Mononuclear cell chemotactic protein-1 chemiluminescence detection kit

Abstract

The invention discloses a kit for detecting monocyte chemotactic protein-1 by a chemiluminescence method. The kit comprises a magnetic bead working solution, an enzyme working solution, an analysis buffer solution and a calibrator, the magnetic bead working solution comprises a mouse anti-human monocyte chemotactic protein-1 specific monoclonal antibody coated with streptavidin magnetic beads, an MCP-1 antibody and a magnetic bead buffer solution, and the concentration of the MCP-1 antibody in the magnetic bead working solution is 0.5 mu g / mL-5 mu g / mL; the enzyme working solution comprises an alkaline phosphatase labeled mouse anti-human monocyte chemotactic protein-1 specific monoclonal antibody and an enzyme buffer solution; the analysis buffer solution contains an animal serum component, and the calibrator contains a monocyte chemotactic protein-1 component. According to the chemiluminiscence detection kit for the monocyte chemotactic protein-1, the MCP-1 antibody is added into the magnetic bead working solution, so that the linear proportion of a working calibration curve is better, and the repeatability of a high-concentration sample is obviously improved.

Description

Technical Field

[0001] This invention relates to the field of chemiluminescence in vitro detection technology, specifically to a chemiluminescence detection kit for mononuclear cell chemotactic protein-1. Background Technology

[0002] Monocyte Chemotactic Protein 1 (MCP-1), also known as CC Motif Ligand 2 (CCL2), is one of the earliest discovered CC family chemokines. Its precursor molecule consists of a signal peptide and a mature peptide, composed of 23 and 76 amino acids respectively. The mature protein is formed by cleaving the signal peptide. The MCP-1 structure contains four cysteine ​​residues linked by two intrachain disulfide bonds; this structure is crucial for MCP-1's function. The mechanism of action of MCP-1 involves its N-terminus binding to a receptor. Studies have reported that the biggest difference between MCP-1 and its inhibitors lies in the absence of the N-terminus, preventing MCP-1 inhibitors from binding to receptors and thus inhibiting MCP-1 activity. Therefore, it is inferred that the N-terminus of MCP-1 may be the key structure for its function. MCP-1 can bind to CCR1, CCR2, CCR4, and CCR5 chemokine receptors, but it has the highest affinity for CCR2. CCR2 is a G protein-coupled receptor, which can be divided into type A and type B, with type B being the predominant type. After MCP-1 binds to CCR2, it induces a receptor allosteric change, activating the guanosine triphosphate (GTP) protein-coupled phosphatidylinositol signaling pathway and activating the messenger system to function. MCP-1 is an important inflammatory chemokine released by various cell types, including endothelial cells, epithelial cells, vascular smooth muscle cells, fibroblasts, and monocytes / macrophages. It plays a role in regulating the migration of inflammatory cells and directing them to sites of tissue damage.

[0003] After binding to its receptor, MCP-1 chemotactically activates and activates immune cells to migrate to inflamed tissues, thus initiating and promoting the inflammatory response. MCP-1 activates intracellular signal transduction pathways, thereby releasing large amounts of oxygen free radicals and cytotoxic enzymes. After being induced, cells express tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), interleukin-6 (IL-6), and adhesion molecules, etc., which positively act on mast cells and basophils, causing a large release of histamine, regulating phagocytic function, and promoting apoptosis. MCP-1 can chemotactically activate monocytes / macrophages to secrete interleukin-12 (IL-12), inducing naïve CD4+ T cells to differentiate into helper T cells type 1 (Th1). Th1 cells produce interleukin-2 (IL-2), TNF-α, and interferon-gamma (IFN-γ), thereby enhancing cellular immunity and strengthening macrophage activity. Helper T cells type 2 (Th2) produce interleukin-4 (IL-4) and interleukin-10 (IL-10), which, by inhibiting T cell proliferation and Th1 cell responses, weaken the activation rate of macrophages and further influence the function of pro-inflammatory factors. Nuclear transcription factor kappa B (NF-κB) is another signaling molecule that participates in cell activation and amyloid upregulation through MCP-1. NF-κB can participate in the occurrence and development of various diseases by regulating MCP-1. As a key chemokine in the inflammatory cascade, MCP-1 can activate the NF-κB signaling pathway and induce the production of various pro-inflammatory factors. It can not only reflect the severity of inflammatory damage, but also affect the progression of sepsis and increase the risk of acute respiratory distress syndrome.

