Immortalized cell combination for screening red blood cell blood group antibody as well as construction method and application of immortalized cell combination

Abstract

The invention provides an immortalized cell combination for screening red blood cell blood group antibodies as well as a construction method and application of the immortalized cell combination. According to the immortalized cell combination, low-interference host cells are screened, interference antigen genes are knocked out through immortalized treatment and a gene editing technology, blood group antigens such as Rh and Kidd are expressed by utilizing a mammalian cell expression system, and an immortalized cell strain or a clone combination of high-expression single antigens is constructed and used for screening blood group antibodies. Compared with a traditional method, the method has the advantages that the detection sensitivity, specificity and stability are remarkably improved, the method has the advantages of authenticity of a red blood cell method and sensitivity of a purified antigen method, and a high-simulation, high-precision, stable and reliable antibody screening solution is provided for clinical blood transfusion.

Description

Technical Field

[0001] The present invention relates to the technical field of red blood cell blood group antibody detection, and in particular to an immortalized cell combination for screening red blood cell blood group antibodies, a construction method thereof, and an application thereof. Background Art

[0002] The current detection methods for red blood cell blood type antibodies are divided into red blood cell detection method and purified blood type antigen detection method according to the antigen presentation carrier.

[0003] The red blood cell test method uses red blood cells identified by cell surface blood type antigens to react with the sample being tested, and then qualitatively detects the corresponding blood type antibodies by detecting cell agglutination reactions, hemolysis reactions, and other methods. Detection methods include anti-human globulin tests, enzymatic tests, polybrene tests, low ionic strength solution tests, agglutination tests, flow cytometry, etc. The red blood cells used in the current red blood cell test method are directly derived from human peripheral blood. Therefore, the antibodies detected have the advantage of being the most realistic reflection of actual antibodies. However, this method also has the following fatal flaws:

[0004] 1) Low sensitivity: Low titer antibodies may be missed for some blood group antigens that are low in expression on the red blood cell surface;

[0005] 2) Poor specificity: In addition to the target antigen, there are many other blood group antigens and polypeptide non-blood group antigens on the surface of red blood cells. The presence of these antigens can easily interfere with the test results;

[0006] 3) Poor stability: Affected by the collection, transportation, storage, and experimental conditions of red blood cells, the activity of red blood cells and the integrity of surface antigens are unstable, resulting in poor stability of test results.

[0007] The purified blood type antigen test method is to coat the purified blood type antigen on a solid phase carrier such as microbeads or plates, react with the sample to be tested, and detect blood type antibodies qualitatively, semi-quantitatively, or quantitatively through chemical color development or fluorescent signal method. Detection methods include ELISA method, single antigen microbead method, etc. The purified antigens used in the current purified blood type antigen test method have the characteristics of clear antigen type, single antigen, and high antigen purity. Therefore, the detected antibodies have the advantages of high sensitivity and good specificity. However, this method also has the following fatal flaws:

[0008] 1) Blood group antigens have varying degrees of fidelity: antigens directly purified from red blood cells, while having the highest fidelity, are susceptible to denaturation. Among recombinantly expressed antigens, those derived from mammalian cell expression systems have the highest fidelity, followed by those from yeast and insect cell expression systems, with those from prokaryotic expression systems having the lowest fidelity.

[0009] 2) Denatured antigens cause serious false positive interference: Regardless of whether the antigen is directly purified from red blood cells or recombinantly expressed, during the preparation of the reagent, it goes through a series of steps such as antigen purification, elution, concentration, coating, fixation, and preservation. These steps often cause a certain degree of antigen denaturation, and denatured antigens can easily lead to the detection of false positive results. Summary of the Invention

[0010] In response to the shortcomings of traditional red blood cell detection methods and purified blood type antigen detection methods, the present invention provides an immortalized cell combination for screening red blood cell blood type antibodies, as well as its construction method and application. Through gene editing technology, an immortalized host cell detection platform with low background interference is constructed to replace the traditional red blood cell detection system, systematically achieve the controllable expression of complete blood type protein antigens, and be used for the detection of red blood cell blood type antibodies in clinical transfusion medicine.

