A complement C1q monoclonal antibody and its application

Abstract

This invention relates to the field of monoclonal antibody technology, and in particular to a complement C1q monoclonal antibody and its applications. The heavy chain sequence is shown in SEQ ID NO:1, and the light chain sequence is shown in SEQ ID NO:2. The complement C1q monoclonal antibody of this invention exhibits high specificity and high sensitivity, maintains stability under various experimental conditions, and is not easily affected by external factors, ensuring the accuracy and reliability of the detection results. This complement C1q monoclonal antibody can be used in the field of in vitro diagnostic detection, achieving rapid, efficient, and highly sensitive clinical detection of the complement C1q antigen.

Description

Technical Field

[0001] This invention relates to the field of monoclonal antibody technology, specifically to a complement C1q monoclonal antibody and its applications. Background Technology

[0002] C1q, or complement C1q, is the core initiator molecule of the classical complement system activation pathway and a key bridge connecting innate and adaptive immunity. Its molecular weight is about 410 kDa, and it has a six-armed snowflake-like configuration. Each subunit consists of three polypeptide chains: A, B, and C. It has two major functional domains: an N-terminal collagen-like region (which can bind C1r and C1s to form the C1 complex) and a C-terminal globular head region (which can recognize immune complexes, apoptotic cells, and pathogen surface targets).

[0003] In terms of biological function, C1q can not only trigger the lysing and target-cell lysis effect and release inflammatory mediators by activating the classical complement pathway, but also mediate phagocytosis of apoptotic cells and immune complexes to maintain immune homeostasis. Simultaneously, it regulates the activity of dendritic cells, B cells, and T cells to balance adaptive immune responses, and participates in tissue damage repair and inflammation regulation. Clinically, abnormal serum C1q levels have significant diagnostic and therapeutic value. Decreased levels are commonly seen in autoimmune diseases such as systemic lupus erythematosus, hereditary complement deficiencies, severe infections, and liver diseases, while elevated levels are often associated with acute inflammatory stress such as bacterial infections, tissue damage, and malignant tumors.

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

[0005] The purpose of this invention is to provide a complement C1q monoclonal antibody and its application.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A complement C1q monoclonal antibody, the heavy chain sequence of which is shown in SEQ ID NO:1 and the light chain sequence of which is shown in SEQ ID NO:2.

[0008] The method for preparing complement C1q antibody of the present invention includes the following steps:

[0009] (1) Immunization animals: Female Balb / c mice aged 6-8 weeks were selected and immunized according to the pre-established immunization protocol. The C1q antigen enters the peripheral immune organs through blood circulation or lymphatic circulation, stimulates the corresponding B lymphocyte clones, activates and proliferates them, and differentiates them into sensitized B lymphocytes.

[0010] (2) Cell fusion: Mice were euthanized by enucleation and bloodletting. The spleen was aseptically removed and crushed in a petri dish to prepare a spleen cell suspension. The prepared syngeneic myeloma cells were mixed with mouse spleen cells at a ratio of 1:10, and polyethylene glycol (PEG) was added as a fusion promoter. Under the action of PEG, various lymphocytes could fuse with myeloma cells to form hybridoma cells.

[0011] (3) Selective culture: Selected hybridoma cells were screened using HAT selective culture medium.

[0012] (4) Screening and Cloning of Hybridoma Positive Clones: Only a small number of hybridoma cells grown in HAT medium secrete monoclonal antibodies of the desired specificity; therefore, screening and cloning are necessary. The limiting dilution method is typically used for hybridoma cell cloning. Sensitive, rapid, and specific immunological methods are employed to screen for positive hybridoma cells capable of producing the desired monoclonal antibodies, and clonal amplification is then performed. After comprehensive identification of the immunoglobulin type, subclass, specificity, affinity, epitope recognizing the antigen, and molecular weight of the secreted monoclonal antibodies, they are promptly cryopreserved.

[0013] (5) Purification of monoclonal antibodies: Complement C1q antibody was purified by affinity purification.

[0014] The complement C1q monoclonal antibody of the present invention can be used to prepare a latex immunoturbidimetric assay kit. The kit includes reagent R1 and reagent R2.

[0015] Reagent R1 comprises 50 mM 2-morpholine ethanesulfonic 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-morpholine ethanesulfonic acid buffer is 6.5.

[0016] The preparation method of reagent R2 is as follows: Take 0.1 mL of PS microspheres, add 5 mL of 50 mM pH 6.5 MES buffer and mix, then add 100 μL of 0.01 g / mL EDC and 10 μL of 0.05 g / mL NHS solution, and mix and stir at room temperature for 15 min; add 0.05 mg of complement C1q monoclonal antibody to the reaction system and stir at room temperature for 1 h; add 20 μL of blocking agent to the reaction system and stir for 1 h; centrifuge at 8000 rpm for 15 min, resuspend in 5 mL of 25 mM pH 7.0 PBS solution, sonicate for 10 min, and store at 2-8℃ for later use.

