Humanized monoclonal antibody against Ri, its application and detection method based thereon

Abstract

The present invention discloses an anti-Ri humanized monoclonal antibody and its application and a detection method based thereon, specifically an anti-Ri monoclonal antibody and its application and a method for detecting Nova-1 protein / polypeptide or Nova-2 protein / polypeptide using the same. The anti-Ri monoclonal antibody of the present invention has high specificity and good affinity. The antibody can be used for other scientific research such as Nova-1 or Nova-2 antigen detection, and can also meet the requirements of anti-Nova-1 / Nova-2 autoantibody detection kits for positive controls. The antibody is easy to store and modify, and is easy to control the quality of the antibody production process. It has very good application value and very important scientific research guidance significance.

Description

Technical Field

[0001] The present invention belongs to the technical field of monoclonal antibody preparation, and relates to humanized anti-Ri monoclonal antibodies and applications thereof and detection methods based thereon, and specifically to humanized chimeric anti-Ri monoclonal antibodies and applications thereof and methods for detecting Nova-1 / Nova-2 proteins / polypeptides using the same. Background Art

[0002] Paraneoplastic neurological syndrome (PNS) is a unique autoimmune disease of the nervous system, characterized by an immune-mediated abscopal effect of tumors. PNS is characterized by multiple autoantibodies, including anti-Hu (also known as anti-neuronal nuclear autoantibody type 1, ANNA-1), anti-Ri (also known as anti-neuronal nuclear autoantibody type 2, ANNA-2), and anti-Yo (also known as Purkinje cell antibody type 1, PCA1, or cerebellar degeneration-related protein CDR2). Anti-Ri autoantibodies are diagnostic for paraneoplastic cerebellar ataxia, primarily seen in disorders of the brain, brainstem, and spinal cord, including ataxia. They are less common in sensory, motor, and motor neuropathies, but are more common in related cancers such as breast cancer, small cell lung cancer, and bladder cancer. Detection of anti-Ri autoantibodies is essential for the prevention and treatment of paraneoplastic cerebellar ataxias. Both Nova-1 and Nova-2 proteins are target antigens for anti-Ri antibodies. Therefore, a positive test for anti-Nova-1, anti-Nova-2, or both anti-Nova-1 and anti-Nova-2 is considered anti-Ri autoantibody positive. The target antigens, Nova-1 and Nova-2, share high sequence homology. Nova-1 is primarily expressed in the cerebellum and spinal cord, while Nova-2 is primarily expressed in the cortex. In vascular endothelial cells, Nova-2 is crucial for angiogenesis, while in adipocytes, Nova-1 is involved in regulating thermogenesis and obesity. In many cancers, Nova-1 and Nova-2 activate pro-oncogenic pathways through interactions with specific miRNAs and other mechanisms, promoting cell proliferation, colony formation, migration, and invasion.

[0003] Currently, autoantibody detection kits for detecting autoimmune diseases of the nervous system are developing rapidly and are experiencing strong market demand. Detection kits for Ri autoantibodies in paraneoplastic syndromes have already been developed. Most tests for Ri autoantibodies focus on Nova-1 or both Nova-1 and Nova-2 autoantibodies. Whether targeting one or both autoantibodies, or whether the test kits are prepared using various methods such as cell-based immunofluorescence, immunospot, colloidal gold, or ELISA, numerous difficulties exist during the product design and development phase. Furthermore, there are no marketed national or industry-standard reference materials for such products. Companies are forced to use positive and negative sera to establish their own reference materials for product performance verification and quality control studies. However, due to the complex composition of serum and the low incidence of these diseases, the small number of positive sera makes collection difficult, making them difficult to meet the requirements of both preliminary research and long-term product quality control.

[0004] Humanized chimeric antibodies are an important achievement in the field of antibody engineering. They are produced by inserting the light chain and heavy chain variable region (V region) genes of heterologous monoclonal antibodies (such as mouse monoclonal antibodies) into an expression vector containing the human antibody constant region (C region) through DNA recombination technology, and then transforming mammalian cells to express the antibody. The V region of this antibody molecule is heterologous, while the C region is human, so it is called a chimeric antibody. While retaining the ability of the parent antibody to specifically bind to the antigen, humanized chimeric antibodies greatly reduce the immunogenicity of heterologous antibodies. Since humanized chimeric antibodies can specifically recognize the corresponding antigen, they can replace the function of positive serum to a certain extent, and the concentration can be determined by protein quantification and the titer can be determined by antibody titer detection, thereby obtaining quantifiable and traceable corporate reference material raw materials.

