A recombinant human monoclonal antibody against wra blood group antigen and a preparation method and application thereof

Abstract

This invention belongs to the field of biotechnology and relates to a recombinant human monoclonal antibody against the Wra blood group antigen, its preparation method, and its application. The invention involves isolating mononuclear cells from peripheral blood of donors with high titers of anti-Wra antibodies, co-incubating them with Wr(a+) erythrocytes, and obtaining single antigen-specific B cells using microfluidic single-cell sorting technology. The antibody variable region gene is amplified by single-cell RT-PCR and nested PCR, cloned into an expression vector containing the human IgG1 constant region, expressed in mammalian cells, and screened by an indirect antiglobulin assay to obtain a recombinant human monoclonal antibody that specifically binds to Wr(a+) erythrocytes and does not react with Wr(a-) erythrocytes. This invention is the first to achieve the in vitro preparation of an anti-Wra recombinant human monoclonal antibody, which can be industrially produced with good batch-to-batch consistency and eliminates the risk of pathogen contamination in human plasma. It can be used to prepare blood typing reagents and kits.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a recombinant human monoclonal antibody against Wra blood group antigen, its preparation method, and its application. Background Technology

[0002] The Wra antigen is a low-frequency blood group antigen located on the erythrocyte membrane, belonging to the Diego blood group system. Its molecular basis lies in a mutation at amino acid position 658 of the erythrocyte band 3 protein (SLC4A1), where glutamic acid is replaced by lysine (E658K). In clinical transfusion practice, anti-Wra antibodies are a common type of irregular erythrocyte antibody, and their presence can lead to serious clinical consequences such as neonatal hemolytic disease and hemolytic transfusion reactions. Therefore, the preparation of high-quality anti-Wra blood typing reagents is of great value in ensuring the safety of clinical transfusions.

[0003] Currently, all commercially available anti-Wra blood typing reagents are prepared from human plasma. The conventional preparation process involves collecting high-titer plasma containing anti-Wra antibodies from donors, performing a series of separation and purification steps, and then using it as a polyclonal blood typing reagent. However, this traditional preparation method has several inherent drawbacks. First, the supply of raw materials is extremely unstable. Due to the extremely low frequency of Wra antigen distribution in the population, donors who naturally produce anti-Wra antibodies are extremely scarce, directly leading to a long-term supply shortage of plasma-derived anti-Wra antibody reagents. Second, there are significant batch-to-batch variations. The titer, affinity, and subtype composition of anti-Wra antibodies in the plasma of different donors vary. Even plasma donated by the same donor at different times will show fluctuations in antibody titers, making standardized production extremely difficult. Third, there are biosafety risks. Human plasma carries the potential risk of carrying pathogens such as hepatitis B virus, hepatitis C virus, and human immunodeficiency virus, which undoubtedly increases the complexity and production cost of subsequent virus inactivation and removal processes. Fourth, this method cannot achieve industrial-scale production. Limited by the amount of human plasma collected and its low antibody content, this traditional method simply cannot meet the needs of large-scale industrial production.

[0004] Single-cell B-cell antibody discovery technology is a well-developed platform for preparing recombinant monoclonal antibodies in recent years. Its basic principle involves isolating antigen-specific single B cells from the peripheral blood of immunized individuals, then amplifying the antibody variable region gene expressed by these B cells using single-cell RT-PCR technology, and finally cloning it into an expression vector for recombinant expression. Compared to traditional hybridoma technology, this technology has the advantages of shorter cycle time, higher efficiency, and preservation of naturally occurring heavy and light chain pairings in vivo. Compared to in vitro antibody library technologies such as phage display, antibodies obtained through single-cell B-cell technology have undergone in vivo affinity maturation and immune tolerance screening, thus exhibiting better druggability and lower immunogenicity. However, to date, there are no reports of applying single-cell B-cell technology to the preparation of anti-Wra recombinant monoclonal antibodies. Summary of the Invention

[0005] This invention aims to solve the technical problems of existing anti-Wra blood typing reagents, which rely entirely on human plasma, resulting in raw material scarcity, large batch-to-batch variability, high biosafety risks, and the inability to scale up production. This invention is the first to apply single-B cell antibody discovery technology to the preparation of anti-Wra antibodies, successfully obtaining recombinant human monoclonal antibodies that can be stably produced in vitro and in large quantities.

[0006] To solve the above-mentioned technical problems, the technical solution proposed in this application is as follows: This invention provides a recombinant human monoclonal antibody against Wra blood group antigen, wherein the recombinant human monoclonal antibody comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:4.

[0007] Furthermore, the recombinant human monoclonal antibody is a complete IgG1 type antibody molecule.

[0008] On the other hand, this application also claims protection for a recombinant human monoclonal antibody against Wra blood group antigen, the recombinant human monoclonal antibody comprising a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region being shown in SEQ ID NO:6, and the amino acid sequence of the light chain variable region being shown in SEQ ID NO:8.

[0009] Furthermore, the recombinant human monoclonal antibody is a complete IgG1 type antibody molecule.

[0010] On the other hand, this application also claims protection for an isolated nucleic acid molecule that encodes the heavy chain variable region, light chain variable region, intact heavy chain, or intact light chain of the aforementioned recombinant human monoclonal antibody.

[0011] Further, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region is shown in SEQ ID NO:1, and the nucleotide sequence of the nucleic acid molecule encoding the light chain variable region is shown in SEQ ID NO:3; or, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region is shown in SEQ ID NO:5, and the nucleotide sequence of the nucleic acid molecule encoding the light chain variable region is shown in SEQ ID NO:7.

