Humanized anti-beta2 glycoprotein I monoclonal antibody quality control product and application thereof

Abstract

The invention relates to a humanized anti-beta2 glycoprotein I monoclonal antibody quality control product which comprises a first humanized anti-beta2 glycoprotein I monoclonal antibody and a second humanized anti-beta2 glycoprotein I monoclonal antibody, the amino acid sequence of a heavy chain variable region of the first humanized anti-beta2 glycoprotein I monoclonal antibody is shown as SEQ ID NO: 37, the amino acid sequence of a light chain variable region of the first humanized anti-beta2 glycoprotein I monoclonal antibody is shown as SEQ ID NO: 38, and the amino acid sequence of the light chain variable region of the second humanized anti-beta2 glycoprotein I monoclonal antibody is shown as SEQ ID NO: 37. The amino acid sequence of a heavy chain variable region of the second humanized anti-beta2 glycoprotein I monoclonal antibody is as shown in SEQ ID NO: 39, and the amino acid sequence of a light chain variable region of the second humanized anti-beta2 glycoprotein I monoclonal antibody is as shown in SEQ ID NO: 40. The invention overcomes the defects of the existing quality control product in the aspects of raw material source, titer, uniformity, inter-batch consistency, linearity and the like, provides a humanized anti-beta2 glycoprotein I monoclonal antibody quality control product with optimized performance, and has important application value in quality control of a detection test of an anti-beta2-GPI antibody.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostic reagents, specifically relating to a quality control product of a humanized anti-β2 glycoprotein I monoclonal antibody and its application. Background Technology

[0002] Antiphospholipid syndrome (APS) is a systemic autoimmune disease characterized by recurrent arteriovenous thrombotic events and / or pathological pregnancies (such as recurrent miscarriage). Diagnosis and risk assessment of this disease heavily rely on laboratory antibody testing, with anti-β2 glycoprotein I (β2-GPI) antibody being a key indicator closely associated with thrombosis and adverse pregnancy outcomes.

[0003] Currently, clinical detection of anti-β2-GPI antibodies primarily employs immunological methods such as enzyme-linked immunosorbent assay (ELISA) or chemiluminescence immunoassay (CLIA). However, standardization in this detection field remains insufficient, with significant differences in results between different reagents and platforms. This directly impacts the consistency of APS diagnosis and the accuracy of risk assessment. The reliability of test results largely depends on the quality of calibrators and quality control materials used in the testing system.

[0004] Currently, internal quality control materials used for anti-β2-GPI antibody detection primarily rely on human serum as their raw material. This type of raw material has several significant drawbacks: First, the source of human serum presents potential biosafety risks and is subject to strict ethical regulations, making collection difficult; second, high-value positive samples that meet the high antibody titer requirements are extremely scarce in clinical practice and difficult to obtain reliably; more importantly, the complex composition of serum from different individuals leads to significant batch-to-batch variability in the raw material itself, making it difficult to control the quality consistency of the final quality control product and directly affecting the reliability and comparability of the test results.

[0005] Therefore, to overcome the shortcomings of existing quality control products in terms of raw material source, potency, uniformity, batch-to-batch consistency, and linearity, a humanized anti-β2 glycoprotein I (β2-GPI) monoclonal antibody quality control product with significantly optimized performance is provided. Summary of the Invention

[0006] The purpose of this invention is to provide a quality control product for a humanized anti-β2 glycoprotein I monoclonal antibody with significantly optimized performance.

[0007] A second aspect of the present invention provides the application of the above-mentioned humanized anti-β2 glycoprotein I monoclonal antibody quality control in anti-β2-GPI antibody detection or anti-β2-GPI antibody detection kits.

