Complement C1q monoclonal antibody and application

Abstract

The invention relates to the technical field of monoclonal antibodies, in particular to a complement C1q monoclonal antibody and application thereof, the heavy chain sequence of the complement C1q monoclonal antibody is shown as SEQ ID NO: 1, and the light chain sequence of the complement C1q monoclonal antibody is shown as SEQ ID NO: 2. The complement C1q monoclonal antibody provided by the invention has high specificity and high sensitivity, can maintain stability under various experimental conditions, is not easily interfered by external factors, and ensures the accuracy and reliability of a detection result. The complement C1q monoclonal antibody provided by the invention can be used in the field of in vitro diagnosis and detection, and realizes rapid, efficient and high-sensitivity clinical detection of the complement C1q antigen.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of monoclonal antibodies, in particular to a complement C1q monoclonal antibody and application. BACKGROUND

[0002] C1q, namely complement C1q, is a core starting molecule of a classical activation pathway of a complement system, and is a key bridge connecting innate immunity and adaptive immunity, has a molecular weight of about 410 kDa, has a six-arm snowflake-shaped structure, each subunit is composed of A, B and C three polypeptide chains, and has two big functional domains of an N-terminal collagen-like region (capable of combining C1r and C1s to form a C1 complex) and a C-terminal spherical head region (capable of recognizing immune complexes, apoptotic cells and pathogen surface targets).

[0003] In biological functions, C1q can not only trigger a lysis target cell effect and release inflammatory mediators by activating a complement classical pathway, but also mediates phagocytes to remove apoptotic cells and immune complexes to maintain immune self-stability, balances adaptive immune responses by regulating dendritic cell, B cell and T cell activities, and participates in tissue damage repair and inflammation regulation. In a clinical aspect, abnormal serum C1q level has important diagnosis and treatment values, a decrease in the serum C1q level is often seen in autoimmune diseases such as systemic lupus erythematosus, genetic complement deficiency diseases, severe infections and liver diseases, and an increase in the serum C1q level is often related to acute inflammatory stress such as bacterial infection, tissue damage and malignant tumor.

[0004] Therefore, it is of great significance to develop a complement C1q antibody with high specificity and high affinity for high-sensitivity detection of the content of C1q antigen in a sample. SUMMARY

[0005] The application aims to provide a complement C1q monoclonal antibody and application.

[0006] To achieve the above object, the application provides the following technical scheme. A complement C1q monoclonal antibody, a heavy chain sequence of which is shown in SEQ ID NO: 1, and a light chain sequence of which is shown in SEQ ID NO: 2.

[0007] A preparation method of the complement C1q antibody, comprising the following steps: (1) Immunizing an animal: 6-8-week-old female Balb / c mice are selected, and immunization injection is performed according to a prepared immunization scheme. C1q antigen enters a peripheral immune organ through blood circulation or lymph circulation, stimulates corresponding B lymphocyte clones, activates, proliferates and differentiates the B lymphocyte clones into sensitized B lymphocyte.

[0008] (2) Cell fusion: the mice were sacrificed by eyeball enucleation and bloodletting, the spleen was taken out under aseptic operation, and the spleen cell suspension was prepared by grinding in a flat dish. The prepared syngeneic myeloma cells were mixed with the mouse spleen cells at a ratio of 1:10, and a fusion promoter polyethylene glycol was added. Under the action of polyethylene glycol, various lymphocytes can fuse with myeloma cells to form hybridoma cells.

[0009] (3) Selective culture: the fused hybridoma cells were screened by using HAT selective culture medium.

[0010] (4) Screening and cloning of hybridoma positive clones: only a small number of hybridoma cells growing in the HAT culture medium are cells secreting predetermined specific monoclonal antibodies, and therefore, screening and cloning are necessary. The cloning culture of hybridoma cells is usually carried out by using the limited dilution method. The positive hybridoma cells capable of producing the required monoclonal antibodies are screened by using sensitive, rapid and specific immunological methods, and are cloned and expanded. After the immunoglobulin type, subclass, specificity, affinity, epitope and molecular weight of the monoclonal antibodies secreted by the hybridoma cells are comprehensively identified, the hybridoma cells are timely frozen.

[0011] (5) Purification of monoclonal antibodies: the complement C1q monoclonal antibodies are purified by affinity purification.

[0012] The complement C1q monoclonal antibodies of the present application can be used for preparing a latex immunoturbidimetric detection kit. The kit comprises reagent R1 and reagent R2.

