A human FGF21 rabbit monoclonal antibody and its application

Abstract

The present invention relates to a human FGF21 rabbit-derived monoclonal antibody and application thereof, which comprises a first monoclonal antibody or a second monoclonal antibody, wherein: the first monoclonal antibody comprises: a heavy chain variable region CDR1GIDLSSNA, a heavy chain variable region CDR2IGITGNR, a heavy chain variable region CDR3VRGVISDNINGI, a light chain variable region CDR1QSLYDNW, a light chain variable region CDR2KVS, and a light chain variable region CDR3AGGYSGNIHA; the second monoclonal antibody comprises: a heavy chain variable region CDR1GFSLSSYR, a heavy chain variable region CDR2ITSSGNT, a heavy chain variable region CDR3ARETYDNYGSRFNI, a light chain variable region CDR1QSISSY, a light chain variable region CDR2AAS, and a light chain variable region CDR3QSNFLITTYGGA.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and bioanalysis, and specifically relates to a human FGF21 rabbit-derived monoclonal antibody and an application thereof. Background Art

[0002] FGF21 is an important growth factor that regulates sugar and lipid metabolism. Clinical studies have found that blood FGF21 levels are significantly increased in patients with metabolic syndrome and individuals with insulin resistance. [1] FGF21 can be used as a biomarker for disease diagnosis (positively correlated with the following diseases: type 2 diabetes, diabetic nephropathy, non-alcoholic fatty liver disease in patients with type 2 diabetes, etc. [2-5] ; negatively correlated with the following diseases: peripheral arterial disease (PAD)) and can be used for disease risk assessment (such as the expected risk of disease progression in prediabetes) [6] Taking type 2 diabetes as an example, studies have shown that plasma FGF21 levels are significantly increased in prediabetic and diabetic patients, and studies from non-diabetic individuals have shown that normal plasma FGF21 levels can predict the development of human diabetes. [2-3] Blood FGF21 levels can also be used as a molecular marker for medication guidance, efficacy and risk prediction in clinical practice, such as the prediction of cardiovascular disease events in patients treated with statins. [7] , and can also be used as a predictor of adverse cardiovascular events, including in patients with type 2 diabetes [8] .

[0003] Blood FGF21 levels also have application value in health management. Serum FGF21 levels are significantly correlated with triglyceride levels, systolic blood pressure, diastolic blood pressure, pulse pressure, body mass index (BMI), age, fasting blood glucose (FPG) and total cholesterol levels, and can be used as a biomarker for metabolic disorders. [9] It has guiding significance in health management related indicators such as three highs management, diet management, adolescent obesity and fatty liver obesity management, and adult regional bone mineralization density (negative correlation). [10-12] .

[0004] In addition, at the cellular level, FGF21 also regulates cell fate. Studies have found that FGF21 is also related to the aging of stem cells. High expression of FGF21 can inhibit the aging phenotype of stem cells.

[13] .

[0005] In summary, FGF21 not only affects individual metabolism and health, but also affects cell phenotype at the microscopic level. It is also a hot protein for drug development.

[14] However, there is currently a lack of standardized detection methods for FGF21.

[15] The patent with publication number CN112005119A discloses a method for detecting and quantifying FGF21 using mouse monoclonal antibodies. When the FGF21 content is determined according to the unoptimized enzyme-linked immunosorbent assay (ELISA), the sensitivity is 5 pg / mL; after the ELISA assay is optimized, the sensitivity can be increased to the optimal 2 pg / mL. According to the patent, if the sensitivity is to be further improved, it is necessary to use the single molecule immunoassay of the Quanterix Simoa HD-1AnalyzerTM single molecule detection instrument, but the single molecule assay is more expensive and less popular than the enzyme-linked immunosorbent assay. At the same time, the analysis method described in the patent application lacks data support for accuracy and precision.

[0006] Therefore, there is still a need to develop FGF21 detection reagents and methods with better accuracy, precision and sensitivity. In particular, there have been no reports of rabbit monoclonal antibodies that can more accurately identify human FGF21 antigen epitopes. The present invention focuses on solving this problem to meet the development needs of FGF21 biological function research, drug development, diagnostic reagents, etc.

[0007] References:

[0008] 1. Zhang X, Yeung DC, Karpisek M, et al. Serum FGF21 levels are increased in obesity and are independently associated with the metabolic syndrome in humans. Diabetes 2008;57:1246–1253.

[0009] 2.Chen C, Cheung BM, Tso AW, et al. High plasma level of fibroblastgrowth factor 21 is an Independent predictor of type 2diabetes: a 5.4-yearpopulation-based prospective study in Chinese subjects. Diabetes Care. 2011Sep; 34(9):2113-5.

[0010] 3.Woo YC,Lee CH,Fong CH,et al.Serum fibroblast growth factor 21is asuperior biomarker to other adipokines in predicting incident diabetes.ClinEndocrinol(Oxf).2017Jan;86(1):37-43.

[0011] 4.Jian WX,Peng WH,Jin J,et al.Association between serum fibroblastgrowth factor 21and diabetic nephropathy.Metabolism.2012Jun;61(6):853-9.

[0012] 5.Bahijri S,Eldakhakhny B,Enani S,et al.Fibroblast Growth Factor 21:AMore Effective Biomarker Than Free Fatty Acids and Other Insulin SensitivityMeasures for Predicting Non-alcoholic Fatty Liver Disease in Saudi ArabianType 2Diabetes Patients.Cureus.2023Dec 14;15(12):e50524.

[0013] 6.Miyazaki Y,Saita E,Kishimoto Y,et al.Low Plasma Levels ofFibroblast Growth Factor-21in Patients with Peripheral Artery Disease.JAtheroscler Thromb.2018Sep 1;25(9):821-828.

[0014] 7.Ong KL,Hui N,Januszewski AS,et al.High plasma FGF21 levels predictsmajor cardiovascular events in patients treated with atorvastatin(from theTreating to New Targets[TNT]Study).Metabolism.2019Apr;93:93-99.

[0015] 8.Ong KL,Januszewski AS,O’Connell R,et al.The relationship offibroblast growth factor 21with cardiovascular outcome events in theFenofibrate Intervention and Event Lowering in Diabetesstudy.Diabetologia.2015;58:464–473.

[0016] 9.Jin QR,Bando Y,Miyawaki K,et al.Correlation of fibroblast growthfactor 21serum levels with metabolic parameters in Japanese subjects.J MedInvest.2014;61(1-2):28-34.