[0008] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: The first aspect of this invention provides a quality control product of a humanized anti-β2 glycoprotein I monoclonal antibody, comprising a first humanized anti-β2 glycoprotein I monoclonal antibody and a second humanized anti-β2 glycoprotein I monoclonal antibody. The amino acid sequence of the heavy chain variable region of the first humanized anti-β2 glycoprotein I monoclonal antibody is: QVQLQQSGAELARPGASLKMSCKASGYTFTTYTMHWVRQRPGQGLEWIGYIIPSSGYTNYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCARRGMGYPDYWGQGTTLTVSS (SEQ ID NO: 37); The amino acid sequence of the light chain variable region of the first humanized anti-β2 glycoprotein I monoclonal antibody is: QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSYPLTFGAGTKLELK (SEQ ID NO: 38). The amino acid sequence of the heavy chain variable region of the second humanized anti-β2 glycoprotein I monoclonal antibody is: EVQLQQSGPELVKPGASVKISCKASGYTFTDYNMYWVKQSHGKSLEWIGYIYPYNGGTGYNQKFKSKATLTVDNSSSTAYMELRSLTSEDSAVYYCARRGGLDHYFDYWGQGTTLTVSS (SEQ ID NO: 39); The amino acid sequence of the light chain variable region of the second humanized anti-β2 glycoprotein I monoclonal antibody is: DIVLTQSPASLAVSLGQRATISCKASQSVDYDDDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPWTFGGGTKLEIK (SEQ ID NO: 40).

[0009] Furthermore, the nucleic acid sequence encoding the heavy chain variable region of the first humanized anti-β2 glycoprotein I monoclonal antibody is shown in SEQ ID NO: 31.

[0010] Furthermore, the nucleic acid sequence encoding the light chain variable region of the first humanized anti-β2 glycoprotein I monoclonal antibody is shown in SEQ ID NO: 32.

[0011] Furthermore, the nucleic acid sequence encoding the heavy chain variable region of the second humanized anti-β2 glycoprotein I monoclonal antibody is shown in SEQ ID NO: 33.

[0012] Furthermore, the nucleic acid sequence encoding the light chain variable region of the second humanized anti-β2 glycoprotein I monoclonal antibody is shown in SEQ ID NO: 34.

[0013] In some specific embodiments, the C-terminus of the heavy chain variable region of the first humanized anti-β2 glycoprotein I monoclonal antibody and the second humanized anti-β2 glycoprotein I monoclonal antibody are respectively connected to the heavy chain constant region of human IgG1; the C-terminus of the light chain variable region of the first humanized anti-β2 glycoprotein I monoclonal antibody and the second humanized anti-β2 glycoprotein I monoclonal antibody are respectively connected to the light chain constant region of human IgG1.

[0014] Specifically, the amino acid sequence of the heavy chain constant region of human IgG1 is: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 41).

[0015] Furthermore, the coding nucleic acid sequence of the heavy chain constant region of the human IgG1 is shown in SEQ ID NO: 35.

[0016] Specifically, the amino acid sequence of the light chain constant region of the human IgG1 is RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 42).

[0017] Furthermore, the coding nucleic acid sequence of the light chain constant region of the human IgG1 is shown in SEQ ID NO: 36.

[0018] In some embodiments, the mass ratio of the first humanized anti-β2 glycoprotein I monoclonal antibody to the second humanized anti-β2 glycoprotein I monoclonal antibody is 1:(30~100), for example 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100.

[0019] More preferably, the mass ratio of the first humanized anti-β2 glycoprotein I monoclonal antibody to the second humanized anti-β2 glycoprotein I monoclonal antibody is 1:(40~60).

[0020] This invention uses antigen immunization to obtain mouse monoclonal antibodies. By screening different clones, mouse monoclonal antibodies targeting different antigen epitopes are obtained. These monoclonal antibodies are then humanized and combined with specific mixing ratios to overcome the problems that monoclonal antibodies only target one epitope of the antigen, cannot replace clinical samples, and have poor consistency and linearity with clinical samples.

[0021] In some embodiments, the concentration of the humanized anti-β2 glycoprotein I monoclonal antibody quality control is 0.1~5.0 mg / mL, for example 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL or 5 mg / mL.