[0005] Currently, there is still a research gap in the development of anti-Ri monoclonal antibodies that can simultaneously recognize Nova-1 and Nova-2 antigens in paraneoplastic syndrome test kits using human-mouse chimeric monoclonal antibodies. Summary of the Invention

[0006] In order to overcome the shortcomings of the above-mentioned prior art, the object of the present invention is to provide an anti-Ri monoclonal antibody and its application and a method for detecting Nova-1 protein / polypeptide or Nova-2 protein / polypeptide using the same. The monoclonal antibody has strong specificity and high sensitivity, can meet the requirements of positive reference materials in diagnostic kits, is easy to store and modify, and the antibody has the ability to recognize the target antigen Ri in the human body.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention discloses an anti-Ri monoclonal antibody, which is named as humanized monoclonal antibody 3F5, comprising a light chain and a heavy chain;

[0009] The heavy chain variable region includes three heavy chain complementary determining regions CDR1, CDR2 and CDR3, the amino acid sequence of the heavy chain complementary determining region CDR1 is shown in SEQ ID NO.1, the amino acid sequence of the heavy chain complementary determining region CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain complementary determining region CDR3 is shown in SEQ ID NO.3;

[0010] The variable region of the light chain includes three light chain complementary determining regions CDR1, CDR2 and CDR3. The amino acid sequence of the light chain complementary determining region CDR1 is shown in SEQ ID NO.4, the amino acid sequence of the light chain complementary determining region CDR2 is shown in SEQ ID NO.5, and the amino acid sequence of the light chain complementary determining region CDR3 is shown in SEQ ID NO.6.

[0011] Preferably, the amino acid sequence of the heavy chain variable region of the humanized monoclonal antibody 3F5 is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.8.

[0012] Further preferably, the nucleotide sequence encoding the heavy chain variable region of the humanized monoclonal antibody 3F5 is shown as SEQ ID NO.9; the nucleotide sequence encoding the light chain variable region of the humanized monoclonal antibody 3F5 is shown as SEQ ID NO.10.

[0013] Preferably, the monoclonal antibody comprises a light chain having a complementarity determining region with an amino acid sequence that is at least 75% identical to the sequence of the variable region of the light chain, and the complementary domain of the light chain retains the ability to bind to the Ri protein; the monoclonal antibody comprises a heavy chain having a complementarity determining region with an amino acid sequence that is at least 75% identical to the sequence of the variable region of the heavy chain, and the complementary domain of the heavy chain retains the ability to bind to the Ri protein.

[0014] The present invention also discloses a vector, which comprises the nucleotide sequence of the anti-Ri monoclonal antibody.

[0015] The present invention also discloses a host cell, which comprises the above-mentioned vector.

[0016] The present invention also discloses the use of the above-mentioned anti-Ri monoclonal antibody, vector or host cell in preparing a reagent / kit for detecting Ri protein.

[0017] The present invention also discloses the use of the above-mentioned anti-Ri monoclonal antibody, vector or host cell in preparing a reagent / kit for diagnosing and / or prognosing human paraneoplastic movement disorders.

[0018] The present invention also discloses a method for detecting Nova-1 protein / peptide or Nova-2 protein / peptide, wherein a biological sample is contacted with the above-mentioned anti-Ri monoclonal antibody to detect the Nova-1 protein / peptide or Nova-2 protein / peptide in the biological sample.

[0019] Preferably, the biological sample includes but is not limited to plasma, whole blood, serum, tissue, cells, or lysates of tissues and cells containing Nova-1 protein / polypeptide or Nova-2 protein / polypeptide.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention has designed and obtained, for the first time, a highly specific and highly affinity anti-Ri monoclonal antibody. This antibody sequence is the core of the invention. This anti-Ri monoclonal antibody can be used for antigen detection and as a positive control in detection kits. This means it can be used in other scientific research, such as Nova-1 or Nova-2 antigen detection. It also meets the positive control requirements of anti-Nova-1 / Nova-2 autoantibody detection kits. It is easy to store and modify, and facilitates quality control during the antibody production process. Specifically, it can be used to detect whether protein expression is upregulated or downregulated. Detecting changes in protein expression is of certain significance for related diseases. Therefore, it has excellent application value and is of great scientific significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The purification results of N-6his protein;

[0023] Figure 2a The results of Nova-1 protein antibody titer determination in immunized mice;

[0024] Figure 2b The results of Nova-2 protein antibody titer determination in immunized mice;

[0025] Figure 3 The results of subtype identification of hybridoma cell line 3F5 are shown. Each group of data in the figure represents total IgG, IgG1, IgG2a, IgG2b, IgG3, IgM, Ig kappa chain, and Ig lambda chain from left to right.

