A matrix metalloproteinase-3 latex detection kit and preparation method thereof

Abstract

The invention discloses a matrix metalloproteinase-3 latex detection kit and a preparation method thereof, which is composed of the following raw materials: reaction solution 1, reaction solution 2 and matrix metalloproteinase-3 calibrator; reaction solution 1 includes the following components: 95-100g buffer, 0.9-1.8g electrolyte, 0.5-1g sensitizer, 0.1-0.5g stabilizer, 0.2-0.8g anti-interference agent and 0.2-0.4g antibacterial agent; reaction solution 2 is 10-20mL of storage solution of latex microspheres containing surface-coupled matrix metalloproteinase-3 monoclonal antibody, and the matrix metalloproteinase-3 monoclonal antibody is sheep monoclonal antibody and / or rabbit monoclonal antibody; the mass fraction of latex microspheres in the storage solution is 0.1%-2%. The kit of the present invention has strong specificity, wide linear range, high sensitivity, high RF anti-interference performance and long-term stability, and is suitable for analysis and detection of biochemical instruments.

Description

Technical Field

[0001] The invention belongs to the technical field of in vitro diagnostic kits, and in particular relates to a matrix metalloproteinase-3 latex detection kit and a preparation method thereof. Background Art

[0002] Matrix metalloproteinases (MMPs) are a family of more than 25 zinc-dependent enzymes that are primarily involved in cell migration, tissue remodeling, cancer invasion, and metastasis. In addition to degrading extracellular matrix proteins, MMPs are also important for the release and activation of growth factors and cytokines. At the same time, they can inactivate inflammatory mediators and enzymes themselves through protein degradation. MMPs are divided into the following categories based on their functions and substrates: (1) Gelatinases: including MMP-2 and MMP-9; (2) Collagenases: MMP-1, MMP-8, and MMP-13; (3) Matrix degrading enzymes: MMP-3, MMP-10, MMP-11, and MMP-7. Among them, MMP-3 has a wide range of substrates and is expressed in many cells, making it an important member of the MMPs family. The MMP-3 gene is part of the MMP gene cluster located on chromosome 11q22.3, with a molecular weight of 53977 Da. MMP-3 is composed of 477 amino acids and is divided into five domains, including a degradable hydrophobic signal peptide region, a propeptide region that is cleaved and modified when activated, a catalytic active region containing a zinc ion binding site, a hinge region rich in proline, and a hemobindone region. The MMP-3 proenzyme removes the propeptide region under the action of the serine protease, trypsin, to form activated MMP-3.

[0003] The pathological development of rheumatoid arthritis (RA) requires the participation of multiple cells, including chondrocytes, T cells, fibroblast-like synoviocytes and macrophages. Among them, fibroblast-like synoviocytes play a key role. Fibroblast-like synoviocytes can release MMP-3 into the synovial fluid, and MMP-3 belongs to the matrix degrading enzyme class that can degrade aggrecan, collagen II, collagen IX, collagen X, and other proteins that make up the joints. By analyzing the degradation products of proteoglycans in the synovial fluid of RA patients, it was found that its core protein target is highly sensitive to MMP-. At the same time, MMP-3 can also activate other MMPs zymogens, such as MMP-1 zymogen, MMP-8 zymogen, MMP-9 zymogen and MMP-13 zymogen, forming an amplified waterfall effect. Therefore, MMP-3 is regarded as an important mediator molecule in the pathological progression of RA.

[0004] In the early stages of RA, a large number of fibroblast-like synoviocytes and chondrocytes synthesize and release MMP-3 into the synovial fluid and blood. Studies have shown that the concentration of MMP-3 in the synovial fluid of RA patients is significantly higher than that of osteoarthritis patients and normal people. At the same time, the concentration of MMP-3 in the blood of RA patients is also higher than that of osteoarthritis patients and normal people. Therefore, MMP-3 can be used as an indicator for monitoring the activity of early RA, which can provide more accurate disease staging; reduce the number of imaging examinations for patients; reflect the efficacy of drug treatment and provide doctors with more accurate disease progression information; dynamic monitoring of MMP-3 levels can effectively predict the progression of the patient's disease. MMP-3 research and application are very extensive, and Japan has already used MMP-3 as a routine screening item for early RA.

[0005] At present, the main indicators for rheumatoid inflammation detection on the market are C reactive protein (CRP), cyclic citrulline polypeptide (CCP), rheumatoid factor (RF) and anti-mutant citrulline vimentin (MCV). Compared with these markers, MMP-3 shows a more sensitive reaction. At present, the commonly used methods for determining MMP-3 are enzyme-linked immunosorbent assay and immuno-latex turbidimetry. The enzyme-linked immunosorbent assay has low specificity and sensitivity, and the operation is cumbersome and the detection time is long. The specificity and sensitivity of latex immuno-turbidimetry are greatly improved, and it has attracted much attention from the market. In order to obtain higher sensitivity and a wide linear range, most manufacturers use monoclonal antibodies and large-sized latex particles (greater than 200nm). Large-sized latex particles will increase the gravity of the latex, causing the reagent to easily aggregate and settle, making the test stability of the reagent very poor. On the other hand, the reagent will have a lot of interference from rheumatoid factors, resulting in inaccurate test results. Currently, there are widespread problems with the stability and anti-rheumatoid factor interference of this project on the market.

[0006] Therefore, it is very important to develop a matrix metalloproteinase-3 latex detection kit with a wide detection range, high sensitivity, good stability and resistance to rheumatoid factor interference for clinical detection. Summary of the invention

[0007] The present invention aims at the deficiencies of the matrix metalloproteinase-3 latex detection kit in the prior art, and provides a stable matrix metalloproteinase-3 latex detection kit.