[0012] On the other hand, this application also claims protection for an expression vector comprising the aforementioned nucleic acid molecule.

[0013] On the other hand, this application also claims protection for a host cell that contains the aforementioned expression vector.

[0014] On the other hand, this application also claims protection for a method for preparing a recombinant human monoclonal antibody against Wra blood group antigen, comprising the following steps: S1: Donor screening, screening for donors with anti-Wra antibody titers ≥1:256 in peripheral blood; S2: Peripheral blood mononuclear cell isolation: Peripheral blood is collected from the donor, and peripheral blood mononuclear cells are separated by density gradient centrifugation; S3: Wra-specific B cell enrichment: The peripheral blood mononuclear cells obtained in step S2 are co-incubated with Wr(a+) erythrocytes to enrich Wra-specific B cells. S4: Single cell sorting. Using microfluidic single cell sorting technology, single B cells that specifically bind to Wr(a+) erythrocytes in step S3 are sorted into microplates containing lysis buffer. S5: Antibody variable region gene amplification: Using the RNA released from the lysate described in step S4 as a template, the antibody heavy chain variable region gene and light chain variable region gene in each single cell are amplified by reverse transcription polymerase chain reaction and nested polymerase chain reaction, respectively. S6: Expression vector construction: The heavy chain variable region gene fragment and the light chain variable region gene fragment obtained in step S5 are cloned into expression vectors containing human IgG1 constant region gene, respectively, to obtain heavy chain expression vector and light chain expression vector. S7: Recombinant antibody expression and purification: The heavy chain expression vector and light chain expression vector obtained in step S6 are co-transfected into mammalian cells for expression. The culture supernatant is harvested and purified by affinity chromatography to obtain recombinant monoclonal antibodies. S8: Antibody screening and identification. The purified antibody obtained in step S7 is tested for its reactivity with Wr(a+) erythrocytes and Wr(a-) erythrocytes by indirect anti-human globulin assay. Positive clones that specifically bind to Wr(a+) erythrocytes and do not react with Wr(a-) erythrocytes are screened out.

[0015] Further, in step S3, the ratio of peripheral blood mononuclear cells to Wr(a+) erythrocytes is 3 × 10⁻⁶. 6 1×10 7 The incubation conditions are 37℃, 5% CO2, and incubation in the dark for 60-120 minutes; in step S4, B cells bound to Wr(a+) erythrocytes are fluorescently labeled with anti-human IgG antibody before sorting; in step S5, the reverse transcription polymerase chain reaction is performed using oligo(dT) primers for reverse transcription, the first round of multiplex polymerase chain reaction is performed using mixed primers covering the human antibody VH, Vκ and Vλ gene families, and the second round of amplification is performed using nested primers with restriction endonuclease sites.

[0016] Furthermore, the indirect anti-human globulin test mentioned in step S8 is the microcolumn gel indirect anti-human globulin test method; the screening also includes the following identification steps: titer determination, retaining clones with a titer ≥1:512; specificity verification, using no less than 20 Wr(a-) erythrocyte profiles of different phenotypes for cross-reactivity detection; and antibody subtype identification.

[0017] Furthermore, the method further includes step S9 after step S8: transfecting the light and heavy chain expression vectors of the positive clones obtained by screening into CHO-S cells, and obtaining stable CHO-S cell lines that stably express anti-Wra recombinant human monoclonal antibodies after pressure screening and limiting dilution cloning.

[0018] On the other hand, this application also claims protection for the use of any of the recombinant human monoclonal antibodies described above in the preparation of blood typing reagents.

[0019] On the other hand, this application also claims protection for a blood typing kit, the kit comprising any of the recombinant human monoclonal antibodies described above.

[0020] Compared with the prior art, the present invention achieves the following beneficial technical effects: This application marks the first successful in vitro preparation of anti-Wra recombinant human monoclonal antibody, completely breaking through the bottleneck of traditional reagents' complete reliance on human plasma. The obtained antibody is a complete IgG1 type, retaining the natural light and heavy chain pairing, exhibiting high specificity and affinity. Establishing a stable cell line enables industrial-scale production, fundamentally eliminating the risk of pathogen contamination, significantly improving batch-to-batch consistency and standardization, and providing a safe, stable, and renewable reagent source for clinical blood typing, possessing significant clinical application value and broad market prospects. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, 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.

[0022] In one embodiment of this application, a recombinant human monoclonal antibody against the Wra blood group antigen is provided. This antibody is a complete IgG1 type antibody molecule. Specifically, the recombinant antibody prepared by the method of the following embodiment was identified as IgG1 type by subtype identification. Its heavy chain and light chain variable regions originate from the same antigen-specific B cell, completely preserving the naturally occurring heavy chain and light chain pairing form in vivo. As determined by the microcolumn gel indirect antiglobulin assay, this antibody exhibits a specific agglutination reaction with Wr(a+) erythrocytes, but no cross-reaction with Wr(a-) erythrocytes, indicating that the antibody has excellent antigen-binding specificity and affinity.

[0023] In one embodiment of this application, an isolated nucleic acid molecule is provided, which encodes the heavy chain variable region, light chain variable region, intact heavy chain, or intact light chain of the aforementioned recombinant human monoclonal antibody. Specifically, from screened positive monoclonal B cells, the heavy chain variable region gene fragment and the light chain variable region gene fragment are amplified by single-cell RT-PCR. Sequencing confirms that their sequences are intact, without frameshift mutations, without premature stop codons, and that the CDR region sequence is intact, consistent with the sequence characteristics of human antibodies. These nucleic acid molecules can be isolated and preserved individually for subsequent genetic engineering operations.

