Anti-glycogen phosphorylase GPBB monoclonal antibody and its application

Abstract

The present invention discloses a monoclonal antibody against glycogen phosphorylase GPBB and its application. In the monoclonal antibody, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.5. The above monoclonal antibody against glycogen phosphorylase GPBB has high specificity and has no cross reaction with isozymes GPMM and GPLL. In addition, the above antibody has high affinity and a titer of 1:25600, and can be used to prepare a product for detecting glycogen phosphorylase GPBB. The detection sensitivity of the fluorescent immunochromatography detection method established by the present invention is as high as 0.156pg / mL.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a monoclonal antibody against glycogen phosphorylase GPBB and an application thereof. Background Art

[0002] Glycogen phosphorylase (GP, EC2.4.1.1) is a key enzyme in the glycogen breakdown metabolism process and an important source of energy for muscle contraction. GP catalyzes the cleavage of the non-reducing end of glycogen to generate glucose-1-phosphate and glycogen with one less glucose unit. Then glucose-1-phosphate undergoes phosphorylation to generate glucose-6-phosphate and enters the glycolysis pathway. Three main types of GP isoenzymes have been found in human tissues, and they are named GPBB (brain type), GPLL (liver type) and GPMM (muscle type) according to their dominant expression tissues. GPMM only exists in adult skeletal muscle tissue; GPLL is mainly expressed by the liver and other tissues except the heart, brain, and skeletal muscle; GPBB is the main isoenzyme in the fetal period, and is mainly expressed in the brain and heart in adulthood. Although the three isoenzymes of GP are encoded by three independent genes located on chromosomes 20, 14, and 11, the sequences are highly homologous. Amino acid sequence analysis found that GPBB has 84% ​​homology with GPMM and 82% homology with GPLL. On the one hand, because GPBB is the main isoenzyme in the fetal period, it can be used as a marker for certain digestive tract tumors, just like alpha-fetoprotein and carcinoembryonic antigen. On the other hand, GPBB is mainly expressed in the brain and heart in adulthood. In the early stage of ischemic myocardial injury or ischemic stroke, GPBB quickly diffuses into the blood, increasing the concentration of GPBB in the blood. Therefore, GPBB is an early diagnostic marker for ischemic myocardial injury and ischemic stroke. Within 4 hours after ischemic injury, its early diagnostic value is significantly better than other biochemical indicators, such as CK, CK-MB, TnT, etc.

[0003] The existing detection methods for GPBB include near-infrared fluorescence and immunological detection methods. Among them, the near-infrared fluorescence method requires special instruments such as spectrometers, which is time-consuming and cannot meet the needs of rapid screening and detection in emergency cases of sudden diseases. Immunological detection technologies such as colloidal gold and enzyme-linked immunosorbent assays have not yet been approved by the State Food and Drug Administration for clinical use. The main reason is that GPBB is highly homologous to GPMM and GPLL, and its highly specific antibodies are difficult to prepare, which seriously hinders the research and development, production and clinical application of GPBB detection reagents.

[0004] Therefore, in order to make up for the above shortcomings, the present invention is designed to compare the amino acid sequence of GPBB with GPMM and GPLL and perform bioinformatics analysis, screen the GPBB-specific amino acid segment containing the dominant antigenic epitope as an immunogen, prepare and screen the highly specific and high-affinity monoclonal antibody that only recognizes GPBB, thereby establishing a highly specific and highly sensitive GPBB detection reagent, so that it can be widely used in clinical practice, early screening and diagnosis of ischemic myocardial injury and ischemic stroke patients, and reducing the incidence of serious diseases and sequelae. Summary of the invention

[0005] To this end, the purpose of the present invention is to first screen and determine the GPBB-specific amino acid segment, analyze the GPBB-specific dominant antigenic epitope therein, and determine the GPBB-specific amino acid segment containing the dominant antigenic epitope; then synthesize a high-purity GPBB-specific dominant epitope peptide as an immunogen, immunize mice to provide a monoclonal antibody against glycogen phosphorylase GPBB prepared by using a fused hybridoma cell line, and the obtained monoclonal antibody can specifically recognize GPBB and has no cross reaction with GPMM and GPLL after experimental verification; in the third step, a large antigen fragment containing the GPBB-specific dominant epitope peptide is prokaryotically expressed as a screening antigen for monoclonal antibodies and an immunogen for preparing polyclonal antibodies, so as to obtain highly specific and high-affinity anti-GPBB monoclonal antibodies and polyclonal antibodies; finally, the highly specific and high-affinity anti-GPBB monoclonal antibodies and polyclonal antibodies are used in combination to establish a glycogen phosphorylase GPBB double antibody sandwich method detection product, which can specifically detect the content of glycogen phosphorylase GPBB in a sample without cross reaction with GPMM and GPLL, and significantly improve the clinical glycogen phosphorylase GPBB detection level.

