A specific latex microsphere, kit, preparation method and application thereof

Abstract

The present invention discloses a specific latex microsphere, a kit, and a preparation method and application thereof. The specific latex microsphere is a colored latex microsphere conjugated with a specific antigen or antibody; the colored latex microsphere has a particle size of 8 - 18 μm; the conjugation method includes EDC-NHS conjugation. The kit provided by the present invention uses the specific latex microsphere as a marker, combines the streptavidin-biotin amplification system and the microcolumn gel immunoassay technology, and can be used to detect pathogenic microorganism antigens or antibodies; the detection method is simple, rapid, and highly sensitive.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunoassay, and particularly relates to a specific latex microsphere, a kit, and a preparation method and application thereof. Background Art

[0002] At present, the detection methods for pathogenic microorganism antigens and antibodies mainly include immunochromatography, enzyme-linked immunosorbent assay (ELISA), agglutination assay, chemiluminescence assay, and microcolumn gel assay based on specific red blood cell indicators. Immunochromatography reagents mainly rely on colloidal gold immunochromatography. This method is simple to operate and the results are easy to interpret, but its sensitivity is relatively lower compared to other methods. ELISA and agglutination assay are cumbersome in the operation process and require high professional requirements for operators. Chemiluminescence assay requires supporting automated equipment and has a high detection cost. The microcolumn gel assay based on specific red blood cell indicators is limited by the stability of red blood cells. The shelf life of such reagents is relatively short, usually 6 - 8 months, and its applicable range is also relatively limited and cannot function when detecting some non-human globulin substances.

[0003] The microcolumn gel assay based on red blood cell indicators also has certain limitations in terms of sensitivity. This method may not be able to effectively detect some low-concentration pathogenic microorganism antigens because its detection limit is relatively higher compared to other methods. When the antigen concentration is below a certain level, the aggregates formed by the antigen-antibody reaction may be less or not obvious enough, resulting in false negative results that are difficult to observe with the naked eye. Therefore, there is still a need to further improve and optimize the microcolumn gel assay. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. For this purpose, the present invention provides a specific latex microsphere, which can be used as a marker for detecting pathogenic microorganism antigens or antibodies, and the detection method is simple, rapid, and highly sensitive.

[0005] The present invention also provides a kit.

[0006] The present invention also provides a preparation method for the above-mentioned kit.

[0007] The present invention also provides a detection method for pathogenic microorganism antigens or antibodies.

[0008] The present invention also provides an application of the above-mentioned kit or the above-mentioned detection method.

[0009] According to a first aspect of the present invention, there is provided a specific latex microsphere, wherein the specific latex microsphere is a colored latex microsphere conjugated with a specific antigen or antibody;

[0010] The colored latex microsphere has a particle size of 8 - 18 μm.

[0011] In some embodiments of the present invention, a blocking agent is further connected to the surface of the specific latex microspheres.

[0012] In some embodiments of the present invention, the blocking agent includes a protein-based blocking agent and / or a non-protein-based blocking agent.

[0013] In some embodiments of the present invention, the protein-based blocking agent includes at least one of bovine serum albumin, casein, and skim milk.

[0014] In some embodiments of the present invention, the non-protein-based blocking agent includes at least one of polyethylene glycol, Tween-20, and ethanolamine.

[0015] In some embodiments of the present invention, the coupling method includes EDC-NHS coupling.

[0016] In some embodiments of the present invention, the particle size of the colored latex microspheres is 9 - 15 μm.

[0017] In some embodiments of the present invention, the colored latex microspheres include any one of red latex microspheres, blue latex microspheres, green latex microspheres, yellow latex microspheres, and purple latex microspheres.

[0018] According to the second aspect of the present invention, a kit is provided, which includes: the specific latex microspheres and the specific microcolumn gel card described in the first aspect of the present invention; the specific latex microspheres are used to be mixed with a test sample and added into the specific microcolumn gel card for detection;

[0019] The specific microcolumn gel card includes a fixing plate and a gel tube, and the gel tube contains a specific gel;

[0020] The specific gel includes (a) - (d):

[0021] (a) Biotinylated specific antigen or antibody;

[0022] (b) Streptavidin;

[0023] (c) Gel particles;

[0024] (d) Gel medium.

[0025] In some embodiments of the present invention, the volume of the specific gel accounts for .

[0026] In some embodiments of the present invention, the volume of the specific gel in each gel tube is 30 - 40 μL.

[0027] In some embodiments of the present invention, the specific microcolumn gel card further includes a sealing film.

[0028] In some embodiments of the present invention, the gel particles are selected from any one of dextran gel particles, glass microspheres, and polyacrylamide gel particles.

[0029] In some embodiments of the present invention, the dextran gel particles are selected from any one of Sephadex-G25, Sephadex-G50, and Sephadex-G100.

[0030] In some embodiments of the present invention, the gel medium is a phosphate buffer containing sodium chloride, bovine serum albumin, surfactant S9, disodium EDTA, PEG20000, sucrose, and sodium azide.

[0031] In some embodiments of the present invention, the gel medium is a 0.02 mol / L phosphate buffer containing the following final concentration components: 7 - 10 g / L sodium chloride, 1 - 3 g / L disodium EDTA, 8 - 12 g / L bovine serum albumin, 0.5 - 2 g / L S9, 0.5 - 2 g / L PEG20000, 40 - 60 g / L sucrose, and 0.1 - 0.3 g / L sodium azide, and the pH value of the phosphate buffer is 7 - 8.

[0032] In some embodiments of the present invention, the mass - volume ratio of the gel particles to the gel medium is 1:(12 - 16) g / mL.

