A composite system for MCP-1 magnetic microparticle chemiluminescence detection, application and product

Abstract

The application belongs to the technical field of biological detection, and particularly relates to a composite system for MCP-1 magnetic microparticle chemiluminescence detection and application and product. The composite system comprises a coupling buffer and a labeling buffer; the coupling buffer is composed of MES, NaCl, PEG 4000, PVP K30, Tween-20, glycerol and water, and pH is 5.5-6.5; the immune labeling buffer is composed of boric acid, CAPS, NaCl, sucrose, PEG 6000, Triton X-100 and water, and pH is 9.0-10.0. The kit containing the composite system has a sensitivity of 2.31 pg / mL, good linear relationship in a concentration range, good accuracy and high repeatability. The kit can be used for researching the pathogenesis of diseases in which MCP-1 signal pathways are involved, screening MCP-1 inhibiting drugs and qualitatively and quantitatively detecting MCP-1.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological detection, and particularly relates to a composite system for MCP-1 magnetic microparticle chemiluminescence detection and application and product. BACKGROUND

[0002] Monocyte chemoattractant protein-1 (MCP-1) as a key chemokine plays an extremely important role in physiological and pathological processes of the human body. In the physiological state, MCP-1 can attract monocytes, macrophages and other immune cells to migrate to the inflammatory site, and occupies a core position in the initiation and regulation of inflammatory response. When the body is invaded by pathogens, immune cells are activated and secrete MCP-1, guiding immune cells to the infection lesion and starting immune defense.

[0003] In the disease process, MCP-1 is involved in the development of various diseases. In the aspect of kidney disease, MCP-1 expression is significantly up-regulated in renal tubulointerstitial lesions, and its level is positively correlated with the severity of the lesions; in the process of renal function impairment, MCP-1 expression is positively correlated with serum creatinine, urea nitrogen and other indicators of renal function impairment, suggesting that it plays a promoting role in the deterioration of renal function. In cardiovascular diseases such as atherosclerosis, high cholesterol promotes the production of a large amount of MCP-1 by medial smooth muscle cells, attracting monocytes to adhere to and infiltrate the arterial wall, and monocytes phagocytose cholesterol to form foam cells, promoting the formation and progression of atherosclerotic plaques. In the tumor microenvironment, MCP-1 is highly expressed in various tumor tissues such as breast cancer and colorectal cancer, which can promote tumor angiogenesis and help tumor cell metastasis, affecting the prognosis of patients. In addition, in autoimmune diseases such as rheumatoid arthritis and multiple sclerosis, MCP-1 mediates immune cell infiltration and tissue damage, exacerbating the disease.

[0004] Given the important role of MCP-1 in the occurrence and development of diseases, accurate detection of its content is of great significance for disease diagnosis, disease assessment and treatment monitoring. There are various existing detection methods. Immunohistochemical staining can directly present the localization and distribution of MCP-1 in tissues, and is realized by the combination of antibody and MCP-1 and color development reaction, but its quantitative capacity is limited. Polyacrylamide gel electrophoresis silver staining can separate and identify MCP-1, and the protein is separated by gel electrophoresis and then silver staining is performed to develop the band, but the operation is complex and the sensitivity is poor. Real-time quantitative PCR quantifies MCP-1 transcription level from gene level, and is realized by amplifying the relevant gene fragment and monitoring in real time, but it cannot directly reflect the protein content.

[0005] Magnetic particle chemiluminescence method is a combination of magnetic separation, chemiluminescence and immunoassay technology, which has obvious advantages in MCP-1 detection. The magnetic particle is used as a solid carrier, and the magnetic particle has magnetic response, low cost, low energy consumption, no pollution, and can fix biological active substances such as enzymes and antibodies through surface functional groups. During detection, the magnetic particle reacts with the sample fully due to the high specific surface area, and the detection process is efficient under the external magnetic field. In view of the obvious advantages of the magnetic particle chemiluminescence method, it has become a trend to develop a composite system and kit for MCP-1 magnetic particle chemiluminescence detection. SUMMARY

[0006] In view of the above problems, the application provides a composite system for MCP-1 magnetic particle chemiluminescence detection and application and products. The composite system comprises a coupling buffer and a labeling buffer; the coupling buffer is composed of MES, NaCl, PEG 4000, PVP K30, Tween-20, glycerol and water, and the pH is 5.5-6.5; the immune labeling buffer is composed of boric acid, 3-(cyclohexylamine)-1-propanesulfonic acid (CAPS), NaCl, sucrose, PEG 6000, Triton X-100 and water, and the pH is 9.0-10.0. The kit containing the composite system of the application has a sensitivity of 2.31 pg / mL; has a good linear relationship in the concentration range; has good accuracy; and has high repeatability. It can be used for studying the pathogenesis of diseases involved in the MCP-1 signal pathway, screening MCP-1 inhibiting drugs and MCP-1 qualitative and quantitative detection.

