A chimeric antibody against NMDAR1 and its application and detection method based thereon

Abstract

The present invention discloses an anti-NMDAR1 chimeric antibody and its application and a detection method based thereon, wherein the monoclonal antibody has high specificity and good affinity. The antibody can be used for other scientific research such as NMDAR1 antigen detection, and can also meet the demand for positive quality control products of anti-NMDAR1 autoantibody detection kits, is easy to store and transform, and is easy to control the quality during the antibody production process, and has very good application value and very important scientific research guiding significance.

Description

Technical Field

[0001] The invention belongs to the technical field of monoclonal antibody preparation, relates to an anti-NMDAR1 chimeric antibody, and specifically relates to an anti-NMDAR1 chimeric antibody and an application thereof and a detection method based thereon. Background Art

[0002] N-methyl-D-aspartate receptor (NMDAR) is an ion channel protein distributed on the surface of the postsynaptic membrane of nerve cells. It is one of the subtypes of ionotropic glutamate receptors and is composed of NR1, NR2 and NR3 subunits. In functionally active NMDAR, its four subunits should have at least one NR1 and one NR2, while the vast majority of NMDARs are composed of two NR1s and two NR2s, and a small number are composed of three subunits. Autoimmune encephalitis (AE) refers to a type of encephalitis mediated by autoimmune mechanisms, among which anti-NMDAR encephalitis is the most common, accounting for about 54% to 80% of AE cases, followed by anti-leucine-rich glioma-inactivated protein 1 (LGI1) antibody-related encephalitis and anti-γ-amino butyric acid type B receptor (GABABR) antibody-related encephalitis. Anti-NMDAR encephalitis patients mainly have specific IgG antibodies against the NR1 subunit of NMDAR. These antibodies are pathogenic and can induce the cross-linking and internalization of NMDA receptors, block NMDAR-dependent synaptic electrical activity, and cause clinical manifestations such as epilepsy, mental and behavioral abnormalities, and impaired consciousness.

[0003] According to the "Expert Consensus on Autoimmune Encephalitis" (2022 edition), an important clinical diagnosis criterion for anti-NMDAR encephalitis is positive anti-NMDAR antibodies. There are already some detection kits for anti-NMDAR antibodies on the market, but because AE is a rare disease, there are no national or industry standards for such antibody detection kits, so there is no gold standard for detection. Most companies often use positive and negative serum / cerebrospinal fluid to establish their own corporate reference products during product performance verification and quality control. However, due to the complex composition of serum / cerebrospinal fluid, it is not easy to obtain, and the number of positive samples is small, which cannot meet the needs of early research and long-term quality control of later products. In addition, due to the lack of quantitative gold standard methods and marketed products, it is also impossible to quantify the content of antibodies in serum / cerebrospinal fluid.

[0004] Chimeric antibodies are antibodies expressed by inserting the light chain and heavy chain variable region (V region) genes of heterologous monoclonal antibodies (such as mouse monoclonal antibodies) into expression vectors containing human antibody constant regions (C regions) through DNA recombination technology, and then transforming mammalian cells to express the antibodies. The antibodies produced in this way reduce the immunogenicity of heterologous antibodies while retaining the ability of the parent antibody to specifically bind to antigens. If this technology is applied to the preparation of anti-NMDAR1 human-mouse chimeric antibodies, the chimeric antibodies can replace positive serum / cerebrospinal fluid as corporate reference products to a certain extent, and are superior to serum / cerebrospinal fluid samples in protein concentration quantification and antibody titer detection, thereby achieving performance verification and quality control of anti-NMDAR1 autoantibody detection kits. Summary of the invention

[0005] In order to overcome the shortcomings of the above-mentioned prior art, the purpose of the present invention is to provide an anti-NMDAR1 chimeric antibody and its application and a detection method based thereon, so as to solve the problem that the prior art method cannot perform quantitative and qualitative analysis due to insufficient reference positive serum. At the same time, the anti-NMDAR1 chimeric antibody of the present invention has good species specificity compared with the commercially available NMDAR1 mouse or rabbit monoclonal antibody / polyclonal antibody.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention discloses a group of monoclonal antibodies to NMDAR1 protein, including light chain and heavy chain, wherein:

[0008] 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.2, the amino acid sequence of the light chain complementary determining region CDR2 is shown in SEQ ID NO.3, and the amino acid sequence of the light chain complementary determining region CDR3 is shown in SEQ ID NO.4;

[0009] The variable region of the heavy chain 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.6, the amino acid sequence of the heavy chain complementary determining region CDR2 is shown in SEQ ID NO.7, and the amino acid sequence of the heavy chain complementary determining region CDR3 is shown in SEQ ID NO.8.