[0026] Figure 4a This is the titer test result of Ri-3F5 human-mouse chimeric antibody;

[0027] Figure 4b The eyeball blood of rat No. 3 was used as a control for the determination of antibody titer;

[0028] Figure 5Dot blot was used to verify the function of anti-Ri-3F5 human-mouse chimeric antibody;

[0029] Figure 6a To verify the function of anti-Ri human-mouse chimeric antibody by immunofluorescence assay;

[0030] Figure 6b This is the comparison result of the positive signal of immunofluorescence assay;

[0031] Figure 7 Western blotting was used to verify the function of the anti-Ri human-mouse chimeric antibody. DETAILED DESCRIPTION

[0032] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0033] It should be noted that the terms "first", "second", etc. in the description and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that the numbers used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0034] The present invention is described in further detail below with reference to the accompanying drawings:

[0035] Unless otherwise specified, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those skilled in the art.

[0036] The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules containing an antigen-binding site that immunologically binds to an antigen. These include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, human-mouse chimeric antibodies, single-chain antibodies, F ab 、F ab’ and F (ab’)2 Fragments, single-chain FV fragments and F ab Expression library.

[0037] "Monoclonal antibody" refers to a population of antibodies derived from a single species that contains a unique light chain gene product and a unique heavy chain gene product. Specifically, it refers to the fact that all molecules in the population share the same complementarity determining regions, enabling them to bind to a specific epitope of an antigen at the antigen-binding site.

[0038] The "antigen binding site" refers to the part of an antibody molecule that participates in antigen binding. The antigen binding site is formed by amino acid residues in the N-terminal variable (V) regions of the heavy (H) and light (L) chains.

[0039] "Chimeric antibody" refers to an antibody molecule that is spliced ​​from the V region gene of other non-human antibodies and the C region gene of a human antibody, which is then inserted into a vector and transfected into myeloma tissue for expression.

[0040] The term "chimeric antibody" refers to an antibody in which the sequences of the H chain variable region and the L chain variable region (VH and VL) are derived from non-human animal species and the constant region sequences (CH and CL) are derived from humans. The sequence of the variable region is preferably derived from an animal species that allows easy preparation of hybridomas, such as mouse, rat, rabbit, etc.

[0041] The term "humanized monoclonal antibody" refers to a humanized monoclonal antibody in which the CDR regions of a human antibody are replaced with CDR regions from non-human animal species antibodies, also known as CDR-grafted antibodies. The CDR sequences of non-human animal species antibodies are preferably derived from, for example, animal species that allow easy preparation of hybridomas, such as mice, rats, rabbits, etc.

[0042] Example 1 Preparation of anti-Ri monoclonal antibody hybridoma cells

[0043] 1. Purification of N-6his protein

[0044] The human Nova-1 gene sequence, NCBI sequence number NM_002515.3, and the human Nova-2 gene sequence, NCBI sequence number NM_002516.4, were obtained from the GenBank sequence database. By comparing the homology of the Nova-1 and Nova-2 protein sequences, it was found that the sequence homology between the 40th to 149th amino acid sequence of the Nova-1 protein and the 23rd to 132th amino acid sequence of the Nova-2 protein was 85.45%. Therefore, the 40th to 149th amino acid sequence of the Nova-1 protein was selected, and a 6his tag sequence was added to the C-terminus of the gene. The gene sequence was synthesized by Jinweizhi Company into the pET-28a vector and named N-6his to obtain the N-6his recombinant vector. The constructed and correctly sequenced plasmid was transformed into Escherichia coli ArcticExpress (DE3) expression competent cells; a single clone was picked and inoculated into LB medium, shaken at 37°C to an OD value of 0.5-1, induced with a final concentration of 0.5mM IPTG, and expressed at 16°C overnight; the cells were collected, ultrasonically disrupted, and the supernatant was collected after centrifugation. The Ri protein expressed by pET-28a was purified using Ni-NTA column affinity chromatography. The purified protein was recorded as N-6his. The purified protein was dialyzed and concentrated to obtain a high-concentration N-6his protein for use as an immune antigen. The purification results are shown in Figure 2. Figure 1 shown.

