A diabetic retinopathy-related detection metabolite and detection kit thereof

Abstract

The present invention provides an enzyme-linked immunosorbent assay (ELISA) kit and detection method for accurately quantifying the content of ANGPTL8 in samples from subjects. The kit and detection method are simple to operate, can rapidly and simultaneously detect a large number of samples, and have the characteristics of high sensitivity and high specificity. It is also the first kit in China and even internationally that can accurately quantify the content of ANGPTL8 in samples from subjects.

Description

Field of the Invention

[0001] The present invention relates to the field of biological detection, and in particular to a diabetic retinopathy-related detection metabolite and a detection kit thereof. Background Art

[0002] Diabetic retinopathy (DR) is the leading cause of blindness in people aged 20 to 70 years. The direct cause of DR remains unclear. In recent years, the view that DR is solely a diabetic microvascular disease has been challenged. Evidence indicates that all cell types within the retina are involved in multiple disease processes, even in the early stages of diabetes. Early clinical features of DR in patients, such as breakdown of the blood-retinal barrier (BRB) and various visual impairments, support this view. Because DR progresses gradually but rapidly, treatment should be initiated before DR reaches an irreversible stage. However, all current treatments target advanced DR, in which pathological studies demonstrate irreversible retinal vascular and neuronal cell death. Some treatments have only limited effects on visual function or vascular leakage, with most reportedly only preventing further damage.

[0003] Currently, the cytokines or proteins associated with DR progression include the following four categories: (1) factors related to regulating natural immunity, such as IL-1b and IL-10; (2) factors that regulate lymphocyte activation, proliferation, and differentiation, such as IL-6 and IL-12; (3) factors related to macrophage activation, such as TNFa and TGFb; and (4) chemokines, such as MCP-1 and SDF-1. Recent studies have shown that inflammation, oxidative stress, and abnormal cytokine expression are associated with the onset and development of DR. However, the onset of DR is a complex process, and its specific pathogenesis remains to be further explored. Furthermore, in specific research, the criteria for judging various markers vary, and there is a lack of gold standard judgment indicators. Furthermore, most of these tests are qualitative, and there is currently a lack of means and methods for quantitative detection.

[0004] Angiopoietin-like protein 8 (ANGPTL8) is a new type of adipokine that is mainly secreted in the human liver and adipose tissue. It can dose-dependently inhibit the activity of lipoprotein lipase (LPL) and thus increase the level of plasma TG. Because ANGPTL8 plays a role in lipid metabolism and blood sugar regulation, it is expected to become an effective target gene for the treatment of blood sugar and blood lipid disorders. In addition, existing technologies have also confirmed that the expression level of ANGPTL8 also varies in different stages of diabetic retinopathy. However, the existing evidence and existing technologies for the detection of this protein are still at the qualitative stage. Therefore, the accuracy and application value of the currently available detection methods still need to be improved, and quantitative detection still has high research value and room for improvement. Summary of the Invention

[0005] Based on this, the present application provides an enzyme-linked immunosorbent assay (ELISA) quantitative detection kit and its detection method, which can accurately quantify the ANGPTL8 content in samples from subjects, and has the characteristics of simple operation, ability to rapidly detect a large number of samples simultaneously, high sensitivity, and high specificity.

[0006] To achieve the above objectives, the present invention first provides a monoclonal antibody M6AD that specifically recognizes human ANGPTL8. The monoclonal antibody can specifically recognize ANGPTL8 in the blood of a subject and is expected to be used for the early diagnosis and prediction of DR.

[0007] Furthermore, the amino acid sequence of the heavy chain variable region of the monoclonal antibody M6AD that specifically recognizes human ANGPTL8 is shown in SEQ ID NO: 1 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2 in the sequence listing.

[0008] The specific sequence is:

[0009] Heavy chain variable region amino acid sequence

[0010] VQLQASGPGLVGILSEVQLQESEPYSITSDYAWNWIRQSPGNKLEWLGYINYWNWIRKFPGNKLRDTSKNQFFLQL NSVYYLQLNSVTTEATATYPCARFITTTVSAAWGAGTTVTVSS(SEQ ID NO.1)

[0011] Light chain variable region amino acid sequence

[0012] DIVMSQSSVSTVSVGEKVTMSCKSSASQSIGYNQKNFESVSGIKPGQSPLLIYWASTRESSGGSGTDSGTDFTLTI SSADYYCQQVYCQQYYSSWTFGGAGTKLELKR(SEQ ID NO.2)

[0013] Furthermore, the antibody may be modified with a label; the label is preferably an enzyme, fluorescein, chemiluminescent substance or fluorescence;

[0014] The monoclonal antibody obtained in this application is derived from a full-length recombinant human ANGPTL8 protein using a classic hybridoma cell line screening method. It combines the advantages of simple immunogen preparation and the ability of the resulting antibody to recognize the native protein. The monoclonal antibody recognizes human ANGPTL8 with a titer exceeding 256,000.