[0022] In some embodiments, the humanized anti-β2 glycoprotein I monoclonal antibody quality control also includes a PBS solution with a pH of 7.2 to 7.4.

[0023] In some embodiments, the humanized anti-β2 glycoprotein I monoclonal antibody quality control product further includes one or more of fetal bovine serum, protein protectants, and preservatives.

[0024] Furthermore, the protein protectant is a commonly used protein protectant in the art, including but not limited to one or more of arginine, trehalose, bovine serum albumin (BSA), and glycerol.

[0025] Furthermore, the preservative is a commonly used preservative in the art, including but not limited to one or more of Proclin 300, Proclin 950, and NaN3.

[0026] Furthermore, the content of the fetal bovine serum in the humanized anti-β2 glycoprotein I monoclonal antibody quality control product is 10% to 50%, for example, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%.

[0027] Further, the content of the protein protectant in the humanized anti-β2 glycoprotein I monoclonal antibody quality control product is 0.5% to 20%, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, or 20%.

[0028] Furthermore, the preservative content in the humanized anti-β2 glycoprotein I monoclonal antibody quality control product is 0.1% to 0.5%, for example, 0.1%, 0.2%, 0.3%, 0.4% or 0.5%.

[0029] The second aspect of this invention provides the application of the aforementioned humanized anti-β2 glycoprotein I monoclonal antibody quality control in the detection of anti-β2 glycoprotein I antibodies.

[0030] Furthermore, the detection method is enzyme-linked immunosorbent assay (ELISA) or chemiluminescence immunoassay (CLIA).

[0031] The third aspect of this invention provides the application of the aforementioned humanized anti-β2 glycoprotein I monoclonal antibody quality control material in an anti-β2 glycoprotein I antibody detection kit.

[0032] Furthermore, the anti-β2 glycoprotein I antibody detection kit is an enzyme-linked immunosorbent assay (ELISA) kit or a chemiluminescent immunoassay kit.

[0033] Compared with the prior art, the present invention has the following advantages: The humanized anti-β2 glycoprotein I monoclonal antibody quality control product of this invention has a clearly identified source. Prepared through genetic engineering, this humanized monoclonal antibody effectively avoids the biosafety and ethical risks associated with traditional raw materials. The selected antibody possesses high affinity and high titer, supporting the preparation of high-concentration quality control products and meeting the quality control requirements for high-concentration samples in clinical testing. The humanized anti-β2 glycoprotein I monoclonal antibody quality control product of this invention exhibits excellent homogeneity, stability, and batch-to-batch consistency, providing a reliable quality control tool for anti-β2-GPI antibody detection experiments. It has significant application value in improving the standardization level and comparability of results in related testing projects. Attached Figure Description

[0034] Figure 1Electrophoresis images of 5G1 humanized monoclonal antibody and 12B3 humanized monoclonal antibody; Figure 2 Linear fitting curve of humanized anti-β2 glycoprotein I monoclonal antibody (clone number: 5G1); Figure 3 Linear fitting curve of humanized anti-β2 glycoprotein I monoclonal antibody (clone number: 12B3); Figure 4 The linear fitting curve is for the mixed anti-β2 glycoprotein I quality control sample. Detailed Implementation

[0035] The present invention will be further described below with reference to embodiments and comparative examples. However, the present invention is not limited to the following embodiments. The implementation conditions used in the embodiments can be further adjusted according to different requirements of specific applications, and the implementation conditions not specified are conventional conditions in the industry. The technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other.

[0036] In the following examples and comparative examples, the implementation conditions and testing methods not specified are standard conditions in the industry, and the reagents not specified are all commercially available products.

[0037] The pH of the PBS solutions used in the following examples and comparative examples is 7.2 to 7.4.

[0038] Example 1: This example provides a humanized anti-β2 glycoprotein I monoclonal antibody quality control product and its preparation method, as detailed below: 1. Material preparation: Purchase natural β2 glycoprotein I (β2-GPI) protein, with a purity >90% as determined by electrophoresis, for later use.