[0013] The reagent R1 comprises 50 mM 2-morpholinoethanesulfonic acid buffer, and further comprises 5% polyethylene glycol 20000, 1% bovine serum albumin, 0.5% polyoxyethylene sorbitan monolaurate, 1% sucrose and 0.8% ProClin 300 preservative. The pH of the 2-morpholinoethanesulfonic acid buffer is 6.5.

[0014] The preparation method of the reagent R2 is as follows: 0.1 mL PS microspheres are taken, mixed with 5 mL 50 mM pH 6.5 MES buffer solution, and then 100 μL 0.01 g / mL EDC and 10 μL 0.05 g / mL NHS solution are added, respectively, and mixed and stirred at room temperature for 15 min; 0.05 mg complement C1q monoclonal antibodies are added to the reaction system, and stirred at room temperature for 1 h; 20 μL blocking agent is added to the reaction system, and stirred for 1 h; centrifuged at 8000 rpm for 15 min, resuspended with 5 mL 25 mM pH 7.0 PBS solution, ultrasonicated for 10 min, and stored at 2-8℃ for standby.

[0015] The blocking agent is 1% BSA and 0.2% glycine aqueous solution.

[0016] Compared with the prior art, the present application has the following beneficial effects: (1) The complement C1q monoclonal antibody of the present application has high specificity and high sensitivity, can maintain stability under various experimental conditions, is not easily disturbed by external factors, and ensures the accuracy and reliability of the detection results; (2) The complement C1q monoclonal antibody of the present application can be used in the field of in vitro diagnostic detection, and realizes rapid, efficient and high-sensitivity clinical detection of the complement C1q antigen. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Figure 1 is a structural schematic diagram of the complement C1q monoclonal antibody.

[0018] Figure 2 Figure 4 is the SDS-PAGE result of the complement C1q monoclonal antibody after purification.

[0019] Figure 3 Figure 5 is the SEC-HPLC spectrum of the complement C1q monoclonal antibody after purification.

[0020] Figure 4 Figure 6 is a clinical correlation test chart.

[0021] Figure 5 Figure 7 is an accelerated stability test chart of H (high value sample) and L (low value sample) under the condition of 37°C. DETAILED DESCRIPTION

[0022] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0023] Example 1 Preparation of complement C1q monoclonal antibody (1) Immunization of animals The immunization scheme is selected according to the characteristics of the antigen. For soluble antigens with weak immunogenicity, an adjuvant is generally added. Commonly used adjuvants are Freund's complete adjuvant and Freund's incomplete adjuvant. The antigen and the adjuvant are required to be mixed together in equal volume and ground into a water-in-oil emulsion.

[0024] The first immunization adopts 50 μg of antigen plus Freund's complete adjuvant for subcutaneous multi-point injection; the second immunization (same dose of antigen plus Freund's incomplete adjuvant) is carried out after 3 weeks; the third immunization is carried out without adjuvant, intraperitoneal injection, and the titer is detected after 7 days; and the last booster immunization (50 μg of antigen intraperitoneal injection) is carried out, and the spleen cells are taken for fusion after 3 days.

[0025] (2) Cell fusion Myeloma cell suspension: select myeloma cell line, isolate myeloma cells from in vivo grown tumor, prepare cell suspension; Spleen cell suspension: take the mouse immunized 3 days ago, orbital blood, separate serum and freeze for later use. Sacrifice the mouse and soak it in 75% alcohol for 3-5 min. Sterilely remove the spleen and place it in a dish containing 5 mL of incomplete medium, wash it, cut off the surrounding connective tissue, and transfer the spleen to another steel mesh containing 5 mL of incomplete medium. First cut it into 3-5 small pieces with scissors, then grind it with a syringe core. Transfer the spleen cell suspension to a 50 mL centrifuge tube, add 50 mL of incomplete medium, centrifuge at 1000 r / min for 5 min, discard the supernatant, and wash and centrifuge again in the same way. Then resuspend the precipitated cells in 10 mL of incomplete medium, count the viable cells, and one mouse can get 0.5-2 x 10 8 Spleen cells.

