Composite system for MCP-1 magnetic particle chemiluminescence detection, application and product

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a composite system for MCP-1 magnetic particle chemiluminescence detection, application and a product. The composite system comprises a coupling buffer solution and a labeling buffer solution, the coupling buffer solution is composed of MES, NaCl, PEG 4000, PVP K30, Tween-20, glycerin and water, and the pH value of the coupling buffer solution is 5.5-6.5; the immunolabeling buffer solution is composed of boric acid, CAPS, NaCl, cane sugar, PEG 6000, Triton X-100 and water, and the pH value of the immunolabeling buffer solution is 9.0-10.0. The sensitivity of the kit containing the composite system can reach 2.31 pg / mL; the linear relation in the concentration range is good; the accuracy is high; the repeatability is high. The method can be used for researching the pathogenesis of the MCP-1 signal channel participating in diseases, screening MCP-1 inhibition drugs and qualitatively and quantitatively detecting the MCP-1.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and particularly relates to a composite system for chemiluminescence detection of MCP-1 magnetic microparticles, and its applications and products. Background Art

[0002] Monocyte chemoattractant protein-1 (MCP-1), as a key chemokine, plays a very important role in the physiological and pathological processes of the human body. Under physiological conditions, MCP-1 can chemotax monocytes, macrophages and other immune cells to migrate to the inflammatory site, and occupies a central position in the initiation and regulation of the inflammatory response. When the body is invaded by pathogens, immune cells are activated and secrete MCP-1, which guides immune cells to the infection focus and initiates immune defense.

[0003] In the disease process, MCP-1 is involved in the development of various diseases. In terms of kidney diseases, when tubulointerstitial lesions occur, the expression of MCP-1 is significantly up-regulated, and its level is positively correlated with the severity of the lesions; during the process of renal function impairment, the expression of MCP-1 is positively correlated with renal function impairment indicators such as serum creatinine and urea nitrogen, indicating its promoting role in the deterioration of renal function. In cardiovascular diseases such as atherosclerosis, hypercholesterolemia promotes a large amount of MCP-1 production by medial arterial smooth muscle cells, attracts monocytes to adhere to and infiltrate the arterial wall, and monocytes phagocytize 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. It can not only promote tumor angiogenesis, but also contribute to 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] In view of the important position of MCP-1 in the occurrence and development of diseases, accurate detection of its content is of great significance for disease diagnosis, condition assessment and treatment monitoring. There are various existing detection methods. Immunohistochemical staining can visually present the localization and distribution of MCP-1 in tissues, which is achieved through the binding of antibodies to MCP-1 and the color reaction, but its quantitative ability is limited. Silver staining of polyacrylamide gel electrophoresis can separate and identify MCP-1, by separating proteins through gel electrophoresis and then silver staining to show bands, however, the operation is complex and the sensitivity is poor. Real-time quantitative PCR quantifies the transcriptional level of MCP-1 at the gene level, which is achieved by amplifying related gene fragments and monitoring in real time, but it cannot directly reflect the protein content.

[0005] The magnetic particle chemiluminescence method combines magnetic separation, chemiluminescence, and immunoassay technologies, and has outstanding advantages in the detection of MCP-1. This method uses magnetic particles as a solid-phase carrier. The magnetic particles have magnetic responsiveness, low cost, low energy consumption, no pollution, and can immobilize bioactive substances such as enzymes and antibodies through surface functional groups. During detection, the magnetic particles react fully with the sample by virtue of their high specific surface area, and combined with an external magnetic field, the detection process is efficient. Given the significant advantages of the magnetic particle chemiluminescence method, developing a composite system and kit for the magnetic particle chemiluminescence detection of MCP-1 has become the current trend. Summary of the Invention

[0006] In view of the above deficiencies, the present invention provides a composite system, application, and product for the magnetic particle chemiluminescence detection of MCP-1. The composite system of the present invention includes 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 adjusted to 5.5 - 6.5; the immunolabeling 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 adjusted to 9.0 - 10.0. The kit containing the composite system of the present invention has a sensitivity of up to 2.31 pg / mL; has a good linear relationship within the concentration range; has good accuracy; and has high repeatability. It can be used to study the pathogenesis of diseases involving the MCP-1 signaling pathway, screen MCP-1 inhibitory drugs, and perform qualitative and quantitative detection of MCP-1.

