NUP85 antigen epitope peptide and application thereof

Abstract

The invention relates to an NUP85 antigen epitope peptide and application thereof. The invention provides an NUP85 antigen epitope peptide, a monoclonal antibody prepared from the NUP85 antigen epitope peptide and a human NUP85 in-vitro diagnostic kit. According to the human NUP85 in-vitro diagnostic kit, an NUP85 antigen is detected by adopting an ELISA (Enzyme-Linked Immunosorbent Assay) double-antibody sandwich method, and the human NUP85 in-vitro diagnostic kit comprises an NUP85 coating antibody and an NUP85 binding antibody. The kit can be used for carrying out specific high-sensitivity detection on NUP85 in human tissues, cells or body fluid based on an immune experiment method.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to NUP85 antigen epitope peptide and application thereof. Background Art

[0002] Hepatocellular carcinoma (HCC) is the most common type of liver cancer, accounting for 75% to 90% of primary liver cancer and the second most deadly tumor after pancreatic cancer. Currently, the best treatment for HCC is surgical resection, but the recurrence rate is extremely high. Unresectable HCC can be treated with local regional therapies such as microwave ablation (MWA) or radiofrequency ablation (RFA), transarterial chemoembolization (TACE), but the prognosis of HCC patients is still poor, with a 5-year survival rate of only 18%. In 2017, sorafenib came out, opening the precedent for targeted drug research. Since then, the emergence of emerging drugs such as stivarga and cabozantinib has pushed targeted therapy into a research hotspot. However, the actual efficacy of these drugs is limited, and the overall survival (OS) of patients is not significantly prolonged. Due to the highly heterogeneous nature of HCC, that is, patients have similar disease phenotypes but may have different molecular etiologies, it is suggested that molecular-based targeted research will help develop more effective treatment options, so finding more stable molecular markers is of major clinical significance.

[0003] In recent years, there is evidence that biomarkers in the HCC tumor immune microenvironment are closely related to the patient's prognosis. Research on key genes related to the immune microenvironment may provide new ideas for HCC treatment. As an essential component for the assembly and maintenance of the nuclear pore complex (NPC), nucleoporin 85 (NUP85) plays an important role in mediating nuclear-cytoplasmic transport and has the function of repairing DNA and regulating gene expression. NUP85 can regulate the chemokine signaling of tumor-associated macrophages (TAMs) to affect tumor development and plays an important role in the rapid proliferation and metastasis of tumor cells. In addition, high expression of NUP85 may also enable HCC tumor cells to escape immune surveillance and killing by inhibiting TAMs. Studies have shown that inhibiting the expression of the NUP85 gene can effectively improve the patient's prognosis, which is well reflected in malignant tumors such as prostate cancer. However, scholars have not yet reported on the role of NUP85 in HCC.

[0004] Biomarker detection methods include nucleic acid detection (such as PCR, gene sequencing, etc.), protein detection (such as immunoassay, mass spectrometry), metabolite detection (such as chromatography, magnetic resonance spectroscopy), cell level detection (such as flow cytometry, immunohistochemistry), imaging detection (such as PET, CT, MRI) and other detection (such as gene chip technology, biosensors) and other types. Among them, immunoassay has high specificity and sensitivity, can be used for the detection of a variety of biomarkers, and the operation is relatively simple and the detection speed is fast, which is suitable for clinical testing and scientific research. At present, the immune experimental methods known to be used for clinical testing mainly include the following: ELISA, chemiluminescence, fluorescence chromatography, colloidal gold immunoassay, etc. The ELISA method includes the following types: double antibody sandwich method for detecting antigens, double antigen sandwich method for detecting antibodies, indirect method for detecting antibodies, competitive method for detecting antibodies, competitive method for detecting antigens, capture coating method for detecting antibodies, etc. Summary of the invention

[0005] 1. Technical issues to be resolved

[0006] In view of this, one of the main purposes of the present invention is to provide a NUP85 antigen epitope peptide and a human NUP85 in vitro diagnostic kit prepared therefrom. The human NUP85 in vitro diagnostic kit uses an ELISA double antibody sandwich method to detect the NUP85 antigen, which comprises a NUP85 coating antibody and a NUP85 binding antibody. The kit can perform specific and highly sensitive detection of NUP85 in human tissues, cells or body fluids based on an immunoassay method.