[0024] In one embodiment of this application, an expression vector is provided, which contains the aforementioned nucleic acid molecules. Specifically, the amplified heavy chain variable region gene fragment and light chain variable region gene fragment are cloned into an expression vector containing the human IgG1 constant region gene, respectively, to obtain a heavy chain expression vector and a light chain expression vector. In a preferred embodiment, the expression vector is a pcDNA3.4 vector or a derivative thereof. The heavy chain variable region gene and light chain variable region gene are inserted into the multiple cloning site of the pcDNA3.4-IgG1 vector, respectively. After sequencing confirms that the insertion direction is correct and the reading frame is correct, a recombinant plasmid that can be used for recombinant antibody expression is obtained.

[0025] In one embodiment of this application, a host cell is provided, which contains the aforementioned expression vector. Specifically, the constructed heavy chain expression vector and light chain expression vector are co-transfected into mammalian cells, allowing the exogenous gene to be transcribed and translated within the host cell, thereby expressing the complete recombinant antibody molecule. In a preferred embodiment, the host cell is a mammalian cell, more preferably HEK293 cells or CHO cells. In the transient expression system, HEK293F cells are used for PEI-mediated transfection; in the stable expression system, CHO-S cells are used to establish a stable cell line to achieve large-scale antibody production.

[0026] The preparation method provided in this application will be described in detail below.

[0027] In one embodiment of this application, a method for preparing recombinant human monoclonal antibodies against the Wra blood group antigen is provided. The method first includes a donor screening step. Since the Wra antigen has an extremely low distribution frequency in the population, naturally occurring anti-Wra antibodies in blood donors are extremely rare; therefore, donor screening is a crucial prerequisite for this method. Specifically, the method involves screening blood donors using a microcolumn gel indirect antiglobulin assay to determine the titer of anti-Wra antibodies in their peripheral blood. The screening criterion is a donor with an anti-Wra antibody titer of not less than 1:256. In a specific embodiment, a 42-year-old male blood donor with an anti-Wra antibody titer of 1:1024 was selected. This donor had a history of blood transfusions. After obtaining informed consent from the donor, 20 mL of his peripheral blood was collected using a heparin sodium anticoagulant tube, and subsequent processing was performed within 2 hours of blood collection to ensure cell viability and the accuracy of experimental results.

[0028] After donor screening, peripheral blood mononuclear cell (PBMC) isolation was performed. The collected PBMCs were diluted with an equal volume of phosphate-buffered saline (PFS) and gently mixed. 20 mL of Ficoll-Paque PLUS separation buffer was added to a 50 mL centrifuge tube. The diluted blood was slowly piled up along the tube wall above the Ficoll buffer, maintaining a clear interface throughout the process. The tube was centrifuged at 800 g for 20 minutes at room temperature, with both acceleration and deceleration set to 0, and the brakes were not used to prevent disruption of the separation interface. After centrifugation, the middle white membrane layer (PBMCs) was carefully aspirated and transferred to a new centrifuge tube. Three volumes of PFS were added, and the tube was centrifuged at 300 g for 10 minutes, discarding the supernatant. The washing process was repeated once. Finally, the cell pellet was resuspended in FACS buffer and counted. In one specific embodiment, approximately 1.5 × 10⁶ cells were successfully obtained. 7 The peripheral blood mononuclear cells were analyzed by trypan blue staining, and the cell viability was 97%, indicating that the cells were in good condition and could be used for subsequent experiments.

[0029] The Wra-specific B cell enrichment step was then performed. The isolated peripheral blood mononuclear cells were co-incubated with erythrocytes of the known Wr(a+) phenotype to enrich B cells capable of specifically recognizing the Wra antigen. Before the specific operation, the Wr(a+) erythrocytes were isolated and identified: 2 mL of anticoagulated blood of the known Wr(a+) phenotype was used, and erythrocytes were isolated using a erythrocyte separation kit according to the product instructions. After separation, the morphology of the erythrocytes was examined under a microscope to confirm the separation effect. Under low magnification, the field of view should be filled with a large number of round, anucleated erythrocytes; under high magnification, the cells should be round with mostly round, lightly stained areas in the center, indicating normal morphology. Subsequently, commercially available anti-Wra standard serum was used to confirm the Wra antigen positivity of the isolated erythrocytes using the microcolumn gel indirect antiglobulin test. The confirmed erythrocytes were stored at 4°C for later use, but the storage time should not exceed 72 hours to avoid a decrease in antigen activity. In a preferred embodiment, the cell ratio of peripheral blood mononuclear cells to Wr(a+) erythrocytes was 3 × 10⁻⁶. 6 1×10 7 The aforementioned number of peripheral blood mononuclear cells and Wr(a+) erythrocytes were washed once with 1× phosphate-buffered saline and gently mixed. The original liquid in the sorting chip was discarded, and the diluted cell suspension was added. The chip was incubated for 10 minutes. After the array was completed, the cell suspension was removed, and the chip was washed with 1× phosphate-buffered saline until no floating cells remained. Subsequently, incubation medium containing fluorescently labeled anti-human IgG antibody was added, and the chip was incubated at 37°C and 5% CO2 in the dark for 60 to 120 minutes to fluorescently label the B cells bound to the erythrocytes. In one specific embodiment, the incubation time was 90 minutes. After incubation, the incubation medium was aspirated, and the chip was washed with 1× phosphate-buffered saline until no floating cells remained.