[0007] The technical solution of the present invention is as follows: In the first aspect, the present invention provides a composite system for the magnetic particle chemiluminescence detection of MCP-1, and the composite system includes: 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 adjusted to 5.5 - 6.5; The immunolabeling buffer is composed of boric acid, CAPS, NaCl, sucrose, PEG 6000, Triton X-100, and water, and the pH is adjusted to 9.0 - 10.0.

[0008] Specifically, the coupling buffer contains 4 - 5 g / L MES, 2.5 - 3.5 g / L NaCl, 12 - 18 g / L PEG4000, 2 - 3 g / L PVP K30, 0.02 - 0.07% v / v Tween-20, and 4 - 5% v / v glycerol.

[0009] 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.

[0010] Preferably, in the composite system, the preparation method of 1 L of coupling buffer includes: adding MES and NaCl into water, stirring until dissolved, adding PEG 4000 and PVP K30, heating and stirring until dissolved, cooling to room temperature, adding Tween-20 and glycerol, adjusting the pH, making up the volume to 1 L with water, and filtering to obtain the coupling buffer.

[0011] 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.

[0012] 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.

[0013] Preferably, the preparation method of 1 L of CAPS buffer in the composite system includes: (1) Adding boric acid into water, stirring to dissolve, and adjusting the pH to obtain a boric acid buffer; (2) Adding CAPS into water, stirring to dissolve, and adjusting the pH to obtain a CAPS buffer; (3) Mixing the boric acid buffer and the CAPS buffer to obtain a composite buffer system; (4) In the composite buffer system, adding NaCl, sucrose, PEG 6000, and Triton X-100, mixing evenly, adjusting the pH, making up the volume to 1 L with water, and filtering to obtain the CAPS buffer.

[0014] Further preferably, the boric acid buffer in step (1) is a 100 mM boric acid buffer with a pH value of 9.0.

[0015] Further preferably, the CAPS buffer in step (2) is a 30 mM CAPS buffer with a pH value of 10.5.

[0016] Further preferably, the volume ratio of the boric acid buffer to the CAPS buffer in step (3) is 5:3.

[0017] Further preferably, the pH described in step (4) is 9.0 - 10.0.

[0018] In a second aspect, the present invention provides the use of the above composite system in the preparation of an MCP-1 magnetic particle chemiluminescence detection kit.

[0019] In a third aspect, the present invention provides a kit for MCP-1 magnetic particle chemiluminescence detection, and the kit contains the above composite system.

[0020] Specifically, the kit includes an MCP-1 antibody magnetic particle complex reagent and an acridinium ester-labeled MCP-1 antibody complex.

[0021] More specifically, the preparation method of the MCP-1 antibody magnetic particle complex reagent includes the following steps: S1. MCP-1 antibody 1 is formulated into an MCP-1 antibody 1 solution with a coupling buffer. S2. The MCP-1 antibody 1 solution is added to the magnetic particle suspension to obtain a premixed solution, and a coupling catalyst is added. After mixing, a coupling reaction is carried out. S3. After the coupling reaction is completed, the supernatant is removed, and the magnetic particles are resuspended with a blocking solution for a blocking reaction. S4. After the blocking reaction is completed, the supernatant is removed. After washing 3 times, a magnetic particle preservation solution is added to obtain the MCP-1 antibody magnetic particle complex reagent.

[0022] Preferably, the concentration of the MCP-1 antibody 1 solution in step S1 or step S2 is 0.5 - 2 mg / L.

[0023] Further preferably, the concentration of the MCP-1 antibody 1 solution in step S1 or step S2 is 1 mg / L.

[0024] Preferably, the concentration of the magnetic particle suspension in step S2 is 10 mg / mL.

[0025] Preferably, the volume ratio of the MCP-1 antibody 1 solution to the magnetic particle suspension in step S2 is 3:25.

[0026] Preferably, the coupling catalyst in step S2 is a boric acid buffer containing 3M (NH4)2SO4.