[0007] (II) Technical solution

[0008] In order to achieve the above object, as one aspect of the present invention, human NUP85 antigen epitope polypeptides I and II are provided, whose amino acid sequences are shown in SEQ ID No.1 and SEQ ID No.2, respectively, specifically:

[0009] (1)AMYGEKRFADAAS (SEQ ID No. 1); and

[0010] (2) MTSRIAPRSFWMT (SEQ ID No. 2).

[0011] As one aspect of the present invention, a method for preparing polypeptides I and II by chemical synthesis is provided, wherein the chemical synthesis method is selected from liquid phase synthesis and solid phase synthesis; the solid phase synthesis method is selected from tBoc solid phase synthesis and Fmoc solid phase synthesis.

[0012] As one aspect of the present invention, a hapten of human NUP85 is provided, wherein the hapten is NUP85 polypeptides I and II.

[0013] As one aspect of the present invention, a complete antigen of human NUP85 is provided, wherein the complete antigen is a hapten-carrier complex formed by chemically coupling NUP85 polypeptides I and II with a carrier protein; the carrier protein is selected from bovine serum albumin (BSA), ovalbumin (OVA), human serum albumin (HSA), and keyhole limpet hemocyanin (KLH). Preferably, bovine serum albumin BSA is not likely to cause cross-reactions as a carrier protein.

[0014] As one aspect of the present invention, anti-human NUP85 monoclonal antibodies I and II or antigen-binding fragments thereof are provided, and the antigen epitopes recognized and bound by the monoclonal antibodies or antigen-binding fragments thereof are shown in SEQ ID No. 1 and SEQ ID No. 2, respectively. The antibodies or antigen-binding fragments thereof can be used as coating antibodies (NUP85 capture antibodies) and NUP85 binding antibodies in the double antibody sandwich method, so as to be used to prepare a human NUP85 in vitro diagnostic kit, which can perform specific and high-sensitivity detection of NUP85 in human tissues, cells or body fluids based on an immunoassay method, preferably detecting NUP85 in blood specimens, especially in serum or plasma.

[0015] As one aspect of the present invention, a human NUP85 in vitro diagnostic kit is provided, the kit comprising a coating antibody, a biotin-labeled binding antibody, and necessary tools and reagents, etc. The reagents comprise horseradish peroxidase labeled with streptavidin, a substrate solution (developer), a stop solution, a calibrator and / or a quality control product. The human NUP85 in vitro diagnostic kit comprises a NUP85 coating antibody (capture antibody) and a NUP85 binding antibody, wherein, when the coating antibody is derived from one of the monoclonal antibodies I and II of the present invention, the binding antibody is derived from the other of the monoclonal antibodies I and II.

[0016] As one aspect of the present invention, a method for preparing the diagnostic kit for quantitatively detecting human NUP85 in a sample according to the present invention is provided, which comprises the following steps:

[0017] (1) providing a first NUP85 antibody as a capture antibody (coating antibody);

[0018] (2) Providing a second NUP85 antibody as a binding antibody.

[0019] As one aspect of the present invention, a human NUP85 in vitro diagnostic kit for liver cancer diagnosis is provided. Also provided is a use of anti-human NUP85 monoclonal antibodies I and II or their antigen-binding fragments in the preparation of liver cancer diagnostic reagents or kits.

[0020] Specifically, according to some embodiments of the present invention, the human NUP85 in vitro diagnostic kit of the present invention preferably detects NUP85 in serum or plasma. The kit of the present invention uses a double antibody sandwich method to detect the human NUP85, wherein:

[0021] The double antibody sandwich method uses a first NUP85 antibody as a capture antibody (coating antibody), and the first NUP85 antibody is derived from one of human NUP85 polypeptides I and II; and the double antibody sandwich method uses a second NUP85 antibody as a binding antibody, and the second NUP85 antibody is derived from the other of human NUP85 polypeptides I and II.

[0022] According to an embodiment of the present invention, the NUP85 antigen epitope peptide of the present invention is synthesized by solid phase method using the American ABI431A type polypeptide automatic synthesizer. The molecular weights of polypeptides I and II of the present invention are 2420.5282 and 1799.9266, respectively, which can be determined by mass spectrometry, and the synthesized polypeptide sequence can be identified by polypeptide sequence determination. The purity of the peptide segment can be evaluated by thin layer chromatography and high performance liquid chromatography, and the concentration of the polypeptide can be determined.