[0010] Preferably, the amino acid sequence of the light chain variable region of the monoclonal antibody is shown as SEQ ID NO.1, and the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO.5.

[0011] Further preferably, the nucleotide sequence encoding the light chain variable region of the monoclonal antibody to the NMDAR1 protein is shown as SEQ ID NO.9; the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody to the NMDAR1 protein is shown as SEQ ID NO.10.

[0012] Preferably, the monoclonal antibody comprises a light chain having a complementarity determining region of an amino acid sequence that is at least 80% 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 NMDAR1 protein; the monoclonal antibody comprises a heavy chain having a complementarity determining region of an amino acid sequence that is at least 80% 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 NMDAR1 protein.

[0013] Further preferably, the monoclonal antibody further comprises a light chain having an amino acid sequence at least 80%, 85%, 90%, 95%, 99% identical to the sequence of the variable region of the light chain; the monoclonal antibody comprises a heavy chain having an amino acid sequence at least 80%, 85%, 90%, 95%, 99% identical to the sequence of the variable region of the heavy chain, and the sequence of the variable region of the light chain and the sequence of the variable region of the heavy chain maintain the ability to bind to the NMDAR1 protein. Or it is a conservative variant obtained by conservative mutation of the above antibody sequence by adding, deleting, replacing, or modifying one or more amino acids.

[0014] The present invention also provides a recombinant vector for expressing the monoclonal antibody, wherein the vector comprises a nucleotide sequence encoding the NMDAR1 monoclonal antibody.

[0015] The present invention also provides a recombinant cell expressing the above monoclonal antibody, wherein the recombinant cell comprises the above recombinant vector.

[0016] Preferably, the base cell of the recombinant cell comprises a eukaryotic cell.

[0017] The present invention also provides a product, which comprises the monoclonal antibody of the present invention, the nucleic acid molecule of the present invention, the vector of the present invention and / or the host cell of the present invention.

[0018] The present invention also provides a method for detecting NMDAR1 protein or polypeptide, which comprises contacting a biological sample with the above monoclonal antibody, thereby detecting the NMDAR1 protein or polypeptide in the sample.

[0019] Preferably, the biological sample comprises plasma, whole blood, serum, tissue, cells or lysate of tissue and cells containing NMDAR1 protein / polypeptide.

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

[0021] The present invention provides a human-mouse chimeric monoclonal antibody against NMDAR1 with high specificity and good affinity. Compared with the commercial antibodies in the prior art, the modified human-mouse chimeric antibody disclosed in the present invention has a more obvious humanized species advantage and more accurately serves as a positive reference product; compared with the positive patient serum / cerebrospinal fluid commonly used in the prior art, the modified human-mouse chimeric antibody can be used for accurate protein quantification and antibody titer determination, and can meet long-term product quality control. Therefore, the antibody can be used for other scientific research such as NMDAR1 antigen detection, and can also meet the needs of anti-NMDAR1 autoantibody detection kits for positive quality control products, is easy to store and modify, and is easy to control the quality during the antibody production process, and has very good application value and very important scientific research guidance significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the result of NMDAR1-6his protein purification;

[0023] Figure 2 The results are the determination results of the binding titer of serum antibodies from three immunized mice to NMDAR1-6his protein;

[0024] Figure 3 The immunofluorescence results of serum antibodies from three immunized mice and NMDAR1-6his overexpressing cell slides;

[0025] Figure 4 This is the typing and identification result of 6A7 mouse monoclonal antibody;

[0026] Figure 5 This is the purification result of NMDAR1-6A7 human-mouse chimeric antibody;

[0027] Figure 6 The titer results of anti-NMDAR1-6A7 human-mouse chimeric antibody were verified by ELISA;

[0028] Figure 7a To verify the function of anti-NMDAR1-6A7 human-mouse chimeric antibody by immunofluorescence assay;

[0029] Figure 7b The magnified image shows typical positive cell morphology. DETAILED DESCRIPTION

[0030] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme 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 described embodiments 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 creative work should fall within the scope of protection of the present invention.