[0007] The technical scheme of the application is as follows:

[0008] In a first aspect, the application provides a composite system for MCP-1 magnetic particle chemiluminescence detection, which comprises a coupling buffer and a labeling buffer.

[0009] The coupling buffer is composed of MES, NaCl, PEG 4000, PVP K30, Tween-20, glycerol and water, and the pH is 5.5-6.5.

[0010] The immune labeling buffer is composed of boric acid, CAPS, NaCl, sucrose, PEG 6000, Triton X-100 and water, and the pH is 9.0-10.0.

[0011] Specifically, the coupling buffer contains 4-5 g / L MES, 2.5-3.5 g / L NaCl, 12-18 g / L PEG 4000, 2-3 g / L PVP K30, 0.02-0.07% v / v Tween-20 and 4-5% v / v glycerol.

[0012] Preferably, the coupling buffer contains 4.88 g / L MES, 2.92 g / L NaCl, 15 g / L PEG 4000, 2.5 g / L PVP K30, 0.05% v / v Tween-20 and 4.5% v / v glycerol.

[0013] Preferably, the preparation method of 1L of the coupling buffer in the complex system comprises: adding MES and NaCl into water and stirring until dissolved, adding PEG 4000 and PVP K30 and stirring until dissolved under heating, after cooling to room temperature, adding Tween-20 and glycerol, adjusting pH, supplementing water to 1L, and filtering to obtain the coupling buffer.

[0014] Specifically, the immunolabeling buffer contains 2.5-3.5 g / L boric acid, 6-7 g / L CAPS, 5.5-6.5 g / L NaCl, 25-26 g / L sucrose, 25-35 g / L PEG 6000 and 0.01-0.03% v / v Triton X-100.

[0015] Preferably, the immunolabeling buffer contains 3.09 g / L boric acid, 6.64 g / L CAPS, 5.85 g / L NaCl, 25.67 g / L sucrose, 30 g / L PEG 6000 and 0.02% v / v Triton X-100.

[0016] Preferably, the preparation method of 1L of the CAPS buffer in the complex system comprises:

[0017] (1) boric acid is added into water and stirred until dissolved, and pH is adjusted to obtain a boric acid buffer;

[0018] (2) CAPS is added into water and stirred until dissolved, and pH is adjusted to obtain a CAPS buffer;

[0019] (3) the boric acid buffer and the CAPS buffer are mixed to obtain a complex buffer system;

[0020] (4) in the complex buffer system, NaCl, sucrose, PEG 6000 and Triton X-100 are added and mixed, pH is adjusted, water is supplemented to 1L, and after filtration, the CAPS buffer is obtained.

[0021] Further preferably, the boric acid buffer in step (1) is a 100 mM boric acid buffer with a pH value of 9.0.

[0022] Further preferably, the CAPS buffer in step (2) is a 30 mM CAPS buffer with a pH value of 10.5.

[0023] Further preferably, the volume ratio of the boric acid buffer and the CAPS buffer in step (3) is 5:3.

[0024] Further preferably, the pH in step (4) is 9.0-10.0.

[0025] In a second aspect, the present application provides use of the above-mentioned complex system in preparation of a MCP-1 magnetic microparticle chemiluminescence detection kit.

[0026] In a third aspect, the present application provides a MCP-1 magnetic microparticle chemiluminescence detection kit, which contains the above-mentioned complex system.

[0027] Specifically, the kit contains a MCP-1 antibody magnetic microparticle complex reagent and an acridinium ester-labeled MCP-1 antibody complex.

[0028] Further specifically, the preparation method of the MCP-1 antibody magnetic microparticle complex reagent comprises the following steps:

[0029] S1, preparing a MCP-1 antibody 1 solution by using a coupling buffer to dilute the MCP-1 antibody 1;

[0030] S2, adding the MCP-1 antibody 1 solution into a magnetic microparticle suspension to obtain a premix, adding a coupling catalyst, mixing uniformly, and then performing a coupling reaction;

[0031] S3, after the coupling reaction is completed, removing the supernatant, adding a blocking solution to resuspend the magnetic microparticles, and performing a blocking reaction;

[0032] S4, after the blocking reaction is completed, removing the supernatant, washing for 3 times, and then adding a magnetic microparticle storage solution, thereby obtaining the MCP-1 antibody magnetic microparticle complex reagent.

[0033] Preferably, the concentration of the MCP-1 antibody 1 solution in step S1 or step S2 is 0.5-2 mg / L.