[0030] After enrichment, a single-cell sorting step is performed. Using microfluidic single-cell sorting technology, individual B cells producing antibodies specifically binding to Wr(a+) erythrocytes are sorted into microplates containing lysis buffer. Specifically, after washing, 2 mL of 1× phosphate buffer is added, and then the microfluidic single-cell sorting device is used. This device can accurately pick individual antigen-specific B cells based on fluorescence signals and cell morphology. In a preferred embodiment, the Cytostar antibody discovery technology platform is used for sorting. The picked individual B cells are then allocated to 96-well polymerase chain reaction (PCR) plates containing lysis buffer. After picking, the plates are sealed and briefly centrifuged to collect the cell lysis buffer, which is then stored at -80°C for later use or directly used in subsequent reverse transcription polymerase chain reaction (RT-PCR) experiments.

[0031] Next, the antibody variable region gene amplification step was performed. Using RNA released from the lysis buffer as a template, the antibody heavy chain variable region gene and light chain variable region gene in each single cell were amplified by reverse transcription polymerase chain reaction (RT-PCR) and nested polymerase chain reaction (NCR), respectively. Specifically, this step consisted of two stages. The first stage was first-strand cDNA synthesis: the 96-well plate containing the lysed cells was removed from -80°C and thawed on ice. 1 μL of oligo(dT) primer, 1 μL of 10 mM dNTP, and 6 μL of enzyme-free water were added to each well, bringing the total volume to 13 μL. The plate was denatured at 65°C for 5 minutes, and then immediately cooled on ice for 2 minutes. Next, 4 μL of 5× first-strand buffer, 1 μL of 0.1 M DTT, 0.5 μL of RNase inhibitor, and 0.5 μL of reverse transcriptase were added to each well, bringing the final total volume to 20 μL. The following program was run on a polymerase chain reaction (PCR) instrument: reverse transcription was performed at 50°C for 60 minutes, followed by enzyme inactivation at 70°C for 15 minutes, and finally incubated at 4°C. The second stage was antibody gene amplification, employing a nested PCR strategy to improve amplification specificity and efficiency. The first round was a multiplex PCR, with 2 μL of synthesized cDNA used as a template in each well to prepare a 20 μL reaction mixture, including 4 μL of 5× polymerase buffer, 0.4 μL of 10 mM dNTPs, 1 μL of mixed primers covering the human antibody VH, Vκ, and Vλ gene families, 0.2 μL of polymerase, and 12.4 μL of enzyme-free water. The PCR program was: pre-denaturation at 98°C for 30 seconds; then 35 cycles, each cycle consisting of denaturation at 98°C for 10 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 30 seconds, and a final extension at 72°C for 5 minutes. The second round was a nested polymerase chain reaction (PCR). 1 μL of the first round PCR product was used as a template, and amplification was performed using nested primers with restriction endonuclease sites. The reaction system was the same as the first round. The PCR program was: 98℃ pre-denaturation for 30 seconds; 30 cycles, each cycle consisting of 98℃ denaturation for 10 seconds, 60℃ annealing for 30 seconds, and 72℃ extension for 30 seconds; and a final extension at 72℃ for 5 minutes. After amplification, the product was identified by agarose gel electrophoresis. Wells showing clear bands at approximately 400 bp of the heavy chain and approximately 350 bp of the light chain were considered positive. The gel at the corresponding positions was excised, and the target gene fragment was recovered and purified for cloning.

[0032] After amplifying the antibody variable region gene, the expression vector construction step is performed. The recovered and purified heavy chain variable region gene fragment and light chain variable region gene fragment are cloned into expression vectors containing the human IgG1 constant region gene, respectively, to obtain heavy chain expression vectors and light chain expression vectors. In a preferred embodiment, the expression vector is the pcDNA3.4-IgG1 vector. Specifically, the recovered VH, Vκ / Vλ gene fragments and the pcDNA3.4-IgG1 expression vector are double-digested with their corresponding restriction endonucleases at 37°C for 2 hours. After digestion, the digestion products are purified using a gel extraction kit. Then, ligation is performed overnight at 16°C using T4 DNA ligase at a molar ratio of insert fragment to vector of 3:1. The ligation product is transformed into DH5α competent cells, plated on LB agar plates containing ampicillin, and cultured at 37°C for 14 to 16 hours. Three single clones are picked from each transformation plate for bacterial polymerase chain reaction (PCR) identification to screen for positive clones containing the target gene fragment. Positive clones were sent for Sanger sequencing. Sequencing results were compared and analyzed using the IMGT / V-QUEST database to confirm that the antibody gene had no frameshift mutations, no premature stop codons, an intact CDR region sequence, and conformed to human antibody sequence characteristics. For correctly sequenced positive clones, endotoxin-free plasmids were extracted, and their A260 / A280 ratio was determined. Quality-compliant plasmids were stored at -20°C for later use. In one specific embodiment, the A260 / A280 ratio of the plasmid was between 1.82 and 1.86.