[0017] 10.Richter MM,Thomsen MN,Skytte MJ,et al.Effect of a 6-WeekCarbohydrate-Reduced High-Protein Diet on Levels of FGF21 and GDF15 in PeopleWith Type 2Diabetes.J Endocr Soc.2024Jan 24;8(4):bvae008.

[0018] 11. Giannini C, Feldstein AE, Santoro N, et al. Circulating levels of FGF-21 in obese youth: associations with liver fat content and markers of liverdamage. J Clin Endocrinol Metab. 2013Jul;98(7):2993-3000.

[0019] 12.Hao RH,Gao JL,Li M,et al.Association between fibroblast growthfactor 21and bone mineral density in adults.Endocrine.2018Feb;59(2):296-303.

[0020] 13. Li

[0021] 14. Geng L, Lam KSL, Xu A. The therapeutic potential of FGF21 inmetabolic diseases: from bench to clinic. Nat Rev Endocrinol. 2020Nov; 16(11):654-667.

[0022] 15. Chinese Association of Women Physicians, Diabetes Committee, “Expert Consensus on Screening and Intervention for High-risk Populations of Diabetes”, 2022. Summary of the invention

[0023] The object of the present invention is to provide a human FGF21 rabbit monoclonal antibody for accurately, precisely and with high sensitivity analyzing and detecting biological samples of human FGF21 protein that meets the requirements of analytical methodology.

[0024] In order to achieve the above object, the technical solution adopted by the present invention is:

[0025] The first aspect of the present invention provides a human FGF21 rabbit monoclonal antibody, which includes a first monoclonal antibody or a second monoclonal antibody, wherein: the first monoclonal antibody includes:

[0026] (1) a heavy chain variable region CDR1 comprising the amino acid sequence GIDLSSNA and conservative substitutions thereof;

[0027] (2) heavy chain variable region CDR2, which comprises the amino acid sequence IGITGNR and conservative substitutions thereof;

[0028] (3) a heavy chain variable region CDR3 comprising the amino acid sequence VRGVISDNINGI and conservative substitutions thereof;

[0029] (4) a light chain variable region CDR1 comprising the amino acid sequence QSLYDNW and conservative substitutions thereof;

[0030] (5) light chain variable region CDR2, which comprises the amino acid sequence KVS and conservative substitutions thereof;

[0031] (6) light chain variable region CDR3, which comprises the amino acid sequence AGGYSGNIHA and conservative substitutions thereof;

[0032] The second monoclonal antibody comprises:

[0033] (1) a heavy chain variable region CDR1 comprising the amino acid sequence GFSLSSYR and conservative substitutions thereof;

[0034] (2) heavy chain variable region CDR2, which comprises the amino acid sequence ITSSGNT and conservative substitutions thereof;

[0035] (3) a heavy chain variable region CDR3 comprising the amino acid sequence ARETYDNYGSRFNI and conservative substitutions thereof;

[0036] (4) a light chain variable region CDR1 comprising the amino acid sequence QSISSY and conservative substitutions thereof;

[0037] (5) light chain variable region CDR2, which comprises the amino acid sequence AAS and conservative substitutions thereof;

[0038] (6) Light chain variable region CDR3, which comprises the amino acid sequence QSNFLITTYGGA and conservative substitutions thereof.

[0039] According to some specific embodiments, the heavy chain constant region of the first monoclonal antibody and the second monoclonal antibody comprises the amino acid sequence of SEQ ID NO: 1 and conservative substitutions thereof, and the light chain constant region comprises the amino acid sequence of SEQ ID NO: 2 and conservative substitutions thereof.

[0040] According to some specific embodiments, the nucleotide sequence encoding the heavy chain constant region is SEQ ID NO: 3 and its synonymous nucleotide sequences, and the nucleotide sequence encoding the light chain constant region is SEQ ID NO: 4 and its synonymous nucleotide sequences.

[0041] According to some specific embodiments, the first monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 5 and conservative substitutions thereof, and the light chain comprises the amino acid sequence of SEQ ID NO: 6 and conservative substitutions thereof.

[0042] According to some specific embodiments, the nucleotide sequence encoding the heavy chain of the first monoclonal antibody is SEQ ID NO: 7 and its synonymous nucleotide sequences, and the nucleotide sequence encoding the light chain of the first monoclonal antibody is SEQ ID NO: 8 and its synonymous nucleotide sequences.

[0043] According to some specific embodiments, the second monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 9 and conservative substitutions thereof, and the light chain comprises the amino acid sequence of SEQ ID NO: 10 and conservative substitutions thereof.

[0044] According to some specific embodiments, the nucleotide sequence encoding the heavy chain of the second monoclonal antibody is SEQ ID NO: 11 and its synonymous nucleotide sequences, and the nucleotide sequence encoding the light chain of the second monoclonal antibody is SEQ ID NO: 12 and its synonymous nucleotide sequences.

[0045] According to some specific embodiments, the human FGF21 rabbit monoclonal antibody is prepared by the following steps:

[0046] (1) Expression and purification of human FGF21 protein antigen;

[0047] (2) multiple animal immunizations of rabbits with human FGF21 protein antigen;

[0048] (3) Rabbit spleen PBMCs were isolated, and goat anti-rabbit IgG-FITC and FGF21-AF647 double-positive cells were screened by flow cytometry and cultured;

[0049] (4) The positive cloned cells are sequenced, and after the sequencing is confirmed to be correct, the plasmid is amplified and transfected into CHO cells, cultured and purified, and the human FGF21 rabbit monoclonal antibody is collected.

[0050] This application screened out monoclonal antibodies with high ELISA signals through screening and comparison of rabbit monoclonal antibodies, and obtained a pair of clones with the highest signal level (25pg / mL FGF21 ELISA detection OD value greater than 1) through combined pairing screening and optimization, which can be used for quantitative analysis of human FGF21 biological samples.

[0051] The second aspect of the present invention provides a kit, which includes a capture antibody and a detection antibody, wherein the capture antibody is one of the first monoclonal antibody and the second monoclonal antibody as described above, and the detection antibody is the other of the first monoclonal antibody and the second monoclonal antibody as described above.

[0052] According to some specific embodiments, the capture antibody is the first monoclonal antibody, and the detection antibody is the second monoclonal antibody.