[0015] Furthermore, the present invention also provides a gene encoding the above-mentioned monoclonal antibody, wherein the gene encoding the heavy chain variable region gene comprises a heavy chain variable region gene; the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO. 3, specifically:

[0016] gtgcagctgcaggcgagcggcccgggcctggtgggcattctgagcgaagtgcagctgcaggaaagcgaaccgtatagcat

[0017] taccagcgattatgcgtggaactggattcgccagagcccgggcaacaaactggaatggctgggctatattaactattgga

[0018] actggattcgcaaatttccgggcaacaaactgcgcgataccagcaaaaaccagttttttctgcagctgaacagcgtgtat

[0019] tatctgcagctgaacagcgtgaccaccgaagcgaccgcgacctatccgtgcgcgcgctttattaccaccaccgtgagcgcggcgtggggcgcgggcaccaccgtgaccgtgagcagc;

[0020] The coding gene also includes a light chain variable region gene; the nucleotide sequence of the light chain variable region is shown in SEQ ID NO.4, specifically:

[0021] gatattgtgatgagccagagcagcgtgagcaccgtgagcgtgggcgaaaaagtgaccatgagctgcaaaagcagcgcgag

[0022] ccagagcattggctataaccagaaaaactttgaaagcgtgagcggcattaaaccgggccagagcccgctgctgatttatt

[0023] gggcgagcacccgcgaaagcagcggcggcagcggcaccgatagcggcaccgattttaccctgaccattagcagcgcggat

[0024] tattattgccagcaggtgtattgccagcagtattatagcagctggacctttggcggcgcgggcaccaaactggaactgaaacgc.

[0025] Furthermore, the present invention also provides a recombinant vector into which the gene encoding the monoclonal antibody is inserted.

[0026] Furthermore, the present invention also provides a host cell, which carries the above-mentioned recombinant vector.

[0027] Furthermore, the present invention also provides a product for quantitatively detecting the content of ANGPTL8 in a sample, wherein the product comprises the aforementioned monoclonal antibody or the aforementioned encoding gene or the aforementioned recombinant vector or the aforementioned host cell.

[0028] The terms "sample" and "specimen" are used interchangeably herein and, when used herein, refer to a composition obtained or derived from a subject (e.g., an individual of interest) that contains cells and / or other molecular entities to be characterized and / or identified based on, for example, physical, biochemical, chemical, and / or physiological characteristics. For example, the phrase "disease sample" or variations thereof refers to any sample obtained from a subject of interest that is expected or known to contain cells and / or molecular entities to be characterized. Samples include, but are not limited to, tissue samples (e.g., tumor tissue samples), primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous humor, lymph fluid, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tumor lysates, and tissue culture fluid, tissue extracts such as homogenized tissue, tumor tissue, cell extracts, and combinations thereof.

[0029] As a preferred embodiment, the sample is selected from blood, serum, and plasma.

[0030] Furthermore, the product is an enzyme-linked immunosorbent assay kit for accurately quantifying the ANGPTL8 content in a sample from a subject to be tested. The enzyme-linked immunosorbent assay kit includes a capture antibody, which is the monoclonal antibody M6AD.

[0031] Furthermore, the enzyme-linked immunosorbent assay kit includes an enzyme-labeled plate coated with a capture antibody, a sample diluent, a standard, a detection antibody, avidin labeled with horseradish peroxidase, a concentrated washing solution, a substrate solution, and a stop solution;

[0032] Wherein, the capture antibody is the monoclonal antibody M6AD;

[0033] The sample diluent is PBS buffer added with a final concentration of 1% BSA, 0.05% Tween-20 and 0.1% Proclin300;

[0034] The standard product is a human ANGPTL8 protein standard product;

[0035] The detection antibody is a 100 μg / mL biotin-labeled detection antibody A7 (PBS, 50% glycerol) solution. The amino acid sequence of the heavy chain variable region of the detection antibody is:

[0036] EVQLVESGPGVSGGAEALVRPGAASGFNIKDTYIHWVRAIYWAKQSHVGLEWVARIYPAIYSVKQSHVDTSHNTAY LQMNSLRAEDGAVYLARPHSEDSAIYHYCVRDLVFYAMDYWGQGTLVLVSS(SEQ ID NO.5)

[0037] Light chain variable region amino acid sequence:

[0038] DIQMAQSPSSLSASLGDRVTHTCRASQDINKYIAWFQHKPGKAPKLIYSASFLYSAVPSRFSGSGRDYSFTLTISL QPEDFATYCLQYDTTPPTFGQGTKVEIKRTV(SEQ ID NO.6)

[0039] The concentrated washing solution is 10× PBS containing a final concentration of 1.0% Tween-20;

[0040] The substrate solution is a TMB colorimetric substrate solution;

[0041] The stop solution is 2M sulfuric acid.

[0042] Furthermore, the present invention also provides a method for accurately quantifying the amount of ANGPTL8 in a sample from a subject using the above-mentioned kit, comprising the following steps:

[0043] 1) Place the sample treatment solution, biotin-labeled detection antibody, standard, horseradish peroxidase-labeled avidin, biotin-labeled antibody diluent, horseradish peroxidase-labeled avidin diluent, concentrated wash solution, substrate solution, and stop solution at 18-25°C and equilibrate for at least 30 minutes;

[0044] 2) Set up standard wells and test sample wells respectively, add the standard or test sample to each well and incubate, then discard the liquid, spin dry, add biotin-labeled detection antibody working solution and incubate, discard the liquid in the wells, wash and spin dry, add horseradish peroxidase-labeled avidin working solution to each well, incubate, discard the liquid in the wells, wash and spin dry, add substrate solution to each well, protect from light and develop color, add stop solution to each well, mix well, and measure the optical density (OD) value of each well at a wavelength of 450 nm using a microplate reader within 5 minutes after the reaction is terminated;

[0045] 3) Data processing: Draw a curve by subtracting the value of well S0 from the standard and sample values. If duplicate wells are set, take the average value and calculate the standard curve with the concentration of the standard as the ordinate and the OD value as the abscissa. According to the sample OD value, the corresponding concentration can be found from the standard curve.

[0046] Furthermore, the detection method provided by the present invention can specifically detect ANGPTL8 in samples with a sensitivity of 13.4 pg / ml, precision: intra-assay CV% <4%, inter-assay CV% <7%, and no cross-reaction with other related proteins.

[0047] Beneficial effects

[0048] The enzyme-linked immunosorbent assay (ELISA) kit and its detection method for accurately quantifying the ANGPTL8 content in samples from test subjects provided by the present invention are simple to operate, can rapidly and simultaneously detect a large number of samples, and have the characteristics of high sensitivity and high specificity. It is also the first kit in China and even internationally that can accurately quantify the ANGPTL8 content in samples from test subjects. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Monoclonal antibody M6AD was purified by Protein A affinity chromatography and detected by SDS-PAGE. Band 1 represents the purified antibody, and band 2 represents BSA protein (control). M represents a protein molecular weight marker. Figure 2The standard curve for ANGPTL8 detection was prepared by multiplying the ANGPTL8 standard concentration by the sample dilution factor (×5) on the ordinate and the corresponding measured OD450 value on the abscissa.

[0050] Figure 3 ANGPTL2, ANGPTL3, ANGPTL4, and ACE-specific assay results. Example

[0051] The present invention is described in detail below with reference to the accompanying drawings and examples. The examples described below are merely preferred embodiments of the present invention. It should be noted that the following description is merely for the purpose of explaining the present invention and does not limit the present invention in any form. Any simple modifications, equivalent changes, and modifications made to the embodiments based on the technical essence of the present invention fall within the scope of the technical solution of the present invention.

[0052] In the following examples, the materials, plasmids, reagents, etc. used were obtained from commercial sources unless otherwise specified.

