Hybridoma cell strain capable of secreting IgG1 type monoclonal antibody resisting human N-type blood group antigen and application of hybridoma cell strain
By preparing the hybridoma cell line N1C8 with IgG1 monoclonal antibody, the problem of complex and easily missed detection of anti-N antibody in the existing technology has been solved, realizing efficient and accurate detection of human N blood group antigen, and avoiding the occurrence of hemolytic transfusion reaction and neonatal hemolytic disease.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-14
- Publication Date
- 2026-04-10
AI Technical Summary
In existing technologies, anti-N antibodies are mostly low-temperature antibodies, and the detection methods are complex and prone to false negatives, leading to inconsistencies in ABO blood typing or crossmatching, which may cause hemolytic transfusion reactions and neonatal hemolytic disease. In particular, IgG type anti-N antibodies are difficult to detect in indirect antiglobulin tests.
The hybridoma cell line N1C8, which secretes IgG1 monoclonal antibodies against human N blood group antigen, was prepared using hybridoma technology. Monoclonal antibodies against human N blood group antigen were prepared using this cell line, and a human N blood group antigen detection reagent was developed. The antibody was then used to react with red blood cells for detection.
This method enables efficient detection of human N blood group antigen, ensuring the accuracy of test results and avoiding hemolytic transfusion reactions and neonatal hemolytic disease caused by missed detection of IgG anti-N antibodies. It provides a scientific and comprehensive method for detecting human N blood group antigen.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of biotechnology and medicine, in particular to a hybridoma cell strain secreting IgG1 type monoclonal antibody against human N type blood group antigen, the monoclonal antibody and application thereof. BACKGROUND
[0002] MN blood group system is the second red blood cell blood group system discovered after ABO blood group system, and its complexity is only next to Rh blood group system. Glycophorin A (GPA) and glycophorin B (GPB) are important components of human red blood cell membrane proteins and important molecular markers for red blood cell differentiation. There are M and N antigenic determinants on GPA, and M and N are complex allelic antigens. Anti-N antibody is mostly naturally occurring IgM type antibody, and IgG type anti-N antibody produced by immunization is extremely rare. Anti-N antibody is mostly cold antibody, and the reaction with N antigen positive red blood cells is relatively strong at 4℃, the reaction is weakened or no reaction at 25℃ or above, and the agglutination reaction disappears at 37℃. Anti-N antibody detected in test tube saline method at 22℃ or below can cause ABO blood group positive and negative typing inconsistency or cross matching incompatibility, which brings troubles to determine the detection results and timely clinical blood transfusion. If a large amount of N antigen positive blood is transfused to a patient, IgG type anti-N antibody can be produced by the immune stimulation of N antigen positive red blood cells in the body, and the antibody can only appear agglutination reaction in the indirect anti-human globulin test. If IgG type anti-N antibody is missed, it can also cause hemolytic transfusion reaction and the occurrence of hemolytic disease of the newborn. MN blood group system causes hemolytic disease of the newborn similar to ABO blood group system, and the first fetus without blood transfusion history can also be affected, and low titer antibody can also cause severe hemolytic disease of the newborn. SUMMARY
[0003] The purpose of the present application is to provide a hybridoma cell strain secreting IgG1 type monoclonal antibody against human N type blood group antigen, to prepare monoclonal antibody against human N type blood group antigen by using the cell strain, and to develop human N blood group antigen detection reagent.
[0004] The present application prepares a hybridoma cell strain secreting IgG1 type monoclonal antibody against human N type blood group antigen by using human O type NN homozygote red blood cells as antigens through hybridoma technology, and prepares monoclonal antibody against human N type blood group antigen by using the cell strain. The human N blood group antigen detection reagent is developed by adding salt and protein in the antibody, and can be applied to the detection of human N blood group antigen, so that the human N blood group antigen detection method is more scientific and perfect.
[0005] The present application provides a hybridoma cell strain secreting IgG1 type monoclonal antibody against human N type blood group antigen, which is named hybridoma cell strain N1C8 (N1C8). Hybridoma cell lineN1C8): preserved in China Center for Type Culture Collection, Wuhan University, Wuhan, China, on August 7, 2025, and the preservation number is CCTCC NO: C2025231.