[0045] The human Nova-1 gene sequence was retrieved using the same method, with the NCBI sequence number NM_002515.3. A GST tag sequence was added to the C-terminus of the gene and synthesized by Jinweizhi Company into the pGEX 4t-1 vector to generate the Nova-1-GST recombinant vector. The Nova-1 protein expressed in pGEX 4t-1 was purified using GSH affinity chromatography, and the purified protein was designated Nova-1. The purified protein was then dialyzed and concentrated to obtain a high-concentration Nova-1 protein, which was used as a detection antigen.

[0046] The same method was used to locate the human Nova-2 gene sequence, NCBI sequence number NM_002516.4. A 6his tag sequence was added to the C-terminus of the gene. This gene sequence was synthesized by Jinweizhi Company and inserted into the pET-28a vector to generate the Nova-2-6his recombinant vector. The Nova-2 protein expressed in pET-28a was purified using Ni-NTA affinity chromatography. The purified protein was designated Nova-2. The purified protein was dialyzed and concentrated to obtain a high-concentration Nova-2 protein, which was used as a detection antigen.

[0047] 2. Immunization of mice

[0048] Immunize three Balb / c mice using 40 μg of the purified N-6his recombinant protein from step 1 as the immunogen. Mix the antigen with Freund's adjuvant in a 1:1 ratio of equal volumes and thoroughly grind to a water-in-oil emulsion. Each mouse is injected with 100 μL of the antigen and Freund's adjuvant mixture (40 μg antigen per mouse). The injection site is the back. A second immunization is performed 14 days after the initial immunization, and a third immunization is performed 14 days after the second immunization. For the subsequent two immunizations, the dosage, method, and route of injection are the same as for the initial immunization.

[0049] 3. Detection of antibody production in immunized mice

[0050] Three days after the immunization, blood was collected from the mouse eyeballs, and the serum was collected by centrifugation. The titer was tested using ELISA, and mice with high titers were selected for booster immunization. The specific operation of ELISA titer detection is as follows: use pH 9.6 carbonate buffer to coat purified Nova-1 protein and Nova-2 protein as detection antigens, with a concentration of 100 ng / well, and coat at 4°C overnight; the next day, wash three times with 0.1M PBST, 3 minutes each time, and pat dry; add blocking protein (2% BSA solution), 200uL / well, incubate at 37°C for 1 hour; wash three times with 0.1M PBST, 3 minutes each time, and pat dry; dilute the serum of three immunized mice and one normal control mouse with PBS in series, at dilutions of 1:50, 1:150, 1:450, 1:1350, 1:4050, 1:12150, 1:36450, and 1:109350, add 100ul to each well, incubate at 37°C for 1 hour; wash three times with 0.1M PBST, 3 minutes each time, and pat dry; use goat anti-mouse IgG-HRP Dilute 1:5000 (manufacturer: Jackson), incubate at 37°C for 30 min; wash three times with 0.1 M PBST, 3 min each time, and pat dry; add TMB for color development for 10 min, stop color development with 2 M H2SO4, and measure absorbance at 450 nm.

[0051] Test results such as Figure 2a and Figure 2b As shown in the figure, a comprehensive comparison of the titers of serum antibodies from the three immunized mice showed that only mouse No. 3 had strong specificity for both Nova-1 and Nova-2 proteins, so mouse No. 3 was selected for subsequent experiments.

[0052] 4. Cell fusion and hybridoma screening

[0053] 4.1 Preparation of Feeder Cells and Myeloma Cells: Macrophages from the ascites of normal mice were cultured as feeder cells. Feeder cells were obtained from the ascites of 4-6 mice and plated onto 10 96-well plates. After 2 days of culture, hybridoma cells were prepared. SP2 / 0 myeloma cells, growing well and in the logarithmic growth phase with a cell density of 90%, were collected from 10 plates. After centrifugation, the supernatant was discarded, and the cells were resuspended in DMEM medium and counted.

[0054] 4.2 Preparation of spleen cells: Mouse No. 3 was boosted with intraperitoneal injection of antigen. Three days later, the eyeballs were removed and blood was collected to separate the serum. The blood was rinsed with running water and placed in a 75% ethanol solution for 10 minutes. The spleen of the mouse was aseptically removed and placed in a 200-mesh disposable sterile filter. The spleen was minced with sterile surgical scissors and ground with the piston handle of a 50 mL sterile syringe to obtain a single spleen cell suspension.