[0004] Currently, the main detection methods for monocyte chemoattractant protein-1 (MCP-1) are flow cytometry and enzyme-linked immunosorbent assay (ELISA). These methods cannot meet the requirements for rapid detection of large quantities of clinical samples, and their accuracy and repeatability do not meet the requirements for clinical use. Therefore, there is an urgent need in this field for a novel chemiluminescence immunoassay kit for MCP-1 that can detect it rapidly and with high accuracy. Summary of the Invention

[0005] To address the aforementioned issues, this application provides a chemiluminescence assay kit for monocyte chemoattractant protein-1 (MCP-1), which offers higher detection speed and accuracy.

[0006] The objective of this invention is achieved through the following technical solution: a chemiluminescent assay kit for monocyte chemoattractant protein-1 (MCP-1), comprising a magnetic bead working solution, an enzyme working solution, an analytical buffer, and a calibrator; the magnetic bead working solution comprises a mouse anti-human MCP-1 specific monoclonal antibody coated with streptavidin magnetic beads, an MCP-1 antibody, and a magnetic bead buffer, wherein the concentration of the MCP-1 antibody in the magnetic bead working solution is 0.5 μg / mL - 5 μg / mL; the enzyme working solution comprises an alkaline phosphatase-labeled mouse anti-human MCP-1 specific monoclonal antibody and an enzyme buffer; the analytical buffer contains animal serum components, and the calibrator contains MCP-1 components.

[0007] The labeling ratio of mouse anti-human monocyte chemoattractant protein-1 specific monoclonal antibody to alkaline phosphatase in the enzyme working solution during the labeling process is 1:1 to 1:5.

[0008] The animal serum component in the analytical buffer is adult bovine serum, and the dilution ratio of the adult bovine serum is 1:4 to 1:49.

[0009] The analytical buffer contains a heterophilic antibody blocking agent, and the amount of the heterophilic antibody blocking agent used is 1 μg / mL to 20 μg / mL.

[0010] The magnetic bead buffer solution comprises the following components: tris(hydroxymethyl)aminomethane, NaCl, hydrochloric acid, sucrose, bovine serum albumin, PC-950, and Tween-20.

[0011] The preparation of the magnetic bead working solution includes the following steps: (1) The mouse anti-human monocyte chemoattractant protein-1 specific monoclonal antibody was labeled with biotinylate and then purified by dialysis to obtain biotinylated mouse anti-human monocyte chemoattractant protein-1 specific monoclonal antibody. (2) Take streptavidin magnetic beads, replenish them with magnetic bead buffer, wash them with a magnetic separator, and bring the volume to a final level; (3) After mixing the biotinylated mouse anti-human monocyte chemoattractant protein-1 specific monoclonal antibody with magnetic beads at a mass ratio of 1:100, the mixture was rolled at room temperature for 0.5 to 3 hours. (4) Add magnetic bead blocking solution to the solution in step (3), the mass ratio of magnetic bead blocking solution to magnetic microparticles is 1:200, and react at room temperature for 30 min; the blocking solution is vitamin H; (5) After separating the immunomagnetic beads from the solution in step (4) using a magnetic separator, the solution is replenished with magnetic bead buffer and washed. After washing, the magnetic particles are resuspended and the working concentration of the magnetic particles is 0.2 mg / mL. (6) Add MCP-1 antibody to the solution in step (5) so that the concentration of MCP-1 antibody in the magnetic bead working solution is 0.5 μg / mL to 5 μg / mL.

[0012] Compared with the prior art, this application has the following beneficial effects: (1) The mononuclear cell chemiluminescence detection kit of the present invention can improve the linearity of the working calibration curve by adding MCP-1 antibody to the magnetic bead working solution, significantly improve the repeatability of high concentration samples, and significantly enhance the performance of the reagent.