[0006] Therefore, one aspect of the present invention relates to a monoclonal antibody or an antigen-binding fragment thereof against glycogen phosphorylase GPBB, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2 and CDR3, and the light chain variable region comprises CDR1, CDR2 and CDR3, wherein:

[0007] The amino acid sequence of the heavy chain CDR1 is the sequence shown in SEQ ID NO.2 or an amino acid sequence having one conservative amino acid substitution compared with the sequence shown in SEQ ID NO.2;

[0008] The amino acid sequence of the heavy chain CDR2 is the sequence shown in SEQ ID NO.3 or an amino acid sequence having one conservative amino acid substitution compared with the sequence shown in SEQ ID NO.3;

[0009] The amino acid sequence of the heavy chain CDR3 is the sequence shown in SEQ ID NO.4 or an amino acid sequence having one conservative amino acid substitution compared with the sequence shown in SEQ ID NO.4;

[0010] The amino acid sequence of the light chain CDR1 is the sequence shown in SEQ ID NO.6 or an amino acid sequence having one conservative amino acid substitution compared with the sequence shown in SEQ ID NO.6;

[0011] The amino acid sequence of the light chain CDR2 is LVS or an amino acid sequence having one conservative amino acid substitution compared to the sequence LVS;

[0012] The amino acid sequence of the light chain CDR3 is the sequence shown in SEQ ID NO.7 or an amino acid sequence having one conservative amino acid substitution compared with the sequence shown in SEQ ID NO.7.

[0013] In a further aspect, the present invention also relates to a monoclonal antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO.5.

[0014] The present invention also relates to the above-mentioned monoclonal antibody or its antigen-binding fragment, wherein the antibody or antigen-binding fragment is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a single-chain antibody or a humanized antibody, and these antibodies or antigen-binding fragments can recognize and bind to human glycogen phosphorylase GPBB because they retain the variable regions of the heavy chain and the light chain.

[0015] In addition, the present invention also relates to a nucleic acid molecule comprising a nucleic acid encoding the above-mentioned antibody or antigen-binding fragment thereof, and an expression vector comprising the above-mentioned nucleic acid molecule, wherein the expression vector is capable of expressing the above-mentioned antibody or antigen-binding fragment thereof. At the same time, the present invention also relates to a recombinant comprising the above-mentioned nucleic acid molecule or the above-mentioned expression vector, which can produce the above-mentioned antibody or antigen-binding fragment thereof.

[0016] On the other hand, the present invention relates to a monoclonal antibody hybridoma cell line against glycogen phosphorylase GPBB, wherein the monoclonal antibody hybridoma cell line secretes the above-mentioned monoclonal antibody. Further, the present invention relates to a monoclonal antibody hybridoma cell line against glycogen phosphorylase GPBB, wherein the monoclonal antibody hybridoma cell line is a mouse hybridoma cell line AC2118, and the deposit number is CGMCC No.45928.

[0017] In another aspect, the present invention relates to the use of the above monoclonal antibody or its antigen-binding fragment in the preparation of a product for determining glycogen phosphorylase GPBB. Further, the present invention relates to a kit for determining the content of glycogen phosphorylase GPBB, the kit comprising the above monoclonal antibody or its antigen-binding fragment for recognizing and binding to human glycogen phosphorylase GPBB.

[0018] Description of biological material deposit

[0019] The monoclonal antibody hybridoma cell line of the present invention: mouse hybridoma cell line AC2118, is deposited in the General Microbiology Center of China National Microbiological Culture Collection (CGMCC), the registration number of the collection center is CGMCC No. 45928, and the deposit date is May 22, 2024. The address of the General Microbiology Center of China National Microbiological Culture Collection is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Postal Code 100101. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is the B cell epitope distribution map of the amino acid sequence of glycogen phosphorylase GPBB.

[0021] Figure 2 The figure shows the SDS-PAGE electrophoresis diagram of the prokaryotic expressed glycogen phosphorylase GPBB carboxyl terminal antigen, wherein the labels are: M is a marker; 1 is the antigen before purification; 2 is the Ni column passthrough; 3 is the antigen after Ni column purification.

[0022] Figure 3 This is the result of the titer determination of rabbit anti-glycogen phosphorylase GPBB carboxyl terminal antigen polyclonal antibody.

[0023] Figure 4 It is the result of the determination of the specificity of the monoclonal antibody against glycogen phosphorylase GPBB.

[0024] Figure 5 This is a subtype identification diagram of anti-glycogen phosphorylase GPBB monoclonal antibody AC2118. DETAILED DESCRIPTION

[0025] The purpose of the present invention is to determine the GPBB-specific dominant epitope peptide sequence by analysis and screening, synthesize a high-purity GPBB-specific dominant epitope peptide as an immunogen, immunize mice to prepare monoclonal antibodies, and provide an anti-glycogen phosphorylase GPBB monoclonal antibody prepared by using a fused hybridoma cell line. First, the amino acid sequences of three isozymes of glycogen phosphorylase, brain-type GPBB, liver-type GPLL and muscle-type GPMM, are obtained from the NCBI website, and the sequences are compared to find the amino acid sequences in which GPBB is different from GPMM and GPLL. Then, the GPBB-specific dominant antigenic epitope is analyzed, and the GPBB-specific amino acid segment containing the dominant antigenic epitope is determined; then, a high-purity GPBB-specific dominant epitope peptide is synthesized as an immunogen, and the immunized mice provide a monoclonal antibody against glycogen phosphorylase GPBB prepared by using a fused hybridoma cell line, and the anti-glycogen phosphorylase GPBB monoclonal antibody can recognize human glycogen phosphorylase GPBB, and has no cross reaction with GPMM and GPLL. At the same time, the large antigen fragment 461-843aa containing the GPBB specific dominant epitope peptide was expressed in prokaryotes and used as an immunogen to prepare polyclonal antibodies.