[0055] 4.3 Cell Fusion: Preheat a 37°C water bath in a clean hood. Add pre-cultured SP2 / 0 cells and spleen cells at a 1:10 ratio (90% cell density) to a 50 mL centrifuge tube and mix thoroughly. Centrifuge at 500 g for 10 min. Aspirate the supernatant and gently tap the bottom of the tube to loosen the cell pellet. Slowly add 1 mL of 45% PEG-1450 solution pre-warmed to 37°C over 90 seconds, gently shaking the tube continuously. Keep the tube in a 37°C water bath throughout the process. Add DMEM medium to the cell mixture dropwise at a constant rate: 1 mL in the first minute, 2 mL in the second minute, 3 mL in the third minute, 4 mL in the fourth minute, and 5 mL in the fifth minute, while shaking in a 37°C water bath. Incubate at 37°C for 15 min, centrifuge at 500 g for 5 min, and discard the supernatant. Add 5 ml of DMEM medium containing HAT to gently resuspend the pelleted cells. Finally, top up with DMED medium containing HAT to approximately 100 ml. Aliquot 100 μL / well into a 96-well cell culture plate seeded with feeder cells and incubate the plate at 37°C in a 5% CO2 incubator. Add 100 μL of HAT medium to each well the day after fusion. Perform a half-change of HAT medium every 2-3 days. Culture for approximately 2 weeks before screening.

[0056] 4.4 Screening of Positive Hybridoma Cells: Observe the growth of hybridoma cells. After 7 days, when the cell culture supernatant turns yellow, aspirate an appropriate amount of cell supernatant for ELISA antibody detection. Based on the ELISA results, select clones with high OD values ​​that are more than twice that of the negative control, plate them on a 96-well plate, and perform the first subclone screening. After 7-10 days, perform ELISA antibody detection again, select clones with high OD values, plate them on a 96-well plate, and perform the second subclone screening, so that there is approximately one cell per well. After 7-10 days of culture, perform ELISA antibody detection again, select clones with high OD values, plate them on a 96-well plate, and perform the third subclone screening, so that there is approximately one cell per well. Select several clones with high OD values ​​for further verification. Finally, one positive clone cell was screened and named: 3F5.

[0057] 4.5 Subtype identification of positive hybridoma cells: Re-coat the purified N-6his protein as an antigen to detect the subtype of 3F5 monoclonal cells. The antigen coating concentration is 100 ng / well and the cells are coated overnight at 4°C. The next day, wash the cells three times with 0.1 M PBST for 3 minutes each time and pat dry. Add blocking protein (2% BSA solution) at 200 uL / well and incubate at 37°C for 1 hour. Wash the cells three times with 0.1 M PBST for 3 minutes each time and pat dry. Add 100 ul / well of hybridoma cell supernatant and use 1:1000 dilution of No. 3 mouse eyeball blood as a positive control. Add 100 ul to each well and incubate at 37°C for 1 hour. Wash the cells three times with 0.1 M PBST for 3 minutes each time and pat dry. Use HRP-labeled secondary antibodies (total IgG / IgG1 / IgG2a / IgG2b / IgG3 / IgM / Kappa chain / lambda chain), antibody diluted 1:5000 (manufacturer: Jackson), incubated at 37°C for 30 min; washed 3 times with 0.1M PBST, 3 min each time, patted dry; added TMB for 5 min, stopped with 2M H2SO4, and measured absorbance at 450 nm. The experimental results are shown in Figure 2. Figure 3 As shown, the heavy chain of the 3F5 monoclonal antibody is IgG1, and the light chain is Ig kappa chain.

[0058] Example 2 Identification of anti-Ri monoclonal antibody sequences

[0059] The 3F5 hybridoma cells screened in Example 1 were cultured and expanded. The cells were lysed with Trizol, and total RNA was extracted from the hybridoma cell lysate (Quick-RNA MicroPrep Kit). RNA was separated by agarose gel electrophoresis and then subjected to 5' RACE to obtain cDNA (Clontech SMARTer RACE 5' / 3' kit), which was used as a template for PCR amplification ( Max DNA Polymerase) was used to extract the antibody variable region cDNA, which was then sequenced by a sequencing company to obtain the amino acid sequence of the heavy chain variable region and the light chain variable region of the 3F5 monoclonal antibody. The Kabat method was used to mark the complementary determining region sequence of the heavy chain variable region and the complementary determining region sequence of the light chain variable region of the 3F5 monoclonal antibody. The relevant sequences are shown in Table 1.