[0011] In order to achieve the above technical objectives, the present invention adopts the following solutions:

[0012] The present invention provides an immortalized cell combination for screening red blood cell blood type antibodies, comprising: immortalizing candidate host cells with the lowest level of surface polymorphic antigen interference and knocking out the interfering antigen gene, expressing different target red blood cell blood type antigens, and screening to obtain immortalized single antigen cell lines or clone combinations that highly express different blood type antigens.

[0013] Preferably, the candidate host cells include: CHO cells or their derivatives, 293 cells or their derivatives, lymphocytes (B cells, T cells), myeloid cells (granulocytes, monocytes), erythroid differentiation precursor cells (primitive erythrocytes, immature erythrocytes, reticulocytes), bone marrow support cells (hematopoietic stem cells, stromal cells), embryonic stem cells or macrophages.

[0014] The present invention also provides a method for constructing an immortalized cell combination for screening red blood cell blood group antibodies, comprising:

[0015] (1) Screening candidate host cells with the lowest interference level of surface polymorphic antigens;

[0016] (2) immortalizing the candidate host cells;

[0017] (3) Knock out the expression genes of all known polymorphic antigens that interfere with blood group antibody detection in immortalized candidate host cells, and establish immortalized host cells with low background interference for expressing single blood group antigens;

[0018] (4) Express different target blood group antigens in immortalized host cells, screen and establish cell lines or clones that highly express different blood group antigens.

[0019] Preferably, in step (1), the candidate host cells include: CHO cells or cells derived therefrom, 293 cells or cells derived therefrom, lymphocytes (B cells, T cells), myeloid cells (granulocytes, monocytes), erythroid precursor cells (primitive erythrocytes, immature erythrocytes, reticulocytes), bone marrow support cells (hematopoietic stem cells, stromal cells), embryonic stem cells or macrophages; the screening method includes: using antibodies specific for blood group antigens or a combination thereof, antibodies specific for tissue cell type polymorphic antigens or a combination thereof, and sera from normal subjects and sensitized patients to screen host cells with non-binding or low binding levels by flow cytometry.

[0020] Preferably, in step (2), the method for performing immortalization treatment includes: a method of transforming with Epstein-Barr virus, a method of introducing a specific immortalization gene (such as hTERT gene) into the host cell genome through gene editing technology, or a method of fusing the host cell with an immortalized cell line (such as myeloma cells).

[0021] Preferably, in step (3), the expression gene of the polymorphic antigen that interferes with blood type antibody detection refers to a polymorphic alloantigen gene, including but not limited to HLA class I / II genes, MICA genes and HPA genes; the knockout method includes: CRISPR / Cas9 gene editing technology, RNA interference (RNAi) technology, zinc finger nuclease (ZFN) technology and transcription activator-like effector nuclease (TALEN) technology.

[0022] Preferably, in step (4), the target blood group antigens are transfected into immortalized host cells via a plasmid vector or a viral vector, and cell lines or clones highly expressing different blood group antigens are screened and established; the blood group antigens include but are not limited to at least two of the Rh antigens D, C, c, E, e, Kidd antigens Jka, Jkb, MNS antigens M, N, S, s, Mur, Mia, Duffy antigens Fya, Fyb, Diego antigens Dia, Dib, and Kell antigens K, k.

[0023] The present invention also provides the use of the above immortalized cell combination or the immortalized cell combination obtained by the above construction method in preparing a red blood cell blood type antibody screening product.

[0024] Preferably, the product is screened for antibodies by flow cytometry, ELISA, or fluorescence detection of cells on a solid support.

[0025] The present invention also provides a red blood cell blood type antibody screening kit, comprising the above immortalized cell combination or the immortalized cell combination obtained by the above construction method.

[0026] The beneficial effects of the present invention are:

[0027] This paper addresses the shortcomings of traditional red blood cell detection methods (low sensitivity, poor specificity, and insufficient stability) and purified blood type antigen detection methods (low antigen fidelity and high false positive rate) by proposing a blood type antibody screening platform based on gene-edited immortalized host cells. Innovations include:

[0028] (1) Construction of a low-background host cell platform: Through polymorphic antigen screening combined with gene knockout technology, interfering antigen genes such as HLA and MICA in host cells are systematically eliminated to eliminate cross-reactions with non-target antigens and improve the specificity of detection.