[0034] Further preferably, the concentration of the MCP-1 antibody 1 solution in step S1 or step S2 is 1 mg / L.

[0035] Preferably, the concentration of the magnetic microparticle suspension in step S2 is 10 mg / mL.

[0036] Preferably, the volume ratio of the MCP-1 antibody 1 solution to the magnetic microparticle suspension in step S2 is 3:25.

[0037] Preferably, the coupling catalyst in step S2 is a boric acid buffer containing 3M (NH4)2SO4.

[0038] Preferably, the volume ratio of the premixed solution to the coupling catalyst in step S2 is 14:1.

[0039] Preferably, the coupling reaction in step S2 is carried out at 35-39℃ for 12-18h.

[0040] Further preferably, the coupling reaction in step S2 is carried out at 37℃ for 14h.

[0041] Preferably, the blocking solution in step S3 is composed of BSA and PBST.

[0042] Further preferably, the blocking solution in step S3 is PBST containing 0.5% BSA.

[0043] Preferably, the blocking reaction in step S3 is carried out at 35-39℃ for 2-4h.

[0044] Further preferably, the blocking reaction in step S3 is carried out at 37℃ for 3h.

[0045] Preferably, the magnetic particle storage solution in step S4 is composed of BSA, ProClean 300 and PBS.

[0046] Further preferably, the magnetic particle storage solution in step S4 is 1x PBS solution containing 0.1% BSA and 0.02% ProClean 300.

[0047] Further specifically, the method for preparing the acridinium ester-labeled MCP-1 antibody complex comprises the following steps:

[0048] a. MCP-1 antibody 2 is prepared into a MCP-1 antibody 2 solution with an immunolabeling buffer;

[0049] b. acridinium ester working solution is added to the MCP-1 antibody 2 solution to perform acridinium ester labeling reaction;

[0050] c. after the reaction is completed, a blocking solution is added to perform blocking reaction, obtaining a reaction solution;

[0051] d. the reaction solution is purified using a gel chromatography column, and the eluate is collected to obtain the acridinium ester-labeled MCP-1 antibody complex.

[0052] Preferably, the concentration of the MCP-1 antibody 2 solution in step a or step b is 0.2mg / mL.

[0053] Preferably, the concentration of the acridinium ester working solution in step b is 0.5mg / mL.

[0054] Preferably, the volume ratio of the MCP-1 antibody 2 solution to the acridinium ester working solution in step b is 35-45:1.

[0055] Further preferably, the volume ratio of the MCP-1 antibody 2 solution to the acridinium ester working solution in step b is 40:1.

[0056] Preferably, the acridinium ester labeling reaction in step b is carried out in the dark at 18-25°C for 45-60 min.

[0057] Further preferably, the acridinium ester labeling reaction in step b is carried out in the dark at 25°C for 50 min.

[0058] Preferably, the blocking solution in step c is a 10% lysine solution.

[0059] Preferably, the blocking reaction in step c is continued at room temperature for 30-45 min.

[0060] Further preferably, the blocking reaction in step c is continued at room temperature for 30 min.

[0061] Further preferably, the gel chromatography column in step d is a SephadexpG-25 gel chromatography column.

[0062] In particular, the kit further comprises a PBST washing solution, a substrate A, a substrate B, a standard, and a quality control.

[0063] Preferably, the substrate A is an acidic luminescent solution, which is composed of HCl, H2O2, and water.

[0064] Further preferably, the substrate A is purified water containing 0.1M HCl and 0.1% H2O2.

[0065] Preferably, the substrate B is an alkaline luminescent solution, which is composed of NaOH, Triton-100, and water.

[0066] Further preferably, the substrate B is purified water containing 0.25M NaOH and 2% Triton-100.

[0067] Preferably, the standard is an MCP-1 antigen standard, and the MCP-1 antigen standard is an MCP-1 antigen standard with a concentration of 5 pg / mL, 20 pg / mL, 50 pg / mL, 100 pg / mL, 200 pg / mL, 500 pg / mL, 1000 pg / mL, and 2000 pg / mL.

[0068] Preferably, the quality control product is MCP-1 antigen with a concentration of 500 pg / mL.

[0069] Specifically, the method for using the kit comprises the following steps:

[0070] ① mixing the sample to be tested, the standard product or the quality control product with the MCP-1 antibody magnetic micro-particle complex reagent and the acridinium ester-labeled MCP-1 antibody complex, and incubating;

[0071] ② removing the supernatant by magnetic separation, adding a luminescent substrate after washing, and determining the relative luminescent intensity;

[0072] ③ calculating the MCP-1 concentration according to the relative luminescent intensity.