[0026] The present invention obtains a mouse hybridoma cell line expressing a high-affinity and high-specificity anti-glycogen phosphorylase GPBB monoclonal antibody, named AC2118. The inventors used the monoclonal antibody as the capture antibody to establish a double antibody sandwich detection method by enzyme-linked immunosorbent assay, with a minimum detection limit of 1.0 pg / mL, which is significantly higher than the 4.0 pg / mL of the commercial antibody, and does not cross-immune react with highly homologous isozymes GPLL and GPMM. On the other hand, the inventors also used the prepared mouse anti-glycogen phosphorylase GPBB monoclonal antibody AC2118 to establish a fluorescent immunochromatography detection method, which has a higher detection sensitivity of 0.156 pg / mL. The above results show that the high-specificity and high-affinity anti-glycogen phosphorylase GPBB monoclonal antibody AC2118 prepared by the present invention can be used for the determination of glycogen phosphorylase GPBB in samples, with very high sensitivity and specificity. The inventor deposited the cell line in the General Microbiology Center of China Culture Collection Administration on May 22, 2024, with the deposit number CGMCC No.45928.

[0027] Subsequently, the inventors sequenced the monoclonal antibody secreted by the mouse hybridoma cell line CGMCC No.45928 and analyzed the immunoglobulin domain sequence, and found that its heavy chain variable region amino acid sequence was: VQLEQSGGGLVKPGGSLKLSCAASGFIFSSYAMSWVRQSPEKRLEWVATIGSGGGYT YYPDSVKGRFTISRDNAKNTLYLQMSSLRPEDTAMYYCGRRSAMDYWGQGTSVTVS S (SEQ ID NO.1), wherein the CDR1 amino acid sequence was GFIFSSYA (SEQ ID NO.2), the CDR2 amino acid sequence was IGSGGGYT (SEQ ID NO.3), and the CDR3 amino acid sequence was GRRSAMDY (SEQ ID NO.4). The amino acid sequence of the light chain variable region is: DIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLE SGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPSWKS (SEQ ID NO.5), wherein the amino acid sequence of CDR1 is KSVSTSGYSY (SEQ ID NO.6), the amino acid sequence of CDR2 is LVS, and the amino acid sequence of CDR3 is QHIRELTR (SEQ ID NO.7).

[0028] It is well known in the art that the heavy chain CDR region and light chain CDR region of an antibody are important amino acid sequence regions for recognizing and binding to the corresponding antigen, and a conservative amino acid substitution in the amino acid sequence of the above CDR region generally does not change the structure of the protein, so a single conservative amino acid substitution in the above region may still have the property of binding to the corresponding antigen. Therefore, the monoclonal antibody or its antigen-binding fragment obtained after a conservative amino acid substitution in the heavy chain CDR1 and / or the heavy chain CDR2 and / or the heavy chain CDR3 and / or the light chain CDR1 and / or the light chain CDR2 and / or the light chain CDR3 can still recognize and bind to human glycogen phosphorylase GPBB. In the patent application of the present invention, conservative amino acid substitution refers to the replacement of a certain amino acid in a protein by another chemically similar amino acid, such as the mutual substitution between aromatic amino acids Phe, Trp, and Tyr, the mutual substitution between aliphatic amino acids Ala, Gly, Leu, Ile, and Val, the mutual substitution between polar amino acids Gln and Asn, the mutual substitution between basic amino acids Lys, Arg, and His, the mutual substitution between acidic amino acids Asp and Glu, and the mutual substitution between hydroxyl amino acids Ser and Thr, etc.

[0029] Those skilled in the art can also prepare various antibody fragments that can bind to human glycogen phosphorylase GPBB from the monoclonal antibody of the present invention, i.e., antigen-binding fragments, such as but not limited to Fab, Fab', and F(ab')2, by using the existing technology in the art. The Fab fragment is a region in the antibody structure that can bind to an antigen, and is composed of a complete heavy chain and a variable region VH of a light chain and a constant region CH1 domain (Fd segment). Both the heavy chain and the light chain have a constant region and a variable region, and there is a disulfide bond link between the light and heavy chains. The antigen-binding fragment can be prepared as follows, for example, after enzymatic hydrolysis with papain, the antibody IgG is degraded into two Fab fragments and an Fc fragment. Under the action of pepsin, the antibody IgG is degraded into a F(ab')2 fragment and a pFc' fragment, and the F(ab')2 fragment is further reduced to form two Fab' fragments. Because the above-mentioned antigen-binding fragment can still bind to the corresponding antigen, it can be used to prepare a product for measuring human glycogen phosphorylase GPBB.

[0030] Those skilled in the art can also prepare single-chain antibodies (scFv) from the monoclonal antibodies of the present invention by the prior art in the art. Single-chain antibodies are antibodies formed by connecting the heavy chain variable region and the light chain variable region of an antibody through a short peptide linker of several amino acids. It has only one chain and is an artificially synthesized antibody. The length and amino acid composition of the short peptide linker are well known in the art, and the short peptide linker that can be used for the monoclonal antibody of the present invention can be determined by simple repeated experiments. Single-chain antibodies can be expressed in, for example, Escherichia coli by genetic engineering technology. The single-chain antibody of the present invention prepared in this way has the characteristic of binding to glycogen phosphorylase GPBB and can be applied to the determination of glycogen phosphorylase GPBB.