[0027] Preferably, the volume ratio of the premixed solution to the coupling catalyst in step S2 is 14:1.

[0028] Preferably, the conditions for the coupling reaction in step S2 are to react at 35 - 39 °C for 12 - 18 h.

[0029] Further preferably, the coupling reaction in step S2 is carried out at 37 °C for 14 h.

[0030] Preferably, the blocking solution in step S3 is composed of BSA and PBST.

[0031] Further preferably, the blocking solution in step S3 is PBST containing 0.5% BSA.

[0032] Preferably, the blocking reaction in step S3 is carried out at 35 - 39 °C for 2 - 4 h.

[0033] Further preferably, the blocking reaction in step S3 is carried out at 37 °C for 3 h.

[0034] Preferably, the magnetic particle preservation solution in step S4 is composed of BSA, ProClean 300 and PBS.

[0035] Further preferably, the magnetic particle preservation solution in step S4 is 1×PBS solution containing 0.1% BSA and 0.02% ProClean 300.

[0036] More specifically, the preparation method of the acridinium ester-labeled MCP-1 antibody complex includes the following steps: a. MCP-1 antibody 2 is formulated into an MCP-1 antibody 2 solution with an immunolabeling buffer. b. An acridinium ester working solution is added to the MCP-1 antibody 2 solution for an acridinium ester labeling reaction. c. After the reaction, a blocking solution is added for a blocking reaction to obtain a reaction solution. d. The reaction solution is purified using a gel filtration column, and the eluate is collected to obtain the acridinium ester-labeled MCP-1 antibody complex.

[0037] Preferably, the concentration of the MCP-1 antibody 2 solution in step a or step b is 0.2 mg / mL.

[0038] Preferably, the concentration of the acridinium ester working solution in step b is 0.5 mg / mL.

[0039] Preferably, the volume ratio of the MCP-1 antibody 2 solution to the acridinium ester working solution in step b is 35 - 45:1.

[0040] Further preferably, the volume ratio of the MCP-1 antibody 2 solution to the acridinium ester working solution in step b is 40:1.

[0041] Preferably, the conditions for the acridinium ester labeling reaction in step b are to react at 18 - 25 °C in the dark for 45 - 60 min.

[0042] Further preferably, the conditions for the acridinium ester labeling reaction in step b are to react at 25°C for 50 min in the dark.

[0043] Preferably, the blocking solution in step c is a 10% lysine solution.

[0044] Preferably, the conditions for the blocking reaction in step c are to continue the reaction for 30 - 45 min at room temperature.

[0045] Further preferably, the conditions for the blocking reaction in step c are to continue the reaction for 30 min at room temperature.

[0046] Further preferably, the gel filtration column in step d is a Sephadex pG - 25 gel filtration column.

[0047] Specifically, the kit further includes a PBST washing solution, substrate A, substrate B, standards, and quality control products.

[0048] Preferably, the substrate A is an acidic luminescent solution, which is composed of HCl, H2O2, and water.

[0049] Further preferably, the substrate A is purified water containing 0.1 M HCl and 0.1% H2O2.

[0050] Preferably, the substrate B is a basic luminescent solution, which is composed of NaOH, Triton - 100, and water.

[0051] Further preferably, the substrate B is purified water containing 0.25 M NaOH and 2% Triton - 100.

[0052] Preferably, the standard is an MCP - 1 antigen standard, and the MCP - 1 antigen standard is an MCP - 1 antigen standard with concentrations 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.

[0053] Preferably, the quality control product is an MCP - 1 antigen with a concentration of 500 pg / mL.

[0054] Specifically, the usage method of the kit includes the following steps: ① Mix the sample to be tested, the standard or the quality control product, the MCP - 1 antibody magnetic particle complex reagent, and the acridinium ester - labeled MCP - 1 antibody complex, and incubate. ② Magnetically separate to remove the supernatant, wash, add the luminescent substrate, and measure the relative luminescence intensity. ③ Calculate the MCP - 1 concentration based on the relative luminescence intensity.

[0055] Preferably, the test sample described in step ① includes, but is not limited to, one or more of: blood sample, plasma sample, tissue sample, and cell sample.