[0029] (2) Highly realistic antigen expression system: Using mammalian cell expression system, especially human cells to express red blood cell blood group antigens, retaining the natural conformation and epitope structure of the antigen, and avoiding the problem of insufficient simulation caused by differences in the expression system of recombinant antibodies.

[0030] (3) Stable expression of immortalized cell lines: Immortalize the host cells and combine them with antigen high expression screening technology to establish a stable cell bank with high expression of blood group antigens, so as to solve the problems of batch differences and low antigen expression in traditional red blood cell sources and ensure the stability and reproducibility of the test.

[0031] (IV) Modular combination detection strategy: Flexibly combine single antigen cell lines of different blood group antigens, and simultaneously screen multiple types of antibodies through flow cytometry, cytological ELISA, and fluorescent signal detection of cell combinations on solid phase carriers. This combines the advantages of antigen authenticity of the red blood cell method and the sensitivity of the purified antigen method, while avoiding the risk of denaturation during the antigen purification process.

[0032] (V) Technology integration and systematic optimization: Integrate host cell screening, gene editing, immortalization, high expression screening and multi-signal detection technologies to build an integrated detection platform to break through the comprehensive limitations of traditional methods in terms of sensitivity, specificity, stability and false positive rate.

[0033] Through the integration of multiple technologies and systematic optimization, the present invention constructs a blood group antibody screening platform that uses low-background immortalized host cells to express a series of blood group antigens. This eliminates interference from non-target antigens, ensures the natural conformation and high expression stability of blood group antigens, significantly improves detection specificity, sensitivity and repeatability, and avoids false positive results caused by antigen denaturation due to antigen purification. It provides a highly realistic, highly accurate, stable and reliable blood group antibody screening solution for clinical blood transfusion. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The results of the B cell surface blood group antigen expression test for five alternative healthy donors are shown.

[0035] Figure 2 Shows the expression level of HLA on the cell surface of host cells before and after HLA antigen gene knockout.

[0036] Figure 3 Shows the expression levels of blood group antigens Rh-D, C, c, E, and e on the host cell surface before and after gene transfection. DETAILED DESCRIPTION

[0037] The present invention will be described in detail below with reference to specific embodiments. The following specific embodiments will help those skilled in the art to further understand the present invention, but will not limit the present invention in any form.

[0038] Example

[0039] During the implementation of this example, commercial immortalized CHO cells, 293 cells, and peripheral blood mononuclear cells (PBMCs) from 20 healthy individuals were first selected as candidate host cells, totaling 22 cells. Next, these candidate host cells were cross-reacted with sera from a group of 30 highly sensitized transplant patients. Then, based on the screening requirement of negative cross-reaction results, five potential host cells (all peripheral blood PBMCs) were screened. Finally, a combination of antibodies against red blood cell blood group antigens was used to preliminarily screen the five candidate host cells, identifying cells that were negative for all known blood group antigens, for use as host cells expressing red blood cell blood group antigens. The materials used in this example were obtained in strict accordance with relevant regulations in the field.

[0040] Below, we detail methods for further screening of candidate host cells based on the initial screening described above, as well as specific steps for immortalization, interfering with gene knockout, blood group antigen gene expression, screening of high-expressing cell lines, and cytological ELISA detection of specific antibodies against Rh antigen cells D, C, c, E, and e.

[0041] 1. Screening of potential B cells for blood group antigen expression

[0042] (1) Peripheral blood samples (10 mL each) were collected from five healthy donors and mononuclear cells (PBMCs) were isolated using Ficoll-Paque Plus.

[0043] (2) Follow the kit instructions to sort B cells using magnetic bead sorting (such as MACS);

[0044] (3) Resuspend B cells in RPMI 1640 medium and adjust the cell concentration to 0.1×10 6 cells / ml;

[0045] (4) Add 1 ml of isolated and purified B cells to the coded 1.5 ml test tubes;

[0046] (5) After centrifugation and removal of the supernatant, add 50 μl of 20 μg / ml monoclonal antibodies or their combinations against the following antigens to each tube: a combination of monoclonal antibodies against HLA class I and II antigens (positive control group - based on the common knowledge that HLA antigens are expressed on the surface of normal B cells); a combination of antibodies against blood group Rh antigens D, C, c, E, e, Kidd antigens Jka, Jkb, MNS antigens M, N, S, s, Mur, Mia, Duffy antigens Fya, Fyb, Diego antigens Dia, Dib, and Kell antigens K, k (blood group antigen detection group); leave one tube without any antibody and replace it with 50 μl of RPMI1640 (negative control);