[0031] A person skilled in the art can design and synthesize nucleic acid molecules encoding the above-mentioned anti-glycogen phosphorylase GPBB monoclonal antibody based on the variable region amino acid sequence of the above-mentioned anti-glycogen phosphorylase GPBB monoclonal antibody, and can also insert the synthesized nucleic acid molecules into a nucleic acid vector to construct an expression vector, which can express a monoclonal antibody or an antigen-binding fragment thereof against glycogen phosphorylase GPBB. A person skilled in the art can also import the synthesized nucleic acid molecules or the constructed expression vectors into organisms such as cells, bacteria, yeast, etc. to obtain a recombinant, and produce the antibody or its antigen-binding fragment of the present invention via the above-mentioned recombinant expression, so that the antibody or its antigen-binding fragment expressed can bind and identify glycogen phosphorylase GPBB, and can be applied to the determination of glycogen phosphorylase GPBB. Therefore, the above-mentioned nucleic acid molecules, expression vectors and recombinants are within the scope of protection of the claims of the present invention. And the above-mentioned technologies all belong to technologies well known in the art, and those skilled in the art can carry out without creative work.

[0032] As described above, the antibody or antigen-binding fragment thereof of the present invention can recognize and bind to glycogen phosphorylase GPBB, and thus can be used to prepare a kit for measuring glycogen phosphorylase GPBB. The kit can be any kit that utilizes the binding reaction between the antibody or antigen-binding fragment thereof of the present invention and glycogen phosphorylase GPBB, such as but not limited to kits for enzyme-linked immunosorbent assay, chemiluminescence, fluorescent immunochromatography, colloidal gold immunochromatography, immunoblotting, and immunohistochemistry.

[0033] In order to explain the technical content, achieved objectives and effects of the technical solution in detail, the following is a description in conjunction with specific embodiments.

[0034] Example 1: Selection of dominant epitope peptides of glycogen phosphorylase GPBB specific sequence

[0035] The amino acid sequences of three isozymes of glycogen phosphorylase were obtained from the National Center for Biotechnology Information (NCBI) website, namely brain-type GPBB (NCBI Reference Sequence: NM_002862.4), liver-type GPLL (NCBI Reference Sequence: NM_002863.5) and muscle-type GPMM (NCBI Reference Sequence: NM_005609.4).Among them, the amino acid sequence of brain-type GPBB is as follows: MAKPLTDSEKRKQISVRGLAGLGDVAEVRKSFNRHLHFTLVKDRNVATPRDYFFALAHTVRDHLVGRWIRTQQHYYERDPKRIYYLSLEFYMGRTLQNTMVNLGLQNACDEAIYQLGLDLEELEEIEEDAGLGNGGLGRLAACFLDSMATLGLAAYGYGIRYEFGIFNQKIVNGWQVEEADDWLRYGNPWEKARPEYMLPVHFYGRVEHTPDGVKWLDTQVVLAMPYDTPVPGYKNNTVNTMRLWSAKAPNDFKLQDFNVGDYIEAVLDRNLAENISRVLYPNDNFFEGKELRLKQEYFVVAATLQDIIRRFKSSKFGCRDPVRTCFETFPDKVAIQLNDTHPALSIPELMRILVDVEKVDWDKAWEITKKTCAYTNHTVLPEALERWPVSMFEKLLPRHLEIIYAINQRHLDHVAALFPGDVDRLRRMSVIEEGDCKRINMAHLCVIGSHAVNGVARIHSEIVKQSVFKDFYELEPEKFQNKTNGITPRRWLLLCNPGLADTIVEKIGEEFLTDLSQLKKLLPLVSDEVFIRDVAKVKQENKLKFSAFLEKEYKVKINPSSMFDVHVKRIHEYKRQLLNCLHVVTLYNRIKRDPAKAFVPRTVMIGGKAAPGYHMAKLIIKLVTSIGDVVNHDPVVGDRLKVIFLENYRVSLAEKVIPAADLSQQISTAGTEASGTGNMKFMLNGALTIGTMDGANVEMAEEAGAENLFIFGLRVEDVEALDRKGYNAREYYDHLPELKQAVDQISSGFFSPKEPDCFKDIVNMLMHHDRFKVFADYEAYMQCQAQVDQLYRNPKEWTKKVIRNIACSGKFSSDRTITEYAREIWGVEPSDLQIPPPNIPRD (SEQ ID NO.8).Using the "Align two or more sequences" function in the Protein BLAST module in the BLAST analysis of the NCBI website, first enter the amino acid sequences of GPBB and GPMM into the sequence box, then click BLAST for comparison analysis to find the difference sequences between GPBB and GPMM; then enter the amino acid sequences of GPBB and GPLL into the sequence box, then click BLAST for comparison analysis to find the difference sequences between GPBB and GPLL; next, compare the two groups of difference sequences to determine the amino acid sequences that are different from GPBB and GPMM and GPLL at the same time, as shown in Table 1. Subsequently, the B cell epitope analysis of GPBB amino acids was performed using Goldkey biological analysis software. First, enter its amino acid sequence, then analyze the B cell epitope, and the resulting epitope distribution map is shown in. Figure 1 According to the distribution of B cell epitopes in GPBB amino acids, the B cell epitopes in the differential amino acid sequences of GPBB, GPMM and GPLL were searched, and the results are shown in Table 1. Among them, there are no B cell epitopes in GPBB specific sequences 1 and 2, and there are B cell epitopes in GPBB specific sequences 3, 4, 5 and 6, which are determined to be GPBB specific sequence dominant epitope peptides, which can be used as immunogens for preparing anti-GPBB monoclonal antibodies.