[0033] In some embodiments of the present invention, the final concentration of streptavidin in the specific gel is 0.2 - 0.8 μg / mL.

[0034] In some embodiments of the present invention, the final concentration of streptavidin in the specific gel is 0.4 - 0.6 μg / mL.

[0035] In some embodiments of the present invention, when the target detected by the detection kit is antigen A, the colored latex microspheres are conjugated with a specific antibody A' against antigen A, and the specific gel contains biotinylated specific antibody A' or other specific antibodies against antigen A.

[0036] In some embodiments of the present invention, when the target detected by the detection kit is antibody B, the colored latex microspheres are conjugated with a specific antigen B' against antibody B, and the specific gel contains biotinylated antigen B' or a heterologous antibody C against antibody B; for example, if antibody B is human - derived IgG, the heterologous antibody C can be an antibody against human IgG from mice.

[0037] According to the third aspect of the present invention, there is provided a method for preparing the kit described in the second aspect of the present invention, and the preparation method includes the following steps:

[0038] S1. Prepare specific latex microspheres: Couple a specific antigen or antibody with a colored latex microsphere and perform a blocking treatment to obtain specific latex microspheres;

[0039] S2. Prepare a biotinylated specific antigen or antibody: Couple a specific antigen or antibody with NHS-biotin to obtain a biotinylated specific antigen or antibody;

[0040] S3. Prepare a specific gel: Mix gel particles, streptavidin, a gel medium, and the biotinylated specific antigen or antibody obtained in step S2 to obtain a specific gel;

[0041] S4. Fill the specific gel obtained in step S3 into a gel tube to obtain a specific microcolumn gel card, and the specific latex microspheres obtained in step S1 and the specific microcolumn gel card obtained in step S4 form a kit.

[0042] In some embodiments of the present invention, the coupling method in step S1 includes NHS-EDC covalent coupling.

[0043] In some embodiments of the present invention, the mass ratio of the colored latex microsphere to the specific antigen or antibody in step S1 is 1:(0.02 - 0.04).

[0044] In some embodiments of the present invention, the particle size of the colored latex microsphere is 8 - 18 μm.

[0045] In some embodiments of the present invention, the particle size of the colored latex microsphere is 9 - 15 μm.

[0046] In some embodiments of the present invention, the colored latex microsphere includes any one of red latex microspheres, blue latex microspheres, green latex microspheres, yellow latex microspheres, and purple latex microspheres.

[0047] In some embodiments of the present invention, the blocking solution used in the blocking treatment in step S1 is a borate buffer solution containing boric acid with a final concentration of 3 - 7 mmol / L, sodium tetraborate decahydrate with a concentration of 10 - 13 mmol / L, Tween-20 with a concentration of 0.03% - 0.07%, BSA with a concentration of 0.5% - 2%, and ethanolamine with a concentration of 0.2% - 0.3%.

[0048] In some embodiments of the present invention, the blocking time of the blocking treatment in step S1 is 1 - 2 h.

[0049] In some embodiments of the present invention, the gel medium in step S3 is a 0.02 mol / L phosphate buffer containing the following components at final concentrations: 7 - 10 g / L sodium chloride, 1 - 3 g / L disodium EDTA, 8 - 12 g / L bovine serum albumin, 0.5 - 2 g / L S9, 0.5 - 2 g / L PEG20000, 40 - 60 g / L sucrose, and 0.1 - 0.3 g / L sodium azide, and the pH value of the phosphate buffer is 7 - 8.

[0050] In some embodiments of the present invention, the mass - to - volume ratio of the gel particles to the gel medium in step S3 is 1:(12 - 16) g / mL.

[0051] In some embodiments of the present invention, the titer of the biotinylated specific antigen or antibody in the mixed system in step S3 is 1:(500 - 1000).

[0052] In some embodiments of the present invention, the final concentration of streptavidin in the specific gel in step S3 is 0.2 - 0.8 μg / mL.

[0053] In some embodiments of the present invention, the final concentration of streptavidin in the specific gel in step S3 is 0.4 - 0.6 μg / mL.

[0054] In some embodiments of the present invention, the gel particles in step S3 are selected from any one of dextran gel particles, glass microspheres, and polyacrylamide gel particles.

[0055] In some embodiments of the present invention, the dextran gel particles are selected from any one of Sephadex - G25, Sephadex - G50, and Sephadex - G100.

[0056] In some embodiments of the present invention, the volume of the specific gel in step S4 accounts for .

[0057] In some embodiments of the present invention, the volume of the specific gel in each gel tube in step S4 is 30 - 40 μL.

[0058] According to the fourth aspect of the present invention, a detection method for a pathogenic microorganism antigen or antibody is provided, and the detection method uses the kit described in the second aspect of the present invention for detection.

[0059] In some embodiments of the present invention, the antibody of the microbial pathogen includes human anti - Mycoplasma pneumoniae IgM antibody.

[0060] In some embodiments of the present invention, the antibody against the microbial pathogen includes human anti-Mycoplasma pneumoniae IgG antibody.

[0061] In some embodiments of the present invention, the detection method includes the following steps:

[0062] Mix the specific latex microspheres with the sample to be tested in equal volume and add them to a gel tube for reaction to obtain a specific latex microsphere complex, then centrifuge the reaction system, and finally interpret the results.

[0063] In some embodiments of the present invention, the temperature of the reaction is 35-40 °C.

[0064] In some embodiments of the present invention, the reaction time is 8-20 min.