[0056] Preferably, the addition amount of the test sample, standard product, or quality control product described in step ① is 50 μL.

[0057] Preferably, the addition amount of the MCP-1 antibody magnetic particle composite reagent product described in step ① is 50 μL.

[0058] Preferably, the addition amount of the acridinium ester-labeled MCP-1 antibody composite product described in step ① is 50 μL.

[0059] Preferably, the incubation described in step ① is carried out at 35 - 39 °C for 20 - 30 min.

[0060] More preferably, the incubation described in step ① is carried out at 37 °C for 20 min.

[0061] Preferably, the luminescent substrate described in step ② is substrate A and substrate B.

[0062] More preferably, the addition amounts of both substrate A and substrate B are 50 μL.

[0063] The beneficial effects of the present invention are as follows: (1) The present invention provides a composite system for MCP-1 magnetic particle chemiluminescence detection, including a coupling buffer solution and a labeling buffer solution. The sensitivity of the kit containing the composite system of the present invention can reach 2.31 pg / mL.

[0064] (2) In the concentration range of 5 - 2000 pg / mL, the relative luminescence intensity of the kit containing the composite system of the present invention has a good linear relationship with the sample concentration, and R 2 > 0.999.

[0065] (3) When the MCP-1 sample concentrations are 200 pg / mL, 500 pg / mL, or 1000 pg / mL, the recovery rates of the kit of the present invention are 96.43 ± 2.23%, 97.48 ± 2.46%, and 96.51 ± 2.68% respectively. The kit provided by the present invention has high accuracy.

[0066] (4) The CV value of the kit containing the composite system of the present invention is 1.93%, showing good repeatability.

[0067] (5) The composite system of the present invention and the kit containing the composite system of the present invention can be used to study the pathogenesis of diseases involving the MCP-1 signaling pathway, screen MCP-1 inhibitory drugs, and qualitatively and quantitatively detect MCP-1. Description of the Drawings

[0068] Figure 1 It is the standard curve of the kit for MCP-1 magnetic particle chemiluminescence detection in Example 1. Detailed Description of the Invention

[0069] The present invention will be further clearly and completely described below through examples. The following examples are only a part of the examples of the present invention, and are not used to limit the present invention, but only to illustrate the present invention. The experimental methods used in the following examples are all conventional experiments unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0070] MCP-1 antibody 1 of the present application is purchased from R&D Systems, with the catalog number MAB279; MCP-1 antibody 2 is purchased from R&D Systems, with the catalog number BAF279; magnetic particles are purchased from Beyotime, ST432-200ml.

[0071] Example 1 A kit for MCP-1 magnetic particle chemiluminescence detection 1. Coupling buffer Coupling buffer: containing 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, with a pH value of 5.5 - 6.5.

[0072] The preparation method of the coupling buffer is as follows: Add 4.88 g of MES and 2.92 g of NaCl to 800 mL of purified water, and stir until completely dissolved. Add 15 g of PEG 4000 and 2.5 g of PVP K30, heat to 37 °C and continuously stir until completely dissolved. After cooling to room temperature, add 0.5 mL of Tween-20 and 45 mL of glycerol, and mix well. Adjust the pH to 5.5 - 6.5, make up the purified water to 1 L, filter with a 0.22 μm filter membrane, and it is the coupling buffer, which is stored at 4 °C.

[0073] 2. Immuno-labeling buffer Immuno-labeling buffer: containing 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, with a pH value of 9.0 - 10.0.

[0074] The preparation method of the immuno-labeling buffer is as follows: (1) 3.09 g of boric acid was added to an appropriate amount of purified water. After stirring until completely dissolved, the pH was adjusted to 9.0, and the purified water was supplemented to 500 mL to obtain a boric acid buffer solution.

[0075] (2) 6.64 g of CAPS was added to an appropriate amount of purified water. After stirring until completely dissolved, the pH was adjusted to 10.5, and the purified water was supplemented to 300 mL to obtain a CAPS buffer solution.

[0076] (3) After mixing 500 mL of the boric acid buffer solution and 300 mL of the CAPS buffer solution, a composite buffer system was obtained.