[0060] Table 1

[0061]

[0062]

[0063] Table 2

[0064]

[0065]

[0066] Example 3 Preparation of anti-Ri human-mouse chimeric antibodies

[0067] The sequenced monoclonal antibody 3F5 heavy chain variable region and human IgG1 heavy chain constant region sequence SEQ ID NO.11 were ligated into the vector pcDNA3.1 and labeled as pcDNA3.1-Ri-VH-3F5. The 3F5 light chain variable region and human IgG1 light chain constant region sequence SEQ ID NO.12 were ligated into the vector pcDNA3.1 and labeled as pcDNA3.1-Ri-VL-3F5. The ligated recombinant plasmids were sent to Sangon Biotechnology for sequencing. The sequenced recombinant plasmids were then extracted and used for cell transfection to produce antibodies.

[0068] The reconstructed pcDNA3.1-Ri-VH-3F5 plasmid and pcDNA3.1-Ri-VL-3F5 plasmid were co-transfected into Expi293F cells for transient expression (transfection reagent PEI, thermo) to obtain cells transfected with the monoclonal antibody 3F5 plasmid. The supernatant was collected 3 days after transfection, purified using protein A filler, and labeled as Ri-3F5. The Ri-3F5 protein concentration was determined to be 0.1 mg / mL using the BCA method.

[0069] Example 4: Detection of the titer of anti-Ri human-mouse chimeric antibodies

[0070] 1. ELISA verification of the function of anti-Ri human-mouse chimeric antibody

[0071] The purified Nova-1 protein and Nova-2 protein were coated as detection antigens using carbonate buffer with a pH of 9.6, with a concentration of 100 ng / well, and coated overnight at 4°C (the coating solution was 25 mL carbonate buffer, pH 9.6); the next day, the wells were washed three times with PBST solution for 3 minutes each time, and patted dry each time; 2% BSA was added to the wells for blocking, and the wells were incubated at 37°C for 1 hour; after incubation, the wells were washed three times with PBST for 3 minutes each time, and the excess water was removed each time; the Ri-3F5 human leukocyte antigen prepared in Example 3 was incubated. Mouse chimeric antibody and eyeball blood of mouse No. 3 were used as positive controls, and the dilution ratios were 1:1, 1:3, 1:9, 1:27, 1:81, 1:243, 1:729, and 1:2187, respectively. 100 μL of the above dilutions were added to each well, incubated at 37°C for 1 hour, washed 3 times with PBST, each time for 3 minutes, and patted dry each time; goat anti-human / goat anti-mouse IgG-HRP secondary antibody was diluted 1:5000 and added to the plate, incubated at 37°C for 30 minutes; after incubation, the plate was washed 3 times with PBST, patted dry, TMB was added for color development for 3 minutes, 2M H2SO4 was used for termination, and the absorbance was measured at 450 nm. The experimental results are shown in Figure 4a and Figure 4b , 3F5 monoclonal antibody can specifically recognize Nova-1 and Nova-2 antigens, and when the antigen coating amount is 100 ng / well, the titer of Ri-3F5 human-mouse chimeric antibody can reach ≥1:2187.

[0072] 2. Dot Blot Verification of the Function of Anti-Ri Human-Mouse Chimeric Antibodies

[0073] Anti-human Fab antibody (manufacturer: Sigma) was diluted to a concentration of 100 ng / μL as a quality control point (Ctrl). Nova-1 protein, Nova-2 protein and Yo control protein were diluted to a concentration of 40 ng / μL as test proteins. 0.8 μL of sample was taken from each sample using a micropipette and bound to the NC membrane in the shape of a right-angled trapezoid in a circular imprint pattern, distributed as follows: Figure 5As shown in the annotation, dry at 37°C for 30 minutes, place in a 24-well plate, and incubate with 0.1 mg / mL Ri-3F5 human-mouse chimeric antibody purified in Example 4 (1:200 dilution), Ri antibody-positive (i.e., Nova-1 antibody and Nova-2 antibody are both positive) patient serum (1:10 dilution), Nova-1 antibody-positive patient serum (1:10 dilution), Nova-2 antibody-positive patient serum (1:10 dilution), No. 3 immune mouse eyeball blood (1:200 dilution), and 0.5 mg / mL Nova-1 commercial antibody (1:1000 dilution, manufacturer: thermo) for 300 minutes. ul / well, incubate on a shaker at room temperature for 30 minutes, wash with 1% NaCl, 4 minutes / time, for a total of 2 washes; dilute AP-labeled goat anti-human / mouse / rabbit IgG secondary antibody 2000 times (manufacturer: Jackson) and add it to the corresponding wells respectively, incubate on a shaker at room temperature for 30 minutes, wash with 1% NaCl, 4 minutes / time, for a total of 2 washes; after removing the washing solution in the well, add 50-fold diluted BCIP / NBT substrate, 300μL / well, stand at room temperature in the dark for 4 minutes, discard the substrate dilution in the well, rinse the test strip with distilled water 1-2 times, take it out to dry or dry it at 37℃-45℃, observe the color development, and the results are as follows Figure 5 shown.