[0053] Example 1 Screening and Preparation of Human ANGPTL8-Specific Monoclonal Antibodies

[0054] Human ANGPTL8 (angiopoietin like 8 [Homo sapiens], GenBank: KAI2588810.1) was amplified and then digested with double enzymes and inserted into the prokaryotic expression vector pET30a(+) for screening and identification. After selecting a positive plasmid that correctly expresses human ANGPTL8, ultrapure water and lysis buffer were added to a nickel affinity chromatography column in sequence to equilibrate the purification column; the sample was loaded onto the equilibrated Ni-NTA purification column, the bottom seal of the column was opened, the flow-through was collected and loaded a second time (retained for later use); the column was washed with wash buffer (50 mmol / L Na2HPO4, 10 mmol / L KH2PO4, 2.7 mmol / L KCl, 137 mmol / L NaCl, 20 mmol / L imidazole, pH 8.0); and the target protein was eluted with elution buffer (50 mmol / L Na2HPO4, 10 mmol / L KH2PO4, 2.7 mmol / L KCl, 137 mmol / L NaCl, 250 mmol / L imidazole, pH 8.0).

[0055] Female BALB / c mice aged 6 to 8 weeks were immunized five times with purified human ANGPTL8 protein. The primary immunization consisted of 100 μg of human ANGPTL8 protein emulsified in an equal volume of Freund's complete adjuvant and injected intraperitoneally. A second immunization was performed one month later, with 60 μg of the immunogen emulsified in an equal volume of Freund's incomplete adjuvant and injected intraperitoneally. Serum was collected from the canthus of the eye one week after injection for titer analysis. A third immunization was performed one month later, and a fourth booster immunization was performed two months later, using the same protocol as the second immunization. Finally, a pulse immunization was performed, with 100 μg of the immunogen injected intraperitoneally per mouse. Three to four days after the injection, spleen cells were harvested and fused with Sp2 / 0-AG14 cells. Female BALB / c mice aged 6 to 8 weeks were sacrificed by cervical dislocation. The peritoneal cavity was aseptically irrigated several times with incomplete RPMI 1640 medium. The irrigated fluid was centrifuged at 1000 rpm for 10 minutes, and the supernatant discarded. Resuspend the cells in complete RPMI 1640 medium and count the cells. Adjust the cell concentration to 2 × 10 5 / mL, added to 96-well plate, 100 μL / well, and incubated in a 37°C, 5% CO2 incubator.

[0056] Take mice 3-4 days after immunization, bleed their eyeballs, and collect pre-fusion serum. Kill the mice by cervical intubation and remove the spleen aseptically. Prepare a single-cell suspension, mix the spleen cells with Sp2 / 0-AG14 cells in the logarithmic growth phase at a ratio of 4:1, centrifuge at 1000rpm for 10 minutes, and discard the supernatant. Resuspend the cells in 40-60mL of complete RPMI 1640 medium and inoculate them into a 96-well plate containing feeder cells at 100μL / well. Add 50μL of 5×HAT to each well on the second day. Incubate in a 37°C, 5% CO2 incubator and observe the cell growth status day by day. On the 5th day after the fusion is completed, use 1×HAT to replace the cell medium, and on the 14th day, use 1×HT to replace the cell medium.

[0057] When the cell clones have grown to 1 / 3 of the visual field, carefully aspirate the cell supernatant and detect antibodies in the supernatant using an indirect ELISA assay with 400 ng / mL ANGPTL8 protein as the coating antigen. Repeatedly clone wells positive for antibody secretion using the limiting dilution method until all monoclonal wells have a 100% antibody-positive supernatant.

[0058] The cloned positive clones were sequentially transferred into 24-well plates, 6-well plates and T25 cell culture flasks, stably cultured for 90 days and then established.

[0059] Hybridoma cells were inoculated into the peritoneal cavity of BABL / c mice pretreated with pristane and the cell density was adjusted to 1-2 × 10 7Each mouse was injected with 500 μL of cell suspension. After 7 to 10 days, ascites was extracted and the monoclonal antibody M6AD was purified by Protein A affinity chromatography using the AKTAexplore100 protein purification system. The purified antibody was detected by SDS-PAGE. Figure 1 As shown, band 1 is the purified antibody, band 2 is the BSA protein (control), and M is a protein molecular weight marker.

[0060] Total RNA was extracted from hybridoma cells in the logarithmic growth phase using Trizol reagent and subjected to 1% agarose gel electrophoresis. The heavy and light chain variable regions of the anti-human ANGPTL8 monoclonal antibody gene were amplified using RT-PCR. Primers were designed based on conserved sequences in the light and heavy chains of mouse IgG, respectively. First-strand cDNA was synthesized using Oligo(d)T primers and SuperScript II reverse transcriptase using the GoldScript cDNA synthesis kit instructions, using total RNA as a template. The product was recovered and purified using a PCR product recovery kit (Omega) and sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing analysis.