[0033] After obtaining the recombinant expression vector, recombinant antibody expression and purification steps were performed. The heavy chain expression vector and the light chain expression vector were co-transfected into mammalian cells for expression. The culture supernatant was harvested and purified by affinity chromatography to obtain the recombinant monoclonal antibody. In a preferred embodiment, HEK293F cells were used for PEI-mediated transient transfection. HEK293F cells were passaged one day before transfection to ensure that the cells were in the logarithmic growth phase on the day of transfection, and the cell density was adjusted to 1×10⁻⁶. 6Cells / mL with a viability of not less than 95%. For example, using a 30mL culture system: 12μg of heavy chain plasmid and 18μg of light chain plasmid are diluted with 1.5mL of serum-free Opti-MEM medium; 90μg of PEI transfection reagent is diluted with 1.5mL of serum-free Opti-MEM medium. After mixing thoroughly, the mixture is allowed to stand at room temperature for 5 minutes. Then, the PEI dilution is added dropwise to the plasmid dilution, gently mixed, and incubated at room temperature for 15 minutes to form the transfection complex. Finally, the transfection complex is added dropwise to the cell suspension, gently shaken, and cultured in a shaker at 37°C, 8% CO2, and 120rpm. In a preferred embodiment, the mass ratio of heavy chain plasmid to light chain plasmid is 1:1 to 1:2, more preferably 1:1.5. On days 5 to 7, when the cell viability drops to 60% to 70%, the culture supernatant is collected. Cell debris is removed by centrifugation at 3000g for 10 minutes, and the supernatant is collected. The antibody was then purified using a Protein A affinity chromatography column. The purified antibody was dialyzed against phosphate buffer and finally concentrated by ultrafiltration. The concentration of the purified antibody was determined using the A280 method.

[0034] After antibody expression and purification, antibody screening and identification steps were performed. The reactivity of the purified antibody with Wr(a+) and Wr(a-) erythrocytes was tested using an indirect antiglobulin assay to screen for positive clones that specifically bind to Wr(a+) erythrocytes but do not react with Wr(a-) erythrocytes. Specifically, a small amount of culture supernatant was first taken, and the agglutination reaction with Wr(a+) erythrocytes was detected using a microcolumn gel indirect antiglobulin assay. Negative and positive controls were set up to screen for clones that could produce a positive reaction. For clones that were initially positive, the culture system was scaled up for transfection and expression. After purification by Protein A affinity chromatography, the purified antibody was systematically characterized. The identification process included: titer determination: the purified antibody was serially diluted starting at a 1:2 ratio, and the reactivity of each dilution with Wr(a+) erythrocytes was tested using a microcolumn gel indirect antiglobulin assay. The highest dilution that produced a Wr(a-) agglutination reaction was defined as the antibody titer, and clones with a titer not lower than 1:512 were retained. Specificity verification: at least 20 erythrocyte profiles from different donors known to have the Wr(a-) phenotype and covering common blood group antigen polymorphisms were cross-reactively tested with the purified antibody. Clones showing no agglutination reaction in any Wr(a-) erythrocytes were deemed to have qualified specificity. Subtype identification: the antibody subtype was identified using a human IgG subtype detection kit. In one specific example, the identified antibody subtype was IgG1κ or IgG1λ. Through the above rigorous screening and identification process, several candidate clones of recombinant human IgG1 monoclonal antibodies with Wr antigen specificity were successfully obtained.

[0035] In one embodiment of this application, two antibodies were obtained by sequencing and named WRa-01 and WRa-02, respectively. The heavy chain variable region nucleotide sequence of antibody WRa-01 is shown in SEQ ID NO:1, and its encoded amino acid sequence is shown in SEQ ID NO:2; the light chain variable region nucleotide sequence is shown in SEQ ID NO:3, and its encoded amino acid sequence is shown in SEQ ID NO:4. The heavy chain variable region nucleotide sequence of antibody WRa-02 is shown in SEQ ID NO:5, and its encoded amino acid sequence is shown in SEQ ID NO:6; the light chain variable region nucleotide sequence is shown in SEQ ID NO:7, and its encoded amino acid sequence is shown in SEQ ID NO:8.

[0036] In one embodiment of this application, the step of establishing a stable expression cell line is included after the above screening step. The light and heavy chain expression vectors of the positive clones obtained from the screening are transfected into CHO-S cells. After pressure screening and limiting dilution cloning, a stable CHO-S cell line expressing the anti-Wra recombinant human monoclonal antibody is obtained. Specifically, the optimal clones selected are chosen, and their heavy and light chain expression vectors are linearized and co-transfected into CHO-S cells using electroporation. Forty-eight hours after transfection, pressure screening is performed using selection medium containing methotrexate to enrich cells that have successfully integrated the foreign gene. The surviving cells are seeded into 96-well plates using limiting dilution to ensure that each well ultimately grows a monoclonal cell derived from one cell. After culturing for 2 to 3 weeks, the supernatant from each monoclonal well is collected, and the antibody expression level is detected by enzyme-linked immunosorbent assay (ELISA). The clone with the highest antibody expression level is screened and subjected to stepwise amplification culture to finally obtain a CHO-S cell line capable of stably and highly expressing the anti-Wra recombinant monoclonal antibody. The cell line was passaged 10 times consecutively, and the antibody expression level was monitored at each passage. The results showed that the antibody expression level remained stable, with a relative standard deviation of less than 15%, demonstrating that the constructed cell line has good genetic and production stability.

[0037] In one embodiment of this application, the application of the above-mentioned recombinant human monoclonal antibody in the preparation of blood typing reagents or blood typing kits is provided. Specifically, the purified anti-Wra recombinant monoclonal antibody is diluted to the working concentration with phosphate buffer containing 1% bovine serum albumin to prepare the working solution of the blood typing reagent. The working concentration is determined by checkerboard titration, and in one specific embodiment, it is 0.5 to 2 μg / mL. During detection, the prepared working solution and the red blood cell suspension to be tested are placed in a microcolumn gel card, and the detection is performed according to the conventional indirect antiglobulin test procedure. Using a commercially available human polyclonal anti-Wra reagent as a control, 100 clinical samples with known phenotypes were tested in parallel. The experimental results show that the detection results of the recombinant monoclonal antibody reagent prepared in this application are completely consistent with those of the commercially available human polyclonal reagent, with both the positive and negative concordance rates reaching 100%. Meanwhile, repeated testing of recombinant monoclonal antibody reagents prepared in the same batch and different batches showed that the test results were highly consistent, with intra-batch coefficient of variation of less than 5% and inter-batch coefficient of variation of less than 10%, which was significantly better than the batch consistency of human polyclonal reagents.