[0077] (4) In the composite buffer system, 5.85 g of NaCl and 25.67 g of sucrose were added, and stirred until completely dissolved. Then 30 g of PEG 6000 was added and heated to 37 °C with continuous stirring until completely dissolved. After cooling to room temperature, 0.2 mL of Triton X-100 was added and mixed evenly. The pH value was 9.0 - 10.0, and the purified water was supplemented to 1 L. It was filtered with a 0.22 μm filter membrane to obtain the labeling buffer solution, which was stored at 4 °C.

[0078] 3. Preparation of MCP-1 antibody magnetic particle composite reagent (1) The supernatant was removed by magnetic separation of the magnetic particles, and the magnetic beads were resuspended with the boric acid buffer solution. After gently pipetting and removing the supernatant, this washing process was repeated 3 times. Then the magnetic particles were formulated into a 10 mg / mL suspension with the boric acid buffer solution to obtain a magnetic particle suspension.

[0079] (2) MCP-1 antibody 1 was formulated into a 1 mg / mL solution with the coupling buffer to obtain an MCP-1 antibody 1 solution; according to the volume ratio of the MCP-1 antibody 1 solution to the magnetic particle suspension of 3:25, the MCP-1 antibody 1 solution was added to the magnetic particle suspension to obtain a premixed solution. According to the volume ratio of 14:1, a boric acid buffer solution containing 3 M (NH4)2SO4 was added to the premixed solution and mixed evenly, and the reaction was carried out at 37 °C for 14 h.

[0080] (3) After the reaction was completed, the supernatant was removed, and 500 μL of the blocking solution was added to resuspend the magnetic particles, and the blocking reaction was carried out at 37 °C for 3 h.

[0081] (4) After the blocking reaction was completed, the supernatant was separated and removed. After washing 3 times with 500 μL of the blocking solution, it was resuspended with the magnetic particle storage solution to obtain a 10 mg / mL MCP-1 antibody magnetic particle composite reagent.

[0082] The described blocking solution is: PBST containing 0.5% BSA with a pH of 7.2; the magnetic particle storage solution is a 1×PBS solution containing 0.1% BSA and 0.02% ProClean 300.

[0083] 4. Preparation of Acridinium Ester-Labeled MCP-1 Antibody Complex (1) Prepare an acridinium ester working solution with a concentration of 0.5 mg / mL using DMSO as the solvent for the acridinium ester.

[0084] (2) Dilute MCP-1 antibody 2 to a concentration of 0.2 mg / mL with immunolabeling buffer to obtain an MCP-1 antibody 2 solution; add the acridinium ester working solution to the MCP-1 antibody 2 solution according to a volume ratio of 40:1 of the MCP-1 antibody 2 solution to the acridinium ester working solution, and mix well. Place it in the dark at 25 °C and react for 45 - 60 min for acridinium ester labeling.

[0085] (3) After the reaction is completed, add 20 μL of 10% lysine solution and mix well. Place it in the dark at room temperature and continue to react for 30 min to obtain a reaction solution.

[0086] (4) Purify the reaction solution using a Sephadex pG-25 gel chromatography column, and collect the eluate to obtain the acridinium ester-labeled MCP-1 antibody complex.

[0087] 5. Kit for Chemiluminescence Detection of MCP-1 Using Magnetic Particles The kit includes an MCP-1 antibody magnetic particle complex reagent, an acridinium ester-labeled MCP-1 antibody complex, a PBST washing solution, substrate A (purified water containing 0.1 M HCl and 0.1% H2O2), and substrate B (purified water containing 0.25 M NaOH and 2% Triton-100), standards (MCP-1 antigen standards A - H with concentrations 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 respectively), and a quality control product (MCP-1 antigen with a concentration of 500 pg / mL).

[0088] Example 2 Method for Using the Kit for Chemiluminescence Detection of MCP-1 Using Magnetic Particles 1. Reagent Equilibration: Take out all the reagents in the kit (MCP-1 antibody magnetic particle complex reagent, acridinium ester-labeled MCP-1 antibody complex, PBST washing solution, substrate A, substrate B, standards, and quality control product), and equilibrate at room temperature for 30 minutes.