[0061] The amino acid sequence of the heavy chain variable region of the monoclonal antibody M6AD is:

[0062] VQLQASGPGLVGILSEVQLQESEPYSITSDYAWNWIRQSPGNKLEWLGYINYWNWIRKFPGNKLRDTSKNQFFLQL NSVYYLQLNSVTTEATATYPCARFITTTVSAAWGAGTTVTVSS(SEQ ID NO.1), light chain variable region amino acid sequence: DIVMSQSSVSTVSVGEKVTMSCKSSASQSIGYNQKNFESVSGIKPGQSPLLIYWASTRESSGGSGTDSGTDFTLTISSAD YYCQQVYCQQYYSSWTFGGAGTKLELKR (SEQ ID NO.2)

[0063] The nucleotide sequence of its heavy chain variable region is:

[0064] gtgcagctgcaggcgagcggcccgggcctggtgggcattctgagcgaagtgcagctgcaggaaagcgaaccgtatagcattaccagcgattatgcgtggaactggattcgccagagcccgggcaacaaactggaatggctgggctatattaactattggaactggattcgcaaatttccg ggcaacaaactgcgcgataccagcaaaaaccagttttttctgcagctgaacagcgtgtattatctgcagctgaacagcgtgaccaccgaa gcgaccgcgacctatccgtgcgcgcgctttattaccaccaccgtgagcgcggcgtggggcgcgggcaccaccgtgaccgtgagcagc(SEQ ID NO.3);

[0065] The nucleotide sequence of the light chain variable region is:

[0066] gatattgtgatgagccagagcagcgtgagcaccgtgagcgtgggcgaaaaagtgaccatgagctgcaaaagcagcgcgagccagagcattggctataaccagaaaaactttgaaagcgtgagcggcattaaaccgggccagagcccgctgctgattta ttgggcgagcacccgcgaaagcagcggcggcagcggcaccgatagcggcaccgattttaccctgaccattagcagcgcggattattattgccagcaggtgtattgccagcagtattatagcagctggacctttggcggcgcgggcaccaaactggaac tgaaacgc (SEQ ID NO. 4).

[0067] Example 2 Establishment of immune ELISA method

[0068] 1. Preparation of ELISA Kit

[0069] An enzyme-linked immunosorbent assay kit for accurately quantifying the ANGPTL8 content in a sample from a subject to be tested, the enzyme-linked immunosorbent assay kit comprising a microporous reaction plate coated with a capture antibody, a sample processing solution, a biotin-labeled detection antibody, an ANGPTL8 standard, avidin labeled with horseradish peroxidase, a diluent, a concentrated washing solution, a substrate solution, a stop solution, and a plate sticker.

[0070] Among them, the capture antibody is the monoclonal antibody M6AD;

[0071] The sample treatment solution was PBS buffer supplemented with a final concentration of 1% BSA, 0.05% Tween-20, and 0.1% Proclin300;

[0072] Biotin-labeled detection antibody: 100 μg / mL biotin-labeled detection antibody A7 (PBS, 50% glycerol) solution;

[0073] The amino acid sequence of the heavy chain variable region of A7 is:

[0074] EVQLVESGPGVSGGAEALVRPGAASGFNIKDTYIHWVRAIYWAKQSHVGLEWVARIYPAIYSVKQSHVDTSHNTAY LQMNSLRAEDGAVYLARPHSEDSAIYHYCVRDLVFYAMDYWGQGTLVLVSS(SEQ ID NO.5)

[0075] Light chain variable region amino acid sequence:

[0076] DIQMAQSPSSLSASLGDRVTHTCRASQDINKYIAWFQHKPGKAPKLIYSASFLYSAVPSRFSGSGRDYSFTLTISL QPEDFATYCLQYDTTPPTFGQGTKVEIKRTV(SEQ ID NO.6)

[0077] Standard: 25 ng of ANGPTL8 protein (lyophilized powder). The recombinant ANGPTL8 protein is obtained by amplifying and sequencing the ANGPTL8 encoding gene, ligating it into the pET28a plasmid after correct identification, transforming the recombinant expression vector pET28a-ANGPTL8 into competent Escherichia coli BL21 (DE3) cells, resuming the culture of the preserved ANGPTL8 expression strain, inducing protein expression with IPTG overnight at 16°C, and purifying the recombinant protein.