[0031] It should be noted that the terms "first", "second", etc. in the specification 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 data 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 that are clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0032] The present invention is further described in detail below in conjunction with the accompanying drawings: Unless otherwise noted, the scientific and technical terms used in the present invention should have the meanings generally understood by ordinary technicians in the field.

[0033] The term "antibody" as used herein refers to immunoglobulin molecules and the immunologically active portion of immunoglobulin molecules, i.e., molecules containing an antigen binding site that immunologically binds to an antigen. It includes, but is not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, human-mouse chimeric antibodies, single-chain, F ab 、F ab’ and F (ab’)2 Fragments, single-chain FV fragments and F ab Expression library.

[0034] "Monoclonal antibody" refers to a population of antibodies from a single species that contains a unique light chain gene product and a unique heavy chain gene product. Specifically, it means that the complementarity determining regions of monoclonal antibodies are the same in all molecules in the population and can bind to the antigen binding site of a specific epitope of an antigen.

[0035] "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) region of the heavy (H) and light (L) chains.

[0036] "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.

[0037] The term "chimeric antibody" means 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 in which the constant region sequences (CH and CL) are derived from humans. The sequence of the variable region is preferably derived from, for example, an animal species that allows easy preparation of hybridomas, such as mice, rats, rabbits, etc.

[0038] Example 1 Preparation of anti-NMDAR1 monoclonal antibody hybridoma cells

[0039] 1. Preparation of Recombinant NMDAR1 Protein

[0040] Referring to the amino acid sequence and structural feature annotation of the accession number Q05586 in the Uniprot database, the cDNA sequence encoding the amino acids 201-549 of the extracellular region of human NMDAR1 was synthesized by Jinweizhi Company based on the sequence numbered NM_007327.4 in GenBank, and a 6his tag sequence was added to the C-terminus of the gene. The gene sequence was synthesized into the pCDNA3.1 vector to obtain the NMDAR1-6his-pCDNA3.1 recombinant vector. The constructed and correctly sequenced recombinant vector was transfected into 293 suspension cells using PEI transfection reagent (manufacturer: thermo). After culturing for 3 days, the cells and supernatant were collected by centrifugation and purified using Ni affinity chromatography. The 100mM imidazole elution sample was collected and dialyzed to 50mM Tris-HCl (pH9.5), and purified by DEAE anion exchange chromatography. 50mM NaCl was used for elution to obtain a protein with high purity. After dialysis and concentration, a high-concentration protein was obtained and labeled as NMDAR1-6his for use as an antigen. The results of protein purification and concentration are shown in Figure 1 As shown. Figure 1 As can be seen, NMDAR1-6his antigen was successfully expressed and purified.

[0041] 2. Antigen Immunization of Mice

[0042] 40 μg of purified NMDAR1-6his recombinant protein in step 1 was used as an immunogen to immunize three Balb / c mice. The antigen was mixed with complete Freund's adjuvant in an equal volume at a ratio of 1:1 and fully ground into a water-in-oil milky liquid. The injection volume for each mouse was 100 μL of a mixture of antigen and Freund's adjuvant (antigen volume was 40 μg / mouse), and the injection site was the thigh. The second immunization was performed 14 days after the first immunization. During the second immunization, the antigen was mixed with an equal volume of incomplete Freund's adjuvant. The injection dose and process were the same as the first immunization. The third immunization was performed 14 days after the second immunization. During the third immunization, the dose, method, and route of the injected antigen were exactly the same as those of the second immunization.