[0038] In one embodiment of this application, a blood typing kit is provided, comprising the aforementioned recombinant human monoclonal antibody. Specifically, purified anti-Wra recombinant monoclonal antibody is formulated into a blood typing reagent working solution, which, together with microcolumn gel cards, red blood cell suspension, positive control, and negative control components, constitutes the blood typing kit. In a preferred embodiment, the kit is a microcolumn gel card kit, in which the antibody working solution is pre-coated in a microcolumn gel card. During use, the antibody is directly added to the red blood cell suspension to be tested for detection, making the operation simple and quick, suitable for routine clinical blood typing.

[0039] In summary, this application successfully achieved stable in vitro production of anti-Wra recombinant antibodies by applying single B-cell antibody discovery technology to the preparation of anti-Wra blood group antigen recombinant human monoclonal antibodies. This method not only solves the technical challenges of scarce raw materials, large batch-to-batch variability, high biosafety risks, and inability to scale up production in traditional human plasma antibody reagents, but also produces recombinant antibodies with excellent affinity, specificity, and batch-to-batch consistency, fully meeting the clinical requirements of blood typing reagents and demonstrating broad application prospects.

[0040] The following is an embodiment of this application: Example 1: Preparation of anti-Wra recombinant human monoclonal antibody 1. Donor screening and sample collection Blood donors with an anti-Wra antibody titer ≥1:256 were screened using the microcolumn gel IAT method. One donor (male, 42 years old, with a history of blood transfusions) with an anti-Wra antibody titer of 1:1024 was selected. After signing the informed consent form, 20 mL of peripheral blood was collected using a heparin sodium anticoagulant tube and processed within 2 hours of collection.

[0041] 2. Isolation and identification of Wr(a+) erythrocytes Take 2 mL of anticoagulated blood with a known Wr(a+) phenotype and separate red blood cells using the IPHASE Red Blood Cells Isolation Kit, following the manufacturer's instructions. Microscopic examination of red blood cell morphology: Under low magnification, the field of view is filled with numerous round, enucleated red blood cells; under high magnification, the cells are round, mostly with a lightly stained area in the center, and their morphology is normal. After confirming the positive status of red blood cell Wra antigen using the microcolumn gel IAT method with commercially available anti-Wra standard serum, store at 4°C for later use (storage time not exceeding 72 hours).

[0042] 3. Isolation of peripheral blood PBMCs 20 mL of peripheral blood was collected and diluted with an equal volume of PBS, then gently mixed. 20 mL of Ficoll-Paque PLUS was added to a 50 mL centrifuge tube. The diluted blood was slowly poured onto the Ficoll solution along the tube wall, maintaining a clear interface. The tube was centrifuged at 800 g for 20 minutes at room temperature, with both acceleration and deceleration set to 0 and the brakes not applied. The intermediate white membrane layer (PBMC layer) was carefully aspirated into a new centrifuge tube, 3 volumes of PBS were added, and the tube was centrifuged at 300 g for 10 minutes. The supernatant was discarded. The cells were washed once more. The cells were resuspended in FACS buffer and counted, yielding approximately 1.5 × 10⁻⁶ cells. 7 The cell viability of PBMCs was 97% as determined by trypan blue staining.

[0043] 4. Wra-specific B cell enrichment and single-cell sorting Take 3×10 6 PBMC and 1×10 7Wr(a+) erythrocytes were washed once with 1×PBS and gently mixed. The original liquid in the CytostarAbD sorting chip was discarded, and the diluted cell suspension was added. The array was run for 10 minutes. After the array was complete, the cell suspension was removed, and the cells were washed with 1×PBS until no floating cells remained. Incubation buffer containing Anti-Human IgG Antibody 488 was added, and the cells were incubated at 37°C in a 5% CO2 incubator in the dark for 90 minutes. After incubation, the incubation buffer was aspirated, and the cells were washed with 1×PBS until no floating cells remained. After washing, 2 mL of 1×PBS was added, and the CytostarAbD single-cell sorting device was used to pick individual antigen-specific B cells into a 96-well PCR plate containing lysis buffer based on fluorescence signal and cell morphology. After picking, the plate was sealed, and the cell lysis buffer was collected by brief centrifugation and stored at -80°C or used directly for subsequent RT-PCR.

[0044] 5. Single-cell RT-PCR amplification of antibody heavy and light chain genes (1) First-strand cDNA synthesis: After removing the 96-well plate from -80℃, thaw it on ice. Add 1 μL of oligo(dT) primer, 1 μL of 10mM dNTP, and 6 μL of enzyme-free water to each well (total volume 13 μL). Denature at 65℃ for 5 minutes, then immediately cool on ice for 2 minutes. Add 4 μL of 5× first-strand buffer, 1 μL of 0.1M DTT, 0.5 μL of RNase inhibitor, and 0.5 μL of SuperScript III reverse transcriptase to each well (total volume 20 μL). PCR program: 50℃ for 60 minutes, 70℃ for 15 minutes, and incubate at 4℃.