[0073] Preferably, the sample to be tested in step ① comprises but is not limited to one or more of the following: a blood sample, a plasma sample, a tissue sample, and a cell sample.

[0074] Preferably, the sample to be tested, the standard product or the quality control product in step ① is added in an amount of 50 μL.

[0075] Preferably, the MCP-1 antibody magnetic micro-particle complex reagent in step ① is added in an amount of 50 μL.

[0076] Preferably, the acridinium ester-labeled MCP-1 antibody complex in step ① is added in an amount of 50 μL.

[0077] Preferably, the incubation in step ① is incubation at 35-39 ℃ for 20-30 min.

[0078] Further preferably, the incubation in step ① is incubation at 37 ℃ for 20 min.

[0079] Preferably, the luminescent substrate in step ② is substrate A and substrate B.

[0080] Further preferably, the substrate A and the substrate B are each added in an amount of 50 μL.

[0081] The present application has the following advantages:

[0082] (1) The present application provides a complex system for MCP-1 magnetic micro-particle chemiluminescence detection, which comprises a coupling buffer and a labeling buffer. The kit containing the complex system of the present application has a sensitivity of 2.31 pg / mL.

[0083] (2) The kit containing the complex system of the present application has a good linear relationship between the relative luminescent intensity and the sample concentration in the concentration range of 5-2000 pg / mL, and the R 2 > 0.999.

[0084] (3) The kit containing the composite system of the application has a recovery rate of 96.43±2.23%, 97.48±2.46%, 96.51±2.68% when the sample concentration of MCP-1 is 200 pg / mL, 500 pg / mL or 1000 pg / mL respectively. The kit provided by the application has high accuracy.

[0085] (4) The kit containing the composite system of the application has a CV value of 1.93%, and has good repeatability.

[0086] (5) The composite system and the kit containing the composite system of the application can be used for researching the pathogenesis of diseases in which MCP-1 signaling pathway is involved, screening MCP-1 inhibiting drugs and qualitative and quantitative detection of MCP-1. BRIEF DESCRIPTION OF DRAWINGS

[0087] Figure 1 Standard curve of the kit for MCP-1 magnetic microparticle chemiluminescence detection in Example 1. DETAILED DESCRIPTION

[0088] The application will be further clarified by the following examples. The following examples are only a part of the application and are not used to limit the application, but only to illustrate the application. The experimental methods used in the following examples are conventional experiments, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0089] The MCP-1 antibody 1 of the application is purchased from R&D Systems with the item number MAB279; the MCP-1 antibody 2 is purchased from R&D Systems with the item number BAF279; the magnetic microparticles are purchased from Biyun Tian with the item number ST432-200ml.

[0090] Example 1: A kit for MCP-1 magnetic microparticle chemiluminescence detection

[0091] 1. Coupling buffer

[0092] The coupling buffer contains 4.88 g / L MES, 2.92 g / L NaCl, 15 g / L PEG 4000, 2.5 g / L PVPK30, 0.05% v / v Tween-20 and 4.5% v / v glycerol, and the pH value is 5.5-6.5.

[0093] The preparation method of the coupling buffer is as follows: 4.88 g of MES and 2.92 g of NaCl are added into 800 mL of purified water, and stirred until completely dissolved. 15 g of PEG 4000 and 2.5 g of PVP K30 are added, heated to 37°C and continuously stirred until completely dissolved. After cooling to room temperature, 0.5 mL of Tween-20 and 45 mL of glycerol are added and mixed. The pH is adjusted to 5.5-6.5, purified water is added to 1 L, and it is filtered with a 0.22 μm filter membrane. The coupling buffer is obtained and stored at 4°C.

[0094] 2. Immunolabeling buffer

[0095] The immunolabeling buffer contains 3.09 g / L of boric acid, 6.64 g / L of CAPS, 5.85 g / L of NaCl, 25.67 g / L of sucrose, 30 g / L of PEG 6000, and 0.02% v / v of Triton X-100, and the pH value is 9.0-10.0.

[0096] The preparation method of the immunolabeling buffer is as follows:

[0097] (1) 3.09 g of boric acid is added to a suitable amount of purified water, stirred until completely dissolved, and then the pH is adjusted to 9.0. Purified water is added to 500 mL to obtain a boric acid buffer.

[0098] (2) 6.64 g of CAPS is added to a suitable amount of purified water, stirred until completely dissolved, and then the pH is adjusted to 10.5. Purified water is added to 300 mL to obtain a CAPS buffer.

[0099] (3) The boric acid buffer and the CAPS buffer are mixed to obtain a composite buffer system.