[0043] 3. Detection of antibody production in immunized mice

[0044] 3.1 Determination of antibody titer in sera of immunized mice: Blood was collected from the tail of mice 3 days after the third immunization, and the serum was collected by centrifugation. The titer was tested by ELISA. The specific operation was as follows: the purified NMDAR1-6his protein was coated as antigen with a concentration of 100 ng / well using carbonate buffer at pH 9.6, and coated overnight at 4°C; the next day, the cells were washed 3 times with 0.1 M PBST, each time for 3 min, and patted dry; blocking protein (2% BSA solution) was added, 200 μL / well, and incubated at 37°C for 1 h; 0.1 M Wash 3 times with PBST, 3 min each time, and pat dry; dilute the sera of 3 immunized mice and one normal control mouse with PBS in multiple ratios, at 1:100, 1:300, 1:900, 1:2700, 1:6300, 1:18900, 1:56700, and 1:170100, add 100 μL to each well, and incubate at 37°C for 1 h; wash 3 times with 0.1 M PBST, 3 min each time, and pat dry; use goat anti-mouse IgG-HRP 1:5000 dilution (manufacturer: Jackson), incubate at 37°C for 1 h; wash 3 times with 0.1 M PBST, 3 min each time, and pat dry; add TMB for color development for 2 min, and use 2 M H 2 SO 4 Stop color development and measure the absorbance at 450nm. The test results are as follows: Figure 2 As shown, the immune effect of mouse serum No. 1 is the best.

[0045] 3.2 Detection of antibody specificity in immune mouse serum: NMDAR1-6his-pCDNA3.1 recombinant vector and empty pCDNA3.1 were transfected into 293T adherent cells using PEI transfection reagent and cultured. After 48 hours, the cells were fixed with acetone to prepare NMDAR1 overexpression cell slides and pCDNA3.1 control slides. The collected mouse tail blood was used for immunofluorescence experiments. The specific operations were as follows: Mouse tail blood was diluted 1:100 with PBST and incubated on NMDAR1 overexpression cell slides and pCDNA3.1 control cell slides. The cells were incubated at room temperature for 1 hour, washed 3 times with PBST for 5 minutes each time, and AlexaFluor594-labeled goat anti-mouse IgG (manufacturer: Jackson) diluted 1:200 was added. The cells were incubated at room temperature for 30 minutes, washed 3 times with PBST for 5 minutes each time. The results were observed under a microscope. Figure 3 As shown, from Figure 3 It can be seen that the fluorescence signal of the serum of rat No. 1 is the strongest and has the best specificity with the overexpressed NMDAR1-6his protein. Based on the titer and specificity data of serum antibodies, rat No. 1 was selected for subsequent experiments.

[0046] 4. Cell fusion and hybridoma cell screening

[0047] 4.1 Preparation of feeder cells and myeloma cells: Take macrophages from the ascites of ordinary mice as feeder cells for culture. Take feeder cells from the ascites of 4-6 mice and plate them on 10 96-well plates. Prepare hybridoma cells after 2 days of culture. Collect myeloma cells SP2 / 0 with good growth and a cell density of 90% in the logarithmic growth phase, a total of 10 dishes. Centrifuge and discard the supernatant, resuspend the cells in DMEM medium and count them.

[0048] 4.2 Preparation of spleen cells: Mouse No. 1 was boosted with intraperitoneal injection of antigen. Three days later, blood was collected from the mouse tail to separate the serum, which was rinsed with running water and placed in 75% ethanol solution for 10 minutes. The mouse spleen was aseptically removed and placed in a 200-mesh disposable sterile filter. The spleen was cut into pieces with sterile surgical scissors and ground with the piston handle of a 50mL sterile syringe to obtain a single spleen cell suspension.

[0049] 4.3 Cell fusion: Prepare a 37℃ water bath in the clean bench and preheat it in advance. Add the SP2 / 0 cells and spleen cells with a cell density of 90% in advance to a 50mL centrifuge tube at a ratio of 1:10 and mix well. Centrifuge at 500g for 10min, aspirate the supernatant, flick the bottom of the centrifuge tube to loosen the cell pellet slightly; slowly drip 1mL of 45% PEG1450 solution preheated to 37℃ within 90s; and shake the centrifuge tube gently continuously; the centrifuge tube is in a 37℃ water bath during the whole process. Then add DMEM culture medium to the cell mixture at a uniform speed, drip 1mL dropwise in the first minute, add 2mL in the second minute, add 3mL in the third minute, add 4mL in the fourth minute, add 5mL in the fifth minute, and shake while adding in a 37℃ water bath. Then incubate at 37℃ for 15min, centrifuge at 500g for 5min, and remove the supernatant. Add 5 mL of DMEM medium containing HAT, gently suspend the precipitated cells, and finally add DMED medium containing HAT to about 100 mL. Dispense into a 96-well cell culture plate with feeder cells, 100 μL / well, and then place the culture plate at 37°C and 5% CO 2 Culture in an incubator. On the second day after fusion, add 100 μL of HAT culture medium to each well, and then change half of the HAT culture medium every 2-3 days. Cultivate for about 2 weeks before screening.