[0017] The sealing agent is an aqueous solution of 1% BSA and 0.2% glycine.

[0018] Compared with the prior art, the beneficial effects of the present invention are:

[0019] (1) The complement C1q monoclonal antibody of the present invention has high specificity and high sensitivity, can maintain stability under various experimental conditions, is not easily affected by external factors, and ensures the accuracy and reliability of the detection results;

[0020] (2) The complement C1q monoclonal antibody of the present invention can be used in the field of in vitro diagnostic detection, realizing rapid, efficient and highly sensitive clinical detection of complement C1q antigen. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the structure of a complement C1q monoclonal antibody.

[0022] Figure 2 The image shows the SDS-PAGE results of the purified complement C1q monoclonal antibody.

[0023] Figure 3 This is the SEC-HPLC chromatogram of the purified complement C1q monoclonal antibody.

[0024] Figure 4 This is a chart for clinical relevance testing.

[0025] Figure 5 The graph shows the accelerated stability test results of H (high value sample) and L (low value sample) at 37℃. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Example 1: Preparation of complement C1q monoclonal antibody

[0028] (1) Immunized animals

[0029] The immunization regimen is selected based on the characteristics of the antigen. For soluble antigens with weak immunogenicity, adjuvants are generally added. Commonly used adjuvants are Freund's complete adjuvant and Freund's incomplete adjuvant. The antigen and adjuvant should be mixed in equal volumes and ground into a water-in-oil emulsion.

[0030] The initial immunization was performed by subcutaneous injection of 50 μg antigen with Freund's complete adjuvant at multiple sites; the second immunization was performed 3 weeks later (same dose of antigen with Freund's incomplete adjuvant); the third immunization was performed by intraperitoneal injection without adjuvant, and the titer was tested 7 days later; the final booster immunization was performed (50 μg antigen intraperitoneal injection), and spleen cells were harvested for fusion 3 days later.

[0031] (2) Cell fusion

[0032] Myeloma cell suspension: Select a good myeloma cell line, isolate myeloma cells from tumors growing in vivo, and prepare a cell suspension;

[0033] Spleen cell suspension: Mice that received a booster 3 days prior underwent orbital bloodletting, and the serum was separated and frozen for later use. The mice were euthanized, and the cells were immersed in 75% ethanol for 3-5 minutes. The spleen was aseptically removed and washed in a petri dish containing 5 mL of incomplete culture medium. The surrounding connective tissue was removed, and the spleen was transferred to a steel mesh in another petri dish containing 5 mL of incomplete culture medium. It was first cut into 3-5 small pieces with scissors, then ground with a syringe core. The spleen cell suspension was transferred to a 50 mL centrifuge tube, 50 mL of incomplete culture medium was added, and the tube was centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the cells were washed and centrifuged again using the same method. The precipitated cells were then resuspended in 10 mL of incomplete culture medium, and the viable cell count was recorded. 0.5-2 × 10⁶ cells were obtained from one mouse. 8 One spleen cell.

[0034] Feeder Cells: After euthanizing, disinfecting, and fixing the mice, use sterile scissors to lift the abdominal skin from the posterior abdomen to expose the peritoneum. Disinfect the peritoneum with an alcohol swab. Inject 10 mL of incomplete culture medium into the peritoneal cavity using a syringe, taking care to avoid perforation of the intestine. Hold the syringe firmly with your right hand, leaving the needle in the peritoneal cavity, and gently massage the abdomen with an alcohol swab in your left hand for 1 minute. Then aspirate the injected culture medium. Centrifuge at 1000 rpm for 10 minutes and discard the supernatant. Resuspend the pelleted cells in 5 mL of HAT medium. Based on the cell count, add more HAT medium to bring the cell concentration to 2 × 10⁶ cells / mL. 5 / mL, for later use.

