Specific latex microsphere, kit and preparation method and application thereof

Abstract

The invention discloses a specific latex microsphere, a kit as well as a preparation method and application of the specific latex microsphere. The specific latex microsphere is a colored latex microsphere coupled with a specific antigen or antibody; the particle size of the colored latex microspheres is 8-18 [mu] m; the coupling mode comprises EDC (Ethylene Diamine Chloride)-NHS (N-Hydroxysuccinimide) coupling. The kit provided by the invention takes specific latex microspheres as a marker, and can be used for detecting pathogenic microorganism antigens or antibodies in combination with a streptavidin-biotin amplification system and a microcolumn gel immune experiment technology; the detection method is simple and rapid to operate and high in sensitivity.

Description

Technical Field

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

[0002] At present, the detection methods of pathogenic microorganism antigens and antibodies mainly include immunochromatography, enzyme-linked immunosorbent assay, agglutination method, chemiluminescence method and microcolumn gel method based on specific red blood cell indicator. Immunochromatography reagents are mainly colloidal gold immunochromatography, which is simple to operate and the results are easy to interpret, but the sensitivity is lower than other methods; the operation process of enzyme-linked immunosorbent assay and agglutination method is cumbersome and requires high professional skills of operators; chemiluminescence method requires supporting automated equipment and has high detection costs; microcolumn gel method based on specific red blood cell indicator is limited by the stability of red blood cells. This type of reagent has a short validity period, usually 6 to 8 months, and its scope of application is relatively limited. It cannot play a role in detecting some non-human globulin substances.

[0003] The microcolumn gel method 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 higher than other methods. When the antigen concentration is lower than a certain level, the agglutination formed by the antigen-antibody reaction may be less or not obvious enough, making it difficult to observe with the naked eye, resulting in false negative results. Therefore, the microcolumn gel method still needs to be further improved and optimized. 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. To this end, the present invention provides a specific latex microsphere, which can be used as a marker to detect pathogenic microorganism antigens or antibodies, and the detection method is simple to operate, rapid and highly sensitive.

[0005] The invention also provides a kit.

[0006] The invention also provides a method for preparing the kit.

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

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

[0009] According to the first aspect of the present invention, a specific latex microsphere is provided, wherein the specific latex microsphere is a colored latex microsphere coupled with a specific antigen or antibody; The particle size of the colored latex microspheres is 8-18 μm.

[0010] In some embodiments of the present invention, a blocking agent is also connected to the surface of the specific latex microspheres.

[0011] In some embodiments of the present invention, the blocking agent includes a proteinaceous blocking agent and / or a non-proteinaceous blocking agent.

[0012] In some embodiments of the present invention, the protein blocking agent includes at least one of bovine serum albumin, casein and skim milk.

[0013] In some embodiments of the present invention, the non-proteinaceous blocking agent comprises at least one of polyethylene glycol, Tween-20 and ethanolamine.

[0014] In some embodiments of the present invention, the coupling method includes EDC-NHS coupling.

[0015] In some embodiments of the present invention, the particle size of the colored latex microspheres is 9 to 15 μm.

[0016] 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.

[0017] According to a second aspect of the present invention, a kit is provided, the kit comprising: the specific latex microspheres and the specific microcolumn gel card according to the first aspect of the present invention; the specific latex microspheres are used to be mixed with a sample to be tested and added to the specific microcolumn gel card for detection; The specific microcolumn gel card comprises a fixing plate and a gel tube, wherein the gel tube contains a specific gel; The specific gel includes (a) to (d): (a) biotinylated specific antigen or antibody; (b) streptavidin; (c) gel particles; (d) Gel medium.

[0018] In some embodiments of the present invention, the volume of the specific gel accounts for the volume of the gel tube. .

[0019] In some embodiments of the present invention, the volume of the specific gel in each gel tube is 30-40 μL.

[0020] In some embodiments of the present invention, the specific micro-column gel card further comprises a sealing film.

[0021] In some embodiments of the present invention, the gel particles are selected from any one of dextran gel particles, glass microbeads and polyacrylamide gel particles.

[0022] In some embodiments of the present invention, the dextran gel particles are selected from any one of Sephadex-G25, Sephadex-G50 and Sephadex-G100.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] In some embodiments of the present invention, the final concentration of the streptavidin in the specific gel is 0.2-0.8 μg / mL.

[0027] In some embodiments of the present invention, the final concentration of the streptavidin in the specific gel is 0.4-0.6 μg / mL.