[0036] Table 1. GPBB specific sequences and B cell epitopes

[0037] Serial number Amino acid sequence and position B cell epitope (score) 1 22-LGDVAEVR-29 No epitope 2 367-EITKK-371 No epitope 3 507-KIGEEFLTDLSQLKKLLPLVS-527 IGEEFLT(1.95); FLTDLSQ(1.97) 4 720-EALDRKGYNAREYYDHLPELKQAVDQI-746 GYNAREY (3.41); PELKQAV (2.40) 5 781-CQAQVDQLYRNPKEWTKK-798 YRNPKEW(3.31) 6 829-EPSDLQIPPPNIPRD-843 EPSDLQI (2.13); QIPPPNI (1.86)

[0038] Example 2: Synthesis and coupling of glycogen phosphorylase GPBB specific sequence dominant epitope peptide

[0039] The GPBB-specific sequence dominant epitope peptides 3, 4, 5 and 6 screened and determined in Example 1 were selected to prepare synthetic peptides as immunogens for the preparation of monoclonal antibodies. First, four GPBB-specific sequence dominant epitope peptides were synthesized and coupled to hemocyanin KLH as immunogens. Peptide synthesis and KLH coupling were commissioned to Shanghai Dechi Biotechnology Co., Ltd., and HPLC purity analysis was greater than 90%.

[0040] Example 3: Preparation of glycogen phosphorylase GPBB carboxyl terminal antigen

[0041] Figure 2 As shown. The analysis found that the GPBB carboxyl terminal 461-843aa antigen expressed by pET-GPBB was mainly expressed in a soluble form with a molecular weight of about 45kDa. The pET-GPBB recombinant plasmid expressed antigen was purified by Ni column, and the protein peaks were collected respectively. The purified products were analyzed by SDS-PAGE electrophoresis. The purity of the purified recombinant expressed antigen was analyzed by Gel-ProR AnalyzerVersion 3.0 software, indicating that the purity of the glycogen phosphorylase GPBB carboxyl terminal antigen expressed by the pET-GPBB recombinant plasmid was 97.04%.

[0042] Example 4: Preparation of anti-glycogen phosphorylase GPBB monoclonal antibody

[0043] The KLH conjugates of the GPBB-specific sequence dominant epitope peptides 3, 4, 5 and 6 prepared in Example 2 were used as immunogens, and 8-week-old BALB / c female mice were used. 100 μg / mouse antigen was added with an equal amount of Freund's complete adjuvant, and the mice were thoroughly emulsified by a blender and injected subcutaneously and intraperitoneally on the back to immunize the mice. The second and third immunizations were performed 4 weeks and 8 weeks later, respectively, and 50 μg / mouse antigen was added with incomplete Freund's adjuvant, and the mice were thoroughly emulsified by a blender and injected subcutaneously and intraperitoneally on the back to immunize the mice. 7 days after the third immunization, blood was collected from the tail vein of the mice, centrifuged, and serum was taken for gradient dilution, and the titer of the immune serum was detected by indirect ELISA. The specific operation was as follows: the glycogen phosphorylase GPBB carboxyl terminal antigen prepared in Example 3 was diluted with carbonate coating buffer to a concentration of 2.0 μg / ml, 100 μl was coated per well, and the plate was kept at 4°C overnight; the plate was washed twice with washing solution; 110 μl / well blocking solution was added to block at room temperature for 6 hours; the plate was washed 5 times with washing solution. Add 100 μl of mouse serum dilutions of different gradients to each well, incubate at room temperature for 30 minutes, and discard the liquid. Wash the plate 5 times, invert the washed ELISA plate on absorbent paper and pat dry, add 100 μl / well HRP-labeled goat anti-mouse IgG antibody, and incubate at room temperature for 30 minutes. Wash the plate 5 times. Add 50 μL of TMB colorimetric solution A and B to each well, and color at room temperature in the dark for 15 minutes. Add 50 μL of 2MH2SO4 stop solution to each well to terminate the reaction. Set the detection wavelength of the microplate reader to 450nm, measure the OD value of each well, and read the value within 10 minutes after termination. The results showed that the titer of the sera of mice immunized with GPBB-specific sequence dominant epitope peptides 4 and 5 was high, and booster immunization could be performed; the titer of the sera of mice immunized with GPBB-specific sequence dominant epitope peptides 3 and 6 was very low, and no subsequent immunization experiments were performed. Therefore, the mice with the highest serum titer in the immunization groups of GPBB-specific sequence dominant epitope peptides 4 and 5 were selected for intraperitoneal injection booster immunization (50 μg / mouse), and spleen cells were obtained for fusion 3 days later.