[0047] (6) After incubation for 30 minutes, wash three times with PBS;

[0048] (7) After centrifugation and removal of the supernatant, appropriate amounts of fluorescently labeled PE secondary antibodies were added and incubated in the dark at room temperature for 30 minutes;

[0049] (8) After centrifugation to remove the supernatant, the cells were washed with PBS and centrifuged to remove unbound secondary antibody;

[0050] (9) Resuspend the cells in 100 μl PBS and prepare for flow cytometry analysis;

[0051] (10) By comparing the differences in the test results of all blood group antibodies and negative controls, such as Figure 1 As shown, none of the five selected healthy donors expressed significant blood group antigens on their B cell surfaces, but exhibited high levels of HLA. One B cell line that was clearly negative for blood group antibody binding was selected for use in the construction of the immortalized cell line described below.

[0052] 2. Establishment of Immortalized B Cell Lines for Blood Group Antigen Expression

[0053] (1) B95-8 cells were cultured to the logarithmic growth phase and the supernatant was collected;

[0054] (2) Concentrate viral particles by ultracentrifugation (100,000 × g, 2 h);

[0055] (3) Resuspend the virus particles in RPMI 1640 medium and adjust the virus titer to 1×10 6 pfu / mL;

[0056] (4) Adjust the purified B cells to 1×10 6 cells / mL, resuspended in RPMI 1640 medium;

[0057] (5) In a sterile operating table, B cells and EBV viral particles were mixed at a volume ratio of 1:1 and gently mixed;

[0058] (6) Incubate the mixture in a 37°C, 5% CO2 incubator for 2 hours;

[0059] (7) Gently shake the mixture every 30 minutes to ensure sufficient contact between cells and virus;

[0060] (8) After incubation, transfer the mixture into RPMI 1640 medium containing 10% FBS and mix gently;

[0061] (9) The cells were cultured in a 37°C, 5% CO2 incubator;

[0062] (10) Replace the culture medium every 3 days and observe cell growth;

[0063] (11) 2 weeks after infection, immortalized B cells usually begin to proliferate significantly;

[0064] (12) Following the kit instructions, label the cells with fluorescently labeled antibodies (anti-CD19-FITC, anti-EBNA-1-PE) to identify whether the cells were successfully transformed.

[0065] 3. Knockout of HLA expression genes in immortalized B cell lines:

[0066] This experiment used CRISPR / Cas9 gene editing technology to knock out genes expressing HLA-I (A, B, C), HLA-II (DR, DP, DQ), and BCR in an immortalized B cell line. This example uses the knockout of the heavy chain of the HLA-I molecule as an example to describe the specific gene knockout and the verification of the knockout effect:

[0067] (1) Cell preparation: Select an immortalized B cell line in the logarithmic growth phase and perform routine culture to ensure that the cells are in good condition. Usually use RPMI 1640 culture medium supplemented with 10% fetal bovine serum (FBS),

[0068] 1% Penicillin / Streptomycin (P / S).

[0069] (2) Plasmid preparation: Select the appropriate lentiviral vector LentiCRISPR v2 carrying Cas9 protein.

[0070] Targeting the heavy chain genes of HLA-I molecules (such as HLA-A, HLA-B, and HLA-C), we selected exon 2 of the HLA-A gene (sequence 5'-AGGCTGAGGCTGAGGCTGAG-3') as the target and designed sgRNA. The target sequence and complementary sequence were synthesized with linkers and annealed to obtain a double-stranded sgRNA.

[0071] (3) Enzyme digestion of the vector: The lentiviral vector LentiCRISPR v2 was digested with enzymes. The digestion system was as follows: 3 μg LentiCRISPR v2, 2 μl 10× NEB Buffer, 2 μl BsmBI, and double-distilled water was added to 20 μl. The reaction conditions were 37°C for 6 hours, followed by termination with Loading Buffer.