[0023] According to an embodiment of the present invention, when the diagnostic kit of the present invention is used, the blood sample to be tested (preferably serum or plasma) is mixed with BSA coupled with purified NUP85 antibody (i.e., capture antibody), and the antigen to be tested (NUP85) in the sample is specifically bound to the capture antibody to form a capture antibody-antigen to be tested complex. Then, the components not bound to the antigen to be tested are washed to remove the capture antibody-antigen to be tested complex. Then, the biotinylated binding antibody of the present invention is added, and the binding antibody specifically binds to the antigen to be tested in the capture antibody-antigen to be tested complex to form a capture antibody-antigen to be tested-binding antibody complex. Then, the unbound components are washed to separate the capture antibody-antigen to be tested-binding antibody complex. Finally, an enzyme-labeled streptavidin complex is added to finally form an antibody-antigen to be tested-biotinylated binding antibody-enzyme-labeled streptavidin complex, and after thorough washing, the color developer A and color developer B of the present invention are then added, and finally the stop solution is added, and the detection is performed using an enzyme reader, and the concentration of NUP85 is calculated according to the absorbance OD value and the standard curve.

[0024] According to some embodiments of the present invention, in the present invention, the capture antibody and the binding antibody are preferably coupled to BSA.

[0025] Preferably, the concentration of the capture antibody is 2 ng / ml to 6 ng / ml, more preferably 3.5 ng / ml to 5 ng / ml, and most preferably 4 ng / ml.

[0026] Preferably, in the diagnostic kit of the present invention, the concentration of the binding antibody is 10 ng / ml to 20 ng / ml, more preferably 12 ng / ml to 18 ng / ml, and most preferably 16 ng / ml.

[0027] Preferably, in the diagnostic kit of the present invention, the usage ratio of the capture antibody and the binding antibody to BSA is (3.2ng-6.5ng):(2mg-5.5mg), more preferably (3.8ng-5.5ng):(3mg-5.2mg), and most preferably 4.5ng:5mg.

[0028] (III) Beneficial effects

[0029] The present invention provides a NUP85 antigen epitope peptide and a human NUP85 in vitro diagnostic kit prepared therefrom. Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1. After a lot of theoretical research and experimental exploration, the inventors of the present invention finally screened out two polypeptides with good affinity, and added A to the N-terminus of the peptide segment to form a NUP85 polypeptide I containing 14 amino acids. Compared with the sequences at other positions of the NUP85 protein, the peptide segment interval and length selected by the present invention are more rational in terms of hydrophilicity and synthesizability.

[0031] 2. The inventors have optimized the various components in the kit of the present invention through a large number of experimental explorations, so that it is possible to obtain results that meet clinical detection requirements for human NUP85, that is, good accuracy and precision, high specificity, good sensitivity, and good stability.

[0032] 3. The inventors of the present invention have optimized the conditions of each step of preparing the diagnostic kit for detecting human NUP85 of the present invention through a large number of experimental explorations, including coupling the capture antibody with BSA and determining the concentrations of the capture antibody and the binding antibody, thereby enabling the diagnostic kit of the present invention to obtain the results required for clinical testing of human NUP85, namely, good accuracy and precision, high specificity, good sensitivity and good stability.

[0033] (IV) Terms and Definitions

[0034] The term "NUP85" used herein refers to human 85 kDa nucleoporin, a protein component encoding the NUP107-160 subunit of the nuclear pore complex, whose amino acid sequence is known in the art and can be found in professional databases such as NCBI.

[0035] The term "antigenic epitope peptide" used herein is also called antigenic determinant, which refers to a special chemical group in an antigen molecule that determines the antigen specificity and is the site that specifically binds to an antibody, T cell receptor or B cell receptor. The "antigenic epitope peptide" referred to herein is a linear epitope peptide composed of a continuous amino acid sequence.

[0036] The term "hapten" as used herein refers to a substance that is only immunoreactive but not immunogenic. A hapten has a small molecular weight and lacks a complex structure and sufficient immune epitopes that can directly stimulate the immune system.