[0089] 2. Sample Processing: Centrifuge the serum / plasma sample to remove the precipitate to obtain the sample to be tested; 3. Immune Reaction: Add 50 μL of the standard / quality control product / sample to be tested, 50 μL of the MCP-1 antibody magnetic particle complex reagent, and 50 μL of the acridinium ester-labeled MCP-1 antibody complex to the reaction cup in sequence, mix well, and incubate at 37 °C for 20 min; 4. Washing: Magnetic separation for 3 minutes, remove the supernatant, add 300 μL PBST washing solution, vortex for 30 seconds, discard the supernatant after magnetic separation, and repeat washing 3 times; 5. Chemiluminescence detection: Add 50 μL of substrate A and 50 μL of substrate B in sequence, put into the chemiluminescence detector, and read the relative luminescence unit (RLU).

[0090] 6. Use the concentration of the standard as the horizontal axis and the corresponding RLU value as the vertical axis to fit the standard curve, and calculate the MCP-1 content in the sample based on the standard curve.

[0091] Comparative Example 1 A kit for chemiluminescence detection of MCP-1 magnetic microparticles The difference between Comparative Example 1 and Example 1 is only that the "coupling buffer" is different.

[0092] The coupling buffer of Comparative Example 1 contained 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, and had a pH value of 5.5-6.5.

[0093] The preparation method of the coupling buffer of Comparative Example 1 is as follows: add 4.88g MES and 2.92g NaCl to 800mL purified water and stir until completely dissolved. Add 2.5g PVP K30, heat to 37°C and continue stirring until completely dissolved, cool to room temperature, add 0.5mL Tween-20 and 45mL glycerol, mix well. Adjust the pH to 5.5-6.5, add purified water to 1L, filter with a 0.22μm filter membrane, and obtain the coupling buffer, which is stored at 4°C.

[0094] Comparative Example 2 A kit for chemiluminescence detection of MCP-1 magnetic microparticles The difference between Comparative Example 2 and Example 1 is only that the "coupling buffer" is different.

[0095] The coupling buffer of Comparative Example 2 contained 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, and had a pH value of 5.5-6.5.

[0096] The preparation method of the coupling buffer in Comparative Example 2 was as follows: 4.88 g of MES and 2.92 g of NaCl were added to 800 mL of purified water, and stirred until completely dissolved. 15 g of PEG 4000 was 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 were added and mixed well. The pH was adjusted to 5.5 - 6.5, and purified water was added to make up to 1 L, and then filtered through a 0.22 μm filter membrane to obtain the coupling buffer, which was stored at 4 °C.

[0097] Comparative Example 3 A kit for chemiluminescence detection of MCP-1 magnetic particles The difference between Comparative Example 3 and Example 1 was only the "coupling buffer".

[0098] The coupling buffer in Comparative Example 3: contained 4.88 g / L of MES, 2.92 g / L of NaCl, 0.05% v / v of Tween-20 and 4.5% v / v of glycerol, and the pH value was 5.5 - 6.5.

[0099] The preparation method of the coupling buffer in Comparative Example 3 was as follows: 4.88 g of MES and 2.92 g of NaCl were added to 800 mL of purified water, and stirred until completely dissolved. 0.5 mL of Tween-20 and 45 mL of glycerol were added and mixed well. The pH was adjusted to 5.5 - 6.5, and purified water was added to make up to 1 L, and then filtered through a 0.22 μm filter membrane to obtain the coupling buffer, which was stored at 4 °C.

[0100] Comparative Example 4 A kit for chemiluminescence detection of MCP-1 magnetic particles The difference between Comparative Example 4 and Example 1 was only the "immunolabeling buffer".

[0101] The immunolabeling buffer in Comparative Example 4: contained 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 the pH value was 9.0 - 10.0.

[0102] The preparation method of the immunolabeling buffer in Comparative Example 4 was as follows: (1) 3.09 g of boric acid was added to an appropriate amount of purified water, stirred until completely dissolved, then the pH was adjusted to 9.0, and purified water was added to make up to 500 mL to obtain the boric acid buffer.