[0072] (4) Ligation and transformation: Ligate the generated sgRNA double strands with the vector fragments recovered by enzyme digestion. The ligation system is: 100ng of the vector recovered by enzyme digestion, 50ng of the sgRNA double strands, 1μl T4 DNA Ligase, 2μl 10×Ligation Buffer, and add double distilled water to 20μl. The ligation reaction conditions are 16℃

[0073] overnight.

[0074] (5) Transform the ligation product into competent E. coli cells (such as DH5α), spread on LB plates containing ampicillin, and culture at 37°C overnight.

[0075] (6) Plasmid verification: Pick single clones, extract plasmids and perform sequencing verification to ensure that the sgRNA sequence is correctly inserted into the vector.

[0076] (7) Transient transfection: Transiently transfect the correctly verified recombinant plasmid LentiCRISPR v2-sgRNA into the immortalized B cell line. Use a transfection reagent such as Lipofectamine 3000 for transfection. Extract genomic DNA from the cells 48 hours after transfection.

[0077] (8) Editing identification: The target region is amplified by PCR, and then T7E1 enzyme digestion is performed to identify the editing effect.

[0078] If two bands appear after T7E1 digestion, it indicates that gene editing has occurred.

[0079] (9) Viral packaging: The recombinant plasmid identified as having editing activity and the packaging plasmid (pMD2.G) were transiently transfected into 293T cells. The viral fluid was collected 48 hours later and the viral titer was determined.

[0080] (10) Cell infection and screening: 0.1 ml of virus solution with a titer of 1E+8 TU / ml was used to infect 1E+6 immortalized B cell lines. Puromycin resistance screening was performed 48 hours after infection until cell clones were formed.

[0081] (11) After obtaining a stable cell line, perform protein expression verification: Detect the expression of HLA-I class molecules on the cell surface by flow cytometry to ensure that their expression is significantly decreased or turns negative. Use an anti-HLA-I class molecule antibody (CB-1) for flow cytometry analysis. Figure 2 The HLA detection levels on the cell surface before and after gene knockout are shown, confirming that HLA was completely knocked out.

[0082] 4. Construction of immortalized B cell lines expressing blood group antigens: In this example, single antigen-expressing cell lines of blood group antigens Rh-D, C, c, E, and e were constructed. The construction method of the Rh-D antigen cell line is now described in detail as an example.

[0083] This experiment used the lentiviral vector pLVX-IRES-ZsGreen1 to construct an EBV-transformed immortalized B cell line stably expressing the blood group antigen Rh-D. The Rh-D gene was amplified by RT-PCR, inserted into the lentiviral vector, packaged, and infected with the target cells. Finally, a cell line stably expressing Rh-D was obtained through puromycin selection and flow cytometry verification. The specific laboratory steps are briefly described below:

[0084] (1) Cell preparation: Select the above-screened immortalized B cell line transformed by EBV with the interferon gene knocked out and in the logarithmic growth phase. Use RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (P / S) for routine culture.

[0085] (2) Plasmid preparation: Select the lentiviral expression vector pLVX-IRES-ZsGreen1. Amplify the cDNA sequence of the Rh-D gene from Rh-D positive cells by RT-PCR. Design specific primers to amplify the Rh-D gene. Primers are designed as follows: Upstream primer:

[0086] 5'-GGATCCATGACCGTGGTGGTGGTGG-3' (BamHI site); downstream primer:

[0087] 5'-GAATTCCTAGGTTAGGTTAGGTTAG-3' (EcoRI site). The amplified Rh-D gene and expression vector pLVX-IRES-ZsGreen1 were then digested separately. Finally, the recovered Rh-D gene fragment was ligated with the vector fragment. The above plasmid preparation techniques are conventional molecular biology techniques and are not described in detail.

[0088] (3) Transformation and screening: The ligation product was transformed into Escherichia coli competent cells DH5α, spread on LB plates containing ampicillin, and cultured at 37°C overnight.

[0089] (4) Single clones were picked, plasmids were extracted and sequenced to verify that the Rh-D gene was correctly inserted into the vector.

[0090] (5) Lentivirus packaging: Transiently transfect 293T cells with the verified recombinant plasmid and packaging plasmid pMD2.G. Collect the viral fluid 48 hours later and measure the viral titer.