[0028] In some embodiments of the present invention, when the target detected by the detection kit is antigen A, the colored latex microspheres are coupled with specific antibodies A' of antigen A, and the specific gel contains biotinylated specific antibodies A' or other specific antibodies against antigen A.

[0029] In some embodiments of the present invention, when the target detected by the detection kit is antibody B, the colored latex microspheres are coupled with the specific antigen B' of 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 IgG, the heterologous antibody C can be a mouse anti-human IgG antibody.

[0030] According to the third aspect of the present invention, a method for preparing the kit according to the second aspect of the present invention is provided, the preparation method comprising the following steps: S1. Preparing specific latex microspheres: coupling specific antigens or antibodies with colored latex microspheres and performing blocking treatment to obtain specific latex microspheres; S2. Preparing a biotinylated specific antigen or antibody: coupling the specific antigen or antibody with NHS-biotin to obtain a biotinylated specific antigen or antibody; S3. Preparation of specific gel: Mixing the gel particles, streptavidin, gel medium and the biotinylated specific antigen or antibody obtained in step S2 to obtain a specific gel; S4. Fill the specific gel obtained in step S3 into a 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 constitute a kit.

[0031] In some embodiments of the present invention, the coupling method in step S1 includes NHS-EDC covalent coupling.

[0032] In some embodiments of the present invention, the mass ratio of the colored latex microspheres to the specific antigen or antibody in step S1 is 1:(0.02-0.04).

[0033] In some embodiments of the present invention, the particle size of the colored latex microspheres is 8-18 μm.

[0034] In some embodiments of the present invention, the particle size of the colored latex microspheres is 9 to 15 μm.

[0035] 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.

[0036] In some embodiments of the present invention, the blocking solution used in the blocking treatment in step S1 is: a borate buffer solution containing a final concentration of 3-7 mmol / L boric acid, 10-13 mmol / L sodium tetraborate decahydrate, 0.03%-0.07% Tween-20, 0.5%-2% BSA and 0.2%-0.3% ethanolamine.

[0037] In some embodiments of the present invention, the sealing time of the sealing treatment in step S1 is 1 to 2 hours.

[0038] In some embodiments of the present invention, the gel medium in step S3 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.

[0039] In some embodiments of the present invention, the mass volume ratio of the gel particles to the gel medium in step S3 is 1: (12-16) g / mL.

[0040] 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).

[0041] In some embodiments of the present invention, the final concentration of the streptavidin in the specific gel in step S3 is 0.2-0.8 μg / mL.

[0042] In some embodiments of the present invention, the final concentration of the streptavidin in the specific gel in step S3 is 0.4-0.6 μg / mL.

[0043] In some embodiments of the present invention, the gel particles in step S3 are selected from any one of dextran gel particles, glass microbeads and polyacrylamide gel particles.

[0044] In some embodiments of the present invention, the dextran gel particles are selected from any one of Sephadex-G25, Sephadex-G50 and Sephadex-G100.

[0045] In some embodiments of the present invention, the volume of the specific gel in step S4 accounts for % of the volume of the gel tube. .

[0046] In some embodiments of the present invention, the volume of the specific gel in each gel tube in step S4 is 30-40 μL.

[0047] According to a fourth aspect of the present invention, a method for detecting antigens or antibodies against pathogenic microorganisms is provided, wherein the detection method uses the kit described in the second aspect of the present invention for detection.

[0048] In some embodiments of the invention, the antibodies to microbial pathogens include human anti-Mycoplasma pneumoniae IgM antibodies.

[0049] In some embodiments of the invention, the antibodies to microbial pathogens include human anti-Mycoplasma pneumoniae IgG antibodies.

[0050] In some embodiments of the present invention, the detection method comprises the following steps: The specific latex microspheres and the sample to be tested are mixed in equal volumes and added into a gel tube for reaction to obtain a specific latex microsphere complex, and then the reaction system is centrifuged and the result is finally interpreted.

[0051] In some embodiments of the present invention, the reaction temperature is 35-40°C.

[0052] In some embodiments of the present invention, the reaction time is 8 to 20 min.

[0053] In some embodiments of the present invention, the centrifugation sequentially comprises a primary centrifugation and a secondary centrifugation.

[0054] In some embodiments of the present invention, the conditions of the first centrifugation are 800-1000 rpm, 1-3 min.

[0055] In some embodiments of the present invention, the secondary centrifugation is performed at 1400-1600 rpm for 2-5 min.