[0053] According to some specific embodiments, the kit also includes an immune marker, a complex of an enzyme that can bind to the immune marker and catalyze the color development of the substrate, and a substrate that can be catalyzed by the enzyme to develop color. Wherein, the immune marker includes but is not limited to biotin, FITC, etc. The complex of the enzyme includes but is not limited to streptavidin coupled to the enzyme. The enzyme includes but is not limited to horseradish peroxidase, β-galactosidase, glucose oxidase or alkaline phosphatase, etc. The substrate includes but is not limited to TMB color developing solution, OPD color developing solution, etc.

[0054] According to some embodiments, the detection antibody is conjugated with biotin. According to some embodiments, the kit includes but is not limited to an enzyme-linked immunosorbent assay kit.

[0055] According to some specific embodiments, the samples to be tested in the kit can be various biological samples, including but not limited to blood, sputum, saliva, pleural effusion, pleural / peritoneal fluid, cerebrospinal fluid, bronchial / pulmonary lavage fluid, secretions (such as nasopharyngeal secretions), excretions (such as urine, feces), cell culture supernatant, etc. Among them, blood can be whole blood, plasma, serum, etc.

[0056] According to some specific embodiments, the sample to be tested in the kit is a plasma sample.

[0057] According to some specific embodiments, the kit is an enzyme-linked immunosorbent assay kit. The present application can overcome the problems of weak signal, low instrument reading and insufficient sensitivity in ELISA detection. The kit of the present application has a strong ELISA detection signal, a high microplate reader reading value, a high standard curve fitting correlation coefficient, high accuracy, precision and sensitivity, and is convenient for sample analysis and detection.

[0058] According to some specific embodiments, the concentration of the capture antibody is 0.1-1 μg / mL, for example, 0.1 μg / mL, 0.15 μg / mL, 0.2 μg / mL, 0.25 μg / mL, 0.3 μg / mL, 0.35 μg / mL, 0.4 μg / mL, 0.45 μg / mL, 0.5 μg / mL, 0.55 μg / mL, 0.6 μg / mL, 0.65 μg / mL, 0.7 μg / mL, 0.75 μg / mL, 0.8 μg / mL, 0.85 μg / mL, 0.9 μg / mL, 0.95 μg / mL or 1 μg / mL.

[0059] According to some specific embodiments, the concentration of the detection antibody is 0.05-0.5 μg / mL, for example, 0.05 μg / mL, 0.06 μg / mL, 0.07 μg / mL, 0.08 μg / mL, 0.09 μg / mL, 0.1 μg / mL, 0.11 μg / mL, 0.15 μg / mL, 0.2 μg / mL, 0.25 μg / mL, 0.3 μg / mL, 0.35 μg / mL, 0.4 μg / mL, 0.45 μg / mL or 0.5 μg / mL.

[0060] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0061] The human FGF21 rabbit monoclonal antibody of the present invention is easy to prepare, and has high expression amount (72 mL can purify about 3 mg of antibody) and titer (titer greater than 50,000). The human FGF21 rabbit monoclonal antibody of the present invention can be used to achieve high sensitivity, high accuracy and high precision FGF21 biological sample analysis that meets the requirements of bioanalysis methodology using only a simple and universal double antibody sandwich ELISA method. The antibody of the present invention is more accurate in recognizing antigen epitopes than mouse monoclonal antibodies, has higher affinity and stability, and uses a low amount of ELISA antibodies, which is cost-effective. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 It is the standard curve of Example 7;

[0063] Figure 2 This is the standard curve of Example 8. DETAILED DESCRIPTION

[0064] It should be noted that, unless otherwise defined, the technical terms or scientific terms used in this application are the usual meanings understood by those skilled in the art. The experimental methods in the following examples are conventional methods unless otherwise specified. The reagents and materials used in the following examples are commercially available products unless otherwise specified. When used herein and in the appended claims, the singular forms "one", "another", and "the" include plural referents unless the context clearly indicates otherwise.

[0065] In the present application, non-limiting examples of conservative substitutions are Ala replaced by Val, Arg replaced by Lys, Asn replaced by Gln, Asp replaced by Glu, Cys replaced by Ser, Gln replaced by Asn, Glu replaced by Asp, Gly replaced by Ala, His replaced by Arg, Ile replaced by Leu, Leu replaced by Ile, Lys replaced by Arg, Met replaced by Leu, Phe replaced by Tyr, Pro replaced by Ala, Ser replaced by Thr, Thr replaced by Ser, Trp replaced by Tyr, Tyr replaced by Phe, and Val replaced by Leu.

[0066] In the research on the biological function of FGF21 and the development of drugs, diagnostic reagents, etc., the key reagents of biological analysis, especially the ligand binding analysis method of biological macromolecules (the most commonly used is ELISA method) are indispensable for the determination of target molecule content in biological samples and pharmacokinetic and toxicokinetic studies. The present invention mainly clarifies the application potential of the screened rabbit monoclonal antibody in the field of biological analysis of FGF21 blood-derived biological samples.

[0067] The present invention mainly adopts the technical route of immunizing New Zealand white rabbits with human FGF21 recombinant protein to screen rabbit monoclonal antibodies, screens rabbit monoclonal antibodies against human FGF21, and performs paired screening to screen out capture antibodies and detection antibodies that can optimally quantitatively detect FGF21, and form a pair of antibody pairs for ELISA detection, meeting the requirements of sensitivity, accuracy and precision analysis methodology (the minimum quantitative limit can reach 0.7629pg / mL, the correlation coefficient of the standard curve is not less than 0.999, and the CV of the back-calculated concentration value of each standard point of the standard curve is within 20%), and confirms the applicability of human plasma samples. Compared with the existing mouse monoclonal antibodies, rabbit monoclonal antibodies are more accurate in antigen epitope recognition and are more suitable for the analysis and detection of FGF21.

[0068] The technical solution provided by the present invention is further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and do not limit the scope of protection of the present invention. The reagents used in the present invention, unless otherwise specified, can be obtained from any supplier and have the quality / purity standards that meet the requirements of the corresponding application.

[0069] Example 1 FGF21 gene synthesis, eukaryotic expression and protein preparation

[0070] 1.1 FGF21 gene sequence codon optimization, gene synthesis and vector construction

[0071] The FGF21 gene sequence optimization and synthesis, and the PATX2 vector construction were commissioned to Anhui General Biotechnology. After the vector FGF21-PATX2 plasmid was amplified in Escherichia coli, 0.93 mg was extracted.

[0072] 1.2 Host CHO cell culture preparation

[0073] According to 0.25×10 6 cells / mL were subcultured and expanded into shake flasks, which were cultured in a shaker at 37°C, 5% CO2, and 130 rpm. The cell viability was 99.82% at the time of inoculation, the subculture volume was 300 mL, and the culture was carried out for 2 days.