[0037] The term "complete antigen" as used herein means that when a hapten is combined with a macromolecular carrier (such as a protein, polypeptide, etc.), it can acquire immunogenicity and become a complete antigen, thereby being able to stimulate the body to produce an immune response. This combination is usually achieved by chemical coupling, where a specific chemical group of the hapten is combined with an active group on the carrier protein to form a hapten-carrier complex.

[0038] The term "antibody" as used herein is a protein secreted by plasma cells (effector B cells) that can specifically bind to the corresponding antigen. The "antibody" referred to herein is a monoclonal antibody or its antigen-binding fragment. A monoclonal antibody refers to an antibody that is highly uniform and directed only against a specific single antigen epitope and is produced by a single B lymphocyte clone. It is an antibody produced by a cell line that can continuously secrete specific antibodies obtained by fusing B lymphocytes of immunized animals with myeloma cells, forming hybridoma cells, and then screening and culturing them. The antigen-binding fragment of a monoclonal antibody refers to a partial structure that can retain the ability to specifically bind to a specific antigen obtained by enzymatic cleavage, recombination or other means of a monoclonal antibody. These antigen-binding fragments generally include, but are not limited to, Fab fragments (antigen-binding fragments), F(ab')2 fragments, single-chain antibodies (scFv), nanobodies, etc.

[0039] The term "capture antibody" as used herein refers to a monoclonal antibody that can specifically recognize and bind to an antigen to be detected in a sample.

[0040] The term "coated antibody" used herein refers to a monoclonal antibody coated on a solid-phase ELISA plate, which can specifically bind to the antigen to be detected.

[0041] The term "binding antibody" used herein refers to another antibody in the kit that can specifically bind to the antigen to be detected, and the antigen to be detected specifically recognizes different epitopes on the coating antibody and the binding antibody respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0043] Figure 1 It is the linear fitting curve of the kit in Example 6. DETAILED DESCRIPTION

[0044] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0045] In order to better understand the technical solution, the technical solution will be described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0046] Unless otherwise specified, the solutions described below are all aqueous solutions and the percentages in the solutions are all volume percentages.

[0047] Example 1: Preparation of NUP85 Epitope Peptides I and II

[0048] The polypeptides were synthesized by the Fmoc solid phase method using an American ABI431A polypeptide automatic synthesizer. The molecular weights of polypeptides I and II of the present invention were 2420.5282 and 1799.9266, respectively, which could be determined by mass spectrometry and the synthesized polypeptide sequences were identified by polypeptide sequence determination.

[0049] The specific preparation steps of NUP85 polypeptides I and II of the present invention are as follows:

[0050] Weigh 135 mg of HMP resin, the substitution equivalent is 1.0 meq, that is, 0.1 mol is placed in the reaction chamber of the American ABI431A automatic peptide synthesizer, and the synthesizer automatically connects specific amino acids in different orders, and the coupling rate reaches 99%. The reaction is as follows:

[0051] (1) Activation of amino acids (HOBt / DCC method)

[0052]

[0053] Fmoc-protected amino acids

[0054]

[0055] (2) Connecting amino acids to the resin

[0056] (3) Removal of Fmoc protecting group from amino acids

[0057]

[0058] (4) Activation of another amino acid

[0059] (5) Coupling

[0060]

[0061] (6) Repeat steps (3) to (5) until the synthesis is completed

[0062] 142 mg of peptide resin of NUP85 peptide segment I and 135 mg of peptide resin of NUP85 peptide segment II were obtained respectively.

[0063] (7) Cleavage of peptide resin

[0064] The peptide chain was cut with TFA (trifluoroacetic acid), and EDT and thioanisole were used as scavengers. The reaction was carried out at room temperature for 4.0-5.0 hours, and the cutting reagent was removed. The crude products of NUP85 peptide segments I and II were obtained, respectively.

[0065] (8) Epitope peptides I and II are purified using existing techniques and identified using existing techniques.

[0066] The obtained peptides I and II are purified in the corresponding steps and then identified, and the identified synthetic peptide is the target product. The crude peptide purification step and the identification step are both existing technologies.

[0067] Example 2: Preparation of NUP85 capture antibody in the kit

[0068] 1. Preparation of NUP85 Antibody

[0069] The NUP85 peptide segments I and II are coupled to the carrier protein BSA using existing technology, and are prepared into monoclonal antibodies I and II.