[0013] (2) The present invention uses chemiluminescence method for detection, which has the advantages of rapid detection, large detection throughput and convenient and simple operation. It can realize the rapid detection of a large number of clinical samples and provide auxiliary diagnosis for diseases such as rheumatoid arthritis. Detailed Implementation

[0014] Example This embodiment discloses a chemiluminescent assay kit for detecting monocyte chemoattractant protein-1 (MCP-1), comprising a magnetic bead working solution, an enzyme working solution, an analytical buffer, and calibrators. The magnetic bead working solution comprises streptavidin-coated mouse anti-human MCP-1 specific monoclonal antibody, MCP-1 antibody, and magnetic bead buffer; the enzyme working solution comprises alkaline phosphatase-labeled mouse anti-human MCP-1 specific monoclonal antibody and enzyme buffer; the analytical buffer contains animal serum components; and the calibrators contain MCP-1 components.

[0015] Specifically, the preparation of the above-mentioned magnetic bead working solution includes the following steps: (1) The mouse anti-human monocyte chemoattractant protein-1 specific monoclonal antibody was labeled with biotinylate and then purified by dialysis to obtain biotinylated mouse anti-human monocyte chemoattractant protein-1 specific monoclonal antibody.

[0016] (2) Measure 10 mg of streptavidin magnetic beads, with a streptavidin concentration of 10 mg / mL; add magnetic bead buffer to 3 mL, then wash three times using a magnetic separator, and finally bring the volume to 3 mL. The magnetic bead buffer consists of 12.11 g / L Tris, 9.00 g / L NaCl, 3.7 mL / L hydrochloric acid (HCl), 10.00 g / L sucrose, 5.00 g / L bovine serum albumin (BSA), 1 mL / L PC-950, and 0.5 mL / L Tween-20.

[0017] (3) After mixing the biotinylated mouse anti-human monocyte chemoattractant protein-1 specific monoclonal antibody with magnetic beads at a mass ratio of 1:100, the mixture was rolled at room temperature for 1 hour.

[0018] (4) Add magnetic bead blocking solution to the solution in step (3), the mass ratio of magnetic bead blocking solution to magnetic microparticles is 1:200, and react at room temperature for 30 min; the blocking solution is vitamin H.

[0019] (5) After separating the immunomagnetic beads from the solution in step (4) using a magnetic separator, add the above magnetic bead buffer to 3 mL and wash 3 times. After washing, resuspend the magnetic particles and make the working concentration of the magnetic particles 0.2 mg / mL.

[0020] (6) Add MCP-1 antibody to the solution in step (5) to make the concentration of MCP-1 antibody in the magnetic bead working solution 2 μg / mL, and obtain the magnetic bead working solution.

[0021] The preparation of the above enzyme working solution includes the following steps: A. Mouse anti-human monocyte chemoattractant protein-1 specific monoclonal antibody was chemically labeled and then bound to alkaline phosphatase at a ratio of 1:2 to obtain alkaline phosphatase-labeled mouse anti-human monocyte chemoattractant protein-1 specific monoclonal antibody.

[0022] B. Mix the alkaline phosphatase-labeled mouse anti-human monocyte chemoattractant protein-1 (MCP-1) specific monoclonal antibody obtained in step A with enzyme buffer to obtain the enzyme working solution. The ratio of the alkaline phosphatase-labeled mouse anti-human MCP-1 specific monoclonal antibody to the enzyme buffer is 1:1000. The enzyme buffer consists of 12.11 g / L Tris, 9.00 g / L NaCl, 3.7 mL / L concentrated HCl, 10.00 g / L trehalose, 5.00 g / L BSA, 5.00 g / L casein, 0.1 mL / L TX-100, 1 mL / L PC-950, 10 mL / L glycerol, 10 mL / L 0.1 M MgCl2, and 10 mL / L 0.1 M ZnCl2.

[0023] The analysis buffer consists of adult bovine serum, PC-950, heterophile antibody blocking agent, and purified water. The adult bovine serum is diluted at a ratio of 1:9, the concentration of PC-950 is 3 mL / L, and the concentration of heterophile antibody blocking agent is 5 μg / mL.