[0039] 2. Animal immunization: BALB / c mice were immunized by intraperitoneal injection of 2.5 mg of commercially available natural β2-GPI protein as an immunogen; blood was collected from the mice via vein, and the antibody titer in the mouse serum was detected by indirect ELISA.

[0040] 3. Cell fusion and screening: 3.1. Cell Fusion and Monoclonal Screening: Spleen cells from immunized mice were mixed with SP2 / 0 mouse myeloma cells in the logarithmic growth phase at a ratio of 10:1, centrifuged, and the supernatant was discarded. The resulting cell mixture was placed in a 37°C water bath, and 50% PEG1500 was added while stirring, over 1-1.5 minutes. The mixture was then allowed to stand for 2 minutes. Then, over 5 minutes, pre-warmed serum-free culture medium (37°C) was added dropwise, centrifuged, the supernatant was discarded, and complete selection medium (20% FBS + HAT) was added. The cells were gently resuspended by pipetting. 200 μL / well was then seeded into 96-well plates containing feeder cells and cultured at 37°C with 5% CO2. After 7-10 days, indirect ELISA screening was performed to select the hybridoma cells from the top 20 positive wells with high OD values.

[0041] 3.2. Twenty maternal clone supernatants were selected and analyzed on a chemiluminescence platform. Biotinylated glycoprotein β2-GPI antigen was bound to streptavidin SA magnetic beads at a concentration of 2 μg / mL. The maternal clone supernatants (containing mouse glycoprotein β2-GPI antibody) were serially diluted with PBS solution to prepare the primary antibody working solution. AP-labeled goat anti-mouse IgG antibody was diluted 2000-fold to prepare the secondary antibody working solution. An indirect fully automated chemiluminescence CLIA indirect assay was performed to test the titer of the monoclonal antibodies, and the five clones with the highest titers were selected.

[0042] 4. Subcloning, scale-up culture, and cryopreservation: 4.1. Five positive cells were selected and counted using a cell counting chamber. The cell suspension was diluted proportionally according to the count and diluted with 10% serum HT medium to achieve a density of 1 cell / well in a 96-well cell culture plate. Each positive clone was placed on a separate cell culture plate and cultured for 10 days. The cells were then detected by enzyme-linked immunosorbent assay (ELISA).

[0043] 4.2. The qualified subclonal supernatant was tested on a chemiluminescence platform. Biotinylated glycoprotein β2-GPI antigen was conjugated with streptavidin SA magnetic beads at a concentration of 0.5 μg / mL. The subclonal supernatant (containing mouse glycoprotein β2-GPI antibody) was serially diluted with PBS solution to prepare the primary antibody working solution. AP-labeled goat anti-mouse IgG antibody was diluted 2000 times to prepare the secondary antibody working solution. The titer of the monoclonal antibody was tested by an indirect fully automated chemiluminescence CLIA assay.

[0044] 5. Cell sequencing: Hybridoma cell lines were subjected to variable region NGS gene sequencing. Two monoclonal antibodies were obtained, as shown in Tables 1 and 2.

[0045] Table 1 Table 2 6. Constructing antibody expression vectors: The heavy chain variable region gene of 5G1 was spliced ​​with the human IgG1 heavy chain constant region gene. Then, the spliced ​​and complete heavy chain gene was ligated with the eukaryotic expression vector pcDNA3.4 to construct the first human-mouse chimeric antibody heavy chain gene recombinant expression plasmid, so that the human IgG1 heavy chain constant region was linked to the C-terminus of the heavy chain variable region of 5G1.

[0046] The light chain variable region gene of 5G1 was spliced ​​with the light chain constant region gene of human IgG1. Then, the spliced ​​and complete heavy chain gene was ligated with the eukaryotic expression vector pcDNA3.4 to construct the first human-mouse chimeric antibody light chain gene recombinant expression plasmid, so that the human IgG1 light chain constant region was linked to the C-terminus of the light chain variable region of 5G1.