[0044] Resuscitate SP20 myeloma cells and culture them until they are in the logarithmic growth phase. Take the spleen of the immunized BALB / c mouse and prepare a spleen cell suspension. Take the above spleen cells and myeloma cells in a ratio of 5:1, mix them in serum-free DMEM medium, centrifuge at 1500rpm for 5 minutes, fully aspirate the supernatant, gently shake the bottom of the centrifuge tube to disperse the cells, add 1mL of preheated 50% PEG fusion cells within 1 minute, gently shake while adding, let stand for 90 seconds after adding, add serum-free DMEM medium to terminate fusion, let stand at 37℃ for 10min, centrifuge at 1500rpm for 5 minutes, suspend the precipitate with HAT medium, dispense into 96-well cell plates containing feeder cells, and culture in a cell culture incubator at 37℃ and 5% CO2. After culturing in the cell culture incubator for 5 days, the medium was replaced once with HAT medium. On the 10th day, the medium was replaced with HAT medium. When the fused cells covered 40%-60% of the bottom of the well, the cell culture supernatant was taken and serially diluted with PBS, and the dilution multiples were 400, 800, 1600, 3200, 6400, 12800 and 25600. The indirect ELISA method was also used to screen positive clones, and the specific operation was as above.

[0045] A total of 13 positive clones were obtained with cell culture supernatants having titers exceeding 1:6400, among which the cell culture supernatants of the AC2118 clone strain immunized with the GPBB-specific sequence dominant epitope peptide 4 and the DF1026 clone strain immunized with the GPBB-specific sequence dominant epitope peptide 5 had the highest titers, reaching 1:25600.

[0046] Example 5: Preparation of anti-glycogen phosphorylase GPBB polyclonal antibody

[0047] Select healthy male big-eared white rabbits, and use the prokaryotic expressed glycogen phosphorylase GPBB carboxyl terminal antigen in Example 3 as an immunogen to prepare polyclonal antibodies. Take 1 mg of antigen and mix it with 1 mL of Freund's complete adjuvant. When the blender is thoroughly emulsified into a milky white viscous water-in-oil emulsion, multiple subcutaneous injections are performed on both sides of the white rabbit spine, and each injection is not less than 0.1 mL. After 4 weeks, take 1 mg of antigen and 1 mL of Freund's incomplete adjuvant respectively, and after thorough emulsification with a blender, select different points at the above-mentioned parts for a second immunization. After 2 weeks, blood is collected from the ear vein, and the polyclonal antibody titer is determined by the above-mentioned indirect ELISA method. If the titer is high, a third booster immunization can be performed. Blood is collected from the heart after 1 week, and after the blood coagulation and clot shrinkage, the serum is separated by centrifugation at 5000 rpm for 15 minutes. The indirect ELISA method for determining the serum titer of polyclonal antibodies was performed as described in Example 4. Rabbit serum was diluted with PBS at dilution ratios of 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000 and 1:512000. 100 μL of sample was added to each well for detection. The results are shown in FIG. Figure 3 As shown in the figure, the titer of the polyclonal antibody prepared by immunizing rabbits with the carboxyl terminal antigen of glycogen phosphorylase GPBB can reach 1:512000, using the rabbit serum diluted 1:2000 before immunization as the negative control.

[0048] Rabbit polyclonal antibodies were purified according to the instructions of the "Montage antibody purification prosep-G kit", and the antibody concentration was adjusted to 5.0 mg / mL with PB buffer. Anti-glycogen phosphorylase GPBB polyclonal antibodies were labeled with horseradish peroxidase (HRP) using the modified sodium periodate method. The specific operation was as follows: 5 mg HRP was dissolved in 0.5 mL deionized water, and 1 mL 0.06 mol / L NaIO4 was added and stirred gently at room temperature in the dark for 30 minutes. The liquid turned yellow-green; 1 mL 0.16 mol / L ethylene glycol was added and stirred gently at room temperature for 1 hour to terminate the oxidation reaction; the solution was placed in a dialysis bag and dialyzed overnight at 4°C with 1000 mL 0.01 mol / L, pH 9.5 carbonate buffer, and the solution was changed 3 times; 1 mL of buffer containing 5 mg polyclonal antibodies was added to 3 mL aldehyde HRP solution, and the solution was stirred gently at room temperature in the dark for 2 to 3 hours to combine; 5 mg NaHB4, 4℃ overnight; put into dialysis bag, dialyze with 1000mL of 0.01mol / L, pH7.2 phosphate buffer at 4℃ for 24 hours, change the liquid 3 times; centrifuge at 3000r / min for 30min, remove the precipitate, add an equal amount of glycerol to the supernatant, divide into small portions, and store at low temperature for later use.

[0049] Example 6: Determination of the specificity of anti-glycogen phosphorylase GPBB monoclonal antibodies

[0050] The mouse anti-glycogen phosphorylase GPBB monoclonal antibodies AC2118 and DF1026 prepared in Example 4 of the present invention were used as coating antibodies, respectively, the commercial anti-GPBB monoclonal antibody (Product No. A34290) of Biospacific was used as the control coating antibody, and the rabbit anti-glycogen phosphorylase GPBB polyclonal antibody was used as the detection antibody to establish a double antibody sandwich detection method for human glycogen phosphorylase GPBB.