[0100] (4) In the composite buffer system, 5.85 g of NaCl and 25.67 g of sucrose are added and stirred until completely dissolved. 30 g of PEG 6000 is added and heated to 37°C while continuously stirring until completely dissolved. After cooling to room temperature, 0.2 mL of Triton X-100 is added and mixed. The pH value is 9.0-10.0. Purified water is added to 1 L, filtered with a 0.22 μm filter membrane to obtain the labeling buffer, and stored at 4°C.

[0101] 3. Preparation of MCP-1 antibody magnetic microsphere complex reagent

[0102] (1) The supernatant is removed by magnetic separation of the magnetic microspheres. The magnetic beads are resuspended with the boric acid buffer, and the supernatant is removed after gentle blowing. This washing is repeated three times. The magnetic microspheres are prepared into a suspension of 10 mg / mL using the boric acid buffer.

[0103] (2) MCP-1 antibody 1 was prepared into a solution of 1 mg / mL with conjugation buffer; the solution of MCP-1 antibody 1 was added into the magnetic particle suspension according to the volume ratio of 3:25, to obtain a premix. The premix was added into the borate buffer containing 3M (NH4)2SO4 according to the volume ratio of 14:1, mixed well, and reacted at 37°C for 14 h.

[0104] (3) After the reaction was completed, the supernatant was removed, 500 μL of blocking solution was added to resuspend the magnetic particles, and a blocking reaction was performed at 37°C for 3 h.

[0105] (4) After the blocking reaction was completed, the supernatant was separated and removed. After being washed with 500 μL of blocking solution for 3 times, the magnetic particles were resuspended with the magnetic particle storage solution to obtain a solution of 10 mg / mL of MCP-1 antibody magnetic particle complex.

[0106] The blocking solution was PBST containing 0.5% BSA at pH 7.2; and the magnetic particle storage solution was a 1x PBS solution containing 0.1% BSA and 0.02% ProClean 300.

[0107] 4. Preparation of acridinium ester-labeled MCP-1 antibody complex

[0108] (1) The acridinium ester was prepared into a working solution of 0.5 mg / mL with DMSO.

[0109] (2) MCP-1 antibody 2 was diluted into a solution of 0.2 mg / mL with immunolabeling buffer; the working solution of acridinium ester was added into the solution of MCP-1 antibody 2 according to the volume ratio of 40:1, mixed well, and reacted in the dark at 25°C for 45-60 min to perform acridinium ester labeling.

[0110] (3) After the reaction was completed, 20 μL of 10% lysine solution was added and mixed well, and the reaction solution was further reacted in the dark at room temperature for 30 min.

[0111] (4) The reaction solution was purified by Sephadex pG-25 gel chromatography column, and the eluate was collected to obtain the acridinium ester-labeled MCP-1 antibody complex.

[0112] 5. Kit for MCP-1 magnetic particle chemiluminescence detection

[0113] The kit comprises MCP-1 antibody magnetic particle complex reagent, acridinium ester labeled MCP-1 antibody complex, PBST washing solution, substrate A (purified water containing 0.1M HCl and 0.1% H2O2) and substrate B (purified water containing 0.25M NaOH and 2% Triton-100), standard (MCP-1 antigen standard A-H, concentrations are 5pg / mL, 20pg / mL, 50pg / mL, 100pg / mL, 200pg / mL, 500pg / mL, 1000pg / mL, 2000pg / mL respectively), quality control (MCP-1 antigen with a concentration of 500pg / mL).

[0114] Example 2 Use method of the kit for MCP-1 magnetic particle chemiluminescence detection

[0115] 1. Reagent equilibration: take out all reagents (MCP-1 antibody magnetic particle complex reagent, acridinium ester labeled MCP-1 antibody complex, PBST washing solution, substrate A, substrate B, standard and quality control) in the kit, and equilibrate at room temperature for 30 minutes.

[0116] 2. Sample processing: centrifuge the serum / plasma sample to remove the precipitate and obtain the sample to be tested;

[0117] 3. Immune reaction: add 50μL standard / quality control / sample to be tested, 50μL MCP-1 antibody magnetic particle complex reagent and 50μL acridinium ester labeled MCP-1 antibody complex in the reaction cup in turn, mix well and incubate at 37℃ for 20min;

[0118] 4. Washing: magnetic separation for 3min, remove the supernatant, add 300μL PBST washing solution, vortex for 30s, discard the supernatant after magnetic separation, and repeat the washing for 3 times;

[0119] 5. Chemiluminescence detection: add 50μL substrate A and 50μL substrate B in turn, put into the chemiluminescence detector, and read the relative light unit (RLU).

[0120] 6. Take the standard concentration as the abscissa and the corresponding RLU value as the ordinate, fit the standard curve, and calculate the MCP-1 content in the sample according to the standard curve.