[0008] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0009] A matrix metalloproteinase-3 latex detection kit, characterized in that it is composed of the following raw materials: reaction solution 1, reaction solution 2, and matrix metalloproteinase-3 calibrator, which provide a stable biochemical environment for the target antigen or antibody to be detected and resist rheumatoid factor interference;

[0010] The reaction solution 1 comprises the following components: 95-100 g of buffer, 0.9-1.8 g of electrolyte, 0.5-1 g of sensitizer, 0.1-0.5 g of stabilizer, 0.2-0.8 g of anti-interference agent and 0.2-0.4 g of antibacterial agent;

[0011] The reaction solution 2 is 10 to 20 mL of a storage solution containing latex microspheres with surface-coupled matrix metalloproteinase-3 monoclonal antibodies, wherein the matrix metalloproteinase-3 monoclonal antibodies are sheep monoclonal antibodies and / or rabbit monoclonal antibodies; the mass fraction of the latex microspheres in the storage solution is 0.1% to 2%; preferably, the reaction solution 2 is a mixed storage solution of latex microspheres with a particle size of 60 to 100 nm and surface-coupled sheep monoclonal antibodies and latex microspheres with a particle size of 150 to 200 nm and surface-coupled rabbit monoclonal antibodies, and the mass fraction of each of the latex microspheres in the storage solution is 0.05% to 1%;

[0012] The matrix metalloproteinase-3 calibrator is prepared from the following ingredients: 95-100 g buffer, 0.9-1.8 g electrolyte, 0.1-0.5 g surfactant, 0.1-0.5 g stabilizer and 0.2-0.4 g antibacterial agent; after mixing, 10-30 μL matrix metalloproteinase-3 is added, and stirred at a speed of 500-1000 rpm for 10-30 min to obtain a calibrator;

[0013] The storage solution is composed of the following components: 95-100 g of 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer with a concentration of 50-100 mmol / L and a pH value of 7.0-7.5, 0.9-1.8 g of sodium chloride, 0.1-0.5 g of Tween 20, 0.1-0.5 g of a stabilizer, 0.2-0.4 g of an antibacterial agent, and 0.1-0.3 g of betaine;

[0014] The buffer is at least one of a tris (hydroxymethyl) aminomethane (Tris) buffer, a 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer, a 2- (N-morpholino)ethanesulfonic acid (MES) buffer, and a 3-morpholine-2-hydroxypropanesulfonic acid (MOPSO) buffer with a concentration of 50 to 100 mmol / L and a pH value of 7.0 to 7.5; the sensitizer is at least one of polyethylene glycol 6000, polyethylene glycol 35000, and dextran; the electrolyte is at least one of sodium chloride, magnesium chloride, and calcium chloride; the surfactant is at least one of Tween-20 and ethylenediaminetetraacetic acid.2 sodium (EDTA.2Na);

[0015] The anti-interference agent is at least one of hexamercaptohexanol, amino acids, and bovine serum albumin, preferably hexamercaptohexanol, which can block the active site of rheumatoid factor, prevent the rheumatoid factor in the serum sample from reacting with the MMP-3 antibody, and improve the anti-RF interference performance of the kit;

[0016] The stabilizer is at least one of an ionic liquid and Triton-100, preferably an ionic liquid; the ionic liquid is one or more of tetramethylguanidine hydrochloride, 1-decyl-3-methylimidazolium chloride, 1-ethyl-3-methylimidazolium trifluoromethanesulfonate, and tributylmethylammonium chloride; more preferably 1-decyl-3-methylimidazolium chloride;

[0017] The antibacterial agent is at least one of sodium azide and polydopamine modified core-shell structure ZnO metal organic framework nanoparticles, preferably ZnO@PDA@ZIF-7 composite nanoparticles;

[0018] The reaction solution 1 is prepared by the following steps: 95-100 g of tris (hydroxymethyl) aminomethane (Tris) buffer, 0.9-1.8 g of electrolyte, 0.1-0.5 g of stabilizer, 0.2-0.8 g of anti-interference agent and 0.2-0.4 g of antibacterial agent are mixed, and the reaction solution 1 is obtained by stirring at a speed of 800-2000 rpm for 20-40 min.

[0019] The reaction solution 2 is prepared by the following steps: adding latex microspheres to a buffer solution and stirring, first adding an activator, then adding a matrix metalloproteinase-3 monoclonal antibody, continuing to stir the reaction, then adding a blocking agent and stirring until the reaction is completed, separating to obtain an antibody-latex microsphere complex, and then adding a storage solution; mixing antibody-latex microspheres with a particle size of 60 to 100 nm and antibody-latex microspheres with a particle size of 150 to 200 nm in a mass ratio of 1: (0.8 to 1.2) and then standing to obtain a reaction solution 2.

[0020] More specifically, the reaction solution 2 is prepared by the following steps:

[0021] (1) Preparation of antibody-latex microspheres: 0.5-1 mL of latex microspheres with a particle size of 60-100 nm are added to 5-10 mL of buffer, and stirred at a speed of 200-1000 rpm for 5-15 min. Then, an appropriate amount of activator is added and stirred for 10-30 min to activate the carboxyl groups on the surface of the latex microspheres; then, 0.1-1 mg of matrix metalloproteinase-3 monoclonal antibody is added, and the stirring reaction is continued for 1-3 h; then, 10-50 uL of blocking agent is added, and the stirring reaction is continued for 0.5-1 h; then, tangential flow filtration is used for separation, and 10-20 mL of storage solution is added to obtain an antibody-latex microsphere storage solution with a particle size of 60-100 nm; the antibody-latex microsphere storage solution with a particle size of 150-200 nm is obtained by the same method;

[0022] (2) Preparation of reaction solution 2: The antibody-latex microsphere stock solution with a particle size of 60 to 100 nm and the antibody-latex microsphere stock solution with a particle size of 150 to 200 nm are mixed and allowed to stand for 12 to 48 hours to obtain reaction solution 2.