[0070] The coupling steps are as follows:

[0071] (1) Dissolve the carrier protein BSA in coupling buffer (0.14 M phosphate, pH 7.4) to a final concentration of 2.0 mg / ml.

[0072] (2) Dissolve the polypeptide to be coupled in the solution of step (1) to a final concentration of 2.0 mg / ml and a molar ratio of polypeptide to carrier protein of 15:0.5 to 30:2.

[0073] (3) Add fresh GA to the above solution to a final concentration of 0.25 mg / ml and react on a magnetic stirrer at 4°C for 1-2 h.

[0074] (4) After the reaction is completed, sodium borohydride is added to the above solution to a final concentration of 4.5 mg / ml, and the mixture is reacted on a magnetic stirrer at 4°C for 1 h.

[0075] (5) Remove uncoupled peptides and GA reagents by dialysis or gel filtration, and replace with PBS solution (0.01 M, pH 7.4) to obtain peptide I-BSA complex immunogens and peptide II-BSA complex immunogens.

[0076] 2. The preparation process of monoclonal antibodies I and II is as follows:

[0077] (1) Preparation of antigen-adjuvant mixture

[0078] a. Adjust the concentration of the antigen protein solution: dissolve the peptide I-BSA complex immunogen in PBS solution (0.01M, pH 7.4) and adjust the concentration to 0.6-0.8 mg / mL.

[0079] b. Prepare CFA: Shake CFA evenly for later use.

[0080] c. Preliminary mixing: Mix the prepared peptide I-BSA complex solution and CFA in a 1:1 volume ratio.

[0081] d. Further emulsification: Shake gently for 5 minutes to form a stable water-oil emulsion.

[0082] e. Storage: The mixed emulsion should be used as soon as possible. If it needs to be stored for a short time, it should be placed at 4°C and the emulsification should be checked again before use.

[0083] (2) Antigen vaccination

[0084] a. Prepare a syringe, draw up 300ul of the prepared antigen injection solution, and slowly push it into the abdominal cavity of the mouse. The immunization time points are as follows: the first immunization on the first day, the first, second, third, and fourth booster immunizations on the 14th, 28th, 35th, and 42nd days, and prepare for sampling on the third day of the fourth booster immunization.

[0085] (3) Preparation of hybridomas

[0086] a. On a sterile operating table, anesthetize the mouse and remove the spleen. Then immediately place it in a 50ml centrifuge tube filled with 4℃ sterile PBS (containing double antibody) and immediately transport it to the cell room for the next experiment.

[0087] b. Preparation of single cell suspension

[0088] Use sterile scissors to cut the spleen into small pieces and then use a cell strainer to filter out single cells.

[0089] In this step, the pipette tip can be used to press the tissue on the filter to separate single spleen cells.

[0090] The separated single cell solution is filtered again to obtain the final single cell suspension.

[0091] Centrifuge the single cell suspension at 300g for 5 min, discard the waste liquid, and then resuspend it in sterile PBS (containing double antibody).

[0092] Repeat the above steps once.

[0093] (4) Use magnetic beads to sort B cells

[0094] (5) Cell fusion

[0095] a. Centrifuge spleen cells and myeloma cells at 300a-500g for 5-8 minutes, discard the waste liquid, and then resuspend with fusion diluent.

[0096] b. Mix spleen cells and myeloma cells at a ratio of 5:1-10:1.

[0097] c. Mix well and add to centrifuge tube. Centrifuge at 300g for 5 minutes and remove waste liquid.

[0098] d. Use the remaining residual liquid to resuspend the cells, hold the cell liquid with your hands to keep its temperature above 28°. Slowly add 1 ml of polyethylene glycol (PEG) (the fusion agent generally uses PEG with a molecular weight of 1000D, 1500D, or 4000D, with a concentration of 30% to 50%), drop by drop, shake evenly, and add for 1-2 minutes.

[0099] e. According to the above steps, slowly add 6 ml of fusion diluent, and control the adding time to 10-12 minutes.

[0100] f. Finally, add 7 ml of fusion diluent, centrifuge at 300 g for 5 min, and remove the waste liquid.