[0024] Calibrator preparation method: Dilute monocyte chemoattractant protein-1 with calibration buffer to prepare calibrators with concentrations of 0 pg / mL, 10 pg / mL, 50 pg / mL, 250 pg / mL, 1000 pg / mL, and 2000 pg / mL. The calibration buffer consists of 12.11 g / L Tris, 9.00 g / L NaCl, approximately 3.7 mL / L concentrated HCl, 10.00 g / L trehalose, 20.00 g / L casein, 0.1 mL / L TX-100, 1 mL / L PC-950, and 10 mL / L glycerol.

[0025] Performance tests were conducted on the magnetic bead working solution prepared by the above method: A control reagent was prepared using a method essentially the same as that used for the magnetic bead working solution, except that it did not contain MCP-1 antibody. The magnetic bead working solution prepared using this method was then used as a validation reagent. The control reagent and validation reagent were used to test calibrators and samples containing reagents from the R&D manufacturer (including high, medium, and low value samples), and the repeatability of high-concentration samples was verified. The luminescence value test results of calibrators at different concentrations are shown in the table below: The following table compares the test results of samples containing reagents from R&D manufacturers: The repeatability verification of high-concentration samples is shown in the table below: The above experiments show that adding free MCP-1 to the magnetic bead working solution can improve the linear gradient of the reagent detection curve, significantly improve the repeatability of high-concentration samples, and enhance the consistency of sample comparison.

[0026] The performance of the chemiluminescence immunoassay kit for monocyte chemoattractant protein-1 prepared by the above method was tested: 1. Establishment of calibration curve: Using the LA3000 fully automated chemiluminescence analyzer (Sichuan Medical Device Registration Certificate No. 20222220095) manufactured by Sichuan Wovente Biotechnology Co., Ltd., the calibration procedure was initiated. The test parameters were set as follows: at 37℃, 20 μL of calibrator solution, 50 μL of magnetic bead working solution, and 50 μL of analysis buffer were added to the sample cup, and incubated for 10 minutes. The sample was then washed three times with Wovente Biotechnology's matching cleaning solution (Sichuan Medical Device Registration No. 20160047), followed by the addition of 150 μL of enzyme working solution and incubation for 10 minutes. This was repeated three times with the cleaning solution. Finally, Wovente Biotechnology's matching substrate solution (Sichuan Medical Device Registration No. 20160048) was added, and the luminescence value was detected. Based on the set calibrator concentration and detection signal, a multi-point calibration curve was obtained using a four-parameter fitting method.

[0027] The luminescence values ​​of different concentrations of monocyte chemoattractant protein-1 calibrators are shown in the table below: 2. Limit of detection: Using calibration buffer as a blank sample, the measurement was repeated 20 times to obtain the RLU (relative luminescence value) of the 20 measurements. The mean (M) and standard deviation (SD) of these 20 measurements were calculated to obtain the RLU value corresponding to M+2SD. The RLU value corresponding to M+2SD was then substituted into the calibration curve to calculate the corresponding concentration value, which is the blank limit.

[0028] 3. Linear: For monocyte chemoattractant protein-1 (MCP-1) low-value samples, high-value samples (close to the upper limit of the measurement range) are diluted to at least five concentrations according to a certain ratio, where the low-value concentration samples must be close to the lower limit of the linear range. The assay is performed according to the kit instructions, with each concentration measured three times. The average value is calculated, and the average result is linearly fitted to the dilution ratio or theoretical concentration using the least squares method. The linear correlation coefficient |r| is calculated, and the linear correlation coefficient r should not be less than 0.9900.

[0029] 4. Accuracy: Add a high concentration of monocyte chemoattractant protein-1 serum sample A to serum sample B. The volume of A added should not exceed 10% of the total volume (A+B). Perform three parallel measurements and calculate the recovery rate R according to the following formula. The result should be in the range of 85%-115%.

[0030] Where R is the recovery rate, V is the volume of sample A, V0 is the volume of sample B, c is the detection concentration of sample B after adding sample A, c0 is the concentration of sample B, and cs is the concentration of sample A.