[0023] The activator is an aqueous solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; the blocking agent is at least one of a CE510 blocking agent and a DB1130 blocking agent; the matrix metalloproteinase-3 monoclonal antibody is a sheep monoclonal antibody and / or a rabbit monoclonal antibody; preferably, latex microspheres with a particle size of 60 to 100 nm are coupled to sheep monoclonal antibodies, and latex microspheres with a particle size of 150 to 200 nm are coupled to rabbit monoclonal antibodies.

[0024] The preparation steps of the antibody-latex microspheres of the present invention can be mainly divided into: activation of the reaction sites of the latex microspheres, coupling of the antibody and the latex microspheres, blocking of the unreacted active sites of the latex microspheres, separation of the unreacted substances and the antibody-microsphere complex, and recovery of the antibody-microsphere complex.

[0025] The reaction solution 1 of the present invention is a diluent, which provides a stable and anti-rheumatoid factor-interference biochemical environment for the target antigen or antibody to be detected, so that the target antigen or antibody maintains a natural conformation and controls the time and rate of the reaction to reach the end point. The reaction solution 2 is a uniform milky white liquid containing monoclonal antibody coated latex microspheres containing matrix metalloproteinase-3, which can specifically react with a certain concentration of target antigen to make the system solution turbid and larger; the size of the latex microspheres will affect the sensitivity and linear range of the detection. Generally, latex microspheres larger than 150nm can provide better sensitivity, and latex microspheres of 60 to 150nm can provide a wider linear range. The surface of the latex microspheres has an appropriate amount of carboxyl groups, which can be chemically coupled with antibodies with amino groups by amide reaction. The matrix metalloproteinase-3 calibrator is an antigen used for comparison and calculation with a sample, containing a certain concentration of natural matrix metalloproteinase-3 antigen and antigen diluent, which can maintain the natural conformation of matrix metalloproteinase-3 and provide a stable reaction environment for antigen and antibody.

[0026] The present invention uses latex microspheres A with a particle size of 80 nm coupled with a sheep monoclonal antibody with a wide linear range and latex microspheres B with a particle size of 150 nm coupled with a rabbit monoclonal antibody with high sensitivity, and combines the characteristics of the latex microspheres themselves with the characteristics of the antibodies to further provide a wide linear range and high detection sensitivity for the kit of the present invention. The present invention uses ionic liquids as stabilizers. Since ionic liquids have unique physical, chemical and biological properties, such as high stability, low volatility, good solubility, high conductivity and good amphiphilicity, compared with the prior art, the kit of the present invention is not only green and environmentally friendly, but also has higher stability and can maintain high precision after being placed for a long time.

[0027] Preferably, the ZnO@PDA@ZIF-7 composite nanoparticles of the present invention are prepared by the following method:

[0028] (1) dispersing 0.1-0.5 g of ZIF-7 nanoparticles in 5-12 g of methanol solution, then adding 0.1-0.5 g of dopamine hydrochloride to form a mixed solution, and then stirring the mixed solution in a water bath at 60-75° C. at a speed of 800-1500 rpm for 8-12 h, centrifuging after completion, washing with ethanol three times, and drying at 80° C. for 15-30 h to obtain PDA@ZIF-7 nanoparticles;

[0029] (2) 0.9-1.8 g of the PDA@ZIF-7 nanoparticles were dispersed in 8-14 g of methanol, and then 12-20 g of 10 mmol / L zinc nitrate methanol solution was added. After stirring at 1000-1500 rpm for 30-60 min, 0.9-1.8 g of 0.01 mol / L sodium hydroxide methanol solution was added, and the reaction was continued for 30-50 min. After the reaction was completed, the mixture was centrifuged, washed with methanol three times, and dried at 60° C. for 20-30 h to obtain ZnO@PDA@ZIF-7 composite nanoparticles.

[0030] Furthermore, the preparation method of the ZIF-7 nanoparticles is: add 0.5-1.5g benzimidazole and 1.5-3.0g zinc nitrate to 8.0-12.0g N,N-dimethylformamide, stir at a speed of 500-1000rpm for 12-36h, centrifuge after reaction, wash three times with methanol, and dry at 80°C for 15-30h to obtain ZIF-7 nanoparticles.

[0031] The metal organic framework ZIF-7 has become the most representative material due to its good biocompatibility, simple synthesis, large loading capacity, and easy modification. Among them, the benzimidazole in ZIF-7 has certain antibacterial activity and can be used as an anti-inflammatory, antibacterial and antifungal material. Dopamine hydrochloride (HDA) is a structural model of mussel adhesion protein with high biocompatibility and strong surface adhesion. HDA has abundant catechol and amino functional groups, and self-polymerizes under alkaline conditions to form a polydopamine (PDA) shell that can adhere to a variety of materials. ZnO nanoparticles have good biocompatibility, high broad-spectrum antibacterial activity and long-term stability under extreme conditions.