[0035] Cell fusion: Mix the prepared myeloma cells and mouse spleen cells at a ratio of 1:10, add 20 mL of PRMI-1640 solution; centrifuge at 1000 r / min for 10 min, discard the supernatant, and aspirate as much supernatant as possible; gently tap the bottom of the centrifuge tube with your finger to disperse the precipitated cells, and place the centrifuge tube in a 37℃ water bath; slowly add 1 mL of 50% PEG pre-warmed at 37℃ into the centrifuge tube over about 45 seconds, stirring gently while adding, and let stand at 37℃ for 5 min; add 20 mL of incomplete culture medium (pre-warmed at 37℃) over 5 min, adding 1 mL in the first minute, 2 mL in the second minute, 5 mL in the third minute, and 6 mL each in the fourth and fifth minutes, while gently rotating the centrifuge tube. The medium should be added along the tube wall, not directly onto the precipitated cells, to prevent dislodging the newly fused cells. Then add PRMI-1640 solution to 50 mL to terminate the PEG effect; centrifuge at 800 r / min for 10 min, discard the supernatant, gently suspend the precipitated cells in the required volume of HAT culture medium, and seed 0.10-0.15 mL per well in a 96-well culture plate. After seeding, place the culture plate in a 37℃ 5% CO2 incubator for culture.

[0036] (3) Selective culture: After inoculating 96-well plates for 5 days, replace half of the medium with HAT medium. After 7-10 days, replace HAT medium with HT medium. After 14 days, ordinary complete medium can be used.

[0037] (4) Screening and cloning of hybridoma-positive clones

[0038] Coating: The antigen was coated with Na2CO3-NaHCO3 buffer at pH 9.6. 20 μg of antigen was added to each microplate, and the coating volume per well was 50 μL. Coating was carried out at 37°C for 2 h.

[0039] Blocking: Discard the antigen, blot dry the liquid in the wells of the ELISA plate, and block with 1% BSA at 37°C for 2 hours.

[0040] Primary antibody: After blocking, discard the liquid in the well, pat dry, add 100 μL of the supernatant of the cells to be tested to each well, and incubate at 37°C for 1 h.

[0041] Secondary antibody: After incubation with primary antibody, discard the liquid in the wells, blot dry, and wash three times with PBS buffer (pH 7.4) for 5 minutes each time. Dilute the secondary antibody according to the instructions, add 100 μL to each well, and incubate at 37°C for 1 hour.

[0042] Color development: After incubation with the secondary antibody, discard the liquid in the wells, pat dry, add the colorimetric reagent, and stop the reaction with the stop solution when the color is the deepest. Read the OD value of each well with a microplate reader, select the positive wells, expand the culture and freeze them.

[0043] (5) Purification of monoclonal antibodies

[0044] Complement C1q monoclonal antibody was purified using affinity purification. Staphylococcal A protein was cross-linked with a carrier, and an affinity chromatography column was prepared to bind the antibody before elution. The antibody was then concentrated to 5 mg / mL. The heavy chain sequence of the purified complement C1q monoclonal antibody is shown in SEQ ID NO:1, and the light chain sequence is shown in SEQ ID NO:2. The structure of the complement C1q monoclonal antibody is shown below. Figure 1 As shown.

[0045] Example 2

[0046] Antibody concentrations were analyzed using SDS-PAEG electrophoresis and SEC-HPLC, respectively. Results are as follows: Figure 2 and Figure 3 As shown.

[0047] SDS-PAEG electrophoresis experimental procedure: First, prepare the separating gel and stacking gel, pour the gels in sequence, and remove the comb after they solidify; mix the protein sample with SDS loading buffer at a ratio of 4:1, heat in a boiling water bath for 5-10 minutes to denature, and then cool; then load the gel into the electrophoresis tank, fill it with buffer, add the prepared sample to the sample wells, and electrophoresis at a constant voltage of 80V until the indicator enters the separating gel, then adjust to 120V until the indicator is close to the bottom of the gel; finally, remove the gel, stain it with Coomassie brilliant blue staining solution with shaking for more than 30 minutes, change the destaining solution and shake several times until the bands are clear and the background is transparent.

[0048] SEC-HPLC experimental procedure: Equilibrate the column with phosphate buffer (PBP) until baseline stability; filter an appropriate amount of sample through a 0.22 μm filter membrane to avoid particulate contamination of the column; set the HPLC instrument parameters (flow rate 0.5-1.0 mL / min, column temperature 30℃), and inject the sample solution (injection volume usually 25 μL) after the system stabilizes; start the experiment; after the experiment, rinse the column with PBP and then seal it with pure solvent. During operation, pay attention to degassing the mobile phase, avoid sudden increases in column pressure, and ensure the system is free of air bubbles.

[0049] Example 3: A complement C1q detection kit (latex immunoturbidimetric assay) was prepared using the complement C1q antibody from Example 1.

[0050] Preparation of reagent R1: 50 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 6.5), and the following additives 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, and 0.8% ProClin 300 preservative; the components work synergistically to form a stable pretreatment environment for the immunoreaction, which can effectively eliminate sample matrix interference and maintain the consistency of subsequent reactions.