[0026] Feeder cells: after the mouse is killed, surface disinfected and fixed, the abdominal skin is lifted up with sterile scissors and forceps from the back, exposing the peritoneum. Wipe the peritoneum with an alcohol cotton ball. Inject 10 mL of incomplete medium into the abdominal cavity with a syringe, taking care not to penetrate the intestinal tube. Hold the syringe with the right hand, leaving the needle in the abdominal cavity, and hold an alcohol cotton ball with the left hand and gently massage the abdomen for 1 min, then suck out the injected medium. Centrifuge at 1000 r / min for 10 min, discard the supernatant. First suspend the precipitated cells with 5 mL of HAT medium, according to the cell count results, add HAT medium to make the cell concentration 2 x 10 5 mL, ready for use.

[0027] Cell fusion: mix the prepared myeloma cells and mouse spleen cells at a ratio of 1:10, add 20 mL of PRMI-1640 liquid; centrifuge at 1000 r / min for 10 min, discard the supernatant, and try to suck it out completely; gently tap the bottom of the centrifuge tube with your fingers to disperse the precipitated cells, and place the centrifuge tube in a 37°C water bath; slowly drop 1 mL of 37°C pre-warmed 50% PEG into the centrifuge tube, drop for about 45 s, gently stir while dropping, and stand still for 5 min at 37°C; add 20 mL of incomplete medium (pre-warmed at 37°C) within 5 min, add 1 mL in the first minute, 2 mL in the second minute, 5 mL in the third minute, and 6 mL in the fourth and fifth minutes, while gently rotating the centrifuge tube while adding, and add along the tube wall, not directly on the precipitated cells, to prevent the just fused cells from being washed away. Then add PRMI-1640 liquid to 50 mL to terminate the PEG action; centrifuge at 800 r / min for 10 min, discard the supernatant, and gently suspend the precipitated cells in the required volume of HAT medium, inoculate 96-well culture plates (0.10-0.15 mL per well), and after inoculation, place the culture plates in a 37°C 5% CO2 incubator for culture.

[0028] (3) Selective culture After inoculating 96-well plates for 5 days, 1 / 2 medium was replaced with HAT medium, and 7-10 days later, HAT medium was replaced with HT medium. On the 14th day, ordinary complete medium can be used.

[0029] (4) Screening and cloning of hybridoma positive clones Coating: Coat the antigen with Na2CO3-NaHCO3 buffer at pH 9.6, add 20 μg of antigen to each enzyme-labeled plate, and coat each well with 50 μL of coating volume at 37°C for 2 h.

[0030] Blocking: Discard the antigen, tap the enzyme-labeled plate well liquid, and block with 1% BSA at 37°C for 2 h.

[0031] Primary antibody: After blocking, discard the well liquid, tap dry, add 100 μL of the cell supernatant to be tested to each well, and incubate at 37°C for 1 h.

[0032] Secondary antibody: After incubating the primary antibody, discard the well liquid, tap dry, and wash with pH 7.4 PBS buffer for 3 times, 5 min each time. Dilute the secondary antibody according to the instructions, add 100 μL to each well, and incubate at 37°C for 1 h.

[0033] Color development: After incubating the secondary antibody, discard the well liquid, tap dry, add color developing agent, and terminate the reaction with a stop solution when the color is the darkest. Read the OD values of each well with an enzyme-labeled instrument, select positive wells, and expand culture and freeze storage.

[0034] (5) Purification of monoclonal antibody The complement C1q monoclonal antibody was purified by affinity purification method, staphylococcal protein A was cross-linked with the carrier to prepare an affinity chromatography column, the antibody was combined and eluted, and the antibody was concentrated to 5 mg / mL. The complement C1q monoclonal antibody heavy chain sequence obtained by purification is shown as SEQ ID NO: 1, and the light chain sequence is shown as SEQ ID NO: 2. The structure of the complement C1q monoclonal antibody is shown as Figure 1 .

[0035] Example 2 The antibody concentration was analyzed by SDS-PAEG electrophoresis and SEC-HPLC, respectively. The results are shown in Figure 2 and Figure 3 .

[0036] SDS-PAEG electrophoresis experimental procedure: first prepare the separation gel and concentrated gel, fill the gel in turn and pull out the comb after it is solidified; mix the protein sample with the SDS loading buffer at a ratio of 4:1, heat in a boiling water bath for 5-10 minutes, then cool; then load the gel into the electrophoresis tank, add enough buffer, add the treated sample to the sample well, first electrophorese at 80V until the indicator enters the separation gel, then adjust to 120V until the indicator approaches the bottom of the gel; finally take out the gel, oscillate with coomassie brilliant blue staining solution for more than 30 minutes, and replace the decolorizing solution several times until the bands are clear and the background is transparent.