[0045] (2) First round of multiplex PCR: Take 2 μL of cDNA as template from each well and prepare a 20 μL reaction system: 4 μL of 5×PhusionBuffer, 0.4 μL of 10 mM dNTP, 1 μL of VH / Vκ / Vλ family primer mixture (10 μM), 0.2 μL of Phusion polymerase, and 12.4 μL of enzyme-free water. PCR program: 98℃ for 30 seconds; 35 cycles (98℃ for 10 seconds, 58℃ for 30 seconds, 72℃ for 30 seconds); 72℃ for 5 minutes.

[0046] (3) Second round of nested PCR: Using 1 μL of the first round PCR product as a template, amplify using nested primers with restriction endonuclease sites. The reaction system is the same as the first round. PCR program: 98℃ for 30 seconds; 30 cycles (98℃ for 10 seconds, 60℃ for 30 seconds, 72℃ for 30 seconds); 72℃ for 5 minutes. Identify the amplified products by 1.5% agarose gel electrophoresis. Wells with clear bands at approximately 400 bp of the heavy chain and approximately 350 bp of the light chain are considered positive. The target fragment is then excised and purified.

[0047] 6. Construction of recombinant antibody expression vector The recovered VH and Vκ / Vλ gene fragments and the pcDNA3.4-IgG1 expression vector were double-digested with their corresponding restriction endonucleases at 37°C for 2 hours. The digestion products were recovered from the gel and ligated overnight at 16°C using T4 DNA ligase at a 3:1 molar ratio of insert fragment to vector. The ligation products were transformed into DH5α competent cells, plated on LB agar plates containing ampicillin, and incubated at 37°C for 14–16 hours. Three clones from each well were picked for colony PCR identification, and positive clones were sent for Sanger sequencing. Sequencing results were compared and analyzed using the IMGT / V-QUEST database to confirm that the antibody gene had no frameshift mutations, no premature stop codons, an intact CDR region, and was consistent with human antibody sequence characteristics. Positive clones with correct sequencing were subjected to endotoxin-free plasmid extraction, and the A260 / A280 ratio was measured (1.82–1.86). The plasmids were stored at -20°C for later use.

[0048] 7. Transient transfection and expression of HEK293F cells HEK293F cells were passaged one day before transfection, and the cell density was adjusted to 1×10⁻⁶ cells on the day of transfection. 6 Cells / mL, viability ≥95%. Taking a 30mL culture system as an example: Dilute 12μg of heavy chain plasmid and 18μg of light chain plasmid (mass ratio 1:1.5) with 1.5mL of serum-free Opti-MEM; dilute 90μg of PEI (PEI to total plasmid mass ratio = 3:1) with 1.5mL of serum-free Opti-MEM. Mix thoroughly and incubate at room temperature for 5 minutes. Add the PEI dilution dropwise to the plasmid dilution, mix gently, and incubate at room temperature for 15 minutes. Add the transfection complex dropwise to the cell suspension, mix gently, and incubate at 37℃, 8% CO2, 120rpm on a shaker. On days 5-7 of culture, when cell viability drops to 60%-70%, centrifuge at 3000g for 10 minutes to collect the culture supernatant and store temporarily at 4℃.

[0049] 8. Antibody purification and initial screening Take 1 mL of culture supernatant and detect its agglutination reaction with Wr(a+) erythrocytes using the microcolumn gel IAT method. Use a negative control (empty vector transfection supernatant) and a positive control (known anti-Wra human plasma) as references to screen positive clones. For the initially screened positive clones, scale up the culture to 100 mL for transfection expression. After centrifugation at 3000 g and filtration through a 0.45 μm filter, purify the culture supernatant using a Protein A affinity chromatography column, dialyze to PBS, and then concentrate by ultrafiltration to determine the concentration (A280 method).

[0050] 9. Identification of positive clonal characteristics (1) Titer determination: The purified antibody was serially diluted starting from 1:2, and the reactivity of each dilution with Wr(a+) erythrocytes was detected by microcolumn gel IAT method. The highest dilution that showed 1+ agglutination reaction was taken as the titer. Clones with a titer ≥1:512 were retained.

[0051] (2) Specificity verification: Cross-reactivity was performed using 20 red blood cell profiles (covering common blood type antigen polymorphisms) from different blood donors with known Wr(a-) phenotypes. Those with no agglutination of all Wr(a-) red blood cells were considered to be specific.

[0052] (3) Subtype identification: The antibody subtype is identified using a human IgG subtype detection kit (such as the Invitrogen Human IgG Subclass ELISA Kit).

[0053] Following the above screening and identification, several candidate clones of recombinant human IgG1 monoclonal antibodies specific to Wra were obtained. Sequencing confirmed that two of these clones were named WRa-01 and WRa-02, respectively, and their variable region sequences are shown in SEQ ID NO:1 to SEQ ID NO:8.

[0054] Example 2: Construction of a stable CHO-S expression cell line The optimal clone obtained in Example 1 (highest titer and strongest specificity) was selected, and its heavy and light chain expression vectors were linearized and co-transfected into CHO-S cells via electroporation. Forty-eight hours after transfection, selection medium containing methotrexate (MTX) was added for pressure selection. Surviving cells were seeded into 96-well plates using limiting dilution. After 2-3 weeks of culture, the supernatant from each well was used to detect antibody expression levels by ELISA. High-expressing clones were screened and amplified stepwise to finally obtain a CHO-S cell line stably expressing high levels of the anti-Wra recombinant monoclonal antibody. After 10 consecutive passages, the antibody expression level remained stable (RSD < 15%), demonstrating the good genetic stability of the cell line.