[0065] In some embodiments of the present invention, the centrifugation sequentially includes primary centrifugation and secondary centrifugation.

[0066] In some embodiments of the present invention, the conditions for the primary centrifugation are 800-1000 rpm, 1-3 min.

[0067] In some embodiments of the present invention, the conditions for the secondary centrifugation are 1400-1600 rpm, 2-5 min.

[0068] In some embodiments of the present invention, the solid content of the specific latex microspheres is 0.5%-1%.

[0069] In some embodiments of the present invention, the solid content of the specific latex microspheres is 0.7%-0.9%.

[0070] In some embodiments of the present invention, the criteria for result interpretation include:

[0071] (1) The specific latex microsphere complex is located on the surface of the specific gel, which is a 4+ strong positive reaction;

[0072] (2) The specific latex microsphere complex is suspended in the specific gel, which is a 1+ to 3+ positive reaction. The closer the specific latex microsphere complex is to the bottom of the gel, the lower the titer;

[0073] (3) The specific latex microsphere complex is all deposited at the bottom of the specific gel, which is a negative result.

[0074] In some embodiments of the present invention, the samples to be tested applicable to the detection method include but are not limited to blood samples, respiratory swab samples, and fecal samples.

[0075] The principle of the detection method includes: The antigen / antibody in the sample to be tested binds to the antibody / antigen linked by streptavidin-biotin in the microcolumn gel medium to form an antigen-antibody immune complex, and then binds to the specific antibody / antigen conjugated on the colored latex microspheres to form a polymerized "latex microsphere conjugated antigen / antibody - antibody / antigen in the sample - antigen / antibody in the gel medium" immune complex. Under a certain centrifugal force, the immune complex of the colored latex microspheres is excluded in the gel surface layer with a three-dimensional network structure or in the gaps between gel particles, which is judged as a positive result; if there is no corresponding antigen-antibody binding, a colored latex microsphere immune complex cannot be formed, and under a certain centrifugal force, the dispersed colored latex microspheres all deposit at the bottom of the gel cavity, which is judged as a negative result.

[0076] According to the fifth aspect of the present invention, there is provided an application of the kit described in the second aspect of the present invention or the detection method described in the fourth aspect of the present invention in the following (I) to (III):

[0077] (I) Preparing a product for blood group identification;

[0078] (II) Preparing a product for detecting autoantibodies in autoimmune diseases;

[0079] (III) Preparing a product for detecting antigens or antibodies of microbial pathogens.

[0080] In some embodiments of the present invention, the antibody of the microbial pathogen includes human anti-Mycoplasma pneumoniae IgM antibody.

[0081] In some embodiments of the present invention, the antibody of the microbial pathogen includes human anti-Mycoplasma pneumoniae IgG antibody.

[0082] The present invention has at least the following beneficial effects:

[0083] The present invention provides a kit based on specific latex microspheres and microcolumn gel. The kit uses specific latex microspheres as markers, combines the streptavidin / biotin amplification system and microcolumn gel immunoassay technology, and can be widely applied to the detection of various antigens or antibodies.

[0084] The present invention provides a detection method for pathogenic microorganism antigens or antibodies based on the above-mentioned kit. This detection method is simple to operate, has higher sensitivity than colloidal gold immunochromatography and existing microcolumn gels based on specific red blood cell indicators, can provide test results within 20 - 30 minutes, has better reagent stability than the microcolumn gel method based on specific red blood cell indicators, and has a shelf life of up to 12 months. It can be operated manually in a simple manner, suitable for primary medical units and on-site rapid detection. It can also be detected using automated equipment, applicable to large institutions such as tertiary hospitals, reducing the risk of iatrogenic infection and improving detection efficiency. The test results can usually be directly judged by visually observing the color development with the naked eye, reducing the dependence on professional analysis equipment, without the need for complex equipment to interpret the results or professional medical staff to explain the results. The test results can be completely independently detected and judged by the tested person by visually observing the agglutination effect in the test area. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] The following further describes the present invention in conjunction with the drawings and embodiments, where:

[0086] Figure 1 It is a structural diagram of a specific microcolumn gel card prepared in Example 1 of the present invention. The upper part is a schematic diagram of the structure, and the lower part is a physical diagram;

[0087] Figure 2 It is a schematic diagram of different result determinations for the detection method of human anti - Mycoplasma pneumoniae IgM antibody in Example 3 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0088] The following will clearly and completely describe the concept of the present invention and the technical effects produced in conjunction with the embodiments to fully understand the purpose, features, and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0089] Materials and Definitions

[0090] In the following embodiments and comparative examples, the purchase information of the materials used is shown in Table 1.

[0091] Table 1 Purchase Information of Materials Used

[0092]

[0093] Example 1

[0094] This example provides a kit based on specific latex microspheres and microcolumn gels. The kit includes specific latex microspheres and a specific microcolumn gel card, and its preparation method specifically includes the following steps:

[0095] 1. Preparation of specific latex microspheres:

[0096] 1) Cleaning of latex microspheres:

[0097] Dilute a 2.5 wt% red latex microsphere suspension (particle size 10 μm) with a coupling buffer (50 mM MES buffer, pH 6.0 ± 0.1) to a 1 wt% latex microsphere suspension, mix well by ultrasound and centrifuge at a rotational speed of 18,000 rpm (any value between 15,000 rpm and 20,000 rpm is acceptable) for 20 min (any value between 15 and 30 min is acceptable); after centrifugation, discard the supernatant, and ultrasonically resuspend the latex microspheres with the coupling buffer to 1 wt% to obtain a washed latex microsphere suspension.