[0056] In some embodiments of the present invention, the solid content of the specific latex microspheres is 0.5% to 1%.

[0057] In some embodiments of the present invention, the solid content of the specific latex microspheres is 0.7% to 0.9%.

[0058] In some embodiments of the present invention, the criteria for result interpretation include: (1) The specific latex microsphere complex is located on the surface of the specific gel, with a 4+ strong positive reaction; (2) The specific latex microsphere complex is suspended in the specific gel, with a positive reaction of 1+ to 3+. 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 completely deposited at the bottom of the specific gel, which is a negative result.

[0059] In some embodiments of the present invention, the samples to be tested that are applicable to the detection method include but are not limited to blood samples, respiratory swab samples and stool samples.

[0060] The principle of the detection method includes: the antigen / antibody in the sample to be tested and the antibody / antigen linked by streptavidin-biotin in the microcolumn gel medium are combined to form an antigen-antibody immune complex, which is then combined with the specific antibody / antigen coupled to the colored latex microspheres to form a polymerized "latex microsphere coupled 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 spatial network structure or in the gaps between the gel particles, which is judged as a positive result; if there is no corresponding antigen or antibody binding, the colored latex microsphere immune complex cannot be formed. Under a certain centrifugal force, the dispersed colored latex microspheres are all deposited at the bottom of the gel pore cavity, which is judged as a negative result.

[0061] According to the fifth aspect of the present invention, the use of the kit according to the second aspect of the present invention or the detection method according to the fourth aspect of the present invention in the following (I) to (III) is proposed: (I) Preparation of products for blood typing; (II) preparing products for use in the detection of autoantibodies in autoimmune diseases; (III) Preparation of antigen or antibody detection products for microbial pathogens.

[0062] In some embodiments of the invention, the antibodies to microbial pathogens include human anti-Mycoplasma pneumoniae IgM antibodies.

[0063] In some embodiments of the invention, the antibodies to microbial pathogens include human anti-Mycoplasma pneumoniae IgG antibodies.

[0064] The present invention has at least the following beneficial effects: The invention provides a kit based on specific latex microspheres and microcolumn gel. The kit uses the specific latex microspheres as markers, combines a streptavidin / biotin amplification system and a microcolumn gel immunoassay technology, and can be widely used in the detection of various antigens or antibodies.

[0065] The present invention provides a detection method for pathogenic microorganism antigens or antibodies based on the above-mentioned kit. The detection method is simple to operate, has a higher sensitivity than colloidal gold immunochromatography and an existing microcolumn gel based on a specific red blood cell indicator, can provide a detection result within 20 to 30 minutes, and has better reagent stability than a microcolumn gel method based on a specific red blood cell indicator, with a validity period of up to 12 months; simple manual operation can be adopted, and the method is suitable for primary medical units and on-site rapid detection; automated equipment can also be adopted for detection, and the method is suitable for institutions with a large number of test samples such as tertiary hospitals, thereby reducing the risk of nosocomial infection and improving detection efficiency; the test result can usually be directly judged by observing the color development with the naked eye, reducing the dependence on professional analysis equipment, and does not require complex equipment to interpret the result or professional medical staff to explain the result, and the result can be completely independently detected and judged by the subject by visually observing the agglutination effect in the detection area. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 The structure diagram of the specific micro-column gel card prepared in Example 1 of the present invention, the upper part is the structural schematic diagram, and the lower part is the actual picture; Figure 2 This is a schematic diagram of different result determinations of the method for detecting human anti-Mycoplasma pneumoniae IgM antibodies in Example 3 of the present invention. DETAILED DESCRIPTION

[0067] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0068] Materials and Definitions In the following examples and comparative examples, the purchase information of the materials used is shown in Table 1.

[0069] Table 1 Purchase information of materials used

[0070] Example 1 This embodiment provides a kit based on specific latex microspheres and microcolumn gels, the kit comprising specific latex microspheres and specific microcolumn gel cards, and the preparation method thereof specifically comprises the following steps: 1. Preparation of specific latex microspheres: 1) Cleaning of latex microspheres: Dilute 2.5 wt% red latex microsphere suspension (particle size 10 μm) with coupling buffer (50 mM MES buffer, pH 6.0±0.1) to a 1 wt% latex microsphere suspension, mix by ultrasonication and centrifuge at a speed of 18,000 rpm (15,000 rpm to 20,000 rpm) for 20 min (15 to 30 min). After centrifugation, discard the supernatant and use coupling buffer to ultrasonically resuspend the latex microspheres to 1 wt% to obtain a washed latex microsphere suspension.