[0121] Comparative Example 1 A kit for MCP-1 magnetic particle chemiluminescence detection

[0122] The difference between Comparative Example 1 and Example 1 is only that the "coupling buffer" is different.

[0123] Coupling buffer of Comparative Example 1: containing 4.88 g / L MES, 2.92 g / L NaCl, 2.5 g / L PVP K30, 0.05% v / v Tween-20 and 4.5% v / v glycerol, pH 5.5-6.5.

[0124] The preparation method of the coupling buffer of Comparative Example 1 is as follows: 4.88 g of MES and 2.92 g of NaCl are added to 800 mL of purified water, and stirred until completely dissolved. 2.5 g of PVP K30 is added, heated to 37°C and continuously stirred until completely dissolved. After cooling to room temperature, 0.5 mL of Tween-20 and 45 mL of glycerol are added, and mixed. The pH is adjusted to 5.5-6.5, purified water is added to 1 L, filtered with a 0.22 μm filter membrane, and the coupling buffer is obtained, which is stored at 4°C.

[0125] Comparative Example 2: A kit for MCP-1 magnetic microparticle chemiluminescence detection

[0126] The difference between Comparative Example 2 and Example 1 is only that the "coupling buffer" is different.

[0127] Coupling buffer of Comparative Example 2: containing 4.88 g / L MES, 2.92 g / L NaCl, 15 g / L PEG 4000, 0.05% v / v Tween-20 and 4.5% v / v glycerol, pH 5.5-6.5.

[0128] The preparation method of the coupling buffer of Comparative Example 2 is as follows: 4.88 g of MES and 2.92 g of NaCl are added to 800 mL of purified water, and stirred until completely dissolved. 15 g of PEG 4000 is added, heated to 37°C and continuously stirred until completely dissolved. After cooling to room temperature, 0.5 mL of Tween-20 and 45 mL of glycerol are added, and mixed. The pH is adjusted to 5.5-6.5, purified water is added to 1 L, filtered with a 0.22 μm filter membrane, and the coupling buffer is obtained, which is stored at 4°C.

[0129] Comparative Example 3: A kit for MCP-1 magnetic microparticle chemiluminescence detection

[0130] The difference between Comparative Example 3 and Example 1 is only that the "coupling buffer" is different.

[0131] Coupling buffer of Comparative Example 3: containing 4.88 g / L MES, 2.92 g / L NaCl, 0.05% v / v Tween-20 and 4.5% v / v glycerol, pH 5.5-6.5.

[0132] The preparation method of the coupling buffer of Comparative Example 3 is as follows: 4.88 g of MES and 2.92 g of NaCl are added into 800 mL of purified water, and stirred until completely dissolved. 0.5 mL of Tween-20 and 45 mL of glycerol are added, and mixed. The pH is adjusted to 5.5-6.5, purified water is added to 1 L, and filtered with a 0.22 μm filter membrane. The coupling buffer is obtained, and stored at 4°C.

[0133] Comparative Example 4: A kit for MCP-1 magnetic microparticle chemiluminescence detection

[0134] Comparative Example 4 differs from Example 1 only in the "immunolabeling buffer".

[0135] The immunolabeling buffer of Comparative Example 4 contains 3.09 g / L boric acid, 5.85 g / L NaCl, 25.67 g / L sucrose, 30 g / L PEG 6000 and 0.02% v / v Triton X-100, and the pH is 9.0-10.0.

[0136] The preparation method of the immunolabeling buffer of Comparative Example 4 is as follows:

[0137] (1) 3.09 g of boric acid is added into a proper amount of purified water, stirred until completely dissolved, and the pH is adjusted to 9.0. Purified water is added to 500 mL to obtain a boric acid buffer.

[0138] (2) 5.85 g of NaCl and 25.67 g of sucrose are added into the boric acid buffer, stirred until completely dissolved, and 30 g of PEG 6000 is added and heated to 37°C while continuously stirring until completely dissolved. After cooling to room temperature, 0.2 mL of Triton X-100 is added and mixed. The pH is 9.0-10.0, purified water is added to 1 L, and filtered with a 0.22 μm filter membrane. The labeling buffer is obtained, and stored at 4°C.

[0139] Comparative Example 5: A kit for MCP-1 magnetic microparticle chemiluminescence detection

[0140] Comparative Example 5 differs from Example 1 only in the "coupling buffer" and "immunolabeling buffer".

[0141] The coupling buffer of Comparative Example 5 contains 4.88 g / L MES, 2.92 g / L NaCl, 0.05% v / v Tween-20 and 4.5% v / v glycerol, and the pH is 5.5-6.5.