[0098] 2) Coupling of latex microspheres with antigen:

[0099] Add freshly prepared Sulfo-NHS solution (stock concentration 50 mg / mL) and EDC solution (stock concentration 50 mg / mL) to the washed latex microsphere suspension obtained in step 1) until the final concentrations of Sulfo-NHS and EDC are both 5 mg / mL; after mixing, place the mixed system on a mixer and rotate and incubate at room temperature for 30 min, then add Mycoplasma pneumoniae antigen until the final concentration is 0.3 mg / mL (any value between 0.2 and 0.4 mg / mL is acceptable), mix well and place on a mixer and rotate and incubate at room temperature for 150 min to obtain a coupling product.

[0100] 3) Blocking of unbound surface groups on latex microspheres:

[0101] Centrifuge the coupling product obtained in step 2) (the centrifugation conditions are the same as those used in step 1) and remove the supernatant, then add a blocking solution (containing boric acid with a final concentration of 5 mmol / L, sodium tetraborate decahydrate with a final concentration of 11.2 mmol / L, 0.05 v / v% Tween-20, 1 w / v% BSA and 0.24 w / v% borate buffer, pH 9.0 ± 0.1) to the centrifuge tube for blocking, and incubate at room temperature for 1 hour to obtain blocked latex microspheres.

[0102] 4) Cleaning and storage of specific latex microsphere markers after coupling:

[0103] The enclosed latex microspheres obtained in step 3) were resuspended in a washing solution (50 mmol / L Tris, 0.5% BSA, 0.05% Tween-20, pH 8.0 ± 0.1) to a concentration of 1% for washing, and the washing was repeated twice. Then the microspheres were resuspended in a preservation solution (a solution containing 25 mmol / L Tris, 15 mmol / L NaCl, 0.05% Tween-20, 1% BSA, 5% trehalose and 0.1% Proclin-300, pH 7.2 ± 0.1) to make the solid content of the latex microspheres in the solution 0.8%, obtaining specific latex microspheres, which were stored at 2 - 8°C.

[0104] 2. Preparation of specific microcolumn gel cards:

[0105] 1) Biotinylation of antibodies:

[0106] The mouse anti-human IgM antibody was reacted with Sulfo-NHS-LC-biotin according to the operation provided in the supplier's instructions to couple the mouse anti-human IgM antibody with biotin, obtaining biotinylated antibodies.

[0107] 2) Preparation of gel medium:

[0108] The following components at the final concentrations were added to 0.02 mol / L phosphate buffer (pH 7.4) and mixed evenly to obtain the gel medium: 8.5 g / L NaCl, 1.5 g / L disodium EDTA, 10 g / L BSA, 1.0 g / L surfactant S9 (Tetronic 1307), 1.0 g / L PEG20000, 50 g / L sucrose and 0.2 g / L sodium azide.

[0109] 3) Preparation of specific gel:

[0110] Sephadex-G50 (dextran gel particles) was selected and mixed with the gel medium prepared in step 2) at a mass-to-volume ratio of 1:14 g / mL (any ratio between 1:12 and 1:16 is acceptable) to prepare a gel; the biotinylated mouse anti-human IgM antibody obtained in step 1) was added to the above gel at a titer of 1:1000 and mixed evenly, and then streptavidin was added at a final concentration of 0.5 μg / mL and mixed evenly to obtain the specific gel.

[0111] 4) Filling and sealing:

[0112] The specific gel obtained in step 3) was added to the gel tubes of the microcolumn gel cards at a filling volume of 34 μL / tube, and heat-press sealing was used for sealing. The sealing film material was a composite aluminum coil, obtaining the specific microcolumn gel cards, the structure of which is as Figure 1 shown.

[0113] Example 2

[0114] This embodiment provides a kit, which includes specific latex microspheres and specific microcolumn gel cards. The difference between this detection kit and Example 1 is that the mouse anti-human IgM antibody in the preparation process of the specific microcolumn gel card is replaced with mouse anti-human IgG antibody, and the titer in the specific gel is 1:500. The other steps are the same.

[0115] Example 3

[0116] This embodiment provides a method for detecting human anti-Mycoplasma pneumoniae IgM antibodies, which uses the kit prepared in Example 1 and specifically comprises the following steps: 1) oscillating and mixing the specific latex microspheres in the kit prepared in Example 1, taking 50 μL of the specific latex microspheres and 50 μL of the plasma sample to be tested into a microcolumn tube, and reacting at 37° C. for 12 min (10 to 15 min is acceptable);

[0117] 2) After the reaction, centrifuge at 900 rpm for 2 min, and then at 1500 rpm for 3 min;

[0118] 3) After centrifugation, the results can be interpreted;

[0119] The results are judged as follows (e.g. Figure 2 shown):

[0120] ①4+ is the latex microsphere-antigen-antibody complex (agglutination) located on the gel surface;

[0121] ②3+: Most of the latex microsphere-antigen-antibody complex (agglutination) is located on the surface of the gel, and a small part is located in the middle of the gel;

[0122] ③2+ means most of the latex microsphere-antigen-antibody complex (agglutination) is located in the middle of the gel, and a small part is located in the upper middle of the gel;

[0123] ④1+ is the latex microsphere-antigen-antibody complex (agglutination) located in the lower middle part of the gel;

[0124] ⑤ Questionable result (±): Most of the latex microspheres are deposited at the bottom of the tube tip, and very few are located near the bottom of the gel;

[0125] ⑥Negative result (-): The latex microspheres are deposited at the bottom of the microcolumn gel tube, and there is no agglutination in the gel.