[0032] The present invention connects ZIF-7 and ZnO, which have good biocompatibility and antibacterial activity, through dopamine with strong adhesion to prepare ZnO@PDA@ZIF-7 composite nanoparticles, which are used as antibacterial agents. The ZnO@PDA@ZIF-7 composite nanoparticles not only have excellent antibacterial properties but also have good biocompatibility, thereby further improving the long-term stability of the test kit.

[0033] The inventive concept of the present invention is: using latex enhanced immunoturbidimetry to quantitatively detect matrix metalloproteinase-3, the detection principle is to use amide reaction to couple antibodies with latex microspheres to form antibody-microsphere complexes; when a certain concentration of matrix metalloproteinase-3 is contained in the sample to be tested, the matrix metalloproteinase-3 antigen will form a larger antigen-antibody-microsphere complex with the antibody-microsphere complex in the reagent; under a certain concentration of electrolyte, the antigen-antibody-microsphere complex will be interwoven with adjacent antibody-latex particles through the electrostatic attraction between oligonucleotides to form a network structure, which can cause a significant change in the turbidity of the reaction system; when the antibody in the reaction system is kept in excess, the change in the turbidity of the system after the reaction is proportional to the amount of matrix metalloproteinase-3, and quantitative analysis can be performed based on the matrix metalloproteinase-3 calibration curve.

[0034] The present invention has the following beneficial effects:

[0035] (1) The present invention adopts tangential flow centrifugation technology, which reduces production costs, improves the efficiency of reagent production, effectively reduces the differences caused by different batches of reaction solutions during the centrifugal blowing process, and greatly reduces the batch difference of reagents. (2) The present invention uses ionic liquid as a stabilizer, which is not only green, environmentally friendly and pollution-free, but also has higher stability. (3) If ZnO@PDA@ZIF-7 composite nanoparticles are used as antibacterial agents, not only are the antibacterial properties excellent, but also the biocompatibility is good, which can further improve the long-term stability of the test kit. The test kit prepared by the present invention has strong specificity, a wide linear range, high sensitivity, high RF anti-interference performance and long-term stability, and is suitable for analysis and detection of biochemical instruments. DETAILED DESCRIPTION

[0036] The present invention is described in detail below with reference to specific embodiments. Those skilled in the art should recognize that the embodiments are only used to illustrate the present invention, and are not intended to limit the present invention, and that any changes or modifications to the embodiments within the scope of the present invention are within the scope of the claims of the present invention.

[0037] All the raw materials used in this implementation are commercially available products.

[0038] Latex microsphere A with a particle size of 80 nm, JSR Co., Ltd., Japan; latex microsphere B with a particle size of 150 nm, JSR Co., Ltd., Japan. (3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, CAS No.: 7084-11-9, Xinnuoda Biotechnology Co., Ltd. Betaine, No.: 16, Shaanxi Chenming Biotechnology Co., Ltd. 1-Decyl-3-methylimidazolium chloride, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences.

[0039] Example 1

[0040] A matrix metalloproteinase-3 latex detection kit is composed of the following raw materials: reaction solution 1, reaction solution 2 and calibration substance:

[0041] Reaction solution 1 was prepared by the following steps: 100 g of 100 mmol / L Tris buffer solution with a pH of 7.4, 0.9 g of sodium chloride, 0.5 g of polyethylene glycol 6000, 0.3 g of a stabilizer and 0.3 g of an antibacterial agent were mixed, and the mixture was stirred at 1000 rpm for 30 min to obtain reaction solution 1.

[0042] Reaction solution 2 was prepared by the following steps: (1) Preparation of antibody-latex microspheres: 0.5 mL of latex microspheres A with a particle size of 80 nm was added to 5 g of Tris buffer with a concentration of 100 mmol / L and a pH of 7.4, and stirred at 500 rpm for 10 min. Then, 50 uL of 10 mg / L 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride aqueous solution was added and stirred for 15 min. Then, 0.5 mg of sheep monoclonal antibody was added and the stirring reaction continued for 2 h. Then, 30 uL of CE510 blocking agent was added and the stirring reaction continued for 30 min. After the reaction was completed, tangential flow filtration was used for separation, and then 10 mL of storage solution was added to obtain sheep antibody-latex microspheres A. The same steps were repeated to obtain sheep antibody-latex microspheres B (particle size 150 nm). (2) Preparation of reaction solution 2: 10 mL of sheep antibody-latex microspheres A and 10 mL The sheep antibody-latex microsphere B was mixed and allowed to stand for 24 hours to obtain reaction solution 2.

[0043] The stock solution is composed of the following materials: 100 g HEPES buffer with a concentration of 100 mmol / L and a pH of 7.4, 1.0 g sodium chloride, 0.2 g Tween 20, 0.4 g stabilizer, 0.5 g antibacterial agent and 0.3 g betaine.

[0044] The calibrator was prepared by the following steps: 100 g of 100 mmol / L Tris buffer with a pH of 7.4, 1.3 g of sodium chloride, 0.3 g of Tween 20, 0.5 g of a stabilizer and 0.3 g of an antimicrobial agent were mixed, and then 10 μL of matrix metalloproteinase-3 was added, and the mixture was stirred at 800 rpm for 20 min to obtain a calibrator.

[0045] The stabilizer is 1-decyl-3-methylimidazolium chloride; the antibacterial agent is sodium azide.