[0047] The heavy chain variable region gene of 12B3 was spliced ​​with the human IgG1 heavy chain constant region gene. Then, the spliced, complete heavy chain gene was ligated with the eukaryotic expression vector pcDNA3.4 to construct a second human-mouse chimeric antibody heavy chain gene recombinant expression plasmid, so that the human IgG1 heavy chain constant region was linked to the C-terminus of the heavy chain variable region of 12B3.

[0048] The light chain variable region gene of 12B3 was spliced ​​with the human IgG1 light chain constant region gene. Then, the spliced, complete heavy chain gene was ligated to the eukaryotic expression vector pcDNA3.4 to construct a second human-mouse chimeric antibody light chain gene recombinant expression plasmid, so that the human IgG1 light chain constant region was linked to the C-terminus of the light chain variable region of 12B3.

[0049] The nucleic acid sequence of the heavy chain variable region gene of 5G1 is: CAGGTCCAGCTGCAGCAGTCTGGGGCTGAACTGGCAAGACCTGGGGCCTCACTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTTACTACCTACACGATGCACTGGGTAAGACAGAGGCCTGGACAGGGTCTGGAATGGATTGGATACATTATTCCTAGCAGTGGTTATACTAATTACAATCAGAAGTTCAAGGACAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAACTGAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAAGGGGGATGGGATACCCTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO: 31); The nucleic acid sequence of the light chain variable region gene of 5G1 is: CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGTACTGGTACCAGCAGAAGCCAGGATCCTCCCCCAGACTCCTGATTTATGACACATCCAACCTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCCGAATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTTACCCACTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA (SEQ ID NO: 32); The nucleic acid sequence of the heavy chain variable region gene of 12B3 is: GAGGTCCAGCTTCAGCAGTCAGGACCTGAGCTGGTGAAACCTGGGGCCTCAGTGAAGATATCCTGCAAGGCTTCTGGATACACATTCACTGACTACAACATGTACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGATATATTTATCCTTACAATGGTGGTACTGGCTACAACCAGAAGTTCAAGAGCAAGGCCACATTGACTGTAGACAATTCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAAGAGGAGGACTGGACCACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO: 33); The nucleic acid sequence of the light chain variable region gene of 12B3 is: GACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAAGGCCAGCCAAAGTGTTGATTATGATGATGATAGTTATATGAACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATGCTGCATCCAATCTAGAATCTGGGATCCCAGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCAAAGTAATGAGGATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA (SEQ ID NO: 34); The nucleic acid sequence of the human IgG1 light chain constant region gene is: AGGACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCAGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAAC AGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGAGCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGC (SEQ ID NO: 36).

[0050] 7. Constructing cell lines: The first human-mouse chimeric antibody heavy and light chain gene recombinant expression plasmid was co-transfected into CHO-S cells, and the second human-mouse chimeric antibody heavy and light chain gene recombinant expression plasmid was also co-transfected into CHO-S cells. The cells were then inoculated at 5 × 10⁶ cells per cell line. 5 Cells / mL were placed in 6-well plates and screened after 48 hours. After obtaining a stable cell pool, monoclonal cells were screened using limiting dilution. Cell supernatants were collected for indirect ELISA detection. Finally, monoclonal cells with the highest expression levels were selected for expansion culture, and the cells were cryopreserved in liquid nitrogen.

[0051] 8. Antibody purification: The cell culture supernatant was purified using a Protein A column according to the manufacturer's instructions to obtain humanized monoclonal antibodies that specifically recognize the glycoprotein β2-GPI. The concentrations were determined using a micro-ultraviolet spectrophotometer: the concentration of the 5G1 humanized monoclonal antibody was 1.04 mg / mL, and the concentration of the 12B3 humanized monoclonal antibody was 3.81 mg / mL. Antibody purity was assessed using SDS-PAGE, and the electrophoresis results are shown below. Figure 1 As shown, lanes 1 and 3 are the non-reduction and reduction electrophoresis patterns of the 5G1 humanized monoclonal antibody; lanes 2 and 4 are the non-reduction and reduction electrophoresis patterns of the 12B3 humanized monoclonal antibody, with a purity of >90%.