[0006] The present application provides a monoclonal antibody against human N blood group antigen, which is secreted by the hybridoma cell strain secreting IgG1 type monoclonal antibody against human N blood group antigen as described above.
[0007] The type of the monoclonal antibody against human N blood group antigen is IgG1 type; the monoclonal antibody against human N blood group antigen has a cell agglutination reaction with human N positive red blood cells and does not have a cell agglutination reaction with human N negative red blood cells.
[0008] The present application provides a blood group detection reagent, which comprises the monoclonal antibody against human N blood group antigen as described above.
[0009] The present application provides a blood group detection method, which comprises the operation step of reacting a sample to be tested with the monoclonal antibody against human N blood group antigen as described above to determine the N blood group of the sample to be tested.
[0010] The present application provides the use of the monoclonal antibody against human N blood group antigen in the preparation of a human N blood group detection reagent.
[0011] The present application has the positive effect that a hybridoma cell strain secreting IgG1 type monoclonal antibody against human N blood group antigen and the application of the monoclonal antibody are disclosed. BALB / c mice are immunized with human O type NN homozygote red blood cell suspension as antigen, and a hybridoma cell strain N1C8 capable of stably passing and secreting monoclonal antibody against human N blood group antigen is obtained through immunization, cell fusion, screening and cloning. The type of the monoclonal antibody against human N blood group antigen secreted by the hybridoma cell strain as described above is IgG1 type. The monoclonal antibody against human N blood group antigen as described above can have a cell agglutination reaction with N positive red blood cells and does not have a cell agglutination reaction with human N negative red blood cells, and is a monoclonal antibody against human N blood group antigen. The cell culture supernatant is detected by tube agglutination test with human N type red blood cells, and the cell supernatant titer can reach 16.
[0012] The above description is only a summary of the technical solutions of the present application, in order to more clearly understand the technical means of the present application, and the content of the specification can be implemented, the following examples are used to describe in detail. The specific embodiments of the present application are given in detail by the following examples. BRIEF DESCRIPTION OF DRAWINGS
[0013] Figure 1 . Tube agglutination titer determination results of monoclonal antibody against human N blood group antigen (clone number: N1C8) and O type NN homozygote cells; Figure 2 Results of in vitro agglutination titer assay of monoclonal antibody against human N blood group antigen (clone number: N1C8) and O-type MN heterozygous cells; Figure 3 Specificity identification results of monoclonal antibody against human N-type blood group antigen (clone number: N1C8); Figure 4 Ig subclass identification results of monoclonal antibody against human N blood group antigen (clone number: N1C8); Figure 5 Chromosome karyotype analysis results of hybridoma cell line (clone number: N1C8); Figure 6 The results of partial sample reactions were detected using a monoclonal antibody against human N blood group antigen (clone number: N1C8) and an anti-N antibody reagent (DIAGEST). Detailed Implementation
[0014] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.
[0015] Example 1 1. Preparation of immunogens The immunogen consisted of fresh human type O NN homozygous red blood cells, washed three times with physiological saline, and prepared into a 10% red blood cell suspension with physiological saline. The immunogen was prepared fresh each time it was used to ensure the freshness of the red blood cells.
[0016] 2. Animal immunization and blood testing.
[0017] BALB / c mice that are 4 weeks old and weigh approximately 20g were immunized with an immunogen at a dose of 500µL per mouse via intraperitoneal injection.
[0018] Vaccinate every 10 days.
[0019] After three immunizations, a booster immunization was administered via the tail vein at an antigen dose of 100 µL per mouse. Three days later, 100 µL of blood was collected from the orbital vein per mouse. The serum was separated by centrifugation at 3500 rpm for 15 min and then analyzed for serum titer. Mouse serum agglutination titers greater than 200 were used for cell fusion assays.
[0020] 3. Cell fusion NS-1 cells and the above-mentioned mouse spleen cells were taken. Five to seven days before fusion, the cells were expanded and their state was adjusted to reach the logarithmic growth phase with a cell viability of over 95%. The logarithmic growth phase NS-1 cells were removed from the incubator, gently agitated, and the cell suspension was collected in a 50 mL centrifuge tube. The cells were centrifuged at 1000 rpm for 10 min, the supernatant was discarded, and the cells were resuspended in 10 mL of 1640 medium for later use.