[0046] Example 2

[0047] The method is basically the same as Example 1, except that the reaction solution 2 is prepared by the following steps:

[0048] (1) Preparation of antibody-latex microspheres: 0.5 mL of latex microspheres A with a particle size of 80 nm was added to 5 g of Tris buffer with a concentration of 100 mmol / L and a pH of 7.4, and stirred at 500 rpm for 10 min. Then, 50 uL of 10 mg / L 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride aqueous solution was added and stirred for 15 min. Then, 0.5 mg of rabbit monoclonal antibody was added and the stirring reaction continued for 2 h. Then, 30 uL of CE510 blocking agent was added and the stirring reaction continued for 30 min. After the reaction, tangential flow filtration was used for separation, and then 10 mL of storage solution was added to obtain rabbit antibody-latex microspheres A. The same steps were repeated to obtain rabbit antibody-latex microspheres B (particle size 150 nm). (2) Preparation of reaction solution 2: 10 mL of rabbit antibody-latex microspheres A and 10 mL of rabbit antibody-latex microspheres B were mixed and allowed to stand for 24 h to obtain reaction solution 2.

[0049] Example 3

[0050] The method is basically the same as Example 1, except that the reaction solution 2 is prepared by the following steps:

[0051] (1) Preparation of antibody-latex microspheres: 0.5 mL of latex microspheres A with a particle size of 80 nm was added to 5 g of Tris buffer with a concentration of 100 mmol / L and a pH of 7.4, and stirred at 500 rpm for 10 min. Then, 50 uL of 10 mg / L 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride aqueous solution was added and stirred for 15 min. Then, 0.5 mg of rabbit monoclonal antibody was added and the stirring reaction continued for 2 h. Then, 30 uL of CE510 blocking agent was added and the stirring reaction continued for 30 min. After the reaction, tangential flow technology was used for separation. Then, 10 mL of storage solution was added to obtain rabbit antibody-latex microspheres A: 0.5 mL of latex microspheres B with a particle size of 150 nm was added to 5 g of Tris buffer with a concentration of 100 mmol / L and a pH of 7.4, and stirred at 500 rpm for 10 min. Then, 50 uL of 10 mg / L The 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride aqueous solution was stirred for reaction for 15 minutes, and then 0.5 mg of sheep monoclonal antibody was added, and the stirring reaction was continued for 2 hours, and then 30 uL CE510 blocking agent was added, and the stirring reaction was continued for 30 minutes. After the reaction, tangential flow technology was used for separation, and then 10 mL of storage solution was added to obtain sheep antibody-latex microspheres B; (2) Preparation of reaction solution 2: 10 mL of rabbit antibody-latex microspheres A and 10 mL of sheep antibody-latex microspheres B were mixed and allowed to stand for 24 hours to obtain reaction solution 2.

[0052] Example 4

[0053] The method is basically the same as Example 1, except that the reaction solution 2 is prepared by the following steps:

[0054] (1) Preparation of antibody-latex microspheres: 0.5 mL of latex microspheres A with a particle size of 80 nm was added to 5 g of 100 mM Tris buffer (pH = 7.4), stirred at 500 rpm for 10 min, and then 50 uL of 10 mg / L 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride aqueous solution was added and stirred for 15 min, then 0.5 mg of sheep monoclonal antibody was added and the stirring reaction continued for 2 h, and then 30 uL of CE510 blocking agent was added and the stirring reaction continued for 30 min. After the reaction, tangential flow technology was used for separation, and then 10 mL of storage solution was added to obtain sheep antibody-latex microspheres A: 0.5 mL of latex microspheres B with a particle size of 150 nm was added to 5 g of 100 mM Tris buffer (pH = 7.4), stirred at 500 rpm for 10 min, and then 50 uL of A 10 mg / L aqueous solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was stirred for reaction for 15 min, and then 0.5 mg of rabbit monoclonal antibody was added and the stirring reaction continued for 2 h. Then 30 uL of CE510 blocking agent was added and the stirring reaction continued for 30 min. After the reaction, tangential flow technology was used for separation, and then 10 mL of storage solution was added to obtain rabbit antibody-latex microspheres B. (2) Preparation of reaction solution 2: 10 mL of sheep antibody-latex microspheres A and 10 mL of rabbit antibody-latex microspheres B were mixed and allowed to stand for 24 h to obtain reaction solution 2.

[0055] Control group 1

[0056] The method is basically the same as Example 4, except that the reaction solution 2 is prepared by the following steps:

[0057] (1) Preparation of antibody-latex microspheres: 0.5 mL of latex microspheres A with a particle size of 80 nm was added to 5 g of 100 mM Tris buffer with a pH of 7.4, and stirred at 500 rpm for 10 min. Then, 50 uL of 10 mg / L 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride aqueous solution was added and stirred for 15 min. Then, 0.5 mg of sheep monoclonal antibody was added and the stirring reaction continued for 2 h. Then, 30 uL of CE510 blocking agent was added and the stirring reaction continued for 30 min. After the reaction, tangential flow technology was used for separation. Then, 10 mL of storage solution was added to obtain sheep antibody-latex microspheres A: 0.5 mL of latex microspheres B with a particle size of 150 nm was added to 5 g of 100 mM Tris buffer with a pH of 7.4, and stirred at 500 rpm for 10 min. Then, 50 uL of A 10 mg / L aqueous solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was stirred for 15 min, and then 0.5 mg of rabbit monoclonal antibody was added, and the stirring reaction continued for 2 h, and then 30 uL of CE510 blocking agent was added, and the stirring reaction continued for 30 min. After the reaction, the mixture was centrifuged at 8000 rpm in a centrifuge, and then 10 mL of storage solution was added to obtain rabbit antibody-latex microspheres B. (2) Preparation of reaction solution 2: 10 mL of sheep antibody-latex microspheres A and 10 mL of rabbit antibody-latex microspheres B were mixed and allowed to stand for 24 h to obtain reaction solution 2.