[0126] Example 4

[0127] This embodiment provides a method for detecting human anti-Mycoplasma pneumoniae IgG antibodies. The detection method uses the detection kit prepared in Example 2, and the specific steps are consistent with those in Example 3.

[0128] Comparative Example 1

[0129] This comparative example provides a detection kit based on indicator red blood cells and microcolumn gel. The detection kit includes specific indicator red blood cells and a specific microcolumn gel card. The preparation method of the above specific microcolumn gel card is the same as that in Example 1; the preparation method of the above specific indicator red blood cells refers to the method described in "Preparation of Indicator Red Blood Cells" in Patent CN104965079A, except that the anti-HIV antibody solution used in Step 2 is replaced with a Mycoplasma pneumoniae antigen solution, and the antigen dosage is the same as that of the specific latex microspheres used in Example 1. Finally, the prepared specific indicator red blood cells are diluted to 0.8 wt% for storage.

[0130] Comparative Example 2

[0131] This comparative example provides a kit, which includes specific latex microspheres and a specific microcolumn gel card. The preparation method of the above specific latex microspheres is the same as that in Example 1; compared with Example 1, the preparation method of the above specific microcolumn gel card has the following differences: the biotinylation treatment in Step 1) is omitted, and the streptavidin in Step 3) is replaced with an equal amount of anti-human globulin antibody, and the remaining steps are the same as those in Example 1.

[0132] Comparative Example 3

[0133] This comparative example provides a kit, which includes specific latex microspheres and a specific microcolumn gel card. The preparation method of the above specific microcolumn gel card is the same as that in Example 1; compared with Example 1, the preparation method of the above specific latex microspheres has the following differences: the particle size of the red latex microspheres is adjusted from 10 μm to 5 μm, and the remaining steps are the same as those in Example 1.

[0134] Comparative Example 4

[0135] This comparative example provides a kit, which includes specific latex microspheres and a specific microcolumn gel card. The preparation method of the above specific microcolumn gel card is the same as that in Example 1; compared with Example 1, the preparation method of the above specific latex microspheres has the following differences: the particle size of the red latex microspheres is adjusted from 10 μm to 20 μm, and the remaining steps are the same as those in Example 1.

[0136] Comparative Example 5

[0137] This comparative example provides a kit, which includes specific latex microspheres and a specific microcolumn gel card. The preparation method of the above-mentioned specific latex microspheres is the same as that in Example 1; compared with Example 1, the preparation method of the above-mentioned specific microcolumn gel card is only different in that: PEG20000 used for preparing the gel medium in step 2) is replaced with an equal amount of S9, that is, the final concentration of S9 in the gel medium is 2.0 g / L, and the remaining steps are the same as those in Example 1.

[0138] Comparative Example 6

[0139] This comparative example provides a kit, which includes specific latex microspheres and a specific microcolumn gel card. The preparation method of the above-mentioned specific latex microspheres is the same as that in Example 1; compared with Example 1, the preparation method of the above-mentioned specific microcolumn gel card is only different in that: S9 used for preparing the gel medium in step 2) is replaced with an equal amount of PEG20000, that is, the final concentration of PEG20000 in the gel medium is 2.0 g / L, and the remaining steps are the same as those in Example 1.

[0140] Comparative Example 7

[0141] This comparative example provides a kit, which includes specific latex microspheres and a specific microcolumn gel card. The preparation method of the above-mentioned specific latex microspheres is the same as that in Example 1; compared with Example 1, the preparation method of the above-mentioned specific microcolumn gel card is only different in that: S9 and PEG20000 used for preparing the gel medium in step 2) are omitted, and the remaining steps are the same as those in Example 1.

[0142] Comparative Example 8

[0143] This comparative example provides a detection kit based on indicator red blood cells and microcolumn gel. The detection kit includes specific indicator red blood cells and a specific microcolumn gel card. The preparation method of the above-mentioned specific microcolumn gel card is the same as that in Example 2; the preparation method of the above-mentioned specific indicator red blood cells is the same as that in Comparative Example 1.

[0144] Comparative Example 9

[0145] This comparative example provides a kit, which includes specific latex microspheres and a specific microcolumn gel card. The preparation method of the above-mentioned specific latex microspheres is the same as that in Example 2; compared with Example 2, the preparation method of the above-mentioned specific microcolumn gel card is only different in that: the biotinylation treatment in step 1) is omitted, and streptavidin in step 3) is replaced with an equal amount of anti-human globulin antibody, and the remaining steps are the same as those in Example 2.

[0146] Comparative Example 10

[0147] This comparative example provides a kit, which includes specific latex microspheres and a specific microcolumn gel card. The preparation method of the above-mentioned specific microcolumn gel card is the same as that in Example 2; compared with Example 2, the preparation method of the above-mentioned specific latex microspheres is only different in that the particle size of the red latex microspheres is adjusted from 10 μm to 5 μm, and the remaining steps are the same as those in Example 2.

[0148] Comparative Example 11

[0149] This comparative example provides a kit, which includes specific latex microspheres and a specific microcolumn gel card. The preparation method of the above-mentioned specific microcolumn gel card is the same as that in Example 2; compared with Example 2, the preparation method of the above-mentioned specific latex microspheres is only different in that the particle size of the red latex microspheres is adjusted from 10 μm to 20 μm, and the remaining steps are the same as those in Example 2.