[0142] The preparation method of the coupling buffer of Comparative Example 5 is as follows: 4.88 g of MES and 2.92 g of NaCl are added into 800 mL of purified water, and stirred until completely dissolved. 0.5 mL of Tween-20 and 45 mL of glycerol are added, and mixed. The pH is adjusted to 5.5-6.5, purified water is added to 1 L, and filtered with a 0.22 μm filter membrane to obtain the coupling buffer, which is stored at 4°C.

[0143] The immunolabeling buffer of Comparative Example 5 contains 3.09 g / L of boric acid, 5.85 g / L of NaCl, 25.67 g / L of sucrose, 30 g / L of PEG 6000 and 0.02% v / v of Triton X-100, and has a pH of 9.0-10.0.

[0144] The preparation method of the immunolabeling buffer of Comparative Example 5 is as follows:

[0145] (1) 3.09 g of boric acid is added into a proper amount of purified water, stirred until completely dissolved, and then the pH is adjusted to 9.0. Purified water is added to 500 mL to obtain the boric acid buffer.

[0146] (2) 5.85 g of NaCl and 25.67 g of sucrose are added into the boric acid buffer, stirred until completely dissolved, and then 30 g of PEG 6000 is added and heated to 37°C while continuously stirring until completely dissolved. After cooling to room temperature, 0.2 mL of Triton X-100 is added and mixed. The pH is 9.0-10.0, purified water is added to 1 L, and filtered with a 0.22 μm filter membrane to obtain the labeling buffer, which is stored at 4°C.

[0147] Experimental Example 1 Performance determination of the kit for MCP-1 magnetic microparticle chemiluminescence detection

[0148] 1. Determination of blank limit

[0149] The blank samples (serum of healthy people) without MCP-1 are determined for relative luminescence intensity by using the kits of Example 1 and Comparative Examples 1-5, respectively, and the method described in Example 2 is repeated for 20 times to obtain the concentration values of 20 test results.

[0150] The concentration value average (X) and the concentration value standard deviation (SD) are calculated according to the concentration values, and the blank limit is calculated according to the formula: blank limit = X + 3SD. The results are shown in Table 1.

[0151] Table 1

[0152]

[0153] 2. Verification of linear range

[0154] ​MCP-1 antigen standard, concentration of 5 pg / mL, 20 pg / mL, 50 pg / mL, 100 pg / mL, 200 pg / mL, 500 pg / mL, 1000 pg / mL, 2000 pg / mL, respectively, using the kit of Example 1, Comparative Examples 1-5, the relative luminescence intensity (n=2) was determined by the method described in Example 2. The standard curve was plotted with the standard concentration as the abscissa and the corresponding relative luminescence intensity average as the ordinate. The relative luminescence intensity of each concentration is shown in Table 2.

[0155] Table 2

[0156]

[0157] The results showed that the blank limit of Example 1 was 2.31 pg / mL, the blank limit of Comparative Example 1 was 3.79 pg / mL, the blank limit of Comparative Example 2 was 3.90 pg / mL, the blank limit of Comparative Example 3 was 4.11 pg / mL, the blank limit of Comparative Example 4 was 2.88 pg / mL, and the blank limit of Comparative Example 5 was 4.48 pg / mL. Among them, the blank limit of Example 1 was the lowest, indicating that the kit of Example 1 had the best sensitivity. Among them, the relative luminescence intensity of Example 1 kit was linearly related to the sample concentration in the concentration range of 5-1000 pg / mL, R 2 > 0.999 ( Figure 1 ).

[0158] 2. Accuracy verification

[0159] Different concentrations of MCP-1 standard were added to the blank sample (healthy human serum) without MCP-1, so that the final concentration of MCP-1 standard was 200 pg / mL, 500 pg / mL or 1000 pg / mL. The relative luminescence intensity of each sample was determined using the kit of Example 1, according to the method described in Example 2. Each concentration was determined 3 times, and the corresponding concentration was calculated according to the relative luminescence intensity and the standard curve regression equation, and the recovery rate was calculated according to the following formula:

[0160]

[0161] The recovery rate determination results of Example 1 are shown in Table 3.

[0162] Table 3

[0163]

[0164] The above results show that when the sample concentration is 200 pg / mL, 500 pg / mL or 1000 pg / mL, the recovery rates of the kit of the present application are 96.43±2.23%, 97.48±2.46%, 96.51±2.68% respectively. The kit provided by the present application has high accuracy.

[0165] 3. Repetitive verification

[0166] The kit of Example 1 is repeatedly tested for the quality control sample (500 pg / mL) for 10 times, and the average value and standard deviation of the results of 10 times of determination are used to calculate the coefficient of variation (CV) according to the following formula.