[0074] The results indicate that the modified Ri-3F5 chimeric antibody and Ri-positive patient serum, when incubated with the same antigens and the same AP-labeled goat anti-human IgG secondary antibody, both effectively recognized Nova-1 and Nova-2 antigens and formed visible stains. However, the serum from immune mouse No. 3 and the commercial Nova-1 antibody required incubation with AP-labeled goat anti-mouse / rabbit IgG secondary antibodies to form visible stains. Furthermore, the serum from immune mouse No. 3 did not recognize anti-human Fab antibodies, failing to meet the quality control requirements. While the commercial Nova-1 antibody recognized anti-human Fab antibodies, the recognition was nonspecific. Due to the different species of incubated secondary antibodies and the poor recognition of quality control points, the serum from immune mouse No. 3 and the commercial Nova-1 antibody could not meet the requirements for patient serum replacement. However, the modified Ri-3F5 chimeric antibody could serve as a replacement for positive serum, meeting the requirements for reference materials in the diagnostic kit described herein.

[0075] 3. Immunofluorescence Verification of the Function of Anti-Ri Human-mouse Chimeric Antibodies

[0076] By PCR or artificial synthesis, the human Nova-1 gene sequence and the human Nova-2 gene sequence were connected to pCDNA3.1 by molecular cloning to obtain the corresponding recombinant vectors. After the constructed recombinant vectors were sequenced correctly, they were extracted for use. 293T cells were cultured in 10% FBS-DMEM high glucose medium at 37°C and 5% CO2 in a cell culture incubator with a 6cm×6cm slide on the bottom of the culture dish. When the cell density reached 30% to 40%, PEI transfection reagent (manufacturer: thermo, catalog number: BMS1003) was used to transfect pcDNA3.1. A3.1-Nova-1 recombinant vector, pcDNA3.1-Nova-2 recombinant vector and empty pCDNA3.1 were transfected into 293T cells and labeled respectively; cells grown for 48 h after transfection were washed twice with PBS, fixed with acetone for 5 min, washed twice with PBS, and dried at 45°C for 30 min. The cell slides were cut into 2.5 mm × 2.5 mm pieces and affixed to glass slides to prepare Nova-1 overexpressing cell slides, Nova-2 overexpressing cell slides and pCDNA3.1 control slides for use as detection chips.

[0077] The anti-Ri-3F5 human-mouse chimeric antibody purified in Example 3 was diluted 1:100 using PBST, and the serum of the Ri-positive patient was diluted 1:10. The cells were incubated with Nova-1-overexpressing cell slides, Nova-2-overexpressing cell slides, and pCDNA3.1 control slides, respectively. The cells were incubated at room temperature for 1 hour, washed three times with PBST, each for 5 minutes. Alexa Fluor 594-labeled goat anti-human IgG (manufacturer: Jackson) was added at a dilution of 1:200, incubated at room temperature for 30 minutes, and washed three times with PBST, each for 5 minutes. The cells were observed under a microscope. The results were as follows: Figure 6a and Figure 6b As shown, from Figure 6a The results show that the modified Ri-3F5 human-mouse chimeric antibody and Ri patient serum can produce obvious fluorescent signals with both Nova-1 overexpressing cell slides and Nova-2 overexpressing cell slides under the same incubation conditions. In addition, compared with positive serum, the fluorescent cell morphology produced by the modified Ri-3F5 human-mouse chimeric antibody is a typical Nova-1 positive signal and a typical Nova-2 positive signal. Figure 6bAs shown, the Ri-3F5 human-mouse chimeric antibody can recognize both overexpressed Nova-1 and overexpressed Nova-2 proteins, demonstrating its significant humanized species advantage and its potential as a serum replacement to meet long-term testing reagent needs. This monoclonal antibody can also aid in the processing of indirect immunofluorescence images using computer artificial intelligence technology. Using this monoclonal antibody in various experiments not only increases the number of positive images but also adds more typical positive cell morphologies, making indirect immunofluorescence results interpreted by computer artificial intelligence technology more accurate.