[0074] Take 250mL of preheated culture medium to a 1L baffled shake flask, then discard the supernatant of the required number of cells after centrifugation, take 50mL of fresh culture medium to resuspend the precipitate, add it to the shake flask, mix well, and place the shake flask in a shaker at 37℃, 5% CO2, 130rpm. Cell viability 99.75%, cell inoculation density 1×10 6 cells / mL, cultured for 1 day.

[0075] 1.3 Transfection and expression

[0076] Take two 50mL centrifuge tubes, vortex the transfection reagent Fecto PRO quickly for 5 seconds, use a pipette to take 333μL Fecto PRO into a new 50mL centrifuge tube, add 33mL culture medium Freestyle CHOexpression medium to another 50mL centrifuge tube, add 167μg plasmid DNA, mix gently and transfer to the centrifuge tube containing 333μL Fecto PRO, mix immediately and incubate at room temperature for 10 minutes. Take out the 1L baffled shake flask containing 300mL cells prepared the day before, wipe it with alcohol, put it in the biosafety cabinet, transfer the Fecto PRO / plasmid DNA mixture to the shake flask with a pipette, and mix it. Use a pipette to draw 167μL of another transfection reagent Fecto PRO Booster into the shake flask, mix it, and put the cells in a shaker, 37℃, 5% CO2, 130rpm for 12 days, and the cell density is 5.06×10 6 cells / mL, cell viability 74.28%.

[0077] 1.4 Protein purification

[0078] Ni resin purification of target protein

[0079] (1) 333 mL of sample was divided into 50 mL centrifuge tubes and then centrifuged at 800 g for 10 min to separate the culture medium and cells;

[0080] (2) The culture medium obtained in the previous step was divided and leveled, and then centrifuged at 12000g for 10 min to separate the supernatant (mixed solution of supernatant protein to be purified), and a small sample of 20 μL was taken, which was IN;

[0081] (3) Load 1 mL of Ni resin into an empty column and equilibrate with pH 7.5 PBS for 10 times column volume (10 mL). Keep the equilibrated resin for later use;

[0082] (4) Add the treated Ni resin filler to the supernatant mixture, incubate in a shaker at 4°C for 30 min, collect the resin, temporarily retain the permeate, and take a small sample of 20 μL, which is the FT;

[0083] (5) Wash the resin with 15 column volumes (15 mL) of pH 7.5 PBS buffer. Temporarily retain the flowthrough and take a 20 μL sample, which is W1.

[0084] (6) Wash the resin with 15 column volumes (15 mL) of pH 7.5 PBS, 30 mM imidazole buffer. The flow-through solution is temporarily retained and a 20 μL sample is taken, which is W2.

[0085] (7) Wash the resin with 10 column volumes (10 mL) of pH 7.5 PBS, 50 mM imidazole buffer. Temporarily retain the flowthrough and take a 20 μL sample, which is W3.

[0086] (8) Wash the resin with 1 column volume (1 mL) of pH 7.5 PBS, 300 mM imidazole buffer. Wash 9 tubes in sequence and take a 20 μL sample from each tube, which is E1-E9;

[0087] (9) SDS-PAGE analysis of samples;

[0088] (10) The protein was dialyzed into 5 L 50 mM Tris pH 8.0, 10% Glycerol, 0.05% Brij 35 for 16 h;

[0089] (11) The dialyzed protein was centrifuged at 12000g for 10 min, the supernatant was concentrated using an ultrafiltration tube, centrifuged at 5000g for 10 min, and then the protein concentration was determined by the Bradford method. At the same time, 2 μg was sampled for SDS-PAGE detection;

[0090] (12) FGF21 antigen packaging: A total of 3.9 mg of protein was obtained. The immunization antigen was in liquid state and placed at -20°C after packaging was completed on the same day. The antigen was packaged into EP tubes according to the amount of antigen for 6 immunizations. 200 μg × 2 tubes for the first immunization, and 100 μg × 10 tubes for the second to sixth immunizations.

[0091] Example 2 Immunization of New Zealand White Rabbits with Human FGF21 Protein

[0092] Two New Zealand white rabbits were immunized with human FGF21 protein antigen:

[0093] (1) First immunization: In a 200μg antigen EP tube, add 1×PBS to make the volume 300μL, then add 100μL adjuvant, mix well and suck into the syringe. Fix the rabbit sideways, disinfect the immunization site on the outside of the thigh, and then perform intramuscular injection, with 2-3 points of hind leg muscle injection. The first immunization cycle is one week.

[0094] (2) Second to fifth immunization: In a 100 μg antigen EP tube, add 1×PBS to make the volume 300 μL, then add 100 μL adjuvant, mix well and suck into the syringe. Fix the rabbit sideways, disinfect the immunization site on the outside of the thigh, and then perform intramuscular injection, with 2-3 points of hind leg muscle injection. Each immunization cycle is one week.

[0095] Example 3 Screening of FGF21 antibody positive clones by single B cells

[0096] 3.1 Blood collection after three immunizations: Fix the rabbit, disinfect the rabbit ear blood collection site, find the ear vein and collect 2mL of blood. Press to stop bleeding and put the rabbit back. Place the collected blood at 37℃ for 30-60min, centrifuge at 2000rpm for 15-20min, extract the upper serum, and store at -20℃.

[0097] 3.2 Serum titer determination after three immunizations:

[0098] FGF21 coated ELISA plate: melt the antigen at room temperature at 5 μg / mL, oscillate and mix well, take the corresponding volume to 10 mL CBS coating buffer to coat the ELISA plate, 100 μL / well, at 4°C overnight.

[0099] ELISA titer determination:

[0100] 1) Blocking: According to the number of coated ELISA plates, prepare 3% BSA-PBST blocking solution at 30 mL / plate, shake the antigen in the plate into the water pool and pat dry, add blocking solution at 300 μL / well, and incubate at 4°C overnight; 2) Washing: Shake the blocking solution in the plate into the water pool, wash 3 times with 1×PBST, 300 μL / well, and pat dry; 3) Primary antibody incubation: The sample to be tested is diluted in multiples (1:2K / 4K / 8K / 16K / 32K / 64K / 128K) starting from 1:2K (K=1000), and PBS is used as a blank control in the last well. The reaction volume is 100 μL / well, 3 Incubate at 7℃ for 1h; 4) Wash the plate: throw the primary antibody in the plate into the water pool, wash with 1×PBST 3 times, 300μL / well, and pat dry; 5) Secondary antibody incubation: HRP-labeled goat anti-rabbit antibody, the reaction volume is 100μL / well, incubate at 37℃ for 0.5h; 6) Wash the plate: throw the secondary antibody in the plate into the water pool, wash with 1×PBST 3 times, 300μL / well, and pat dry; 7) Color development: add TMB at 100μL / well, incubate at 37℃ for 5min; 8) Stop and read: add 2M HCl at 50μL / well to stop the reaction and read immediately. Read the data at 450-620nm wavelength on the microplate reader, export and save the data.