[0050] 4.4 Screening of positive hybridoma cells: Observe the growth of hybridoma cells. After 7-10 days, when the cell culture supernatant turns yellow, take an appropriate amount of cell supernatant for ELISA antibody detection. According to the ELISA results, select clones with high OD values, which are more than twice higher than the negative control, and spread on 96-well plates for the first subclone screening. After 7-10 days, perform ELISA to detect antibodies again, select clones with high OD values, spread on 96-well plates, and perform the second subclone screening, so that there is about 1 cell per well; after 7-10 days of culture, perform ELISA to detect antibodies again, select clones with high OD values, spread on 96-well plates, and perform the third subclone screening, so that there is about 1 cell per well. Select several clones with high OD values ​​for further verification. Finally, a positive clone cell was screened out, named: 6A7 hybridoma cell, and the monoclonal antibody secreted by its cells was named 6A7 mouse monoclonal antibody.

[0051] 4.5 Identification of typing of antibodies secreted by positive hybridoma cells: Re-coat the purified NMDAR1-6his protein as antigen to detect the typing of 6A7 mouse monoclonal antibody, with an antigen coating concentration of 100 ng / well, and coat overnight at 4°C; the next day, wash 3 times with 0.1M PBST, 3 min each time, and pat dry; add blocking protein (2% BSA solution), 200 μL / well, incubate at 37°C for 1 h; wash 3 times with 0.1M PBST, 3 min each time, and pat dry; add 100 μL / well of monoclonal 6A7 cell supernatant, and use mouse tail blood No. 1 diluted 1:1000 as a positive control, add 100 μL to each well, incubate at 37°C for 1 h; wash 3 times with 0.1M PBST, 3 min each time, and pat dry; use HRP-labeled secondary antibodies (total IgG / IgG1 / IgG2a / IgG2b / IgG3 / IgM / Ig Kappachain / Ig lambda chain), antibody diluted 1:5000 (manufacturer: Jackson), incubated at 37°C for 30 min; washed 3 times with 0.1 M BST, 3 min each time, patted dry; TMB was added for 5 min, and 2 M H 2 SO 4 Stop color development and measure absorbance at 450nm. Figure 4 As shown, the heavy chain of 6A7 mouse monoclonal antibody is IgG1, and the light chain is Ig Kappa chain. Then, 6A7 mouse monoclonal antibody was purified from the culture supernatant of positive hybridoma cells as a control, and the protein concentration was 0.91 mg / mL measured by BCA method. Subsequent experiments were continued with 6A7 hybridoma cells.

[0052] Example 2 Identification of anti-NMDAR1 monoclonal antibody sequences

[0053] The 6A7 hybridoma cells screened in Example 1 were cultured and 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 5'RACE was performed 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 6A7 mouse monoclonal antibody. The CDR region of the amino acid sequence of the 6A7 mouse monoclonal antibody was marked using the Kabat method. SEQ ID NO.9 is the nucleotide sequence of the light chain variable region of the 6A7 mouse monoclonal antibody, and SEQ ID NO.10 is the nucleotide sequence of the heavy chain variable region of the 6A7 mouse monoclonal antibody.