[0078] The concentrated washing solution is 10× PBS containing a final concentration of 1.0% Tween-20;

[0079] The substrate solution is a TMB colorimetric substrate solution;

[0080] The stop solution is 2M sulfuric acid.

[0081] Horseradish peroxidase-labeled avidin: 1:40 horseradish peroxidase-labeled avidin stock solution (Jinkairui Company), working solution diluted 1:100;

[0082] Diluent: 0.1% NaN3 0.1 mL, 1% BSA 1 g, 0.05% Tween 20 0.05 mL, and 1× PBS to a volume of 100 mL.

[0083] Concentrated washing solution: 10× PBS containing 1.0 wt% Tween-20;

[0084] Substrate solution: 0.5 mL of 2 mg / mL TMB in anhydrous ethanol, 10 mL of substrate buffer, and 32 μL of 30% H2O2. Prepare immediately.

[0085] Stop solution: 1M H2SO4.

[0086] 2. Sample Collection and Processing

[0087] A total of 182 patients with type 2 diabetes mellitus admitted to the Affiliated Hospital of Qingdao University in Shandong Province from January 2022 to December 2023 were selected as the T2DM group, and 84 healthy subjects who underwent physical examinations during the same period were selected as the control group. The T2DM group included 93 males and 89 females, aged 28 to 87 years, with a body mass index of 16.23 to 35.01 kg / m 2 The control group included 44 males and 40 females, aged 26 to 80 years, with a body mass index of 18.23 to 25.64 kg / m2. 2 . There was no statistically significant difference in age and body mass index between the two groups (P>0.05), and they were comparable. Inclusion criteria: ① Complete clinical data; ② T2DM met the diagnostic criteria of the "Guidelines for the Prevention and Treatment of Type 2 Diabetes in China (2020 Edition)" [8]. Exclusion criteria: ① Other types of diabetes; ② Acute and chronic infections in the past 3 months; ③ Combined hematopoietic, immune, and nervous system damage and malignant tumors; ④ Age <18 years; ⑤ Combined with other endocrine diseases such as thyroid, parathyroid, and adrenal glands; ⑥ History of immunosuppressant and hormone use in the past 6 months; ⑦ Previous eye diseases, congenital amblyopia, and intraocular surgery history. This study was approved by the hospital ethics committee (

[2020] 027).

[0088] 3 ml of fasting venous blood was collected from all study subjects upon admission, aliquoted, and stored at -80°C to avoid repeated freezing and thawing. Prior to testing, the samples were equilibrated at room temperature for half an hour and mixed thoroughly with vortexing. An appropriate amount of patient plasma was diluted 5-fold with diluent.

[0089] Dilute the coating antibody M6AD (1 μg / ml) in coating solution (Na2CO3 and NaHCO3) and add the coating antibody to a 96-well microtiter plate (100 μl / well) for overnight incubation at 4°C. Wash the plate three times with PBST, then add 100 μl of 3% bovine serum blocking solution to each well and block at 37°C for 1 hour.

[0090] Add 100 μl of diluted plasma sample to be tested and serially diluted soluble ANGPTL8 protein standard (starting from 1 ng / ml, with a total of 8 concentrations (1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625, 0 ng / ml), perform 3 replicates for each gradient, and incubate at 37°C for 2 h.

[0091] The plate was washed three times with PBST solution, and HRP-labeled detection antibody A7 (100 μl / well) was added at a ratio of 1:2000, and incubated in a 37°C incubator for 1 h.

[0092] The plate was washed six times with PBST solution, and the color developing solution TMB (100 μl / well) was added. The plate was incubated at room temperature in the dark for 10 min, and then the stop solution (50 μl / well) was added to terminate the color reaction. The OD450 value of each well was measured by a microplate reader.

[0093] The standard curve was prepared by multiplying the concentration of ANGPTL8 standard by the dilution factor of the sample (×5) as the vertical axis and the corresponding measured OD450 value as the horizontal axis (e.g. Figure 2 ), and obtain the calculation formula, and calculate the content of soluble ANGPTL8 in the sample according to the OD450 value of the sample to be tested.

[0094] The linear equation of the standard curve is y=0.3543x+0.1942,

[0095] Standard curve linearity: R 2 =0.988; minimum detection limit ≤13.4pg / ml.