[0058] Example 6

[0059] It is basically the same as Example 4, except that the antibacterial agent is ZIF-7 nanoparticles.

[0060] The ZIF-7 nanoparticles are prepared as follows:

[0061] 1.0 g of benzimidazole and 2.0 g of zinc nitrate were added to 10 g of N,N-dimethylformamide, and the mixture was stirred at 800 rpm for 24 h. After the reaction, the mixture was centrifuged, washed three times with methanol, and dried at 80° C. for 24 h to obtain ZIF-7 nanoparticles.

[0062] Example 7

[0063] It is basically the same as Example 4, except that the antibacterial agent is ZnO nanoparticles.

[0064] The ZnO nanoparticles are prepared as follows:

[0065] 1.5 g of 10 mmol / L zinc nitrate methanol solution was stirred at 1200 rpm for 50 min, and then 0.2 g of 0.01 mol / L sodium hydroxide methanol solution was added. The reaction was continued for 40 min, and then centrifuged. The mixture was washed with methanol three times and dried at 60° C. for 24 h to obtain ZnO nanoparticles.

[0066] Example 8

[0067] It is basically the same as Example 4, except that the antibacterial agent is ZnO@PDA@ZIF-7 composite nanoparticles.

[0068] The ZnO@PDA@ZIF-7 composite nanoparticles are prepared as follows:

[0069] (1) 1.0 g of benzimidazole and 2.0 g of zinc nitrate were added to 10 g of N,N-dimethylformamide, and the mixture was stirred at 800 rpm for 24 h. After the reaction, the mixture was centrifuged, washed three times with methanol, and dried at 80° C. for 24 h to obtain ZIF-7 nanoparticles;

[0070] (2) dispersing 0.5 g of ZIF-7 nanoparticles obtained in step (1) in 10 g of methanol solution, and then adding 0.2 g of dopamine hydrochloride to form a mixed solution, and then stirring the mixed solution in a 70° C. water bath at a speed of 1000 rpm for 10 h, centrifuging after completion, washing with ethanol three times, and drying at 80° C. for 24 h to obtain PDA@ZIF-7 nanoparticles;

[0071] (3) 0.3 g of PDA@ZIF-7 nanoparticles obtained in step (2) were dispersed in 10 g of methanol, and then 15.0 g of 10 mmol / L zinc nitrate methanol solution was added. After stirring at 1200 rpm for 50 min, 0.2 g of 0.01 mol / L sodium hydroxide methanol solution was added, and the reaction was continued for 40 min. After completion, the mixture was centrifuged, washed three times with methanol, and dried at 60° C. for 24 h to obtain ZnO@PDA@ZIF-7 composite nanoparticles.

[0072] Example 9

[0073] The method is basically the same as Example 8, except that 0.2 g of hexamercaptohexanol anti-interference agent is added to the reaction solution 1.

[0074] Control group 2

[0075] It is basically the same as Example 4, except that: there is no stabilizer in the reaction solution 1, and there is no stabilizer in the storage solution in the reaction solution 2.

[0076] The following performance tests and evaluations were performed on Examples 1-9 and Control Groups 1-2, respectively:

[0077] Experimental instrument: HITACHI7180, MMP-3 reagent detection parameters are shown in Table 1.

[0078] (1) The appearance of reaction solution 2 should be a milky white solution without precipitation.

[0079] (2) Reagent blank absorbance: At the corresponding dominant wavelength, the reagent blank absorbance (A) ≤ 1.5.

[0080] (3) Sensitivity: The difference (△A) between the absorbance when testing 100 ng / mL of the analyte and the absorbance when testing the blank reagent.

[0081] (4) Linear range: The reagent is within the specified linear interval: calculate the linear correlation coefficient (r), generally r ≥ 0.990.

[0082] (5) Batch variation: When reagents from different batches are used to test the same sample, the relative range (R) between batches is calculated. Generally, R is ≤ 10.0. The smaller the R, the smaller the batch variation.

[0083] (6) Precision: Repeat the test on a sample 10 times and calculate the coefficient of variation (CV). Generally, CV is ≤10.0. The smaller the CV, the better the precision.

[0084] (7) Opening stability: After opening the vial, place the test kit in the reagent compartment of a biochemical analyzer (2-8°C) for more than 45 days. Test the quality control product every 7 days, three times each time, and calculate the relative deviation between the average value and the value on day 0. Generally, it should be ≤10.0.

[0085] (8) Thermally accelerated stability: The reagents were placed at 37°C for 8 days. The reagents were then taken out and the appearance of the reaction solution 2, reagent blank absorbance, sensitivity, linear range, batch difference and precision were evaluated.

[0086] (9) Long-term stability: Place the reagent at 2-8°C for 28 months and evaluate the R2 reagent properties, reagent blank absorbance, sensitivity, linear range, batch difference and precision every 4 months.

[0087] (10) Anti-rheumatoid factor interference test: Add different concentrations of RF to the serum samples, test each concentration sample three times, calculate the average value and make a relative deviation with the sample with RF added concentration of 0, which should generally be ≤10.0%.

[0088] Table 1 MMP-3 reagent detection parameters

[0089]

[0090]

[0091] Data results and analysis: The reagent performance of the embodiment at month 0 is shown in Table 2. The stability of the bottle opening of different embodiments is shown in Table 3. The thermal acceleration stability of different embodiments is shown in Table 4. The blank absorbance of long-term stability of different embodiments is shown in Table 5. The long-term stability (sensitivity) of different embodiments is shown in Table 6. The long-term stability (batch difference) of different implementation cases is shown in Table 7. The long-term stability (precision) of different embodiments is shown in Table 8. The long-term stability (linearity) of different implementation cases is shown in Table 9. The interference of anti-rheumatoid factor of different embodiments is shown in Table 10.