[0054] Example 3 Construction of anti-NMDAR1 monoclonal antibody sequence recombinant vector

[0055] The nucleotide sequence of the light chain variable region of the 6A7 mouse monoclonal antibody determined by sequencing (SEQ ID NO.9) was amplified and connected to the vector pcDNA3.1 with the human IgG1 light chain constant region sequence SEQ ID NO.11, marked as pcDNA3.1-NMDAR1-VL. The nucleotide sequence of the heavy chain variable region of the 6A7 mouse monoclonal antibody (SEQ ID NO.10) was amplified and connected to the vector pcDNA3.1 with the human IgG1 heavy chain constant region sequence SEQ ID NO.12, marked as pcDNA3.1-NMDAR1-VH. The connected recombinant plasmid was sent to Sangon Biotechnology for sequencing, and the sequenced recombinant plasmid was extracted and used for cell transfection to prepare antibodies.

[0056] The related sequences described in Example 2 and Example 3 are shown in Table 1 below.

[0057] Table 1

[0058]

[0059]

[0060]

[0061] Example 4 Preparation of anti-NMDAR1 human-mouse chimeric antibody

[0062] The pcDNA3.1-NMDAR1-VL and pcDNA3.1-NMDAR1-VH recombinant vectors were co-transfected into Expi 293F cells for transient expression (transfection reagent PEI, thermo) to prepare anti-NMDAR1 human-mouse chimeric antibodies. The supernatant was collected 3 days after transfection and purified using protein A filler. The protein concentration was determined using a BCA kit. The concentration of NMDAR1-6A7 human-mouse chimeric antibody was 0.63 mg / mL. The antibody purification results are shown in Figure 5 shown.

[0063] Example 5 Application of anti-NMDAR1 human-mouse chimeric antibody

[0064] 1. ELISA to verify the function of anti-NMDAR1 human-mouse chimeric antibody

[0065] The purified NMDAR1-6his protein was used to coat the ELISA plate, 100 ng / well, overnight at 4°C (the coating solution was 25 mL carbonate buffer, pH 9.6); the next day, the plate was washed with PBST solution for 3 times, 3 min each time, and patted dry each time; 2% BSA was added to the wells for blocking, and the plate was incubated at 37°C for 1 hour; after the incubation, the plate was washed with PBST for 3 times, 3 min each time, and the plate was patted to remove excess water each time; the NMDAR1-6A7 human-mouse chimeric antibody prepared in Example 4 and the 6A7 mouse monoclonal antibody prepared in Example 1 were incubated separately as As a positive control, normal control mouse serum was used as a negative control. The dilution ratios were 1:3, 1:9, 1:27, 1:81, 1:243, 1:729, 1:2187, and 1:6561, respectively. 100 μL of the above dilutions were added to each well, incubated at 37°C for 1 hour, washed 3 times with PBST, 3 minutes each time, 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, washed 3 times with PBST, patted dry, TMB was added for color development for 3 minutes, and 2M H 2 SO 4 Stop and measure the absorbance at 450 nm.

[0066] The experimental results are as follows Figure 6 The modified NMDAR1-6A7 human-mouse chimeric antibody can specifically recognize the NMDAR1 antigen, and the activity of the modified NMDAR1-6A7 human-mouse chimeric antibody is basically consistent with that of the 6A7 mouse monoclonal antibody before modification.

[0067] 2. Verification of the function of anti-NMDAR1 human-mouse chimeric antibody by indirect immunofluorescence assay

[0068] The anti-NMDAR1-6A7 human-mouse chimeric antibody (adjusted to 0.5 mg / mL) purified in Example 4 and the commercial NMDAR1 antibody (manufacturer: Abcam, adjusted to 0.5 mg / mL) were diluted at a ratio of 1:200 using PBST, and the serum of NMDAR1-positive patient 1 was diluted 1:10, and the cerebrospinal fluid of positive patient 2 was diluted 1:1, and incubated with the NMDAR1-6his overexpressing cell slides and pCDNA3.1 control slides prepared in Example 1, respectively, incubated at room temperature for 1 hour, washed 3 times with PBST, each time for 5 minutes, added with 1:200 diluted Alexa Fluor 594-labeled goat anti-human / goat anti-mouse IgG (manufacturer: Jackson), incubated at room temperature for 30 minutes, and washed 3 times with PBST, each time for 5 minutes; observed under a microscope, according to Figure 7a The results showed that the modified NMDAR1-6A7 human-mouse chimeric antibody was incubated with NMDAR1 patient serum / cerebrospinal fluid under the same incubation conditions (the labeled secondary antibodies used were all goat anti-human IgG). The fluorescence signal intensity of the NMDAR1-6A7 human-mouse chimeric antibody at a dilution of 1:200 was basically consistent with that of the serum with a titer of 100 at a dilution of 1:10, or the cerebrospinal fluid sample with a titer of 10 at a dilution of 1:1. The typical morphology of positive cells was consistent (showing branched polymorphism, see Figure 7b As shown in the figure, it can be used as a substitute for positive serum / cerebrospinal fluid to meet the needs of diagnostic kits. Compared with commercial antibodies, the modified human-mouse chimeric antibody has a more obvious humanized species advantage and can more accurately serve as a positive reference; compared with positive patient serum / cerebrospinal fluid, the modified human-mouse chimeric antibody can accurately quantify protein and determine antibody titer, which can meet long-term product quality control. This monoclonal antibody can also help computer artificial intelligence technology to process indirect immunofluorescence images. Using this monoclonal antibody for a variety of experiments can not only enrich the number of positive images, but also supplement more typical positive cell morphologies, making the indirect immunofluorescence results interpreted by computer artificial intelligence technology more accurate.