[0150] Comparative Example 12

[0151] This comparative example provides a kit, which includes specific latex microspheres and a specific microcolumn gel card. The preparation method of the above-mentioned specific latex microspheres is the same as that in Example 2; compared with Example 2, the preparation method of the above-mentioned specific microcolumn gel card is only different in that the PEG20000 used for preparing the gel medium in step 2) is replaced with an equal amount of S9, that is, the final concentration of S9 in the gel medium is 2.0 g / L, and the remaining steps are the same as those in Example 2.

[0152] Comparative Example 13

[0153] This comparative example provides a kit, which includes specific latex microspheres and a specific microcolumn gel card. The preparation method of the above-mentioned specific latex microspheres is the same as that in Example 2; compared with Example 2, the preparation method of the above-mentioned specific microcolumn gel card is only different in that the S9 used for preparing the gel medium in step 2) is replaced with an equal amount of PEG20000, that is, the final concentration of PEG20000 in the gel medium is 2.0 g / L, and the remaining steps are the same as those in Example 2.

[0154] Comparative Example 14

[0155] This comparative example provides a kit, which includes specific latex microspheres and a specific microcolumn gel card. The preparation method of the above-mentioned specific latex microspheres is the same as that in Example 2; compared with Example 2, the preparation method of the above-mentioned specific microcolumn gel card is only different in that the S9 and PEG20000 used for preparing the gel medium in step 2) are omitted, and the remaining steps are the same as those in Example 2.

[0156] Test Example

[0157] This test example tested the sensitivity, specificity, sensitivity, and long-term storage stability of the kits provided in Examples 1-2 and Comparative Examples 1-14. The specific test methods and test results are as follows:

[0158] 1. Sensitivity:

[0159] The kits provided in Example 1 and Comparative Examples 1-7 were used to detect human anti-Mycoplasma pneumoniae IgM antibodies (MP-IgM) with different titers. The MP-IgM standard was diluted with negative serum or negative plasma; each titer was detected 3 times, and the detection method was referred to the description in Example 3; the detected titer gradient and detection results are shown in Table 2.

[0160] Table 2 Detection sensitivity of MP-IgM

[0161]

[0162] Note: "+" indicates a positive test result, and "-" indicates a negative test result.

[0163] The kits provided in Example 2 and Comparative Examples 8-14 were used to detect human anti-Mycoplasma pneumoniae IgG antibodies (MP-IgG) with different titers. The MP-IgG standard was diluted with negative serum or negative plasma; each titer was detected 3 times, and the detection method was referred to the description in Example 4; the detected titer gradient and detection results are shown in Table 3.

[0164] Table 3 Detection sensitivity of MP-IgG

[0165]

[0166] Note: "+" indicates a positive test result, and "-" indicates a negative test result.

[0167] From the results in Table 2, it can be seen that:

[0168] 1) Compared with Example 1, in Comparative Example 1, specific indicator red blood cells were used to replace specific latex microspheres, and its detection sensitivity was basically no different from that of Example 1;

[0169] 2) Compared with Example 1, in Comparative Example 2, due to the omission of the streptavidin-biotin amplification system, the overall size of the latex microsphere-antigen-antibody complex decreased, so the sensitivity of Comparative Example 2 was significantly lower than that of Example 1;

[0170] 3) Compared with Example 1, in Comparative Example 3, due to the reduction of the particle size of the colored latex microspheres, the overall size of the latex microsphere-antigen-antibody complex also decreased, so the sensitivity of Comparative Example 3 was significantly lower than that of Example 1;

[0171] 4) Compared with Example 1, in Comparative Example 4, due to the increase in the particle size of the colored latex microspheres, the overall size of the latex microsphere-antigen-antibody complex increased. Therefore, Comparative Example 4 was able to form a larger-sized colloidal microsphere-antigen-antibody complex, and its sensitivity was comparable to that of Example 1;

[0172] 5) Compared with Example 1, in the gels of Comparative Example 5 and Comparative Example 6, due to the lack of PEG20000 or S9, the specific aggregation of the latex microspheres was affected, further resulting in a decrease in the retention rate of the latex microsphere-antigen-antibody complex. PEG20000 can reduce the surface tension of the gel medium, increase the kinetics of the antigen-antibody reaction, and promote the approach of antigens and antibodies to each other; while S9 changes the surface charge distribution of antigens and antibodies, enabling the active protein binding sites of antigens and antibodies to be fully exposed; when the specific antigen-antibody active binding sites are fully exposed or the distance becomes closer, the binding probability increases, and the size of the agglomerates also increases; therefore, the sensitivity of Comparative Example 5 and Comparative Example 6 is lower than that of Example 1;

[0173] 6) Compared with Example 1, in Comparative Example 7, due to the omission of PEG20000 and S9, the specific aggregation of the latex microspheres was affected, further reducing the retention rate of the latex microsphere-antigen-antibody complex. There is neither PEG20000 to reduce the surface tension of the gel medium, increase the kinetics of the antigen-antibody reaction, and promote the approach of antigens and antibodies to each other, nor S9 to change the surface charge distribution of antigens and antibodies, enabling the active protein binding sites of antigens and antibodies to be fully exposed, resulting in a lower antigen-antibody binding probability than that of Example 1 with the addition of PEG20000 and S9; therefore, the sensitivity of Comparative Example 7 is significantly lower than that of Example 1.

[0174] According to the results in Table 3, the changing trends of the detection results corresponding to Example 2 and Comparative Examples 8 to 14 are consistent with the changing trends of the detection results shown in Table 2, and the result analysis can refer to the above analysis of Table 2.