[0092] Table 2 Reagent performance at month 0 of different embodiments

[0093]

[0094] Table 3 Bottle opening stability of different embodiments

[0095]

[0096]

[0097]

[0098] Table 4 Thermal acceleration stability of different embodiments

[0099]

[0100]

[0101] Table 5 Long-term stability of different embodiments (blank absorbance)

[0102]

[0103]

[0104] Table 6 Long-term stability (sensitivity) of different embodiments

[0105]

[0106]

[0107] Table 7 Long-term stability of different implementation cases (batch difference)

[0108]

[0109] Table 8 Long-term stability (precision) of different embodiments

[0110]

[0111]

[0112] Table 9 Long-term stability (linearity) of different embodiments

[0113]

[0114]

[0115] Table 10 Anti-rheumatoid factor interference in different embodiments

[0116]

[0117]

[0118] It can be seen from the data in Table 2 that compared with Example 4, no stabilizer was added to reaction solution 1 and reaction solution 2 in control group 2, and precipitation occurred in reaction solution 2. The sensitivity, batch difference and precision were very poor, which did not meet the requirements, indicating that the addition of ionic liquid stabilizer greatly improved the stability of the reagent; the difference between Examples 1-4 is that the antibodies coupled to each latex particle are different, and no precipitation occurred in the R2 reagent, but the sensitivity, batch difference, precision and linear range of Example 4 are the best, indicating that the latex particles A and sheep monoclonal antibody are coupled, and the latex particles B and rabbit monoclonal antibody are coupled The performance is the best; the difference between Example 4 and Control Group 1 is that the particle dispersion method is different. Control Group 1 adopts the method of centrifugation + blowing, and Example 4 adopts the dispersion method of tangential flow. Its batch difference is lower than the batch difference of Control Group 1, indicating that the dispersion method of tangential flow can effectively reduce the batch difference; the difference between Implementation Cases 4 and 6-9 is that the antibacterial agents added are different, and the performance of Examples 8 and 9 is the best, indicating that the addition of ZnO@PDA@ZIF-7 composite nanoparticles can effectively inhibit bacteria and improve the performance of the reagent. From the data in Table 10, it can be concluded that the group in Example 9 adding hexamercaptohexanol can resist 400 IU / mL of RF interference, while the control group and other case groups can only resist 200 IU / mL of RF interference, indicating that the latex reagent of the present invention has the best ability to resist RF interference.

[0119] From the reagent opening data in Table 3, it can be concluded that the opening stability of the control group 2 is very poor, and the deviation of the control group 1 and other embodiment groups can meet the requirements after 49 days of opening. The opening stability of implementation cases 8 and 9 is the best, and the relative deviation is generally less than 3%, indicating that the addition of ionic liquid and ZnO@PDA@ZIF-7 composite nanoparticles can improve the opening stability of the reagent. It shows that the stability performance of the latex reagent provided by the present invention is better.

[0120] From the thermal acceleration data in Table 4, it can be concluded that the performance of the control group after thermal acceleration is the worst, the R2 reagent precipitates, the blank background increases rapidly, and the sensitivity, batch difference, precision and linearity cannot meet the requirements. The performance of implementation cases 8 and 9 not only meets the requirements, but also has the best performance in each indicator.

[0121] From the data in Tables 5-9, it can be concluded that the R2 properties, blank absorbance, sensitivity, batch difference, precision and linearity of the reagents in each embodiment after 32 months of storage can basically meet the requirements, among which the relative deviations of batch difference and precision of Examples 8 and 9 are generally less than 3%, and the performance is the best. The performance of the control group 1 is only poor in batch difference, and the other performances can basically meet the requirements, and the above performances of the control group 2 cannot meet the requirements.

[0122] In summary, the matrix metalloprotein-3 assay kit (latex immunoturbidimetry) of the present invention has good opening stability, heating stability, long-term stability and anti-RF interference performance, and the physical properties, blank absorbance, sensitivity, batch difference, precision, linearity and stability of the reagent can meet the requirements of clinical use.

Claims

1. A matrix metalloproteinase-3 latex detection kit, characterized in that: It is composed of the following raw materials: reaction solution 1, reaction solution 2 and matrix metalloproteinase-3 calibrator; The reaction solution 1 includes the following components: 95-100 g buffer, 0.9-1.8 g electrolyte, 0.5-1 g sensitizer, 0.3 g stabilizer, 0.2 g anti-interference agent and 0.3 g antibacterial agent; The reaction solution 2 is 10-20 mL of a storage solution containing latex microspheres with surface-coupled matrix metalloproteinase-3 monoclonal antibodies; the latex microspheres with surface-coupled matrix metalloproteinase-3 monoclonal antibodies are a mixture of latex microspheres with a particle size of 80 nm and surface-coupled sheep monoclonal antibodies or rabbit monoclonal antibodies and latex microspheres with a particle size of 150 nm and surface-coupled rabbit monoclonal antibodies or sheep monoclonal antibodies in a mass ratio of 1: (0.8-1.2), and the mass fraction of each of the latex microspheres in the storage solution is 0.05%-1%; The storage solution is composed of the following components: 95-100 g of 4-hydroxyethylpiperazineethanesulfonic acid buffer with a concentration of 50-100 mmol / L and a pH value of 7.0-7.5, 0.9-1.8 g of sodium chloride, 0.1-0.5 g of Tween 20, 0.4 g of a stabilizer, 0.5 g of an antibacterial agent, and 0.3 g of betaine; The anti-interference agent is hexamercaptohexanol; the stabilizer is 1-decyl-3-methylimidazole chloride; and the antibacterial agent is ZnO@PDA@ZIF-7 composite nanoparticles.