[0037] SEC-HPLC experimental procedure: equilibrate the chromatographic column with the mobile phase phosphate buffer until the baseline is stable; filter an appropriate amount of sample through a 0.22μm filter to avoid particle contamination of the chromatographic column; set the HPLC instrument parameters (flow rate 0.5-1.0 mL / min, column temperature 30℃), and after the system is stable, inject the sample solution (usually 25μL); start running, after the experiment is completed, flush the chromatographic column with the mobile phase phosphate buffer, and then seal it with pure solvent. Pay attention to degassing the mobile phase and avoiding sudden increase in column pressure to ensure that the system is bubble-free.

[0038] Example 3 Preparation of complement C1q test kit (latex immunoturbidimetry) using complement C1q antibody in Example 1 Preparation of reagent R1: 50 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 6.5), and the following components based on the total mass of the system: 5% polyethylene glycol 20000 (PEG20000), 1% bovine serum albumin (BSA), 0.5% polyoxyethylene sorbitan monolaurate (Tween 20), 1% sucrose, 0.8% ProClin 300 preservative; the components synergistically form a stable immune reaction pretreatment environment, which can effectively eliminate sample matrix interference and maintain the consistency of subsequent reactions.

[0039] Preparation of reagent R2: take 0.1 mL PS microspheres (Nanjing Lideng Medical Technology Co., Ltd., Catalog No.: LD102016) and add 5 mL 50 mM pH 6.5 MES buffer, then add 100μL 0.01g / mL EDC and 10μL 0.05g / mL NHS solution (EDC and NHS are prepared and used immediately), mix and stir at room temperature for 15 min; add 0.05mg complement C1q monoclonal antibody prepared in Example 1 to the reaction system, stir at room temperature for 1h; add 20μL blocking agent (blocking agent is 1% BSA and 0.2% glycine aqueous solution) to the reaction system, stir for 1h; centrifuge at 8000rpm for 15min, resuspend with 5 mL 25 mM pH 7.0 PBS solution, ultrasonic for 10min, store at 2-8℃ for standby.

[0040] Detection instrument: Hitachi 7180 automatic biochemical analyzer.

[0041] Parameter: sample amount 2 μL, reagent R1 180 μL, reagent R2 60 μL.

[0042] In order to highlight the beneficial effects of the present application, the following comparative example experiments are exemplified.

[0043] The comparative example adopts the complement C1q monoclonal antibody of ThermoFisher Company to prepare a complement C1q detection kit (latex immunoturbidimetry) Preparation of reagent R1: 50 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 6.5), and the following components are added based on the total mass of the system: 5% polyethylene glycol 20000 (PEG20000), 1% bovine serum albumin (BSA), 0.5% polyoxyethylene sorbitan monolaurate (Tween 20), 1% sucrose, 0.8% ProClin 300 preservative; Each component cooperates to form a stable immune reaction pretreatment environment, which can effectively eliminate sample matrix interference and maintain the consistency of subsequent reactions.

[0044] Preparation of reagent R2: Take 0.1 mL PS microspheres (Nanjing Liding Medical Technology Co., Ltd., Item No.: LD102016) and add 5 mL 50 mM pH 6.5 MES buffer, then add 100 μL 0.01 g / mL EDC and 10 μL 0.05 g / mL NHS solution (EDC and NHS are prepared and used immediately), and mix and stir at room temperature for 15 min; 0.05 mg of the complement C1q monoclonal antibody prepared in Example 1 (Item No.: MA1-40311, ThermoFisher Company) is added to the reaction system, and stirred at room temperature for 1 h; 20 μL of blocking agent (the blocking agent is a 1% BSA and 0.2% glycine aqueous solution) is added to the reaction system, and stirred for 1 h; centrifuged at 8000 rpm for 15 min, resuspended with 5 mL 25 mM pH 7.0 PBS solution, ultrasonicated for 10 min, and stored at 2-8°C for standby.

[0045] Detection instrument: Hitachi 7180 automatic biochemical analyzer.

[0046] Parameter: sample amount 2 μL, reagent R1 180 μL, reagent R2 60 μL.