[0055] Example 3: Application of recombinant anti-Wra monoclonal antibody in blood typing kits The purified anti-Wra recombinant monoclonal antibody obtained in Example 1 or 2 was diluted with PBS containing 1% BSA to a working concentration (0.5-2 μg / mL as determined by checkerboard titration) to prepare a blood typing reagent working solution. The prepared working solution and the red blood cell suspension to be tested were tested in microcolumn gel cards according to standard IAT procedures. Using a commercially available human polyclonal anti-Wra reagent as a control, 100 clinical samples with known phenotypes (including 5 Wr(a+) and 95 Wr(a-)) were tested. The results showed that the recombinant monoclonal antibody reagent prepared in this invention was completely consistent with the detection results of the commercially available human polyclonal reagent (100% positive concordance rate and 100% negative concordance rate). Furthermore, the detection results of the recombinant monoclonal antibody reagent were highly consistent between different batches (intra-batch CV < 5%, inter-batch CV < 10%), significantly better than the batch consistency of the human polyclonal reagent.

[0056] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A recombinant human monoclonal antibody against Wra blood group antigen, characterized in that, The recombinant human monoclonal antibody comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

4.

2. The recombinant human monoclonal antibody according to claim 1, characterized in that, The recombinant human monoclonal antibody is a complete IgG1 antibody molecule.

3. A recombinant human monoclonal antibody against Wra blood group antigen, characterized in that, The recombinant human monoclonal antibody comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

8.

4. The recombinant human monoclonal antibody according to claim 3, characterized in that, The recombinant human monoclonal antibody is a complete IgG1 antibody molecule.

5. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the heavy chain variable region, light chain variable region, complete heavy chain, or complete light chain of the recombinant human monoclonal antibody according to claim 1 or 3.

6. The nucleic acid molecule according to claim 5, characterized in that, The nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region is shown in SEQ ID NO:1, and the nucleotide sequence of the nucleic acid molecule encoding the light chain variable region is shown in SEQ ID NO:3; or, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region is shown in SEQ ID NO:5, and the nucleotide sequence of the nucleic acid molecule encoding the light chain variable region is shown in SEQ ID NO:

7.

7. An expression carrier, characterized in that, The expression vector comprises the nucleic acid molecule as described in claim 5 or 6.

8. A host cell, characterized in that, The host cell comprises the expression vector of claim 7.

9. A method for preparing recombinant human monoclonal antibodies against Wra blood group antigens, characterized in that, Includes the following steps: S1: Donor screening, screening for donors with anti-Wra antibody titers ≥1:256 in peripheral blood; S2: Peripheral blood mononuclear cell isolation: Peripheral blood is collected from the donor, and peripheral blood mononuclear cells are separated by density gradient centrifugation; S3: Wra-specific B cell enrichment: The peripheral blood mononuclear cells obtained in step S2 are co-incubated with Wr(a+) erythrocytes to enrich Wra-specific B cells. S4: Single cell sorting. Using microfluidic single cell sorting technology, single B cells that specifically bind to Wr(a+) erythrocytes in step S3 are sorted into microplates containing lysis buffer. S5: Antibody variable region gene amplification: Using the RNA released from the lysate described in step S4 as a template, the antibody heavy chain variable region gene and light chain variable region gene in each single cell are amplified by reverse transcription polymerase chain reaction and nested polymerase chain reaction, respectively. S6: Expression vector construction: The heavy chain variable region gene fragment and the light chain variable region gene fragment obtained in step S5 are cloned into expression vectors containing human IgG1 constant region gene, respectively, to obtain heavy chain expression vector and light chain expression vector. S7: Recombinant antibody expression and purification: The heavy chain expression vector and light chain expression vector obtained in step S6 are co-transfected into mammalian cells for expression. The culture supernatant is harvested and purified by affinity chromatography to obtain recombinant monoclonal antibodies. S8: Antibody screening and identification. The purified antibody obtained in step S7 is tested for its reactivity with Wr(a+) erythrocytes and Wr(a-) erythrocytes by indirect anti-human globulin assay. Positive clones that specifically bind to Wr(a+) erythrocytes and do not react with Wr(a-) erythrocytes are screened out.

10. The method according to claim 9, characterized in that, In step S3, the ratio of peripheral blood mononuclear cells to Wr(a+) erythrocytes is 3 × 10⁻⁶. 6 1×10 7 The incubation conditions are 37℃, 5% CO2, and incubation in the dark for 60-120 minutes; in step S4, B cells bound to Wr(a+) erythrocytes are fluorescently labeled with anti-human IgG antibody before sorting; in step S5, the reverse transcription polymerase chain reaction is performed using oligo(dT) primers for reverse transcription, the first round of multiplex polymerase chain reaction is performed using mixed primers covering the human antibody VH, Vκ and Vλ gene families, and the second round of amplification is performed using nested primers with restriction endonuclease sites.

11. The method according to claim 9, characterized in that, The indirect antiglobulin test mentioned in step S8 is the microcolumn gel indirect antiglobulin test method; the screening also includes the following identification steps: titer determination, retaining clones with a titer ≥1:512; Specificity verification was performed by cross-reactivity testing using no fewer than 20 Wr(a-) erythrocyte profiles of different phenotypes; and antibody subtype identification was also conducted.

12. The method according to claim 9, characterized in that, The method further includes step S9 after step S8: transfecting the light and heavy chain expression vectors of the positive clones obtained by screening into CHO-S cells, and obtaining stable CHO-S cell lines that stably express anti-Wra recombinant human monoclonal antibodies after pressure screening and limiting dilution cloning.

13. The use of the recombinant human monoclonal antibody according to any one of claims 1 to 4 in the preparation of blood typing reagents.

14. A blood typing kit, characterized in that, The kit contains the recombinant human monoclonal antibody as described in any one of claims 1 to 4.