2. The matrix metalloproteinase-3 latex detection kit according to claim 1, characterized in that: The matrix metalloproteinase-3 calibrator is prepared from the following ingredients: 95-100 g buffer, 0.9-1.8 g electrolyte, 0.1-0.5 g surfactant, 0.1-0.5 g stabilizer and 0.2-0.4 g antibacterial agent; after mixing, 10-30 μL matrix metalloproteinase-3 is added, and stirred at a rotation speed of 500-1000 rpm for 10-30 minutes to obtain a calibrator.

3. The matrix metalloproteinase-3 latex detection kit according to claim 2, characterized in that: The buffer is at least one of tris(hydroxymethylaminomethane) buffer, 4-hydroxyethylpiperazineethanesulfonic acid buffer, 2-(N-morpholino)ethanesulfonic acid buffer, and 3-morpholine-2-hydroxypropanesulfonic acid buffer with a concentration of 50 to 100 mmol / L and a pH value of 7.0 to 7.5; the sensitizer is at least one of polyethylene glycol 6000, polyethylene glycol 35000, and dextran; the electrolyte is at least one of sodium chloride, magnesium chloride, and calcium chloride; and the surfactant is at least one of Tween-20 and sodium ethylenediaminetetraacetic acid.

2.

4. The matrix metalloproteinase-3 latex detection kit according to claim 1, characterized in that: The ZnO@PDA@ZIF-7 composite nanoparticles are prepared as follows: (1) Dispersing 0.1-0.5 g of ZIF-7 nanoparticles in 5-12 g of methanol solution, then adding 0.1-0.5 g of dopamine hydrochloride to form a mixed solution, and then stirring the mixed solution in a 60-75 ° C water bath at a speed of 800-1500 rpm for 8-12 h, centrifuging after completion, washing with ethanol three times, and drying at 80 ° C for 15-30 h to obtain PDA@ZIF-7 nanoparticles; (2) Disperse 0.9-1.8 g of the PDA@ZIF-7 nanoparticles in 8-14 g of methanol, then add 12-20 g of a 10 mmol / L zinc nitrate methanol solution, stir at 1000-1500 rpm for 30-60 min, add 0.9-1.8 g of a 0.01 mol / L sodium hydroxide methanol solution, continue the reaction for 30-50 min, centrifuge after completion, wash three times with methanol, and dry at 60° C. for 20-30 h to obtain ZnO@PDA@ZIF-7 composite nanoparticles.

5. The matrix metalloproteinase-3 latex detection kit according to claim 1, characterized in that: The reaction solution 1 is prepared by the following steps: 95-100 g of tris(hydroxymethyl)aminomethane buffer, 0.9-1.8 g of electrolyte, 0.3 g of stabilizer, 0.2 g of anti-interference agent and 0.3 g of antibacterial agent, mixing the above ingredients, and stirring at a speed of 800-2000 rpm for 20-40 min to obtain the reaction solution 1.

6. The matrix metalloproteinase-3 latex detection kit according to claim 1, characterized in that: The reaction solution 2 is prepared by the following steps: adding latex microspheres into a buffer solution and stirring, first adding an activator, then adding a matrix metalloproteinase-3 monoclonal antibody, continuing to stir the reaction, then adding a blocking agent and stirring until the reaction is completed, separating to obtain an antibody-latex microsphere complex, and then adding a storage solution; mixing antibody-latex microspheres with a particle size of 80 nm and antibody-latex microspheres with a particle size of 150 nm in a mass ratio of 1: (0.8-1.2) and then standing to obtain a reaction solution 2.

7. The matrix metalloproteinase-3 latex detection kit according to claim 6, characterized in that: The reaction solution 2 is prepared by the following steps: (1) Preparation of antibody-latex microspheres: add 0.5-1 mL of latex microspheres with a particle size of 80 nm to 5-10 mL of buffer, stir at a speed of 200-1000 rpm for 5-15 min, add an appropriate amount of activator and stir for 10-30 min to activate the carboxyl groups on the surface of the latex microspheres; then add 0.1-1 mg of matrix metalloproteinase-3 monoclonal antibody and continue to stir and react for 1-3 h; then add 10-50 uL of blocking agent and continue to stir and react for 0.5-1 h; then separate by tangential flow filtration, and then add 10-20 mL of storage solution to obtain antibody-latex microsphere storage solution with a particle size of 80 nm; the same method is used to obtain antibody-latex microsphere storage solution with a particle size of 150 nm; latex microspheres with a particle size of 80 nm are coupled to goat monoclonal antibodies or rabbit monoclonal antibodies, and latex microspheres with a particle size of 150 nm are coupled to rabbit monoclonal antibodies or goat monoclonal antibodies; (2) Preparation of reaction solution 2: Mix the antibody-latex microsphere stock solution with a particle size of 80 nm and the antibody-latex microsphere stock solution with a particle size of 150 nm, and let stand for 12 to 48 hours to obtain reaction solution 2.

8. The matrix metalloproteinase-3 latex detection kit according to claim 7, characterized in that: The activator is an aqueous solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; and the blocking agent is at least one of a CE510 blocking agent and a DB1130 blocking agent.

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