[0101] It was determined that the serum titer was 32,000 after three immunizations.

[0102] 3.3 After four immunizations, the serum titer was measured to be greater than 64,000.

[0103] 3.4 After five immunizations, the serum titer was measured to be greater than 128,000.

[0104] 3.5 Antigen-specific B cell isolation and culture

[0105] (1) Isolate spleen PBMCs into two centrifuge tubes and centrifuge them. Resuspend the cells in one tube with 5 mL PBS and count the cells at a density of 1.76 × 107 cells / mL, with a cell viability of 46.24%, for flow cytometry sorting; another tube was resuspended in 4.5 mL of B cell complete medium plus 0.5 mL of DMSO, and the cells were counted at 2.44 × 10 7 cells / mL, viability 42.91%, 5 tubes of 1mL each for PBMC cell cryopreservation backup. Use relevant antibodies for staining, set up blank tubes and single staining tubes respectively, incubate on ice for 30 minutes, wash twice with PBS, centrifuge at 300g for 5 minutes each time, and resuspend the cells. Staining scheme: Goat anti-rabbit lgG-FITC (1:300), FGF21-AF647 (1:50), the latter is coupled to FGF21 using ThermoFisher's Alexa Fluor 647C2 maleimide dye.

[0106] (2) Before loading, filter the cells with a sterile flow tube with a filter. Use the blank control and single-staining control to draw gates and adjust the voltage. 5 minutes before loading, add 2% 7-AAD for staining to eliminate dead cells.

[0107] (3) Lymphocytes were selected using FSC-A / SSC-A, adherent cells were eliminated using FSC-W / FSC-A, and double positive cells were selected. Through flow cytometry, one FITC and AF647 positive cell was added to each well of the 96-well plate prepared in the previous step, and the 96-well plate was centrifuged at 300g for 5 minutes, 37°C 5% CO2, and cultured for 7-10 days before detection. A total of 20 96-well plates were inoculated with double positive clones.

[0108] (4) ELISA screening of B cell culture supernatant

[0109] Melt the antigen at room temperature and shake to mix well. Take 143.38 μL of the antigen and add it to 48.75 mL of CBS coating buffer. Coat 5 384-well plates at 25 μL / well, 2 μg / mL, and incubate at 4°C overnight. ELISA test on the second day:

[0110] 1) Blocking: According to the number of coated ELISA plates, prepare 5% skim milk-PBST blocking solution at 39mL / plate, shake the antigen in the plate into the water pool and pat dry, add blocking solution at 100μL / well, and incubate at 37℃ for 1.5h; 2) Washing: Shake the blocking solution in the plate into the water pool, wash 3 times with 1×PBST, 100μL / well, and pat dry; 3) Primary antibody incubation: Use an automatic pipetting station to take 25μL / well sample from the 96-well culture plate to the corresponding position of the 384-well plate, record the number and position of the 384-well plate, and incubate at 37℃ 1h; 4) Washing: throw the primary antibody in the plate into the water pool, wash 3 times with 1×PBST, 100μL / well, and pat dry; 5) Secondary antibody incubation: add HRP-labeled goat anti-rabbit secondary antibody, the reaction volume is 25μL / well, incubate at 37℃ for 45min; 6) Washing: throw the secondary antibody in the plate into the water pool, wash 3 times with 1×PBST, 100μL / well, and pat dry; 7) Color development: add TMB at 25μL / well, incubate at 37℃ for 10min; 8) Stop and read: add 2M HCl at 12μL / well to stop the reaction, and read immediately. Read at 450-620nm wavelength on the microplate reader, export and save the data; 9) Use 384-well plate data conversion software to automatically convert 384-well detection data into data corresponding to 96-well plates. 20 96-well plates screened 11 positive candidate clones (screening standard OD value greater than 0.2).

[0111] (5) Five frozen PMSCs were used to screen double-positive clones and plated on 20 96-well plates. 76 ELISA-positive candidate clones were obtained by 384-well screening (screening standard OD value greater than 0.2).

[0112] (6) PCR amplification of B cell antibody light and heavy chain gene fragments

[0113] After culturing the 11 positive clones of B cells in the first round of screening for 12 days, the supernatant of the positive B clones was removed, the positive clones were lysed, reverse transcription reaction was performed, and the cDNA template was frozen at -80°C; 50 μL of amplification system was prepared using conventional heavy and light chain primers, and cDNA was used as a template. After preparation, the PCR tube was gently mixed and briefly centrifuged to aggregate at the bottom of the tube, and then placed in a PCR instrument for amplification.

[0114] Example 4 Antibody expression and titer detection

[0115] The four clones with the highest OD values ​​in the first batch of 11 positive clones detected by ELISA were selected for sequencing. The heavy and light chain fragments of the antibody were subcloned into expression vectors and transformed into DH5α strains for sequencing analysis and subsequent expression. After colony PCR, the correct positive clones were selected according to the length of the heavy and light chain fragments of the antibody, and the corresponding activated bacterial solution was sent to the sequencing company for sequencing. After sequencing was confirmed to be correct, the amplified plasmid was transfected into CHO cells. After purification, one of them had good activity, and the remaining one clone would be transfected; the 10 positive clones with the highest OD values ​​in the second round of flow ELISA screening were PCR amplified, transformed and sequenced. After sequencing was confirmed to be correct, the amplified plasmid was transfected into CHO cells, and the volume of 72mL was 1×10 6 The cells / mL density was inoculated and cultured for 11 days (16H5 clone was cultured for 13 days). The supernatant was taken for preliminary ELISA test and 5 high-titer antibodies were screened for purification. The expression supernatants of 16H5, 14D9, 5F6, 20D8 and 20C3 clones were purified by GE MabSelect resin to obtain 3.35 mg, 6.48 mg, 3.024 mg, 3.312 mg and 3.822 mg of protein.