[0051] The specific steps are to coat the ELISA plate with mouse anti-glycogen phosphorylase GPBB monoclonal antibody at a concentration of 2.0 μg / mL, coat 100 μL per well, and keep overnight at 4°C; wash the plate twice with washing solution, 200 μL / well; add 150 μL / well blocking solution to block at room temperature for 6 hours; wash the plate 5 times with washing solution, 200 μL / well. 100 μL of human myocardial GPBB antigen (Biospacific, catalog number J37020, concentration of 10 μg / mL), human liver GPLL antigen, and human skeletal muscle GPMM antigen were added to each well. The preparation of GPLL antigen from human liver and GPMM antigen from human skeletal muscle is to cut 0.5g human liver tissue and human skeletal muscle tissue into pieces, add 500μL Tris-HCl buffer (10mM, pH7.4) containing 0.2mM CaCl2 respectively, prepare tissue homogenate under ice bath, centrifuge the homogenate at 10000g for 10 minutes to separate the supernatant, use spectrophotometer to measure the supernatant OD280nm for protein quantification, and adjust the protein concentration to 100μg / mL. Incubate at room temperature for 1.5 hours and discard the solution. Wash the plate 5 times with washing solution, 200μL per well. Add 100μL HRP-rabbit anti-GPBB polyclonal antibody enzyme conjugate to each well and incubate at room temperature for 45min. Wash the plate 5 times, pat dry, add 50μL of TMB colorimetric solution A and B to each well, and color at room temperature in the dark for 15min. Add 50μL 2M H2SO4 stop solution to each well to terminate the reaction. The OD450 nm value of each well was measured by microplate reader within 10 minutes. Figure 4 As shown, the three GPBB monoclonal antibodies are highly specific to GPBB derived from myocardium, and have no cross-reaction with GPLL antigen derived from human liver and GPMM antigen derived from human skeletal muscle.

[0052] Example 7: Detection sensitivity of human glycogen phosphorylase GPBB double antibody sandwich ELISA

[0053] The detection sensitivity of the three glycogen phosphorylase GPBB double antibody sandwich ELISAs established using the monoclonal antibodies prepared by the present invention and the commercial monoclonal antibodies in Example 6 was evaluated. The specific operation steps are as described in Example 6. The GPBB antigen derived from human myocardium (Biospacific, Article No. J37020) was tested after gradient serial dilution. The results are shown in Table 2. The average value + 3SD of the PBS antigen dilution was used as the Cutoff value. The monoclonal antibody AC2118 of the present invention was used as the coating antibody, and the rabbit anti-glycogen phosphorylase GPBB polyclonal antibody was used as the detection antibody. The minimum detectable concentration of the human glycogen phosphorylase GPBB antigen by the double antibody sandwich ELISA was 1.0 pg / mL, which was higher than the minimum detectable concentration of 4.0 pg / mL of the monoclonal antibody DF1026 of the present invention and the control monoclonal antibody A34290.

[0054] Table 2. Glycogen phosphorylase GPBB double antibody sandwich ELISA detection sensitivity (OD450nm)

[0055] Antigen concentration (pg / mL) AC2118 DF1026 A34290 64 1.325±0.114 0.939±0.107 0.891±0.132 32 0.906±0.102 0.802±0.110 0.764±0.108 16 0.694±0.075 0.515±0.064 0.533±0.081 8 0.430±0.058 0.300±0.046 0.297±0.066 4 0.221±0.039 0.142±0.037 0.154±0.038 2 0.143±0.032 0.106±0.039 0.095±0.027 1 0.109±0.024 0.078±0.018 0.066±0.023 0 0.046±0.017 0.051±0.021 0.063±0.019 Cutoff 0.97 0.114 0.12

[0056] Example 8: Detection sensitivity of human glycogen phosphorylase GPBB double antibody sandwich fluorescence immunochromatography

[0057] Based on the double antibody sandwich principle, the mouse anti-glycogen phosphorylase GPBB monoclonal antibody AC2118 prepared by the present invention is labeled with fluorescent microspheres, and the rabbit anti-glycogen phosphorylase GPBB polyclonal antibody prepared by the present invention is coated on a nitrocellulose membrane. The content of human glycogen phosphorylase GPBB in the sample is quantitatively determined by fluorescent immunochromatography to evaluate the detection sensitivity. The specific operation is: take 80 μL of serially diluted human glycogen phosphorylase GPBB antigen, vertically drip it to the sample addition position of the detection card, and place it at room temperature for reaction for 15 minutes. The antigen in the sample and the monoclonal antibody labeled with fluorescent microspheres combine to form a GPBB antigen-GPBB monoclonal antibody immune complex. The immune complex moves forward along the nitrocellulose membrane with the chromatography. When it reaches the detection area (T line), the reaction complex is captured by the rabbit anti-GPBB polyclonal antibody on the nitrocellulose membrane detection line, and the excess fluorescent microsphere-labeled anti-glycogen phosphorylase GPBB monoclonal antibody is captured by the goat anti-mouse IgG antibody coated on the nitrocellulose membrane in the quality control area (C line). The ratio of the fluorescence value of the T line to the fluorescence value of the C line (T / C) was calculated, and the amount of GPBB captured in the sample was positively correlated with the signal intensity of the fluorescent antibody. The cutoff value was three times the T / C value of the antigen diluent, and the AC2118 monoclonal antibody of the present invention was used as the coating antibody. The minimum detectable concentration of human glycogen phosphorylase GPBB determined by the double antibody sandwich fluorescent immunochromatography was 0.156 pg / mL, and the detection sensitivity was significantly higher than that of the enzyme-linked immunosorbent assay. The results are shown in Table 3.