[0071] 2) Coupling of latex microspheres and antigens: Add freshly prepared Sulfo-NHS solution (stock concentration of 50 mg / mL) and EDC solution (stock concentration of 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 to a final concentration of 0.3 mg / mL (0.2-0.4 mg / mL), mix well, place the mixed system on a mixer and rotate and incubate at room temperature for 150 min to obtain a coupling product.

[0072] 3) Blocking of unbound surface groups on latex microspheres: The coupling product obtained in step 2) was centrifuged (the centrifugation conditions were the same as those used in step 1) and the supernatant was removed. Then, blocking solution (containing a final concentration of 5 mmol / L boric acid, 11.2 mmol / L sodium tetraborate decahydrate, 0.05 v / v% Tween-20, 1 w / v% BSA and 0.24 w / v% borate buffer, pH 9.0±0.1) was added to the centrifuge tube for blocking, and incubated at room temperature for 1 hour to obtain blocked latex microspheres.

[0073] 4) Cleaning and storage of specific latex microsphere markers after coupling: The blocked latex microspheres obtained in step 3) were resuspended to a concentration of 1% with washing solution (50 mmol / L Tris, 0.5% BSA, 0.05% Tween-20, pH 8.0±0.1) and washed. The washing was repeated twice, and then the microspheres were resuspended in a preservation solution (containing a solution with a final concentration of 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%, to obtain specific latex microspheres, which were stored at 2~8°C.

[0074] 2. Preparation of specific microcolumn gel cards: 1) Biotinylation of antibodies: The mouse anti-human IgM antibody was reacted with Sulfo-NHS-LC-biotin according to the instructions of the supplier to couple the mouse anti-human IgM antibody with biotin to obtain a biotinylated antibody.

[0075] 2) Preparation of gel medium: The following components with final concentrations were added to 0.02 mol / L phosphate buffer (pH 7.4) and mixed to obtain the gel medium: 8.5 g / L NaCl, 1.5 g / L EDTA-disodium, 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.

[0076] 3) Preparation of specific gel: Sephadex-G50 (dextran gel particles) is selected and mixed with the gel medium obtained in step 2) at a mass volume ratio of 1:14 g / mL (1:12-1:16 is acceptable) to prepare a gel; the biotinylated mouse anti-human IgM antibody obtained in step 1) is added to the above gel at a titer of 1:1000 and mixed evenly, and then streptavidin is added at a final concentration of 0.5 μg / mL and mixed evenly to obtain a specific gel.

[0077] 4) Filling and sealing: The specific gel obtained in step 3) is added into the gel tube of the microcolumn gel card at a filling volume of 34 μL / tube, and sealed with a hot-pressed sealing film, the sealing film material of which is a composite aluminum coil, to obtain a specific microcolumn gel card, the structure of which is as follows: Figure 1 shown.

[0078] Example 2 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.

[0079] Example 3 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); 2) After the reaction, centrifuge at 900 rpm for 2 min, and then at 1500 rpm for 3 min; 3) After centrifugation, the results can be interpreted; The criteria for judging the results are as follows (e.g. Figure 2 shown): ①4+ is the latex microsphere-antigen-antibody complex (agglutination) located on the gel surface; 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; 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; ④1+ is the latex microsphere-antigen-antibody complex (agglutination) located in the lower middle part of the gel; 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; ⑥Negative result (-): The latex microspheres are deposited at the bottom of the microcolumn gel tube, and there is no agglutination in the gel.

[0080] Example 4 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.

[0081] Comparative Example 1 This comparative example provides a detection kit based on indicator red blood cells and microcolumn gel, which includes specific indicator red blood cells and specific microcolumn gel cards. The preparation method of the above-mentioned specific microcolumn gel card is the same as that in Example 1; the preparation method of the above-mentioned specific indicator red blood cells refers to the method described in "Preparation of indicator red blood cells" in patent CN104965079A, the only difference is that the anti-HIV antibody solution used in step 2 is replaced by a Mycoplasma pneumoniae antigen solution, the amount of antigen used is the same as that of the specific latex microspheres used in Example 1, and the specific indicator red blood cells finally prepared are diluted to 0.8 wt% for storage.