[0069] The above contents are 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-NMDAR1 chimeric antibody, characterized in that Includes light and heavy chains; 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.2, the amino acid sequence of the light chain complementary determining region CDR2 is shown in SEQ ID NO.3, and the amino acid sequence of the light chain complementary determining region CDR3 is shown in SEQ ID NO.4; The variable region of the heavy chain 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.6, the amino acid sequence of the heavy chain complementary determining region CDR2 is shown in SEQ ID NO.7, and the amino acid sequence of the heavy chain complementary determining region CDR3 is shown in SEQ ID NO.

8.

2. The anti-NMDAR1 chimeric antibody according to claim 1, characterized in that The amino acid sequence of the light chain variable region of the anti-NMDAR1 chimeric antibody is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain variable region of the anti-NMDAR1 chimeric antibody is shown in SEQ ID NO.

5.

3. A nucleotide encoding the anti-NMDAR1 chimeric antibody according to claim 2, characterized in that: The nucleotide sequence encoding the light chain variable region of the anti-NMDAR1 chimeric antibody is shown in SEQ ID NO.9; the nucleotide sequence encoding the heavy chain variable region of the anti-NMDAR1 chimeric antibody is shown in SEQ ID NO.

10.

4. A recombinant vector expressing the anti-NMDAR1 chimeric antibody according to claim 1 or 2, characterized in that: The recombinant vector comprises nucleotides encoding the anti-NMDAR1 chimeric antibody.

5. A recombinant cell expressing the anti-NMDAR1 chimeric antibody according to claim 1 or 2, characterized in that: The recombinant cell comprises the recombinant vector according to claim 4.

6. Use of the anti-NMDAR1 chimeric antibody according to claim 1 or 2, the nucleotide encoding the anti-NMDAR1 chimeric antibody according to claim 3, the recombinant vector according to claim 4 or the recombinant cell according to claim 5 in preparing a reagent for detecting NMDAR1 protein.

7. Use of the anti-NMDAR1 chimeric antibody according to claim 1 or 2, the nucleotide encoding the anti-NMDAR1 chimeric antibody according to claim 3, the recombinant vector according to claim 4 or the recombinant cell according to claim 5 in preparing a kit for detecting NMDAR1 protein.

8. A method for detecting NMDAR1 protein, characterized in that: The biological sample is contacted with the anti-NMDAR1 chimeric antibody according to claim 1 or 2 to detect the NMDAR1 protein in the biological sample.

9. The method for detecting NMDAR1 protein according to claim 8, characterized in that: The biological sample includes plasma, whole blood, serum, tissue, cell or tissue and cell lysate containing NMDAR1 protein.

10. A method for detecting NMDAR1 polypeptide, characterized in that: The biological sample is contacted with the anti-NMDAR1 chimeric antibody according to claim 1 or 2 to detect the NMDAR1 polypeptide in the biological sample.

11. The method for detecting NMDAR1 polypeptide according to claim 10, characterized in that: The biological sample includes plasma, whole blood, serum, tissue, cell or lysate of tissue and cell containing NMDAR1 polypeptide.

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