[0096] 3. Accuracy inspection:

[0097] 1) Intraplate accuracy analysis: In the same experiment, five samples with known concentrations (500, 250, 125, 62.5, and 31.25 pg / mL) were set up in 10 replicate wells for testing to analyze the accuracy of the kit.

[0098] 2) Inter-plate accuracy analysis: In different batches of experiments, five samples with known concentrations (500, 250, 125, 62.5, and 31.25 pg / mL) were set up in 10 replicate wells for testing to analyze the accuracy of the kit.

[0099] Precision: Intra-assay CV% <4%, inter-assay CV% <7%, demonstrating that the assay has good accuracy.

[0100] 4. Specificity Investigation

[0101] ANGPTL2, ANGPTL3, ANGPTL4 and ACE were serially diluted to different concentrations. M6AD was adjusted to 5 μg / ml with carbonate buffer (0.01M CBS, pH 9.3) and coated on the ELISA plate at 4°C overnight. Washed 3 times with PBS (containing 0.1% Tween20) and blocked with 3% BSA at room temperature for 1 hour. After washing 3 times with PBS, the above-mentioned diluted commercial protein was added and reacted at room temperature for 2 hours, and washed 3 times with PBS. Then, HRP-labeled monoclonal antibody A7 (1:2000, 100 μl / well) was added and the reaction continued at room temperature for 1 hour. After washing 6 times with PBST, HRP reaction substrate TMB (100 μl / well) was added and reacted at room temperature for 10-15 minutes. The reaction was terminated with 2 mol / L H2SO4, and OD450nm was measured with an enzyme reader. Three replicate wells were set for each sample. The specificity analysis results are shown in the figure. Figure 3 shown.

[0102] The results showed that the above-mentioned detection kit can specifically identify ANGPTL8 protein and has no positive reaction with other ANGPTL proteins.

[0103] Clinical stability test:

[0104] The test kits produced in the same batch were randomly divided into two parts, one part was placed in a 4°C refrigerator, and the other part was placed in a 37°C constant temperature box. After 10 days, it was taken out and placed in a 4°C refrigerator for equilibrium overnight. The stability test was performed using a positive quality control product. The test results of the two were the same, confirming that the test kit of this application has good stability.

[0105] The above-described embodiments merely illustrate several embodiments of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make various modifications and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A monoclonal antibody M6AD that specifically recognizes human ANGPTL8, characterized in that: The amino acid sequence of the heavy chain variable region of the monoclonal antibody M6AD that specifically recognizes human ANGPTL8 is shown in SEQ ID NO: 1 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2 in the sequence listing.

2. A gene encoding the monoclonal antibody M6AD according to claim 1, characterized in that: The encoding gene includes a heavy chain variable region gene; the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO.3; the encoding gene also includes a light chain variable region gene; the nucleotide sequence of the light chain variable region is shown in SEQ ID NO.

4.

3. A recombinant vector, wherein the coding gene according to claim 2 is inserted into the recombinant vector. A host cell carrying the recombinant vector according to claim 3 .

5. A product for quantitatively detecting the content of ANGPTL8 in a sample, comprising the monoclonal antibody of claim 1, the encoding gene of claim 2, the recombinant vector of claim 3, or the host cell of claim 4.

6. The product according to claim 5, which is an enzyme-linked immunosorbent assay kit for accurately quantifying the ANGPTL8 content in a sample from a subject, wherein the enzyme-linked immunosorbent assay kit comprises a capture antibody, which is the monoclonal antibody M6AD.

7. The enzyme-linked immunosorbent assay kit according to claim 6, comprising an ELISA plate coated with a capture antibody, a sample diluent, a standard, a detection antibody, avidin labeled with horseradish peroxidase, a concentrated washing solution, a substrate solution, and a stop solution; in, The capture antibody is the monoclonal antibody M6AD; The sample diluent is PBS buffer added with a final concentration of 1% BSA, 0.05% Tween-20 and 0.1% Proclin300; The standard product is a human ANGPTL8 protein standard product; The detection antibody is a 100 μg / mL biotin-labeled detection antibody A7 solution, and the amino acid sequence of the heavy chain variable region of the detection antibody is shown in SEQ ID NO.5; The amino acid sequence of the light chain variable region is shown in SEQ ID NO.

6. The solution is a PBS solution containing 50% glycerol; The concentrated washing solution is 10× PBS containing a final concentration of 1.0% Tween-20; The substrate solution is a TMB colorimetric substrate solution; The stop solution is 2M sulfuric acid.

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