[0082] Comparative Example 2 This comparative example provides a kit, which includes specific latex microspheres and specific microcolumn gel cards. The preparation method of the above-mentioned specific latex microspheres is the same as that in Example 1; the preparation method of the above-mentioned specific microcolumn gel card is different from that in Example 1, except that: the biotinylation treatment in step 1) is omitted, and the streptavidin in step 3) is replaced by an equal amount of anti-human globulin antibody, and the remaining steps are consistent with Example 1.

[0083] Comparative Example 3 This comparative example provides a kit, which includes specific latex microspheres and specific microcolumn gel cards. The preparation method of the specific microcolumn gel cards is the same as that in Example 1. The preparation method of the specific latex microspheres is different from that in Example 1, except that the particle size of the red latex microspheres is adjusted from 10 μm to 5 μm, and the remaining steps are consistent with those in Example 1.

[0084] Comparative Example 4 This comparative example provides a kit, which includes specific latex microspheres and specific microcolumn gel cards. The preparation method of the specific microcolumn gel cards is the same as that of Example 1. The preparation method of the specific latex microspheres is different from that of Example 1, except that the particle size of the red latex microspheres is adjusted from 10 μm to 20 μm, and the remaining steps are consistent with those of Example 1.

[0085] Comparative Example 5 This comparative example provides a kit, which includes specific latex microspheres and specific microcolumn gel cards. The preparation method of the above-mentioned specific latex microspheres is the same as that in Example 1; the preparation method of the above-mentioned specific microcolumn gel card is different from that in Example 1, except that: PEG20000 used in preparing the gel medium in step 2) is replaced by 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 consistent with Example 1.

[0086] Comparative Example 6 This comparative example provides a kit, which includes specific latex microspheres and specific microcolumn gel cards. The preparation method of the above-mentioned specific latex microspheres is the same as that in Example 1; the preparation method of the above-mentioned specific microcolumn gel card is different from that in Example 1, except that: the S9 used for preparing the gel medium in step 2) is replaced by 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 consistent with Example 1.

[0087] Comparative Example 7 This comparative example provides a kit, which includes specific latex microspheres and specific microcolumn gel cards. The preparation method of the above-mentioned specific latex microspheres is the same as that in Example 1; the preparation method of the above-mentioned specific microcolumn gel card is different from that in Example 1, except that: S9 and PEG20000 used in preparing the gel medium in step 2) are omitted, and the remaining steps are consistent with those in Example 1.

[0088] Comparative Example 8 This comparative example provides a detection kit based on indicator red blood cells and microcolumn gel, which includes specific indicator red blood cells and specific microcolumn gel cards. The preparation method of the above-mentioned specific microcolumn gel card is the same as that of Example 2; the preparation method of the above-mentioned specific indicator red blood cells is the same as that of Comparative Example 1.

[0089] Comparative Example 9 This comparative example provides a kit, which includes specific latex microspheres and specific microcolumn gel cards. The preparation method of the above-mentioned specific latex microspheres is the same as that of Example 2; the preparation method of the above-mentioned specific microcolumn gel card is different from that of Example 2, except that: the biotinylation treatment in step 1) is omitted, and the streptavidin in step 3) is replaced by an equal amount of anti-human globulin antibody, and the remaining steps are consistent with Example 2.

[0090] Comparative Example 10 This comparative example provides a kit, which includes specific latex microspheres and specific microcolumn gel cards. The preparation method of the above-mentioned specific microcolumn gel cards is the same as that of Example 2; the preparation method of the above-mentioned specific latex microspheres is different from that of Example 2, except that the particle size of the red latex microspheres is adjusted from 10 μm to 5 μm, and the remaining steps are consistent with Example 2.

[0091] Comparative Example 11 This comparative example provides a kit, which includes specific latex microspheres and specific microcolumn gel cards. The preparation method of the specific microcolumn gel cards is the same as that of Example 2. The preparation method of the specific latex microspheres is different from that of Example 2, except that the particle size of the red latex microspheres is adjusted from 10 μm to 20 μm, and the remaining steps are consistent with those of Example 2.

[0092] Comparative Example 12 This comparative example provides a kit, which includes specific latex microspheres and specific microcolumn gel cards. The preparation method of the above-mentioned specific latex microspheres is the same as that in Example 2; the preparation method of the above-mentioned specific microcolumn gel card is different from that in Example 2, except that: PEG20000 used in preparing the gel medium in step 2) is replaced by 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 consistent with Example 2.