[0052] 9. Antibody evaluation: The purified antibody was detected using an anti-β2-glycoprotein I antibody chemiluminescence detection kit (Haobo) and its matching SMART 6500 fully automated chemiluminescence analyzer. The corresponding reagents included R1 (0.8 μg / mL biotinylated glycoprotein β2-GPI antigen), M (0.6 mg / mL SA magnetic beads: 1 μm streptavidin magnetic beads), R2 (enzyme-labeled secondary antibody: 0.1 μg / mL alkaline phosphatase-labeled mouse anti-human IgG), chemiluminescent substrate (AMPPD), and sample diluent: 8 mM phosphate buffer at pH 7.2. The purified humanized anti-β2-glycoprotein I monoclonal antibody was serially diluted using the sample diluent to prepare the test samples. The procedure was the same as described in patent CN 119846224 B. The instrument's software automatically fitted a standard curve with the concentrations of different calibrators as the x-axis and the luminescence value as the y-axis, and automatically output the test concentration value of the test sample. Based on the dilution factor, the titer of the humanized anti-β2 glycoprotein I monoclonal antibody (clone number: 5G1) was calculated to be 10,793,308 RU / mL; the titer of the humanized anti-β2 glycoprotein I monoclonal antibody (clone number: 12B3) was calculated to be 4,516,664 RU / mL. Linearity correlation was evaluated for the two humanized monoclonal antibodies, and the results are shown in Tables 3 and 4.

[0053] Table 3. Results of serial dilution assays for humanized anti-β2 glycoprotein I monoclonal antibody (clone number: 5G1) Table 4. Results of serial dilution assays for humanized anti-β2 glycoprotein I monoclonal antibody (clone number: 12B3) 10. Preparation of quality control samples: Humanized anti-β2 glycoprotein I monoclonal antibody (clone number: 5G1) and humanized anti-β2 glycoprotein I monoclonal antibody (clone number: 12B3) were diluted to 1 mg / mL, then mixed according to Table 5, and linearity was evaluated to select the optimal mixing ratio.

[0054] Table 5 Table 6. Validation results for different mixing ratios The optimal ratio (5G1:12B3=1:50) was selected, and 20 v / v% fetal bovine serum, 0.5 wt% BSA, 10 v / v% glycerol, and 0.1 v / v% Proclin 950 were added sequentially as the quality control for the humanized anti-β2 glycoprotein I monoclonal antibody. Linearity confirmation and stability evaluations were performed, and the results are shown in Table 7. Figure 4 As shown in Table 8.

[0055] Table 7 Table 8. Accelerated stability at 37℃ Table 9. Freeze-thaw stability at -20℃ As can be seen from the above results, the humanized anti-β2 glycoprotein I monoclonal antibody quality control product of the present invention is prepared by genetic engineering, with a clear and reliable source, effectively avoiding the biosafety and ethical risks involved in traditional raw materials. At the same time, it has good uniformity and batch-to-batch consistency, high potency, and excellent stability.

[0056] Furthermore, the quality control of the humanized anti-β2 glycoprotein I monoclonal antibody of the present invention and the linear R of serially diluted clinical samples 2 A value >0.99 and a slope k between 0.85 and 1.15 indicate good consistency between the quality control product and clinical results. In addition, the humanized anti-β2 glycoprotein I monoclonal antibody stock solution of this invention has a titer as high as 150 wRU / mL, which can be used to prepare high-value quality control products and is suitable for the detection of high-value positive samples.

[0057] In summary, the humanized anti-β2 glycoprotein I monoclonal antibody quality control product of the present invention has important application value in the quality control of the detection test of anti-β2-GPI antibody.