[0091] (6) Cell infection: The collected virus particles with a titer of 1E+8TU / ml were used to infect the immortalized B cell line transformed by EBV in which the above-mentioned interference gene was confirmed to be knocked out ((cell 10E+6) / (1E+8)×1000=100μL virus volume) at a multiplicity of infection (MOI) of 10. Puromycin resistance screening was performed 48 hours after infection until cell clones were formed.

[0092] (7) Verification at the genomic level: Extract genomic DNA from stable cell lines and verify the insertion of the Rh-D gene by sequencing.

[0093] (8) Protein expression verification: Detect the expression of Rh-D antigen on the cell surface by flow cytometry to ensure its positive expression. Figure 3 It is a comparison of the expression levels of Rh-D, C, c, E, and e blood group antigen genes on the host cell surface before and after transfection. The results show the expression levels of the corresponding antigens on the cell surface of host cells transfected with each blood group antigen gene after high-expressing cell clones were screened and amplified by the limiting dilution method.

[0094] 5. Cytological ELISA method for screening corresponding blood group antibodies using a combination of immortalized B cell lines expressing blood group antigens Rh-D, C, c, E, and e:

[0095] (1) Cell preparation

[0096] Cell culture: Rh antigen cells (negative control, D, C, c, E, e) were inoculated into 96-well cell culture plates, and an appropriate amount of cell suspension (about 1×10 4 cells / well).

[0097] Culture the cells in a CO2 incubator and allow them to adhere to the wall, which usually takes 24 hours.

[0098] (2) Sample preparation

[0099] Dilute samples: Dilute anti-D, anti-C, anti-C, anti-E, and anti-E monoclonal antibodies to appropriate concentrations (e.g., 1:100) with PBS. Dilute serum samples (6-25) to 1:100 with PBS.

[0100] (3) Cell fixation and blocking

[0101] Fixed cells:

[0102] 1) Wash the cell plate 3 times with PBS, 3 minutes each time.

[0103] 2) Add 4% paraformaldehyde solution to each well and fix at room temperature for 15 minutes.

[0104] 3) Wash the cell plate 3 times with PBS, 3 minutes each time.

[0105] Closed:

[0106] 1) Add 200 μL of blocking solution (5% skim milk powder or BSA) to each well and block at 37°C for 1 hour.

[0107] 2) Wash the cell plate 3 times with PBS, 3 minutes each time.

[0108] (4) Primary antibody incubation

[0109] 1) Add primary antibodies: According to the experimental design, add diluted anti-D, anti-C, anti-c, anti-E, anti-e monoclonal antibodies and serum samples to the corresponding wells, adding 100 μL to each well.

[0110] 2) Incubate at 37°C for 1 hour.

[0111] 3) Washing: Wash the cell plate 3 times with PBS, 3 minutes each time.

[0112] (5) Secondary antibody incubation

[0113] 1) Adding secondary antibody: Add 100 μL of diluted secondary antibody to each well (anti-mouse Ig-HRP for the monoclonal antibody group and anti-human IgG-HRP for the serum group, with a dilution ratio of 1:1000).

[0114] 2) Incubate at 37°C for 30 minutes.

[0115] 3) Washing: Wash the cell plate 3 times with PBS, 3 minutes each time.

[0116] (6) Substrate color development

[0117] 1) Add substrate:

[0118] ① Add 100 μL TMB substrate solution to each well.

[0119] ②Incubate at room temperature in the dark for 10-15 minutes and observe the color development.

[0120] 2) Termination of reaction: Add 50 μL of stop solution (2 M H2SO4) to each well to terminate the reaction.

[0121] (7) Result detection

[0122] Microplate reader reading:

[0123] The absorbance of each well was read using a microplate reader at a wavelength of 450 nm.

[0124] Record data and analyze them.

[0125] (8) Data Analysis

[0126] 1) Calculate the absorbance value:

[0127] ① Compare the absorbance values ​​of each well to determine whether there are antibodies against specific Rh antigens in the sample.

[0128] ②The absorbance value of the negative control well should be lower, while the absorbance value of the positive well should be significantly higher than that of the negative control.

[0129] 2) Result judgment:

[0130] ①If the absorbance value of a certain well is significantly higher than that of the negative control, it means that the sample contains antibodies against the Rh antigen.