[0116] Antibody titer detection after purification: (1) FGF21 coating: prepare 5 μg / mL FGF21, 100 μL / well, incubate at 4°C overnight; (2) Blocking: add 3% BSA-PBST 300 μL / well, incubate at 37°C for 90 min; (3) Washing: wash 3 times with PBST 300 μL / well; (4) Primary antibody incubation: gradient diluted antibody (1 μg / mL, 2-fold gradient dilution, 7 gradients) 100 μL / well, incubate at 37°C for 60 min; (5) Washing: wash 3 times with PBST 300 μL / well; (6) Secondary antibody incubation: HRP-labeled goat anti-rabbit antibody Goatanti Rabbit (H+L)-HRP 100 μL / well, incubate at 37°C for 30 min; (7) Washing: wash with PBST 300 μL / well, wash the plate 3 times; (8) Color development: add TMB substrate solution 100 μL / well, incubate at 37°C for 10 min; (9) Termination: 2M HCl, 50 μL / well to terminate the reaction; (10) Read the plate: read the plate with an ELISA reader at a wavelength of 450-620 nm, and obtain 5 antibodies with higher detection titers (titers are all higher than 50,000).

[0117] Example 5 Detection of FGF21 protein antibody matching array experiment comparison screening

[0118] (1) Antibody coating: CBS diluted antibody to 1 μg / ml, 100 μL / well, 4°C overnight; (2) Blocking: 3% BSA-PBST, 300 μL / well, incubate at 37°C for 90 min; (3) Washing: PBST, 300 μL / well, wash 3 times; (4) Antigen incubation: CBS solution diluted FGF21 to 0.1 μg / mL, 100 μL / well, incubate at 37°C for 1 h; (5) Washing: PBST, 300 μL / well, wash 3 times; (6) Biotinylated antibody incubation: PBS diluted to 0.5 μg / mL, 100 μ L / well, incubate at 37℃ for 1h; (7) Wash: PBST, 300μL / well, wash 3 times; (8) Streptavidin-HRP incubation: PBS1:10000 dilution, 100μL / well, incubate at 37℃ for 30 minutes; (9) Wash: PBST, 300μL / well, wash 3 times; (10) TMB color development: 100μL / well, incubate at 37℃ for 10min; (11) Stop: 2M HCl, 50μL / well. (12) Reading: Read the plate with a wavelength of 450nm-620nm on an ELISA reader. The experimental results of the paired array ELISA detection of FGF21 are shown in Tables 1 and 2 below, where Table 2 is the experimental results of the negative control. The antibody pairing screening was completed, and a total of 3 pairs of antibodies with high signal values ​​were obtained.

[0119] Table 1

[0120] Antigen 16H5 14D9 5F6 20C3 20D8 16H5-Biotin 0.33 0.24 0.24 0.29 0.26 5F6-Biotin 0.20 0.19 0.44 2.83 0.99 20C3-Biotin 0.41 0.39 2.84 0.76 0.30 20D8-Biotin 0.26 0.21 2.72 0.21 0.25

[0121] Table 2

[0122] PBS 16H5 14D9 5F6 20C3 20D8 16H5-Biotin 0.30 0.21 0.20 0.25 0.25 5F6-Biotin 0.17 0.12 0.32 0.13 0.16 20C3-Biotin 0.34 0.22 0.24 0.56 0.24 20D8-Biotin 0.22 0.17 0.18 0.21 0.24

[0123] Among them, the heavy chain amino acid sequence of 5F6 is shown in SEQ ID NO:5, and the light chain amino acid sequence is shown in SEQ ID NO:6.

[0124] The amino acid sequence of the heavy chain of 20C3 is shown in SEQ ID NO:9, and the amino acid sequence of the light chain is shown in SEQ ID NO:10.

[0125] The heavy chain amino acid sequence of 16H5 is shown in SEQ ID NO:13, and the light chain amino acid sequence is shown in SEQ ID NO:14.

[0126] The heavy chain amino acid sequence of 14D9 is shown in SEQ ID NO:15, and the light chain amino acid sequence is shown in SEQ ID NO:16.

[0127] The heavy chain amino acid sequence of 20D8 is shown in SEQ ID NO:17, and the light chain amino acid sequence is shown in SEQ ID NO:18.

[0128] Among them, the nucleotide sequence encoding the 5F6 heavy chain is shown in SEQ ID NO:7, and the nucleotide sequence encoding the 5F6 light chain is shown in SEQ ID NO:8.

[0129] The nucleotide sequence encoding the 20C3 heavy chain is shown in SEQ ID NO:11, and the nucleotide sequence encoding the 20C3 light chain is shown in SEQ ID NO:12.

[0130] The nucleotide sequence encoding the 16H5 heavy chain is shown in SEQ ID NO:19, and the nucleotide sequence encoding the 16H5 light chain is shown in SEQ ID NO:20.

[0131] The nucleotide sequence encoding the 14D9 heavy chain is shown in SEQ ID NO:21, and the nucleotide sequence encoding the 14D9 light chain is shown in SEQ ID NO:22.

[0132] The nucleotide sequence encoding the 20D8 heavy chain is shown in SEQ ID NO:23, and the nucleotide sequence encoding the 20D8 light chain is shown in SEQ ID NO:24.

[0133] Example 6 Preliminary comparative screening of antibody pair performance

[0134] (1) Antibody coating: dilute the antibody to 0.2 μg / mL in CBS solution, 100 μL / well, incubate at 4°C overnight; (2) Blocking: 3% BSA-PBST, 300 μL / well, incubate at 37°C for 90 min; (3) Washing: PBST, 300 μL / well, wash 3 times; (4) Antigen incubation: CBS gradient dilute FGF21 to 400 pg / mL-6.25 pg / mL, 100 μL / well, incubate at 37°C for 1 h; (5) Washing: PBST, 300 μL / well, wash 3 times; 6) Biotin-labeled antibody: diluted to 0.1 μg / mL in PBS, 100 μL / well, incubated at 37°C for 1 h; (7) Washing: PBST, 300 μL / well, wash 3 times; (8) Streptavidin-HRP incubation: PBS1:10000 diluted, 100 μL / well, incubated at 37°C for 30 min; (9) Washing: PBST, 300 μL / well, wash 3 times; (10) TMB color development: 100 μL / well, incubated at 37°C for 10 min; (11) Stop: 2M HCl, 50 μL / well; (12) Reading: The plate was read using an ELISA reader at a wavelength of 450-620 nm. The test results are shown in Table 3 below. The standard curve of the antibody pair was determined. Among the three antibody pairs, 5F6 and 20C3 clones had the best pairing effect and the highest signal value. With 5F6 as the capture antibody and 20C3-Biotin as the detection antibody, the detection limit could reach 6.25 pg / mL, and the preliminary standard curve range could reach 400-6.25 pg / mL.