[0093] Comparative Example 13 This comparative example provides a kit, which includes specific latex microspheres and specific microcolumn gel cards. The preparation method of the above-mentioned specific latex microspheres is the same as that in Example 2; the preparation method of the above-mentioned specific microcolumn gel card is different from that in Example 2, except that: the S9 used to prepare the gel medium in step 2) is replaced by 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 consistent with Example 2.

[0094] Comparative Example 14 This comparative example provides a kit, which includes specific latex microspheres and specific microcolumn gel cards. The preparation method of the above-mentioned specific latex microspheres is the same as that in Example 2; the preparation method of the above-mentioned specific microcolumn gel card is different from that in Example 2, except that: S9 and PEG20000 used in preparing the gel medium in step 2) are omitted, and the remaining steps are consistent with Example 2.

[0095] Test example This test example tests the sensitivity, specificity, sensitivity and long-term storage stability of the kits provided in Examples 1 to 2 and Comparative Examples 1 to 14. The specific test methods and test results are as follows: 1. Sensitivity: The human anti-Mycoplasma pneumoniae IgM antibodies (MP-IgM) of different titers were detected using the kits provided in Example 1 and Comparative Examples 1 to 7. The MP-IgM standard was diluted with negative serum or negative plasma. Each titer was tested 3 times. The detection method was described in Example 3. The titer gradient and the detection results are shown in Table 2.

[0096] Table 2 MP-IgM detection sensitivity

[0097] Note: “+” indicates a positive test result, and “-” indicates a negative test result.

[0098] The human anti-Mycoplasma pneumoniae IgG antibodies (MP-IgG) of different titers were detected using the kits provided in Example 2 and Comparative Examples 8 to 14. The MP-IgG standard was diluted with negative serum or negative plasma. Each titer was tested 3 times. The detection method was described in Example 4. The titer gradient and the detection results are shown in Table 3.

[0099] Table 3 MP-IgG detection sensitivity

[0100] Note: “+” indicates a positive test result, and “-” indicates a negative test result.

[0101] From the results in Table 2, we can see that: 1) Compared with Example 1, Comparative Example 1 uses specific indicator red blood cells instead of specific latex microspheres, and its detection sensitivity is basically the same as that of Example 1; 2) Compared with Example 1, Comparative Example 2 omitted the streptavidin-biotin amplification system, resulting in a reduction in the overall size of the latex microsphere-antigen-antibody complex, so the sensitivity of Comparative Example 2 was significantly lower than that of Example 1; 3) Compared with Example 1, since the particle size of the colored latex microspheres is reduced in Comparative Example 3, the overall size of the latex microsphere-antigen-antibody complex is also reduced, so the sensitivity of Comparative Example 3 is significantly lower than that of Example 1; 4) Compared with Example 1, Comparative Example 4 increases the particle size of the colored latex microspheres, resulting in an increase in the overall size of the latex microsphere-antigen-antibody complex. Therefore, Comparative Example 4 can form a larger volume of latex microsphere-antigen-antibody complex, and its sensitivity is equivalent to that of Example 1; 5) Compared with Example 1, the gels of Comparative Examples 5 and 6 are affected in their specific latex microsphere aggregation due to the lack of PEG20000 or S9, which further leads 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 antigen-antibody to approach each other; while S9 changes the surface charge distribution of the antigen-antibody, so that the antigen-antibody active protein binding site is fully exposed; when the active binding site of the specific antigen-antibody is fully exposed or the distance becomes closer, the binding probability becomes greater and the size of the agglutination block also increases; therefore, the sensitivity of Comparative Examples 5 and 6 is lower than that of Example 1; 6) Compared with Example 1, Comparative Example 7 omits PEG20000 and S9, which affects the aggregation of specific latex microspheres and further reduces the retention rate of latex microsphere-antigen-antibody complex. There is no PEG20000 to reduce the surface tension of the gel medium, increase the kinetics of the antigen-antibody reaction, and promote the approach of the antigen and antibody to each other. There is also no S9 to change the surface charge distribution of the antigen and antibody, so that the active protein binding sites of the antigen and antibody are fully exposed, resulting in a lower antigen-antibody binding probability than in Example 1 with the addition of PEG20000 and S9. Therefore, the sensitivity of Comparative Example 7 is significantly lower than that of Example 1.

[0102] According to the results in Table 3, the variation trends of the corresponding test results of Example 2 and Comparative Examples 8 to 14 are consistent with the variation trends of the test results shown in Table 2. The analysis of the results can refer to the analysis of Table 2 above.