[0047] Figure 4The clinical correlation test results are presented, it can be seen that the absorbance of both increases obviously with the increase of sample concentration, and under the same sample concentration condition, the absorbance value corresponding to example 3 is obviously higher than that of comparative example 1, which means that the signal response of example 3 to sample concentration change is stronger, and can produce more significant absorbance signal under the same concentration. At the same time, the determination coefficients R 2 (0.9821 for example 3 and 0.9804 for comparative example 1) are very close to 1, indicating that there is a strong linear correlation between the absorbance and the sample concentration of the two schemes, and the concentration correlation performance of the detection results is excellent; the R 2 of example 3 is slightly higher than that of comparative example 1, which means that the linear fitting effect of its detection data and concentration is better, and combined with stronger signal response, it performs more outstanding in the consistency of concentration-signal correlation and signal recognition degree in clinical detection.

[0048] Figure 5 The accelerated stability test results of H (high value sample) and L (low value sample) under 37℃ condition can be seen, the relative deviation of H and L samples of example 3 always maintains within 5%, the overall deviation amplitude is small and the fluctuation is gentle, and the time stability performance is excellent; the relative deviation of H and L samples of comparative example 1 gradually increases with the increase of days, especially the relative deviation of H (comparative example 1) in the later stage is close to 10%, and the fluctuation amplitude is significantly greater than that of example 3. This shows that under the accelerated aging condition of 37℃, the reagent stability of example 3 is obviously better than that of comparative example 1, and the time consistency of the detection results is more reliable.

[0049] The above is only a preferred embodiment of the present application, and does not limit the present application in any form, although the present application has been disclosed as above with a preferred embodiment, however, it is not intended to limit the present application, any person skilled in the art, without departing from the scope of the technical scheme of the present application, can make some changes or modifications to the above disclosed technical content to make equivalent embodiments with equivalent changes, but as long as it does not deviate from the technical scheme content of the present application, any simplification, modification, equivalent change and modification of the above embodiments according to the technical essence of the present application, all still belong to the scope of the technical scheme of the present application.

Claims

1. A complement Clq monoclonal antibody, characterized in that: The heavy chain sequence is shown as SEQ ID NO: 1, and the light chain sequence is shown as SEQ ID NO:

2.

2. The complement C1q monoclonal antibody of claim 1 is used for preparing a latex immunoturbidimetry detection kit.

3. The use of the complement C1q monoclonal antibody according to claim 2 in the preparation of latex immunoturbidimetric test kits, characterized by the fact that it is used in the preparation of a latex immunoturbidimetric test kit for the detection of rheumatoid factor (RF) in serum samples. The kit comprises reagent R1 and reagent R2.

4. The use of the complement C1q monoclonal antibody according to claim 3 in the preparation of latex immunoturbidimetric test kits, characterized by the fact that it is used in the preparation of a latex immunoturbidimetric test kit for the detection of rheumatoid factor (RF) in serum samples. The reagent R1 comprises 50 mM 2-morpholinoethanesulfonic acid buffer, and further comprises 5% polyethylene glycol 20000, 1% bovine serum albumin, 0.5% polyoxyethylene sorbitan monolaurate, 1% sucrose, and 0.8% ProClin 300 preservative.

5. The use of the complement C1q monoclonal antibody according to claim 4 for the preparation of a latex immunoturbidimetric test kit, characterized by the fact that it is used for the detection of anti-C1q antibodies in the serum of patients with SLE. The pH of the 2-morpholinoethanesulfonic acid buffer is 6.

5.

6. The use of the complement C1q monoclonal antibody according to claim 3 in the preparation of a latex immunoturbidimetric test kit, characterized by the fact that it is used in the preparation of a latex immunoturbidimetric test kit for the detection of anti-C1q antibodies in the serum of patients with SLE. The preparation method of the reagent R2 is as follows: 0.1 mL PS microspheres are taken, mixed with 5 mL 50 mM pH 6.5 MES buffer, and then 100 μL 0.01 g / mL EDC and 10 μL 0.05 g / mL NHS solution are added respectively, and mixed and stirred at room temperature for 15 min; 0.05 mg complement C1q monoclonal antibody is added to the reaction system, and stirred at room temperature for 1 h; 20 μL blocking agent is added to the reaction system, and stirred for 1 h; centrifuged at 8000 rpm for 15 min, resuspended with 5 mL 25 mM pH 7.0 PBS solution, ultrasonicated for 10 min, and stored at 2-8℃ for standby.

7. The use of the complement C1q monoclonal antibody according to claim 6 for the preparation of a latex immunoturbidimetric test kit, characterized by the fact that it is used for the detection of anti-C1q antibodies in the serum of patients with SLE. The blocking agent is 1% BSA and 0.2% glycine aqueous solution.

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