[0135] Table 3

[0136] Coating Ab 5F6 5F6 20C3 FGF21 (pg / mL) / / / 400 2.46 1.45 2.16 200 1.26 0.82 1.16 100 0.77 0.46 0.63 50 0.40 0.25 0.37 25 0.27 0.12 0.24 12.5 0.20 0.09 0.18 6.25 0.12 0.08 0.10 0 0.09 0.05 0.08 Detection Ab 20C3-Biotin 20D8-Biotin 5F6-Biotin

[0137] Example 7 5F6 and 20C3-Biotin Antibodies Standard Curve and Sensitivity Methodology Verification

[0138] The methodological optimization of Example 6 was carried out, and the corresponding detection method was also adjusted. The specific steps were as follows: (1) Antibody coating: CBS solution diluted the antibody to 0.2 μg / mL, 100 μL / well, 4°C overnight; (2) Blocking: 3% BSA-PBST, 300 μL / well, incubated at 37°C for 90 min; (3) Washing: PBST, 300 μL / well, wash the plate 3 times; (4) Antigen incubation: 0.5% BSA-PBST gradient dilution of FGF21 to 781.3 pg / mL-0.7629 pg / mL, blank wells were 0.5% BSA-PBST diluent, 100 μL / well, incubate at 37°C for 90 min; (5) Washing: PBST, 300 μL / well, wash 3 times; (6) Biotin-labeled antibody: PBS diluted to 0.1 μg / mL, 100 μL / well, incubate at 37°C for 1 h; (7) Washing: PBST, 300 μL / well, wash 3 times; (8) Streptavidin-HRP incubation: PBS1:32250 dilution, 100 μL / well, incubate at 37°C for 30 min; (9) Washing: PBST, 300 μL / well, wash 3 times; (10) TMB color development: 100 μL / well, incubate at room temperature in the dark for 4 min; (11) Stop: 2 M HCl, 50 μL / well; (12) Reading: Read the plate at a wavelength of 450-620 nm using an ELISA reader. When fitting all data curves, subtract the blank well data. The method was validated methodologically, including standard curve and detection sensitivity.

[0139] According to the concentration of FGF21 human plasma samples in the literature and the methodological requirements of the research purpose, the acceptable standards for the ELISA methodology using antibody pairs for detection are determined as follows: the standard curve is fitted using the Logistic-four-parameter method, and within the range of the standard curve, the back-calculated concentration CV(%) of each standard point duplicate well is ≤20.0% (the highest limit of quantification ULOQ and the lowest limit of quantification LLOQ ≤25.0%), and the Bias(%) between the back-calculated concentration and the theoretical concentration (Bias(%) = (|back-calculated concentration-theoretical concentration|) / theoretical concentration × 100%) should be within 20.0% (ULOQ, LLOQ within 25.0%). Total error = Bias% + CV%, the total error of each standard point is ≤30.0% (ULOQ, LLOQ ≤40.0%). The standard curve is as follows Figure 1 The relevant indicators are shown in Table 4 below. The data show that the standard curve can well meet the above methodological standards, and the lowest sensitivity can reach 0.7629pg / mL, which is sufficient for sample detection. The standard curve adopts Logistic-four-parameter fitting: y=6.52055+((-0.00014935-6.52055) / (1+(x / 687.034) ^ 0.982126)), R 2=1.

[0140] Table 4

[0141] Theoretical concentration of standard point (pg / mL) Back-calculated concentration (pg / mL) Detection of duplicate well concentration CV (%) Bias(%) Total error (%) 781.3 782.9 5.167 0.2048 5.371 390.6 388.5 4.914 0.5376 5.452 195.3 197.7 3.231 1.229 4.460 97.66 97.48 0.2175 0.1843 0.4018 48.83 47.14 3.561 3.461 7.022 24.41 25.00 3.733 2.417 6.150 12.21 12.50 1.233 2.375 3.608 6.104 6.463 2.755 5.881 8.636 3.052 3.136 16.20 2.752 18.95 1.526 1.626 10.94 6.553 17.49 0.7629 0.7613 8.845 0.2097 9.055

[0142] Example 8 Determination of human plasma FGF21

[0143] (1) Transfer the blood from the sodium heparin blood collection tube to a 50 mL centrifuge tube;

[0144] (2) Carefully pour 20-30 mL of blood into two centrifuge tubes containing 15 mL of lymphocyte separation solution.

[0145] (3) Centrifuge at 800 g for 20 min at room temperature, with the centrifuge speed slowly increasing and decreasing. If the blood is stored for more than 2 hours, the centrifugation time is increased to 30 min.

[0146] (4) After centrifugation, the blood is separated into four layers, consisting of a plasma layer (top layer), a mononuclear cell layer (second layer), a separation fluid layer (third layer), and a red blood cell layer (bottom layer);

[0147] (5) Use a pipette to collect 2 mL of plasma into a new centrifuge tube and store it at -80°C until it is taken out before testing;

[0148] (6) According to the method in Example 7, the concentration of the standard was diluted 2 times in sequence, and the standard points of the standard curve were: 768.0pg / mL-1.500pg / mL, and the blank well was 0.5% BSA-PBST diluent. According to the actual requirements of the sample concentration range for the sensitivity of the ELISA method, the ULOQ of the standard curve was set to 768pg / mL and the LLOQ was set to 6pg / mL. 100μL of two human plasma samples were added, and two technical replicates were added to each sample. At the same time, 0.5% BSA-PBST was used to dilute the samples 2 times and 3 times (this dilution factor was obtained based on the previous preliminary experimental data) for detection and comparison to clarify whether there was a matrix effect, and quality control samples (QC) were added to the diluted samples. After the quality control samples were added, the final concentration was set to 3 levels, HQC = 75%, ULOQ = 576pg / mL, MQC = 192pg / mL, and LQC = 15pg / mL.