[0103] (2)In a boric acid buffer solution, add 5.85 g of NaCl, 25.67 g of sucrose, stir until completely dissolved, then add 30 g of PEG 6000, heat to 37 °C and continue stirring until completely dissolved. After cooling to room temperature, add 0.2 mL of Triton X-100 and mix well. Adjust the pH value to 9.0 - 10.0, make up to 1 L with purified water, and filter through a 0.22 μm filter membrane to obtain the labeling buffer solution, which is stored at 4 °C.

[0104] Comparative Example 5 A kit for chemiluminescence detection of MCP-1 magnetic particles The difference between Comparative Example 5 and Example 1 lies only in the "coupling buffer solution" and the "immunolabeling buffer solution".

[0105] The coupling buffer solution of Comparative Example 5: contains 4.88 g / L of MES, 2.92 g / L of NaCl, 0.05% v / v of Tween-20 and 4.5% v / v of glycerol, and the pH value is 5.5 - 6.5.

[0106] The preparation method of the coupling buffer solution of Comparative Example 5 is as follows: add 4.88 g of MES and 2.92 g of NaCl to 800 mL of purified water, stir until completely dissolved. Add 0.5 mL of Tween-20 and 45 mL of glycerol, and mix well. Adjust the pH to 5.5 - 6.5, make up to 1 L with purified water, and filter through a 0.22 μm filter membrane to obtain the coupling buffer solution, which is stored at 4 °C.

[0107] The immunolabeling buffer solution 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 the pH value is 9.0 - 10.0.

[0108] The preparation method of the immunolabeling buffer solution of Comparative Example 5 is as follows: (1) Add 3.09 g of boric acid to an appropriate amount of purified water, stir until completely dissolved, then adjust the pH to 9.0, make up to 500 mL with purified water to obtain the boric acid buffer solution.

[0109] (2) In the boric acid buffer solution, add 5.85 g of NaCl, 25.67 g of sucrose, stir until completely dissolved, then add 30 g of PEG 6000, heat to 37 °C and continue stirring until completely dissolved. After cooling to room temperature, add 0.2 mL of Triton X-100 and mix well. Adjust the pH value to 9.0 - 10.0, make up to 1 L with purified water, and filter through a 0.22 μm filter membrane to obtain the labeling buffer solution, which is stored at 4 °C.

[0110] Experimental Example 1 Performance determination of a kit for chemiluminescence detection of MCP-1 magnetic particles 1. Confirmation of blank limit Blank samples without MCP-1 (healthy human serum) were respectively assayed for relative luminescence intensity using the kits of Example 1 and Comparative Examples 1-5, and the method described in Example 2 was referred to. The detection was repeated 20 times, and the concentration values of the 20 test results were obtained.

[0111] Based on the concentration values, calculate: average concentration value ( ), and standard deviation of concentration value (SD). According to the formula: blank limit = , calculate the blank limit. The results are shown in Table 1:[[]]END]] Table 1

[0112] 2. Verification of linear range MCP-1 antigen standard products with concentrations 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 were respectively assayed for relative luminescence intensity using the kits of Example 1 and Comparative Examples 1-5, and the method described in Example 2 was referred to (n = 2). Taking the standard product concentration as the abscissa and the average value of the corresponding relative luminescence intensity as the ordinate, a standard curve was plotted. The relative luminescence intensities at each concentration are shown in Table 2.

[0113] Table 2

[0114] The measurement 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, within the concentration range of 5 - 1000 pg / mL for the kit of Example 1, the linear relationship between the relative luminescence intensity and the sample concentration was good, and R 2 > 0.999 ( Figure 1 ).

[0115] 2. Accuracy verification Different concentrations of MCP-1 standard products were added to blank samples without MCP-1 (healthy human serum) to make the final concentrations of the MCP-1 standard products 200 pg / mL, 500 pg / mL, or 1000 pg / mL. The kits of Example 1 were used to assay the relative luminescence intensity of each sample, and the method described in Example 2 was referred to. Each concentration was assayed 3 times. According to the relative luminescence intensity and the regression equation of the standard curve, the corresponding concentration was calculated, and the recovery rate was calculated according to the following formula:

[0116] The recovery rate measurement results of Example 1 are shown in Table 3 as follows.