[0167]

[0168] The determination results are shown in Table 4, and the CV value of the kit of Example 1 of the present application is 1.93%, which has good repeatability.

[0169] Table 4

[0170]

[0171] The above results show that the kit of the present application has the characteristics of high sensitivity, high accuracy and good repeatability for MCP-1 detection.

[0172] The above detailed description is a specific description of one of the feasible embodiments of the present application, and the embodiment is not used to limit the patent scope of the present application. It should be noted that any equivalent implementation or change without departing from the present application should be included in the scope of the technical solutions of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A complex system for MCP-1 magnetic microparticle chemiluminescence detection, characterized in that, The complex system comprises a coupling buffer and a labeling buffer. The coupling buffer is composed of MES, NaCl, PEG 4000, PVP K30, Tween-20, glycerol and water, and has a pH of 5.5-6.5; the coupling buffer contains 4-5 g / L MES, 2.5-3.5 g / L NaCl, 12-18 g / L PEG 4000, 2-3 g / L PVP K30, 0.02-0.07% v / v Tween-20 and 4-5% v / v glycerol. The immunolabeling buffer is composed of boric acid, CAPS, NaCl, sucrose, PEG 6000, Triton X-100 and water, and has a pH of 9.0-10.0; the immunolabeling buffer contains 2.5-3.5 g / L boric acid, 6-7 g / L CAPS, 5.5-6.5 g / L NaCl, 25-26 g / L sucrose, 25-35 g / L PEG 6000 and 0.01-0.03% v / v Triton X-100.

2. The composite system of claim 1, wherein, The coupling buffer contains 4.88 g / L MES, 2.92 g / L NaCl, 15 g / L PEG 4000, 2.5 g / L PVP K30, 0.05% v / v Tween-20 and 4.5% v / v glycerol.

3. The composite system of claim 1, wherein, The immunolabeling buffer contains 3.09 g / L boric acid, 6.64 g / L CAPS, 5.85 g / L NaCl, 25.67 g / L sucrose, 30 g / L PEG 6000 and 0.02% v / v Triton X-100.

4. Use of the complex system of any one of claims 1-3 in the preparation of a MCP-1 magnetic microparticle chemiluminescence detection kit.

5. A kit for MCP-1 magnetic microparticle chemiluminescent assay, characterized by, The kit contains the complex system of any one of claims 1-3.

6. The kit of claim 5, wherein The kit comprises a MCP-1 antibody magnetic microparticle complex reagent and an acridinium ester-labeled MCP-1 antibody complex; The preparation method of the MCP-1 antibody magnetic microparticle complex reagent comprises the following steps: S1, the MCP-1 antibody 1 is prepared into a MCP-1 antibody 1 solution with the coupling buffer; S2, the MCP-1 antibody 1 solution is added into a magnetic microparticle suspension to obtain a premix, a coupling catalyst is added, and the mixture is uniformly mixed and then subjected to a coupling reaction; S3, after the coupling reaction is completed, the supernatant is removed, a blocking solution is added to resuspend the magnetic microparticles, and a blocking reaction is performed; S4, after the blocking reaction is completed, the supernatant is removed, the magnetic microparticles are washed for 3 times, and then a magnetic microparticle storage solution is added, thereby obtaining the MCP-1 antibody magnetic microparticle complex reagent; The preparation method of the acridinium ester-labeled MCP-1 antibody complex comprises the following steps: a. the MCP-1 antibody 2 is prepared into a MCP-1 antibody 2 solution with the immunolabeling buffer; b. the acridinium ester working solution is added into the MCP-1 antibody 2 solution to perform an acridinium ester labeling reaction; c. after the reaction is completed, a blocking solution is added to perform a blocking reaction, thereby obtaining a reaction solution. d. The reaction solution is purified by using a gel chromatography column, and the eluate is collected to obtain the acridinium ester-labeled MCP-1 antibody complex.

7. The kit of claim 6, wherein The method for using the kit comprises the following steps: ①incubating the sample to be detected, the standard or the quality control with the MCP-1 antibody magnetic microparticle complex reagent and the acridinium ester-labeled MCP-1 antibody complex; ②removing the supernatant by magnetic separation, adding the luminescent substrate after washing, and determining the relative luminescent intensity; ③calculating the MCP-1 concentration according to the relative luminescent intensity.

8. Use of the composite system according to any one of claims 1 to 3 or of the agent according to any one of claims 5 to 7, characterized in that, The application is used for non-disease diagnosis and treatment purposes, and the application comprises the following any aspect: (1) screening of MCP-1 inhibiting drugs; (2) qualitative and quantitative detection of MCP-1.

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