[0058] The present invention has been described in detail above, with the aim of enabling those skilled in the art to understand and implement the invention. However, this description should not be construed as limiting the scope of protection of the invention. All equivalent changes or modifications made in accordance with the spirit and essence of the invention should be included within the scope of protection of the invention.

Claims

1. A quality control product for a humanized anti-β2 glycoprotein I monoclonal antibody, characterized in that, It includes a first humanized anti-β2 glycoprotein I monoclonal antibody and a second humanized anti-β2 glycoprotein I monoclonal antibody. The amino acid sequence of the heavy chain variable region of the first humanized anti-β2 glycoprotein I monoclonal antibody is: QVQLQQSGAELARPGASLKMSCKASGYTFTTYTMHWVRQRPGQGLEWIGYIIPSSGYTNYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCARRGMGYPDYWGQGTTLTVSS; The amino acid sequence of the light chain variable region of the first humanized anti-β2 glycoprotein I monoclonal antibody is: QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSYPLTFGAGTKLELK, The amino acid sequence of the heavy chain variable region of the second humanized anti-β2 glycoprotein I monoclonal antibody is: EVQLQQSGPELVKPGASVKISCKASGYTFTDYNMYWVKQSHGKSLEWIGYIYPYNGGTGYNQKFKSKATLTVDNSSSTAYMELRSLTSEDSAVYYCARRGGLDHYFDYWGQGTTLTVSS; The amino acid sequence of the light chain variable region of the second humanized anti-β2 glycoprotein I monoclonal antibody is: DIVLTQSPASLAVSLGQRATISCKASQSVDYDDDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPWTFGGGTKLEIK.

2. The humanized anti-β2 glycoprotein I monoclonal antibody quality control product according to claim 1, characterized in that, The C-terminus of the heavy chain variable region of the first humanized anti-β2 glycoprotein I monoclonal antibody and the second humanized anti-β2 glycoprotein I monoclonal antibody are respectively linked to the heavy chain constant region of human IgG1; The C-terminus of the light chain variable region of the first humanized anti-β2 glycoprotein I monoclonal antibody and the second humanized anti-β2 glycoprotein I monoclonal antibody are respectively linked to the light chain constant region of human IgG1.

3. The humanized anti-β2 glycoprotein I monoclonal antibody quality control product according to claim 2, characterized in that, The amino acid sequence of the heavy chain constant region of human IgG1 is: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; The amino acid sequence of the light chain constant region of human IgG1 is: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

4. The humanized anti-β2 glycoprotein I monoclonal antibody quality control product according to claim 1, characterized in that, The mass ratio of the first humanized anti-β2 glycoprotein I monoclonal antibody to the second humanized anti-β2 glycoprotein I monoclonal antibody is 1:(30~100).

5. The humanized anti-β2 glycoprotein I monoclonal antibody quality control product according to claim 4, characterized in that, The mass ratio of the first humanized anti-β2 glycoprotein I monoclonal antibody to the second humanized anti-β2 glycoprotein I monoclonal antibody is 1:(40~60).

6. The humanized anti-β2 glycoprotein I monoclonal antibody quality control product according to claim 1, characterized in that, The concentration of the humanized anti-β2 glycoprotein I monoclonal antibody quality control is 0.1~10 mg / mL.

7. The humanized anti-β2 glycoprotein I monoclonal antibody quality control product according to claim 1, characterized in that, It also includes PBS solutions with a pH of 7.2 to 7.

4.

8. The humanized anti-β2 glycoprotein I monoclonal antibody quality control product according to claim 7, characterized in that, It also includes one or more of fetal bovine serum, protein protectants, and preservatives.

9. The use of the humanized anti-β2 glycoprotein I monoclonal antibody quality control material as described in any one of claims 1 to 8 in the detection of anti-β2 glycoprotein I antibodies.

10. The use of the humanized anti-β2 glycoprotein I monoclonal antibody quality control material as described in any one of claims 1 to 8 in the anti-β2 glycoprotein I antibody detection kit.

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