[0117] Table 3

[0118] The above results show that when the sample concentrations are 200 pg / mL, 500 pg / mL or 1000 pg / mL, the recovery rates of the kit of the present invention are 96.43±2.23%, 97.48±2.46%, 96.51±2.68% respectively. The kit provided by the present invention has high accuracy.

[0119] 3. Repeatability verification The kit of Example 1 was used to repeatedly test the quality control product (500 pg / mL) 10 times. The mean value and standard deviation of the 10 measurement results were used to calculate the coefficient of variation (CV) according to the following formula.

[0120]

[0121] The measurement results are shown in Table 4. The CV value of the kit of Example 1 of the present invention is 1.93%, indicating good repeatability.

[0122] Table 4

[0123] The above results show that the kit of the present invention has the characteristics of high sensitivity, high accuracy and good repeatability for MCP-1 detection.

[0124] The above detailed description is a specific description of one feasible embodiment of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or changes made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention shall be subject to the appended claims.

Claims

1. A composite system for MCP-1 magnetic particle chemiluminescence detection, characterized in that, The described composite system includes: coupling buffer and labeling buffer; The coupling buffer is composed of MES, NaCl, PEG 4000, PVP K30, Tween-20, glycerol and water, with the pH adjusted to 5.5 - 6.5; The immunolabeling buffer is composed of boric acid, CAPS, NaCl, sucrose, PEG 6000, Triton X-100 and water, with the pH adjusted to 9.0 - 10.

0.

2. The composite system according to claim 1, characterized in that, 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.

3. The composite system according to claim 2, characterized in that, 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.

4. The composite system according to claim 1, characterized in that, 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.

5. The composite system according to claim 4, characterized in that, 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.

6. Use of the composite system according to any one of claims 1 - 5 in the preparation of an MCP-1 magnetic particle chemiluminescence detection kit.

7. A kit for chemiluminescence detection of MCP-1 magnetic microparticles, characterized in that, The described kit contains the composite system according to any one of claims 1 - 5.

8. The kit according to claim 7, characterized in that, The described kit includes an MCP-1 antibody magnetic particle composite reagent and an acridinium ester-labeled MCP-1 antibody complex; The preparation method of the described MCP-1 antibody magnetic particle composite reagent includes the following steps: S1. MCP-1 antibody 1 is formulated into an MCP-1 antibody 1 solution with the coupling buffer; S2. The MCP-1 antibody 1 solution is added to the magnetic particle suspension to obtain a premixed solution, and a coupling catalyst is added. After mixing, a coupling reaction is carried out; S3. After the coupling reaction ends, the supernatant is removed, and the magnetic particles are resuspended with a blocking solution for a blocking reaction; S4. After the blocking reaction ends, the supernatant is removed. After washing 3 times, a magnetic particle preservation solution is added to obtain the MCP-1 antibody magnetic particle composite reagent; The preparation method of the described acridinium ester-labeled MCP-1 antibody complex includes the following steps: a. MCP-1 antibody 2 is formulated into an MCP-1 antibody 2 solution with the immunolabeling buffer; b. An acridinium ester working solution is added to the MCP-1 antibody 2 solution for an acridinium ester labeling reaction; c. After the reaction ends, a blocking solution is added for a blocking reaction to obtain a reaction solution; d. The reaction solution was purified using a gel filtration column, and the eluate was collected to obtain the acridinium ester-labeled MCP-1 antibody complex.

9. The kit according to claim 7, wherein, The method for using the kit comprises the following steps: ① Mix the test sample, standard or quality control product with the MCP-1 antibody magnetic particle complex reagent and the acridinium ester-labeled MCP-1 antibody complex, and incubate. ② Magnetically separate to remove the supernatant, wash, add the luminescent substrate, and measure the relative luminescence intensity. ③ Calculate the MCP-1 concentration based on the relative luminescence intensity.

10. Use of the composite system according to any one of claims 1-5 or the reagent according to any one of claims 7-9, characterized in that, The application includes any of the following aspects: (1) Studying the pathogenesis of diseases involved in the MCP-1 signaling pathway; (2) Screening for MCP-1 inhibitory drugs; (3) Qualitative and quantitative detection of MCP-1.

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