[0175] 2. Specificity and sensitivity:

[0176] Using the kits provided in Example 1 and Comparative Examples 1 to 7, 500 clinical samples known to be negative for MP-IgM and 200 clinical samples known to be positive for MP-IgM were tested, and each sample was tested once. The testing method can be referred to the description in Example 3; among them, sensitivity (true positive rate) = the number of detected positive samples / the total number of known positive samples × 100%, specificity (true negative rate) = the number of detected negative samples / the total amount of known negative samples × 100%; total coincidence rate = (the number of true positive samples + the number of true negative samples) / the total number of samples × 100%; the obtained results are shown in Table 4.

[0177] Table 4 Specificity and sensitivity of MP-IgM detection

[0178]

[0179] Using the kits provided in Example 2 and Comparative Examples 8-14, 500 clinical samples known to be MP-IgG negative and 200 clinical samples known to be MP-Ig positive were tested once per sample. The testing method can be referred to the description in Example 4. Among them, sensitivity (true positive rate) = the number of positive samples detected / the total number of known positive samples × 100%, specificity (true negative rate) = the number of negative samples detected / the total number of known negative samples × 100%; total coincidence rate = (the number of true positive samples + the number of true negative samples) / the total number of samples × 100%. The obtained results are shown in Table 5.

[0180] Table 5 Specificity and Sensitivity of MP-IgG Detection

[0181]

[0182] As can be seen from the results in Table 4:

[0183] 1) Compared with Example 1, in Comparative Example 1, specific indicator red blood cells were used to replace specific latex microspheres, and there was basically no difference in the specificity and sensitivity of the detection compared with Example 1.

[0184] 2) Compared with Example 1, in Comparative Example 2, due to the omission of the streptavidin-biotin signal amplification system, the overall size of the latex microsphere-antigen-antibody complex decreased. Therefore, the sensitivity of Comparative Example 2 was significantly lower than that of Example 1.

[0185] 3) Compared with Example 1, in Comparative Example 3, due to the reduction of the particle size of the colored latex microspheres, the overall size of the latex microsphere-antigen-antibody complex also decreased. Therefore, the sensitivity of Comparative Example 3 was significantly lower than that of Example 1.

[0186] 4) Compared with Example 1, in Comparative Example 4, due to the increase in the particle size of the colored latex microspheres, the overall size of the latex microsphere-antigen-antibody complex increased. Therefore, Comparative Example 4 could detect a lower titer of the target antibody, and its sensitivity was higher than that of Example 1, but the probability of false positives would increase. Therefore, the specificity was lower than that of Example 1.

[0187] 5) Compared with Example 1, in Comparative Examples 5 and 6, the gels lacked PEG20000 or S9, which affected the aggregation of specific latex microspheres, further leading to a decrease in the retention rate of the latex microsphere - antigen - antibody complex; PEG20000 can reduce the surface tension of the gel medium, increase the kinetics of the antigen - antibody reaction, and promote the approach of antigens and antibodies to each other; while S9 changes the surface charge distribution of antigens and antibodies, enabling the active protein - binding sites of antigens and antibodies to be fully exposed; when the active binding sites of specific antigens and antibodies are fully exposed, the closer the distance, the greater the binding probability, and the larger the size of the agglomerates; therefore, the sensitivities of Comparative Examples 5 and 6 are lower than that of Example 1;

[0188] 6) Compared with Example 1, in Comparative Example 7, the omission of PEG20000 and S9 led to an impact on the aggregation of specific latex microspheres, further reducing the retention rate of the latex microsphere - antigen - antibody complex. PEG20000 can reduce the surface tension of the gel medium, increase the kinetics of the antigen - antibody reaction, and promote the approach of antigens and antibodies to each other; while S9 changes the surface charge distribution of antigens and antibodies, enabling the active protein - binding sites of antigens and antibodies to be fully exposed; when the active binding sites of specific antigens and antibodies are fully exposed, the closer the distance, the greater the binding probability, and the larger the size of the agglomerates; therefore, the sensitivity of Comparative Example 7 is significantly lower than that of Example 1.

[0189] According to the results in Table 5, the changing trends of the detection results corresponding to Example 2 and Comparative Examples 8 - 14 are consistent with the changing trends of the detection results shown in Table 4, and the result analysis can refer to the above analysis of Table 4.

[0190] 3. Long - term storage stability:

[0191] The kits provided in Example 1 and Comparative Examples 1 - 7 were stored at 2 - 8 °C for 14 months. During this period, samples M1, M2, M3, and M4 were detected using the kits of Example 1 and Comparative Examples 1 - 7 at 8 time points respectively. Among them, samples M1, M2, and M3 were derived from the samples with titers of 1:128, 1:1024, and 1:4096 in the sensitivity detection of MP - IgM in Test Example, and M4 was one of the MP - IgM negative samples in the clinical sample verification; the detection time points and corresponding results are shown in Table 6.

[0192] Table 6 Stability of MP - IgG Detection Kit

[0193]

[0194] The kits provided in Example 2 and Comparative Examples 8 to 14 were stored at 2-8 °C for 14 months. During this period, samples G1, G2, G3 and G4 were detected using the kits of Example 2 and Comparative Examples 8 to 14 at 8 time points respectively. Among them, samples G1, G2, and G3 were derived from the samples with titers of 1:64, 1:512, and 1:2048 in the sensitivity detection of MP-IgG in the test example, and G4 was one of the MP-IgG negative samples in the clinical sample verification; the detection time points and corresponding results are shown in Table 7.