[0021] Extract spleen cells from qualified BALB / c mice, mix them at a ratio of 1:5 to 1:8, centrifuge at 1000 rpm for 15 min, and discard the supernatant.
[0022] Use 50% PEG-4000 as a fusion agent for fusion, fusion for 2 min, add 50 mL of 1640 culture medium to terminate the reaction, centrifuge at 800 rpm for 5 min, and discard the supernatant.
[0023] The cell pellet was resuspended in HAT medium, and the cells were evenly added to a 96-well culture plate and incubated at 37°C in a 5% CO2 incubator.
[0024] 4. Screening and cloning of hybridoma cells Preparation of detection antigen The detection antigen consists of fresh human type O N-positive and type O N-negative red blood cells, washed three times with physiological saline, and then prepared into a red blood cell suspension of the required concentration using physiological saline. The detection antigen is prepared immediately before use to ensure the freshness of the red blood cells.
[0025] Screening and cloning ① After the fused cells are cultured in an incubator for 5 to 7 days, about a hundred cell clones are screened.
[0026] ② Transfer 100 µL of hybridoma cell supernatant to a 96-well plate. Add 40 µL of each test sample to two wells of a U-shaped plate. Prepare a 0.3% suspension of two types of O-type N-positive and O-type N-negative red blood cells with physiological saline, and add 40 µL to each well of the test sample. Mix the U-shaped plate with antibodies and cells using a plate shaker, centrifuge at 300g for 30 seconds, and then shake again to observe for agglutination reaction.
[0027] ③ Cells that agglutinate with type O N-positive red blood cells but do not agglutinate with type O N-negative red blood cells are screened, cloned, and cultured.
[0028] A hybridoma cell line (clone number: N1C8) stably secreting monoclonal antibodies against human blood group N antigen was obtained. After multiple passages and freeze-thaw cycles, the cell line grew well and stably secreted antibodies. After expansion culture, the cells were cryopreserved in liquid nitrogen. The cell line is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on August 7, 2025, with accession number CCTCC NO: C2025231, and named hybridoma cell line N1C8. Hybridoma cell line N1C8).
[0029] Example 2
[0030] The cell line (clone number: N1C8) obtained in Example 1 was expanded and cultured in 8% fetal bovine serum complete 1640 medium. The cell culture supernatant was collected and sterilized to obtain a monoclonal antibody against human N blood group antigen. The agglutination titer of the antibody reached 16 using the saline tube method.
[0031] Salt and protein are added to a monoclonal antibody against human N blood group antigen (clone number: N1C8) to prepare a monoclonal antibody reagent against human N blood group antigen (clone number: N1C8). This reagent can be used to detect human N blood group antigen.
[0032] Example 3
[0033] The titer of the monoclonal antibody (clone number: N1C8) prepared in Example 2 against human N-type blood group antigen was determined by saline tube method and reacted with type O N-positive red blood cells.
[0034] (1) Arrange the test tubes in two rows, with 6 tubes in each row. Starting from the second tube, add 100 μL of physiological saline to each tube precisely.
[0035] (2) Accurately add 100 μL of the reagent to be tested to the first test tube. Accurately add 100 μL of the reagent to be tested to the second test tube and mix well. Accurately pipette 100 μL of the mixture from the second test tube and add it to the third test tube, and mix well. Serially dilute the reagent to be tested by 2 times, and discard 100 μL of the mixture from the last tube.
[0036] (3) Accurately add 100 μL of 2% corresponding type O NN homozygous erythrocyte suspension to each tube in column 1 and mix well. Accurately add 100 μL of 2% corresponding type O MN heterozygous erythrocyte suspension to each tube in column 2, mix well, and incubate at room temperature for 15 min. Simultaneously, prepare a erythrocyte suspension control. Centrifuge at 900 g / min for 15 s and visually observe the results to determine the titer.
[0037] Experimental results are as follows Figure 1 and Figure 2As shown, the monoclonal antibody against human N-type blood group antigen (clone number: N1C8) prepared in Example 2 had an agglutination titer of 16 with O-type NN homozygous erythrocyte suspension and an agglutination titer of 8 with O-type MN heterozygous erythrocyte suspension.