[0103] 2. Specificity and Sensitivity: 500 clinical samples known to be MP-IgM negative and 200 clinical samples known to be MP-IgM positive were tested using the kits provided in Example 1 and Comparative Examples 1 to 7. Each sample was tested once. The detection method is described in Example 3; wherein, sensitivity (true positive rate) = number of positive samples detected / total number of known positive samples × 100%, specificity (true negative rate) = number of negative samples detected / total number of known negative samples × 100%; total compliance rate = (number of true positive samples + number of true negative samples) / total number of samples × 100%; the results are shown in Table 4.

[0104] Table 4 Specificity and sensitivity of MP-IgM detection

[0105] 500 clinical samples known to be MP-IgG negative and 200 clinical samples known to be MP-Ig positive were tested using the kits provided in Example 2 and Comparative Examples 8 to 14. Each sample was tested once. The detection method is described in Example 4; wherein, sensitivity (true positive rate) = number of positive samples detected / total number of known positive samples × 100%, specificity (true negative rate) = number of negative samples detected / total number of known negative samples × 100%; total compliance rate = (number of true positive samples + number of true negative samples) / total number of samples × 100%; the results are shown in Table 5.

[0106] Table 5 Specificity and sensitivity of MP-IgG detection

[0107] From the results in Table 4, we can see that: 1) Compared with Example 1, Comparative Example 1 uses specific indicator red blood cells instead of specific latex microspheres, and its detection specificity and sensitivity are basically the same as those of Example 1; 2) Compared with Example 1, since the streptavidin-biotin signal amplification system is omitted in Comparative Example 2, the overall size of the latex microsphere-antigen-antibody complex is reduced, and therefore the sensitivity of Comparative Example 2 is significantly lower than that of Example 1; 3) Compared with Example 1, since the particle size of the colored latex microspheres is reduced in Comparative Example 3, the overall size of the latex microsphere-antigen-antibody complex is also reduced, so the sensitivity of Comparative Example 3 is significantly lower than that of Example 1; 4) Compared with Example 1, Comparative Example 4 increases the particle size of the colored latex microspheres, resulting in an increase in the overall size of the latex microsphere-antigen-antibody complex. Therefore, Comparative Example 4 can detect a lower titer of target antibody, and its sensitivity is higher than that of Example 1, but the probability of false positives will increase, so the specificity is lower than that of Example 1; 5) Compared with Example 1, the gels of Comparative Examples 5 and 6 are affected in their specific latex microsphere aggregation due to the lack of PEG20000 or S9, which further leads 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 antigen-antibody to approach each other; and S9 changes the surface charge distribution of the antigen-antibody, so that the antigen-antibody active protein binding site can be fully exposed; when the active binding site of the specific antigen-antibody is fully exposed, the closer the distance, the greater the binding probability, and the size of the agglutination block also increases; therefore, the sensitivity of Comparative Examples 5 and 6 is lower than that of Example 1; 6) Compared with Example 1, Comparative Example 7 omits PEG20000 and S9, which affects the aggregation of specific latex microspheres and further reduces 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 antigen-antibody to approach each other; while S9 changes the surface charge distribution of the antigen-antibody, so that the active protein binding sites of the antigen-antibody are fully exposed; when the active binding sites of the specific antigen-antibody are fully exposed, the closer the distance, the greater the probability of binding, and the size of the agglutination block also increases; therefore, the sensitivity of Comparative Example 7 is significantly lower than that of Example 1.

[0108] According to the results in Table 5, the variation trends of the corresponding test results of Example 2 and Comparative Examples 8 to 14 are consistent with the variation trends of the test results shown in Table 4. The analysis of the results can refer to the analysis of Table 4 above.

[0109] 3. Long-term storage stability: The test kits provided in Example 1 and Comparative Examples 1 to 7 were stored at 2 to 8° C. for 14 months. During this period, samples M1, M2, M3 and M4 were tested at 8 time points using the test kits of Example 1 and Comparative Examples 1 to 7, respectively. Among them, samples M1, M2 and M3 were derived from samples with titers of 1:128, 1:1024 and 1:4096 in the test example MP-IgM sensitivity test, respectively, and M4 was an MP-IgM negative sample in the clinical sample verification. The test time points and corresponding results are shown in Table 6.