[0195] Table 7 Stability of MP-IgG Detection Kit

[0196]

[0197] From the results in Table 6, it can be seen that the detection kits provided in Examples 1 and 2 of the present invention have long-term stability, and the shelf life at 2-8 °C can be up to 14 months, while the shelf life of Comparative Example 1 and Comparative Example 8 is only 8 months, and the shelf life of Comparative Example 5, Comparative Example 7, Comparative Example 12 and Comparative Example 14 is 12 months; while in Comparative Example 4 and Comparative Example 11, as the storage time increased, latex microspheres aggregated at the 10th month, resulting in false positive results. Therefore, the Mycoplasma pneumoniae IgM / IgG antibody detection kits provided in Examples 1 and 2 of the present invention not only have the advantages of high sensitivity, strong specificity and high sensitivity, but also can maintain excellent detection performance after long-term storage.

[0198] In summary, the detection kit based on specific latex microspheres and microinjection gel provided by the present invention has good sensitivity, sensitivity and specificity in the detection of MP-IgM and MP-IgG, and its comprehensive performance is significantly better than that of the comparative examples; the streptavidin-biotin coupling system provided by the present invention has a better amplification effect compared with the existing anti-human globulin conjugated human immunoglobulin; the selected latex microspheres with a particle size of 10 μm in the present invention are the optimal scheme, and their sensitivity, sensitivity and specificity are comprehensively optimal, and the total coincidence rate is the highest; the gel medium provided by the present invention also improves the detection performance of Mycoplasma pneumoniae IgM / IgG antibodies, and S9 and PEG20000 in the gel medium have a synergistic effect in improving sensitivity.

[0199] The embodiments of the present invention have been described in detail above with reference to the drawings, but the present invention is not limited to the above embodiments. Various changes can be made without departing from the spirit of the present invention within the knowledge scope of those of ordinary skill in the art. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A kit, characterized in that, The kit includes: specific latex microspheres and a specific microcolumn gel card; The specific latex microspheres are colored latex microspheres conjugated with a specific antigen or antibody; the particle size of the colored latex microspheres is 8 - 18 μm; a blocking agent is also connected to the surface of the specific latex microspheres; the conjugation method includes EDC-NHS conjugation; The specific latex microspheres are used to be mixed with a test sample and added into the specific microcolumn gel card for detection; The specific microcolumn gel card includes a fixing plate and a gel tube, and the gel tube contains a specific gel; The specific gel includes (a) - (d): (a) Biotinylated specific antigen or antibody; (b) Streptavidin; (c) Gel particles; (d) Gel medium.

2. The kit according to claim 1, wherein The volume of the specific gel accounts for ; The gel particles are selected from any one of dextran gel particles, glass microspheres, and polyacrylamide gel particles; The dextran gel particles are selected from any one of Sephadex-G25, Sephadex-G50, and Sephadex-G100; The gel medium is a phosphate buffer solution containing sodium chloride, bovine serum albumin, surfactant S9, disodium EDTA, PEG20000, sucrose, and sodium azide.

3. The kit according to claim 1 or 2, characterized in that, The usage method of the kit includes the following steps: Mix the specific latex microspheres with the test sample in equal volume and add them into the gel tube for reaction to obtain a specific latex microsphere complex, then centrifuge the reaction system, and finally perform result interpretation; The temperature of the reaction is 35 - 40 °C; The time of the reaction is 8 - 20 min.

4. The kit according to claim 3, characterized in that, The criteria for result interpretation include: (1) The specific latex microsphere complex is located on the surface of the specific gel, which is a 4+ strong positive reaction; (2) The specific latex microsphere complex is suspended in the specific gel, which is a 1+ to 3+ positive reaction. The closer the specific latex microsphere complex is to the bottom of the gel, the lower the titer; (3) The specific latex microsphere complex is all deposited at the bottom of the specific gel, which is a negative result.

5. A method for preparing the kit according to claim 1 or 2, characterized in that, The preparation method includes the following steps: S1. Prepare specific latex microspheres: Conjugate a specific antigen or antibody with colored latex microspheres and perform a blocking treatment to obtain specific latex microspheres; S2. Prepare biotinylated specific antigen or antibody: Conjugate a specific antigen or antibody with NHS-biotin to obtain biotinylated specific antigen or antibody; S3. Prepare specific gel: Mix gel particles, streptavidin, gel medium, and the biotinylated specific antigen or antibody obtained in step S2 to obtain specific gel; S4. Fill the specific gel obtained in step S3 into the gel tube to obtain a specific microcolumn gel card. The specific latex microspheres obtained in step S1 and the specific microcolumn gel card obtained in step S4 form a kit.

6. The preparation method according to claim 5, characterized in that, The conjugation method described in step S1 includes NHS-EDC covalent conjugation; The mass ratio of the colored latex microspheres to the specific antigen or antibody in step S1 is 1:(0.02 - 0.04); The gel medium described in step S3 is a 0.02 mol / L phosphate buffer containing the following components at the final concentrations: 7 - 10 g / L sodium chloride, 1 - 3 g / L disodium EDTA, 8 - 12 g / L bovine serum albumin, 0.5 - 2 g / L S9, 0.5 - 2 g / L PEG20000, 40 - 60 g / L sucrose, and 0.1 - 0.3 g / L sodium azide. The pH value of the phosphate buffer is 7 - 8; The mass - to - volume ratio of the gel particles to the gel medium described in step S3 is 1:(12 - 16) g / mL.

7. Use of the kit according to claim 1 or 2 in the following (I) - (III): (I) Preparation of a product for blood group identification; (II) Preparation of a product for detecting autoantibodies in autoimmune diseases; (III) Preparation of a product for detecting antigens or antibodies of microbial pathogens.

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