[0101] (6) Hybridoma cell culture

[0102] Hybridoma culture medium formula: RPMI 1640 medium + L-Gln + 10% FBS + double antibody + 1% Ultroser G

[0103] Hybridoma culture medium formula containing HAT: RPMI 1640 medium + L-Gln + 10% FBS + double antibody + 1% Ultroser G + 1X HAT supplement

[0104] a. Plating: Add 2 ml of hybridoma cells to each well of a 24-well plate.

[0105] b. Add HAT: After 48 hours, when the cells are well attached to the bottom, replace with 2 ml of hybridoma culture medium containing HAT supplement.

[0106] c. Medium replacement: Replace the cells in the wells with new hybridoma culture medium containing HAT every 2 days.

[0107] d. Collect the cell supernatant after 10 days.

[0108] (7) Screening positive wells

[0109] The collected supernatant was stored at 4°C, and the antibody quality was tested by Elisa to screen out the cell wells that could secrete antibodies. The wells were further diluted to the limit, and after the Elisa method was completed, the hybridoma cells that produced antibodies were amplified and frozen.

[0110] (8) Purification of antibodies

[0111] a. Prepare the ammonium sulfate solution first, then slowly add it dropwise to the antibody supernatant, stirring rapidly during the addition process to avoid excessive local concentration of ammonium sulfate and precipitation of impurity proteins. After the addition is completed, stir overnight at 2-8℃ to fully precipitate the antibody.

[0112] b. Centrifuge the solution at 10,000 g for 45-50 minutes, collect the precipitated antibodies, discard the supernatant, and redissolve the antibodies in sterile PBS. Use a dialysis device to dialyze overnight to remove excess ammonium sulfate and impurity proteins.

[0113] c. After dialysis, continue centrifugation at 10,000 g for 45 min to obtain relatively pure monoclonal antibodies I and II.

[0114] 3. Preparation of NUP85 antibody microwell ELISA plate

[0115] Select any one of antibodies I or II, and prepare it into a solution of 2-6 ng / ml with 1× PBS buffer at pH=7.4, then measure a certain volume of the solution into a microwell ELISA plate, and then place the microwell ELISA plate in a 37-degree incubator for 2 hours, take it out, block it with 2% BSA, take it out, wash it with 1× PBS at pH=7.4, and then dry it for 2 hours before taking it out for use.

[0116] Example 3: Method for preparing NUP85 binding antibodies in a kit

[0117] 1. Conjugation of Antibodies to Biotin

[0118] When the coating antibody is derived from one of Antibodies I and II, the antibody coupled to biotin is derived from the other of Antibodies I and II.

[0119] The principle formula for coupling antibodies to biotin is as follows:

[0120]

[0121] 2. The steps for coupling the antibody to biotin are as follows:

[0122] (1) Mix 1-2 ml of 1 mg / ml-3 mg / ml diluted antibody with 20 ul-40 ul (2 umol / L-8 mmol / L) biotin and rotate for 2 hours;

[0123] (2) After the reaction is completed, the solution in the reactor is poured into a dialysis bag and dialyzed in 1× PBS buffer (pH=7.4) at 4°C for 48 h. The dialysis solution is changed 8 times, and then the solution in the dialysis bag is collected to obtain the NUP85-binding antibody.

[0124] 3. The steps for preparing the binding antibody are as follows:

[0125] (1) Prepare the detection antibody storage solution: weigh 2.93 g of NaHCO3, 1.59 g of Na2CO3, and 0.12-0.15 g of tween20, mix them with distilled water and prepare the storage solution.

[0126] (2) The obtained high concentration binding antibody is prepared into a solution of 10-20 ng / ml using a storage solution for later use.

[0127] Example 4: Preparation of other components in the kit

[0128] 1. Preparation method of streptavidin-labeled horseradish peroxidase

[0129] The streptavidin-labeled horseradish peroxidase in the kit was purchased from Sangon Biotechnology Co., Ltd. with a concentration of 300U / ml. When used, it needs to be diluted with a specific enzyme labeling preservation solution at a dilution ratio of 1:5000 to 1:20000. The formula ingredients of 1L of the specific enzyme labeling preservation solution are as follows: 1.21 to 1.42 mol / L glycerol, 7.15 to 8.25 mmol / L sodium phosphate, 15 to 20 umol / L bovine serum albumin, and 12 to 16 umol / L cytochrome C.