[0031] The results are as follows: 5. Repeatability: Using two samples of different concentrations, the measurements were repeated 10 times. The mean M and standard deviation SD of the 10 measurements were calculated, and the coefficient of variation (CV) was also calculated. The results should meet the standards.

[0032] 6. Sample comparison: The samples were compared with those of a commercially available product (R&D, Human CCL2 / MCP-1 Quantikine ELISA Kit, DCP00) to jointly test 150 serum and plasma samples.

[0033] Unit: pg / mL As can be seen from the above experiments, the performance of the monocyte chemoattractant protein-1 chemiluminescence detection kit prepared by the method of this embodiment meets the requirements, and the sample detection is highly consistent with commercially available reagents. However, the kit of this embodiment uses chemiluminescence detection, which can realize the rapid detection of a large number of clinical samples and provide auxiliary diagnosis for diseases such as rheumatoid arthritis.

[0034] It should be noted that all features disclosed in this specification, or all steps in all methods or processes disclosed, may be combined in any way, except for mutually exclusive features and / or steps.

Claims

1. A monocyte chemoattractant protein- 1 chemiluminescent assay kit characterized in that, The kit comprises a magnetic bead working solution, an enzyme working solution, an analysis buffer and a calibrant; the magnetic bead working solution comprises streptavidin magnetic beads coated with a mouse anti-human monocyte chemotactic protein-1 specific monoclonal antibody, an MCP-1 antibody and a magnetic bead buffer, wherein the concentration of the MCP-1 antibody in the magnetic bead working solution is 0.5 μg / mL-5 μg / mL; the enzyme working solution comprises an alkaline phosphatase-labeled mouse anti-human monocyte chemotactic protein-1 specific monoclonal antibody and an enzyme buffer; the analysis buffer contains an animal serum component, and the calibrant contains a monocyte chemotactic protein-1 component.

2. The MCP-1 chemiluminescence assay kit according to claim 1, wherein, The labeling ratio of the mouse anti-human monocyte chemotactic protein-1 specific monoclonal antibody to the alkaline phosphatase in the labeling process is 1:1-1:

5.

3. The MCP-1 chemiluminescence assay kit according to claim 1, wherein The animal serum component in the analysis buffer is fetal bovine serum, and the dilution ratio of the fetal bovine serum is 1:4-1:

49.

4. The MCP-1 chemiluminescence assay kit according to claim 3, wherein, The analysis buffer contains a heterophil antibody blocker, and the amount of the heterophil antibody blocker used is 1 μg / mL-20 μg / mL.

5. The MCP-1 chemiluminescence assay kit according to claim 1, wherein The magnetic bead buffer comprises the following components: tris-hydroxymethyl aminomethane, NaCl, hydrochloric acid, sucrose, bovine serum albumin, PC-950 and Tween-20.

6. The MCP-1 chemiluminescence assay kit according to claim 1, wherein The preparation of the magnetic bead working solution comprises the following steps: (1) the mouse anti-human monocyte chemotactic protein-1 specific monoclonal antibody is labeled with a biotin ester and then purified by dialysis to obtain a biotinylated mouse anti-human monocyte chemotactic protein-1 specific monoclonal antibody; (2) streptavidin magnetic beads are taken, supplemented with the magnetic bead buffer, washed with a magnetic separator, and then fixed to volume; (3) the biotinylated mouse anti-human monocyte chemotactic protein-1 specific monoclonal antibody is mixed with the magnetic beads at a mass ratio of 1:100, and then rolled at room temperature for 0.5-3 h; (4) the solution in step (3) is added with a magnetic bead blocking solution, the mass ratio of the magnetic bead blocking solution to the magnetic particles is 1:200, and then reacted at room temperature for 30 min; the blocking solution is vitamin H; (5) the solution in step (4) is separated from the immunomagnetic beads using a magnetic separator, supplemented with the magnetic bead buffer and then washed, the magnetic particles are resuspended after washing, and the working concentration of the magnetic particles after resuspension is 0.2 mg / mL; (6) the solution in step (5) is added with the MCP-1 antibody, and the concentration of the MCP-1 antibody in the magnetic bead working solution is 0.5 μg / mL-5 μg / mL.

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