[0051] Preparation of reagent R2: Take 0.1 mL of PS microspheres (Nanjing Liding Medical Technology Co., Ltd., catalog number: LD102016) and add 5 mL of 50 mM pH 6.5 MES buffer. After mixing, add 100 μL of 0.01 g / mL EDC and 10 μL of 0.05 g / mL NHS solution (EDC and NHS should be prepared fresh for use). Mix and stir at room temperature for 15 min. Add 0.05 mg of complement C1q monoclonal antibody prepared in Example 1 to the reaction system and stir at room temperature for 1 h. Add 20 μL of blocking agent (blocking agent is 1% BSA and 0.2% glycine aqueous solution) to the reaction system and stir for 1 h. Centrifuge at 8000 rpm for 15 min, resuspend in 5 mL of 25 mM pH 7.0 PBS solution, sonicate for 10 min, and store at 2-8℃ for later use.

[0052] Testing instrument: Hitachi 7180 fully automated biochemical analyzer.

[0053] Parameters: Sample volume 2 μL, reagent R1 180 μL, reagent R2 60 μL.

[0054] To highlight the beneficial effects of the present invention, the following comparative experiments are provided.

[0055] Comparative studies used a complement C1q monoclonal antibody prepared from Thermo Fisher Scientific to prepare a complement C1q detection kit (latex immunoturbidimetric assay).

[0056] Preparation of reagent R1: 50 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 6.5), and the following additives 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, and 0.8% ProClin 300 preservative; the components work synergistically to form a stable pretreatment environment for the immunoreaction, which can effectively eliminate sample matrix interference and maintain the consistency of subsequent reactions.

[0057] Preparation of reagent R2: Take 0.1 mL of PS microspheres (Nanjing Liding Medical Technology Co., Ltd., catalog number: LD102016) and add 5 mL of 50 mM pH 6.5 MES buffer. After mixing, add 100 μL of 0.01 g / mL EDC and 10 μL of 0.05 g / mL NHS solution (EDC and NHS should be prepared fresh for use). Mix and stir at room temperature for 15 min. Add 0.05 mg of complement C1q monoclonal antibody (catalog number: MA1-40311, Thermo Fisher Scientific) prepared in Example 1 to the reaction system and stir at room temperature for 1 h. Add 20 μL of blocking agent (blocking agent is 1% BSA and 0.2% glycine aqueous solution) to the reaction system and stir for 1 h. Centrifuge at 8000 rpm for 15 min, resuspend in 5 mL of 25 mM pH 7.0 PBS solution, sonicate for 10 min, and store at 2-8℃ for later use.

[0058] Testing instrument: Hitachi 7180 fully automated biochemical analyzer.

[0059] Parameters: Sample volume 2 μL, reagent R1 180 μL, reagent R2 60 μL.

[0060] Figure 4 The results presented are from a clinical relevance test. It can be seen that the absorbance of both samples increases significantly with increasing sample concentration. Furthermore, under the same sample concentration conditions, the absorbance value of Example 3 is significantly higher than that of Comparative Example 1, indicating that Example 3 has a stronger signal response to changes in sample concentration and can produce a more significant absorbance signal at the same concentration. Simultaneously, their coefficients of determination R... 2 (The values ​​for Example 3 and Comparative Example 1 are 0.9821 and 0.9804 respectively), both very close to 1, indicating a strong linear correlation between absorbance and sample concentration in both methods, demonstrating excellent concentration correlation in the detection results; Specifically, the R value for Example 3 is... 2 Slightly higher than the comparative example 1, it means that its detection data has a better linear fit with concentration, and combined with a stronger signal response, it performs better in terms of the consistency of concentration-signal correlation and signal identification in clinical detection.

[0061] Figure 5 The accelerated stability test results for H (high-value sample) and L (low-value sample) at 37℃ show that the relative deviations of H and L samples in Example 3 remained within 5%, with small overall deviation amplitude and smooth fluctuations, demonstrating excellent temporal stability. In contrast, the relative deviations of H and L samples in Comparative Example 1 gradually increased with the number of days, especially with H (Comparative Example 1) approaching 10% in the later stages, exhibiting significantly greater fluctuations than Example 3. This indicates that under accelerated aging conditions at 37℃, the reagent stability of Example 3 is significantly better than that of Comparative Example 1, and the temporal consistency of the test results is more reliable.

[0062] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

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; 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 preparation method of the reagent R2 is as follows: 0.1 mL PS microspheres are 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.

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 pH of the 2-morpholinoethanesulfonic acid buffer is 6.

5.

5. 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 latex immunoturbidimetric test kits for the detection of rheumatoid factor (RF) in serum samples. The blocking agent is 1% BSA and 0.2% glycine aqueous solution.

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