[0058] Table 3. Detection sensitivity of glycogen phosphorylase GPBB double antibody sandwich fluorescence immunochromatography

[0059]

[0060]

[0061] Example 9: Subtype analysis of anti-glycogen phosphorylase GPBB monoclonal antibody AC2118

[0062] Take the anti-glycogen phosphorylase GPBB monoclonal antibody AC2118 and use the mouse antibody subtype rapid test card of Antaiji (Beijing) Biotechnology Co., Ltd. to identify the heavy chain and light chain subtypes of mouse antibodies. First, dilute the antibody to 1μg / mL with PBS, then add 100μl of the diluted antibody to each well, let it stand for 5-10 minutes, and then observe and record the results. The results are as follows Figure 5 As shown, the anti-glycogen phosphorylase GPBB monoclonal antibody AC2118 is of mouse IgG1 subtype, and the antibody light chain is of Igκ subtype.

[0063] Example 10: Determination of variable region sequence of anti-glycogen phosphorylase GPBB monoclonal antibody AC2118

[0064] The anti-glycogen phosphorylase GPBB monoclonal antibody AC2118 hybridoma cell line CGMCC No.45928 was taken, and the total RNA of the hybridoma cells was extracted by Trizol method. After reverse transcription of cDNA by Thermo Fisher High Capacity cDNA Rever Transcription Kit, the heavy and light chain primers of the antibody were designed and synthesized by Beijing Qingke Biotechnology Co., Ltd. according to the mouse monoclonal antibody primer sequence in Modern Immunology Experimental Technology (Hubei Science and Technology Press, 2nd Edition), PCR amplification (amplification program: 95℃ preheating for 1min, 30 cycles (95℃30s, 58℃30s, 72℃45s), and finally 72℃ extension for 5min), connected to pMD18-T vector, expressed in Escherichia coli JM109, and positive clones were selected for sequencing. The determined sequence was compared and analyzed on the BLAST website (https: / / www.ncbi.nlm.nih.gov / igblast / ) for the CDR region sequence of the mouse monoclonal antibody.

[0065] After sequence analysis, it was found that the amino acid sequence of the heavy chain variable region was 114 amino acids, and its sequence was as follows: VQLEQSGGGLVKPGGSLKLSCAASGFIFSSYAMSWVRQSPEKRLEWVATIGSGGGYT YYPDSVKGRFTISRDNAKNTLYLQMSSLRPEDTAMYYCGRRSAMDYWGQGTSVTVS S (SEQ ID NO.1), wherein CDR1 was located at 25-32aa, and the amino acid sequence was GFIFSSYA (SEQ ID NO.2), CDR2 was located at 50-57aa, and the amino acid sequence was IGSGGGYT (SEQ ID NO.3), and CDR3 was located at 96-103aa, and the amino acid sequence was GRRSAMDY (SEQ ID NO.4). The amino acid sequence of the light chain variable region is 109 amino acids, and its sequence is as follows: DIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLE SGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPSWKS (SEQ ID NO.5), wherein CDR1 is located at 27-36aa, and the amino acid sequence is KSVSTSGYSY (SEQ ID NO.6); CDR2 is located at 54-56aa, and the amino acid sequence is LVS; CDR3 is located at 93-100aa, and the amino acid sequence is QHIRELTR (SEQ ID NO.7).

Claims

1. An anti-glycogen phosphorylase GPBB monoclonal antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3, and the light chain variable region comprises a light chain CDR1, a light chain CDR2 and a light chain CDR3, characterized in that: The amino acid sequence of the heavy chain CDR1 is the sequence shown in SEQ ID NO.2; The amino acid sequence of the heavy chain CDR2 is the sequence shown in SEQ ID NO.3; The amino acid sequence of the heavy chain CDR3 is the sequence shown in SEQ ID NO.4; The amino acid sequence of the light chain CDR1 is the sequence shown in SEQ ID NO.6; The amino acid sequence of the light chain CDR2 is LVS; The amino acid sequence of the light chain CDR3 is the sequence shown in SEQ ID NO.

7.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO.

5.

3. The monoclonal antibody according to claim 2, characterized in that It is secreted by the mouse hybridoma cell line AC2118 with a deposit number of CGMCC No. 45928.

4. The monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The antibody or antigen-binding fragment is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a single-chain antibody or a humanized antibody.

5. A nucleic acid molecule, characterized in that The nucleic acid comprises a nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

6. An expression vector, characterized in that: The expression vector comprises the nucleic acid molecule of claim 5.

7. A recombinant, characterized in that The recombinant comprises the nucleic acid molecule according to claim 5 or the expression vector according to claim 6.

8. A hybridoma cell line of anti-glycogen phosphorylase GPBB monoclonal antibody, characterized in that: The monoclonal antibody hybridoma cell line is a mouse hybridoma cell line AC2118, and its deposit number is CGMCC No. 45928.

9. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 in the preparation of a product for detecting glycogen phosphorylase GPBB.

10. A kit for detecting glycogen phosphorylase GPBB, characterized in that: Comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

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