[0131] ②If the absorbance value of a well is close to that of the negative control, it means that the sample does not contain antibodies against the Rh antigen.

[0132] 3) Experimental results:

[0133]

[0134] Interpretation of the results:

[0135] ① Negative OD benchmark: The mean OD value of Rh antigen-negative cells was 0.103±0.018, providing a reliable background threshold for positive determination.

[0136] ② Monoclonal antibody specificity: Monoclonal antibodies are highly bound to the target antigen (OD>1.2), and the reaction value of non-target antigen is close to the negative level (<0.14).

[0137] ③ Anti-D, C, c, E, and e antibodies were detected in 1, 1, 3, 8, and 1 cases, respectively, in serum samples.

[0138] Obviously, the above embodiments of the present invention are merely examples to more clearly illustrate the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is impossible to enumerate all implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. An immortalized cell combination for screening red blood cell blood group antibodies, characterized in that: include: After immortalizing the candidate host cells with the lowest level of surface polymorphic antigen interference and knocking out the interfering antigen gene, different target red blood cell blood type antigens are expressed and screened to obtain immortalized single antigen cell lines or clone combinations that highly express different blood type antigens.

2. The immortalized cell combination according to claim 1, characterized in that The candidate host cells include: CHO cells or cells derived therefrom, 293 cells or cells derived therefrom, lymphocytes, myeloid cells, erythroid differentiation precursor cells, bone marrow support cells, embryonic stem cells or macrophages.

3. A method for constructing an immortalized cell panel for screening red blood cell blood group antibodies, comprising: (1) Screening candidate host cells with the lowest interference level of surface polymorphic antigens; (2) immortalizing the candidate host cells; (3) Knock out the expression genes of all known polymorphic antigens that interfere with blood group antibody detection in immortalized candidate host cells, and establish immortalized host cells with low background interference for expressing single blood group antigens; (4) Express different target blood group antigens in immortalized host cells, screen and establish cell lines or clones that highly express different blood group antigens.

4. The construction method according to claim 3, characterized in that In step (1), the candidate host cells include: CHO cells or cells derived therefrom, 293 cells or cells derived therefrom, lymphocytes, myeloid cells, erythroid differentiation precursor cells, bone marrow support cells, embryonic stem cells or macrophages; the screening method includes: using blood group antigen-specific antibodies or a combination thereof, antibodies or a combination thereof to tissue cell type-specific polymorphic antigens, and sera from normal subjects and sensitized patients to screen host cells that do not bind or have low binding levels by flow cytometry.

5. The construction method according to claim 3, characterized in that In step (2), the immortalization treatment method includes: a method of transforming with Epstein-Barr virus, a method of introducing a specific immortalization gene into the host cell genome by gene editing technology, or a method of fusing the host cell with an immortalized cell line.

6. The construction method according to claim 3, characterized in that: In step (3), the expression gene of the polymorphic antigen that interferes with blood type antibody detection refers to a polymorphic alloantigen gene, including HLA class I / II genes, MICA genes and HPA genes; the knockout methods include: CRISPR / Cas9 gene editing technology, RNA interference technology, zinc finger nuclease technology and transcription activator-like effector nuclease technology.

7. The construction method according to claim 3, characterized in that: In step (4), the target blood group antigens are transfected into immortalized host cells via a plasmid vector or a viral vector, and cell lines or clones that highly express different blood group antigens are screened and established; the blood group antigens include at least two of the Rh antigens D, C, c, E, e, Kidd antigens Jka, Jkb, MNS antigens M, N, S, s, Mur, Mia, Duffy antigens Fya, Fyb, Diego antigens Dia, Dib, and Kell antigens K, k.

8. Use of the immortalized cell combination according to claim 1 or 2, or the immortalized cell combination obtained by the construction method according to any one of claims 3 to 7, in the preparation of a product for screening red blood cell blood type antibodies.

9. The use according to claim 8, characterized in that The product realizes antibody screening by flow cytometry fluorescence detection method, cytological ELISA colorimetric method, or fluorescence detection method of cells on solid phase carriers.

10. A red blood cell blood type antibody screening kit, comprising the immortalized cell combination according to claim 1 or 2 or the immortalized cell combination obtained by the construction method according to any one of claims 3 to 7.

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