, with a coating volume of 0.5-1 μL / cm; Step S3: preparing a conjugate pad: mixing 5-50 mM Tris-HCl, 0.2-1 mg / mL trehalose, 0.5-2 mL / L Tween-20 and 0.2-2 mL / L ProClin300, adjusting the pH to 7.4±1, spraying the mixture onto a blank glass fiber, and drying the mixture for later use; Step S4: preparing a sample pad: mixing 5-50 mM Tris-HCl, 0.2-1 mg / mL trehalose, 0.5-2 mL / L Tween-20, 0.1-0.8 g / L erythrocyte monoclonal antibody, 0.2-1 g / L blocking agent and 0.2-2 mL / L ProClin 300, adjusting the pH to 7.4±1, spraying on a blank glass fiber, and drying for later use; Step S5: preparing a sample diluent: adding a buffer solution with a final concentration of 5-100 mM, a NaCl solution with a final concentration of 0.5-1 mg / mL, and a ProClin 300 solution with a volume content of 0.05-0.5% into a container, mixing them evenly, fixing the volume, and adjusting the pH to 6-8 after mixing to obtain a sample diluent; the buffer solution is one of a phosphate buffer solution, a Tris buffer solution, and a citrate buffer solution, or any combination thereof; Step S6: preparing a reagent strip: pasting the nitrocellulose membrane prepared in step S2, the conjugate pad prepared in step S3, the sample pad and the absorbent pad prepared in step S4 to form a large plate, and cutting and assembling the large plate into a reagent strip; Step S7: preparing a test kit: packaging the sample diluent prepared in step S5, the customized gun tip or reaction cup prepared in step S1, and the reagent strip prepared in step S6 to prepare a test kit for the antigen to be tested.

10. An immunochromatographic detection kit obtained by the preparation method of the immunochromatographic detection kit according to claim 9, comprising a reagent strip and a sample diluent, wherein the reagent strip comprises a sample pad, a conjugate pad, a nitrocellulose membrane and a water-absorbing pad stacked and connected in sequence from left to right, characterized in that: The kit also includes a customized gun tip or a reaction cup, wherein the customized gun tip or the reaction cup is coated with microspheres labeled with the antigen antibody to be tested and microspheres labeled with the quality control antibody.

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