[0149] The acceptance criteria for the analysis batch are as follows: the acceptance criteria for the standard curve are the same as those in Example 7, the concentration replicate CV (%) of all samples is ≤20.0%; the concentration recovery rate of the quality control sample (recovery = QC measured value × 100% / QC theoretical value) should be 80% ≤ recovery ≤ 120%, of which the recovery rate of LQC should be 75% ≤ recovery ≤ 125%. Each plate should contain at least 2 sets of 3 levels (low, medium and high concentration) of replicate quality control samples. In the validation of the test sample testing process, the number of replicates of the quality control samples should be consistent with the test sample analysis, both with 2 replicates. At least 80% of the quality control samples on each plate should meet the standard of accuracy of 80% ≤ recovery ≤ 120%, of which the recovery rate of LQC should be 75% ≤ recovery ≤ 125%, and at least 50% of the quality control samples at each concentration level meet the above standards.

[0150] Standard curve see Figure 2 , after fitting using the Logistic-four-parameter method, the formula is: y = 5.9687 + ((0.0116429-5.9687) / (1 + (x / 519.174) ^ 1.08562)), R 2 = 1. As shown in Table 5, the standard point index of the standard curve fully meets the same acceptance criteria described in Example 7, and the concentration replicates of all samples also meet CV (%) ≤ 20.0%.

[0151] The test results of three detection methods for human plasma samples (experimental group 1 without dilution, experimental group 2 with 2-fold dilution, and experimental group 3 with 3-fold dilution) are shown in Table 6 below. The detection concentration CV (%) of experimental group 2 and experimental group 3 is ≤10.0%. It can be considered that diluting the sample by 2 times or more can effectively remove the influence of the matrix effect. The sample detection results are taken from the experimental results of 3-fold dilution (CV is more stable and the QC sample recovery rate is better). The concentrations of samples 1 and 2 are 207.6 pg / mL and 119.1 pg / mL, respectively.

[0152] The recovery data of the quality control samples are shown in Table 7, which meets the above-mentioned analytical batch acceptance criteria. The data also show that the recovery rate of the sample diluted 3 times is better overall, suggesting that 3-fold dilution is most appropriate for sample determination.

[0153] Conclusion: The data show that the antibody pair of the present invention can detect FGF21 in human plasma samples with high accuracy, high precision and high sensitivity. The present invention is currently the method with the highest sensitivity while ensuring the accuracy and precision of biological analysis.

[0154] Table 5

[0155] Theoretical concentration of standard point (pg / mL) Back-calculated concentration (pg / mL) Detection of duplicate well concentration CV (%) Bias(%) Total error (%) 768.0 761.7 0.2922 0.8203 1.113 384.0 395.5 1.113 2.995 4.108 192.0 183.0 0.3915 4.688 5.080 96.00 97.20 2.122 1.250 3.372 48.00 49.81 2.058 3.771 5.829 24.00 25.44 1.618 6.000 7.618 12.00 13.20 1.222 10.00 11.22 6.000 6.266 5.243 4.433 9.676 3.000 3.250 8.973 8.333 17.31 1.500 1.359 0.6972 9.400 10.10

[0156] Table 6

[0157]

[0158] Table 7

[0159]

[0160]

Claims

1. A human FGF21 rabbit monoclonal antibody, characterized in that: It comprises a first monoclonal antibody or a second monoclonal antibody, wherein: The first monoclonal antibody comprises: (1) Heavy chain variable region CDR1, whose amino acid sequence is GIDLSSNA; (2) Heavy chain variable region CDR2, whose amino acid sequence is IGITGNR; (3) Heavy chain variable region CDR3, whose amino acid sequence is VRGVISDNINGI; (4) light chain variable region CDR1, whose amino acid sequence is QSLYDNW; (5) Light chain variable region CDR2, whose amino acid sequence is KVS; (6) light chain variable region CDR3, whose amino acid sequence is AGGYSGNIHA; The second monoclonal antibody comprises: (1) Heavy chain variable region CDR1, whose amino acid sequence is GFSLSSYR; (2) Heavy chain variable region CDR2, whose amino acid sequence is ITSSGNT; (3) heavy chain variable region CDR3, whose amino acid sequence is ARETYDNYGSRFNI; (4) light chain variable region CDR1, whose amino acid sequence is QSISSY; (5) Light chain variable region CDR2, whose amino acid sequence is AAS; (6) Light chain variable region CDR3, whose amino acid sequence is QSNFLITTYGGA.

2. The human FGF21 rabbit monoclonal antibody according to claim 1, characterized in that: The heavy chain constant region of the first monoclonal antibody and the second monoclonal antibody has an amino acid sequence as shown in SEQ ID NO:1, and the light chain constant region has an amino acid sequence as shown in SEQ ID NO:

2.

3. The human FGF21 rabbit monoclonal antibody according to claim 1 or 2, characterized in that: The nucleotide sequence encoding the heavy chain constant region is SEQ ID NO: 3 and its synonymous nucleotide sequence, and the nucleotide sequence encoding the light chain constant region is SEQ ID NO: 4 and its synonymous nucleotide sequence.

4. The human FGF21 rabbit monoclonal antibody according to claim 1, characterized in that: The first monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain has an amino acid sequence as shown in SEQ ID NO:5, and the light chain has an amino acid sequence as shown in SEQ ID NO:

6.

5. The human FGF21 rabbit monoclonal antibody according to claim 1 or 4, characterized in that: The nucleotide sequence encoding the heavy chain of the first monoclonal antibody is SEQ ID NO: 7 and its synonymous nucleotide sequence, and the nucleotide sequence encoding the light chain of the first monoclonal antibody is SEQ ID NO: 8 and its synonymous nucleotide sequence.

6. The human FGF21 rabbit monoclonal antibody according to claim 1, characterized in that: The second monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain has an amino acid sequence as shown in SEQ ID NO:9, and the light chain has an amino acid sequence as shown in SEQ ID NO:

10.

7. The human FGF21 rabbit monoclonal antibody according to claim 1 or 6, characterized in that: The nucleotide sequence encoding the heavy chain of the second monoclonal antibody is SEQ ID NO: 11 and its synonymous nucleotide sequence, and the nucleotide sequence encoding the light chain of the second monoclonal antibody is SEQ ID NO: 12 and its synonymous nucleotide sequence.

8. A kit, characterized in that: It comprises a capture antibody and a detection antibody, wherein the capture antibody is one of the first monoclonal antibody and the second monoclonal antibody according to any one of claims 1 to 7, and the detection antibody is the other of the first monoclonal antibody and the second monoclonal antibody according to any one of claims 1 to 7.

9. The kit according to claim 8, characterized in that: The capture antibody is the first monoclonal antibody, and the detection antibody is the second monoclonal antibody.

10. The kit according to claim 8 or 9, characterized in that: The kit is an enzyme-linked immunosorbent assay kit.

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