[0110] Table 6 Stability of MP-IgG detection kit

[0111] The kits provided in Example 2 and Comparative Examples 8 to 14 were stored at 2 to 8° C. for 14 months. During this period, samples G1, G2, G3 and G4 were tested at 8 time points using the kits provided in Example 2 and Comparative Examples 8 to 14, respectively. Samples G1, G2 and G3 were derived from samples with titers of 1:64, 1:512 and 1:2048 in the MP-IgG sensitivity test of the test example, respectively, and G4 was an MP-IgG negative sample in the clinical sample verification. The test time points and corresponding results are shown in Table 7.

[0112] Table 7 Stability of MP-IgG detection kit

[0113] As shown in Table 6, 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 Examples 1 and 8 is only 8 months, and Comparative Examples 5, 7, 12 and 14 have 12 months; and Comparative Examples 4 and 11 have aggregation of latex microspheres in the 10th month as the storage time increases, 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.

[0114] In summary, the detection kit based on specific latex microspheres and microinjection gel provided by the present invention has good sensitivity, specificity and specificity in detecting MP-IgM and MP-IgG, and its comprehensive performance is significantly better than that of the comparative example; the streptavidin-biotin coupling system provided by the present invention has better amplification effect than the existing anti-human globulin coupled human immunoglobulin; the latex microspheres with a particle size of 10 μm selected by the present invention are the optimal solution, and their sensitivity, specificity and specificity are optimal, and the total compliance 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.

[0115] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. 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 specific latex microsphere, characterized in that: The specific latex microspheres are colored latex microspheres coupled with specific antigens or antibodies; The particle size of the colored latex microspheres is 8-18 μm.

2. The specific latex microspheres according to claim 1, characterized in that: The surface of the specific latex microspheres is also connected to a blocking agent; And / or, the coupling method includes EDC-NHS coupling.

3. A kit, characterized in that: The kit comprises: the specific latex microspheres and the specific microcolumn gel card according to claim 1 or 2; the specific latex microspheres are used to be mixed with the sample to be tested and added to the specific microcolumn gel card for detection; The specific microcolumn gel card comprises a fixing plate and a gel tube, wherein the gel tube contains a specific gel; The specific gel includes (a) to (d): (a) biotinylated specific antigen or antibody; (b) streptavidin; (c) gel particles; (d) Gel medium.

4. The kit according to claim 3, characterized in that The volume of the specific gel accounts for the volume of the gel tube. ; The gel particles are selected from any one of dextran gel particles, glass microbeads 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 containing sodium chloride, bovine serum albumin, surfactant S9, disodium EDTA, PEG20000, sucrose and sodium azide.

5. The method for preparing the kit according to claim 3 or 4, characterized in that: The preparation method comprises the following steps: S1. Preparing specific latex microspheres: coupling specific antigens or antibodies with colored latex microspheres and performing blocking treatment to obtain specific latex microspheres; S2. Preparing a biotinylated specific antigen or antibody: coupling the specific antigen or antibody with NHS-biotin to obtain a biotinylated specific antigen or antibody; S3. Preparation of specific gel: Mixing the gel particles, streptavidin, gel medium and the biotinylated specific antigen or antibody obtained in step S2 to obtain a specific gel; S4. Fill the specific gel obtained in step S3 into a 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 constitute a kit.

6. The preparation method according to claim 5, characterized in that: The coupling method in step S1 includes NHS-EDC covalent coupling; 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 in step S3 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; The mass volume ratio of the gel particles to the gel medium in step S3 is 1: (12-16) g / mL.

7. A method for detecting antigens or antibodies against pathogenic microorganisms, characterized in that: The detection method adopts the kit described in claim 3 or 4 for detection.

8. The detection method according to claim 7, characterized in that: The detection method comprises the following steps: The specific latex microspheres and the sample to be tested are mixed in equal volumes and added into a gel tube for reaction to obtain a specific latex microsphere complex, and then the reaction system is centrifuged and the result is finally interpreted; The reaction temperature is 35-40°C; The reaction time is 8 to 20 min.

9. The detection method according to claim 8, characterized in that: The criteria for interpretation of the results include: (1) The specific latex microsphere complex is located on the surface of the specific gel and is a 4+ strong positive reaction; (2) The specific latex microsphere complex is suspended in a specific gel, with a positive reaction of 1+ to 3+, and 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 completely deposited at the bottom of the specific gel, which is a negative result.

10. Use of the kit according to claim 3 or 4 or the detection method according to any one of claims 7 to 9 in the following (I) to (III): (I) Preparation of products for blood typing; (II) preparing products for use in the detection of autoantibodies in autoimmune diseases; (III) Preparation of antigen or antibody detection products for microbial pathogens.

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