[0038] Example 4
[0039] The monoclonal antibody (clone number: N1C8) against human blood group antigen prepared in Example 2 was specifically identified using the saline tube method. The specific steps are as follows: Take O-type NN homozygous erythrocytes, O-type MN heterozygous erythrocytes, and O-type MM homozygous erythrocytes, wash them three times with physiological saline, centrifuge at 2000 rpm for 5 min, remove the supernatant, and prepare a 2% erythrocyte suspension with physiological saline.
[0040] Arrange the test tubes in two rows of three, and label them. Add 50 μL of the sample to be tested to each tube in the first row, and then add 50 μL of 2% suspension of type O NN homozygous red blood cells, type O MN heterozygous red blood cells, and type O MM homozygous red blood cells to each tube. Use the red blood cell suspension control in the second row of test tubes, that is, replace the sample to be tested with physiological saline solution, and then add 50 μL of the above 2% red blood cell suspension to each tube. Incubate at room temperature for 15 minutes, centrifuge at 900g / min for 15s, gently shake the test tubes, and observe for agglutination with the naked eye.
[0041] Under the premise that no agglutination occurs in the red blood cell suspension control group, observe and record the agglutination results of the monoclonal antibody (clone number: N1C8) against human M blood group antigen prepared in Example 2 with the corresponding red blood cells. Conversely, if there is an agglutination reaction in the control group, the experiment is invalid and needs to be repeated.
[0042] The results are as follows Figure 3 As shown, the monoclonal antibody against human N blood group antigen (clone number: N1C8) agglutinated human type O NN homozygous erythrocytes and type O MN heterozygous erythrocytes, but did not agglutinate human type O MM homozygous erythrocytes. No hemolysis or other difficult-to-distinguish phenomena occurred.
[0043] Example 5
[0044] The monoclonal antibody Ig subtypes against human N-type blood group antigen prepared in Example 2 were identified using a commercially available kit (mouse monoclonal antibody Ig class / subclass / identification ELISA kit, manufacturer: Beijing Bio-Long Immunotherapy Co., Ltd.).
[0045] Experimental results are as follows Figure 3As shown, the monoclonal antibody against human N blood group antigen (clone number: N1C8) is of type IgG1. (Note: 1, 2, 3, 4 are controls (physiological sodium chloride solution); 5, 6, 7, 8 are monoclonal antibodies against human N blood group antigen (clone number: N1C8); 1, 5 are O-type NN homozygous cells; 2, 6 are O-type MN heterozygous cells; 3, 7 are O-type MM homozygous cells 1; 4, 8 are O-type MM homozygous cells 2).
[0046] Example 6
[0047] After passage and culturing the hybridoma cell line (clone number: N1C8) obtained in Example 1 for 48 hours, colchicine was added to achieve a final colchicine concentration of 0.8 μg / mL, and the cells were cultured at 37°C for another 4–6 hours. The cells were then centrifuged at 1000 rpm for 10 min, and the supernatant was discarded.
[0048] Add 8 mL of 0.5% potassium chloride hypotonic solution to the cell pellet, mix well with a pipette, and incubate at 37°C for 30 min. Centrifuge at 1000 rpm for 10 min and discard the supernatant.
[0049] Slowly add 5 mL of fixative (methanol:acetic acid = 3:1) to the cell pellet and mix well. Fix in a 37°C water bath for 20 min, centrifuge at 1000 rpm for 10 min, and discard the supernatant.
[0050] Add 5 mL of fixative to the cell pellet, centrifuge at 1000 rpm for 5 min, discard the supernatant, leaving a small amount of fixative, and gently mix the cells.
[0051] Use a pipette to aspirate cells onto a pre-cooled glass slide, pass them over a flame several times, and allow them to air dry. Stain with Giemsa stain for 20 minutes, rinse thoroughly with running water, allow to air dry, observe chromosome number under a microscope, select coverslips with well-dispersed chromosomes, abundant metaphase, and regular morphology, mount them, and photograph and record the results.