[0078] 4. Western blotting to verify the function of anti-Ri human-mouse chimeric antibody

[0079] One dish of 293T cells overexpressing Nova-1, one dish of 293T cells overexpressing Nova-2, and one dish of empty pCDNA3.1 cells were collected, lysed using RIPA lysis buffer, centrifuged, and the supernatants were labeled as Nova-1-pCDNA3.1, Nova-2-pCDNA3.1, and pCDNA3.1. The protein concentrations were determined using the BCA method. The whole cell lysis solutions of the prokaryotic Nova-1 and Nova-2 constructed in Example 1 before and after induction were then taken, and the above four proteins were subjected to gel electrophoresis. After the electrophoresis, the membrane was transferred at 300 mA. Wet transfer was performed for 90 minutes; 5% skim milk powder was blocked at room temperature for 1 hour; Ri-3F5 human-mouse chimeric antibody (concentration of 0.1 mg / mL) and commercial Nova-2 antibody (manufacturer: proteintech, concentration of 0.6 mg / mL) were incubated on the transferred PVDF membrane respectively at a dilution of 1:1000, and incubated overnight at 4°C; the next day, TBST was washed 3 times, 5 minutes each time; HRP-labeled goat anti-human / goat anti-rabbit secondary antibody (manufacturer: Jackson) was added and incubated at room temperature for 1 hour; TBST was washed 3 times, 5 minutes each time; ECL chemiluminescence solution was added for color development and photography was performed. The results are shown as follows Figure 7 shown.

[0080] The results show that the modified Ri-3F5 human-mouse chimeric antibody can recognize both Nova-1 and Nova-2 proteins in overexpressed cells, and can also recognize Nova-1 and Nova-2 proteins after prokaryotic induced expression. Compared with commercial antibodies, it has obvious humanized species advantages and can be used as a serum substitute to meet the needs of long-term detection reagents.

[0081] The monoclonal antibody of the present invention can be used for both quantification of protein concentration and determination of antibody titer, and the incubation conditions in the immunoblotting method are exactly the same as those of the sample to be tested, thereby solving the technical problem that current mouse monoclonal / polyclonal antibodies cannot be used as reference products in the diagnostic kit.

[0082] The above content is only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. An anti-Ri monoclonal antibody, characterized in that Designated as humanized monoclonal antibody 3F5, it includes a light chain and a heavy chain; The heavy chain variable region includes three heavy chain complementary determining regions CDR1, CDR2 and CDR3, the amino acid sequence of the heavy chain complementary determining region CDR1 is shown in SEQ ID NO.1, the amino acid sequence of the heavy chain complementary determining region CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain complementary determining region CDR3 is shown in SEQ ID NO.3; The variable region of the light chain includes three light chain complementary determining regions CDR1, CDR2 and CDR3. The amino acid sequence of the light chain complementary determining region CDR1 is shown in SEQ ID NO.4, the amino acid sequence of the light chain complementary determining region CDR2 is shown in SEQ ID NO.5, and the amino acid sequence of the light chain complementary determining region CDR3 is shown in SEQ ID NO.

6.

2. The anti-Ri monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the humanized monoclonal antibody 3F5 is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.8; the nucleotide sequence encoding the heavy chain variable region of the humanized monoclonal antibody 3F5 is shown in SEQ ID NO.9; and the nucleotide sequence encoding the light chain variable region of the humanized monoclonal antibody 3F5 is shown in SEQ ID NO.

10.

3. A carrier, characterized in that The vector comprises a polynucleotide encoding the anti-Ri monoclonal antibody according to claim 1 .

4. A host cell, characterized in that The host cell comprises the vector according to claim 3.

5. Use of the anti-Ri monoclonal antibody according to any one of claims 1 to 2, the vector according to claim 3, or the host cell according to claim 4 in the preparation of a reagent / kit for detecting Ri protein.

6. Use of the anti-Ri monoclonal antibody according to any one of claims 1 to 2, the vector according to claim 3, or the host cell according to claim 4 in the preparation of a reagent / kit for the diagnosis and / or prognosis of human paraneoplastic movement disorders.

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