[0052] The number of chromosomes in hybridoma cells is close to the sum of the number of chromosomes in the two parental cells, namely the sum of the number of chromosomes in mouse spleen cells (40) and the number of chromosomes in mouse myeloma cells NS-1 (54-64).
[0053] Example 1: Chromosome number results obtained from hybridoma cell line (clone number: N1C8) are as follows: Figure 4 As shown, the number of chromosomes is between 94 and 103.
[0054] Example 7
[0055] The affinity of the monoclonal antibody (clone number: N1C8) against human type N blood group antigen prepared in Example 2 for reaction with human type O NN homozygous erythrocytes and human type O MN heterozygous erythrocytes was determined by slide agglutination assay. The specific steps are as follows: (1) Take human type O NN homozygous red blood cells and human type O MN heterozygous red blood cells and prepare a 10% red blood cell suspension with physiological saline. Take 50µL and drop it onto a glass slide.
[0056] (2) Take another 50µL of the antibody to be tested and drop it onto the glass slide. Immediately contact the 10% red blood cell suspension with the antibody to be tested and mix well. Record the time when the agglomerate is visible to the naked eye.
[0057] The experimental results are shown in Table 1. In Example 2, the monoclonal antibody against human N-type blood group antigen showed an affinity of 9.67 seconds for human O-type NN homozygous erythrocytes and an affinity of 18.40 seconds for O-type MN heterozygous erythrocytes.
[0058] Table 1. Affinity assay results of monoclonal antibody against human N-type blood group antigen (clone number: N1C8): .
[0059] Example 8
[0060] Ninety-seven samples with known blood types were collected and tested using tube agglutination assays. Simultaneously, the collected clinical samples were tested using a monoclonal antibody reagent against human N-type blood group antigen (clone number: N1C8) and an anti-N antibody reagent from DIAGEST. The results of blood group antigen determination were recorded and compared. The specific steps are as follows: Prepare a 2% red blood cell suspension using physiological saline from a sample of red blood cells with known blood type.
[0061] Take two test tubes and label them. Add 50 μL of monoclonal antibody reagent against human N blood group antigen (clone number: N1C8) to one test tube and add anti-N antibody reagent from DIAGEST to the other test tube. Then add 2% red blood cell suspension of the sample to be tested to each test tube and mix well.
[0062] Centrifuge immediately at 900g / min for 15s, interpret and record the reaction results.
[0063] Experimental results are as follows Figure 6 As shown in Table 2: Table 2. Test results of known blood type samples
[0064] Conclusion: The monoclonal antibody reagent against human N blood group antigen (clone number: N1C8) agglutinated human N-type erythrocytes but did not agglutinate human N-negative erythrocytes. The detection results were consistent with those of the anti-N antibody reagent from DIAGEST.
Claims
1. A hybridoma cell line that secretes an IgG1 monoclonal antibody against human N-type blood group antigen, characterized in that: The cell line is deposited at the China Center for Type Culture Collection (CCTCC) on August 7, 2025, with accession number CCTCC NO: C2025231, and named hybridoma cell line N1C8. Hybridoma cell line N1C8).
2. A monoclonal antibody against human N-type blood group antigen, characterized in that: The monoclonal antibody against human N-type blood group antigen is secreted by the hybridoma cell line N1C8 that secretes IgG1 type monoclonal antibody against human N-type blood group antigen as described in claim 1.
3. The monoclonal antibody against human N-type blood group antigen according to claim 2, characterized in that: The monoclonal antibody against human N-type blood group antigen is of type IgG1. The monoclonal antibody against human N-type blood group antigen reacts with human N-type and MN-type erythrocytes, but does not react with human M-type erythrocytes (i.e., N-negative erythrocytes).
4. Use of a monoclonal antibody against human N-type blood group antigen as described in claim 2 or 3 in the preparation of human N-type blood group detection reagents.
5. A blood typing reagent, characterized in that, Including monoclonal antibodies against human N-type blood group antigen as described in claim 2 or 3.
6. A blood type detection method, characterized in that, The method includes a detection step, in which the sample to be tested is reacted with a monoclonal antibody against human N-type blood group antigen as described in claim 2 or 3, in order to determine the N-type blood group of the sample to be tested.