[0130] 2. Preparation of substrate solution (color developer) and stop solution

[0131] The formula of 1L substrate A solution (color developer A) is as follows: 12.4g sodium acetate, 1.5g citric acid, 0.32ml~0.40ml 30% hydrogen peroxide.

[0132] The formula of 1L substrate B solution (color developer B) is as follows: 0.18g disodium ethylenediaminetetraacetic acid, 0.15-0.24g citric acid, 24-36ml glycerol, and 0.15-0.18g TMB.

[0133] Stop solution formula: dilute concentrated sulfuric acid with distilled water to a 2 mol / L solution.

[0134] 3. Preparation of Calibrators and Quality Control Products

[0135] The NUP85 calibrator and quality control materials in the kit were purchased from a nearshore company, and the high-value protein was diluted to the following concentrations: 1500ng / ml, 1000ng / ml, 500ng / ml, 250ng / ml, and 120ng / ml, and the concentration of the quality control material was 1200ng / ml.

[0136] Example 5: Concentration screening of capture antibody and binding antibody

[0137] In order to determine the appropriate capture antibody concentration and binding antibody concentration, the capture antibody concentration was set to 4.5ng / ml, the NUP85 calibrator concentration was set to 1500ng / ml, 1000ng / ml, 500ng / ml, 250ng / ml, 125ng / ml, and the binding antibody concentration was 10ng / ml, 12ng / ml, 16ng / ml, and 18ng / ml in the range of 10ng / ml to 20ng / ml. These concentrations were tested, and the test data are shown in Table 1. Finally, it was determined that the optimal concentration of the binding antibody was 16ng / ml. Similarly, the capture antibody concentration was set to 2ng / ml, 4ng / ml, and 6ng / ml for comparison. The test data are shown in Table 2, and the capture antibody concentration was finally determined. From the comparison results, it can be seen that the capture antibody concentration is preferably 2ng / ml to 6ng / ml, and the most preferred is 4ng / ml.

[0138] Table 1 Screening of binding antibody concentration

[0139]

[0140] Table 2 Screening of capture antibody concentration

[0141]

[0142] Example 6: Verification of various performances of the kit

[0143] In order to verify the quality of the kit, various properties of the kit with the best concentration screened in Example 5 were tested. The main properties of the kit include: the lowest detection line of the kit, relative sensitivity, relative specificity, compliance rate, linear range, accuracy, repeatability, stability, and batch difference. The detection methods of each performance are as follows:

[0144] 1. Minimum detection limit

[0145] Fifty negative sera were tested using the established method, and the detailed test results are shown in Table 3.

[0146] Table 3 ELISA test results of 50 negative sera

[0147]

[0148]

[0149] According to statistical principles, the positive and negative critical values ​​CP=X+3SD=61.61 and CN=X+2SD=57.63 are determined, that is, the detection value of the sample to be tested ≥61.61pg / mL is positive, the detection value of the sample to be tested ≤57.63pg / mL is negative, and the value between 57.63 and 61.61pg / mL is suspicious. That is, the minimum detection limit is 57.63-61.61pg / mL.

[0150] 2. Relative sensitivity, relative specificity and overall compliance rate

[0151] The established method was used to detect 50 serum samples with different antibody titers (real samples from the hospital, including 35 positive samples and 15 negative samples). The results were compared with the actual patient conditions given by the hospital to analyze the relative sensitivity, relative specificity and total compliance rate of the method. The calculation formula is as follows:

[0152] Relative specificity (%) = {number of negatives / (number of negatives + number of false positives)}×100%.

[0153] The established method was used to test 50 serum samples with different antibody titers. The test results were 33 positive and 17 negative. The specific data are shown in Table 4. The relative sensitivity, relative specificity and overall compliance rate were 94.28%, 91.67% and 94%, respectively, which met the requirements of the kit.

[0154] Table 4 Test results of 50 serum samples (pg / mL)

[0155]

[0156]

[0157] 3. Linear range

[0158] The high-value sample with a NUP85 antigen concentration of 1.5 ng / mL was diluted into 5 concentrations by multiple ratios. Each concentration sample was tested 3 times, and the average concentration was calculated. The average result and the dilution ratio were linearly fitted, and the linear correlation coefficient r was calculated. The result should be no less than 0.9950. The test results are shown in Table 3. The linear curve is as follows Figure 1 As shown in the figure, the linearity of the kit meets the requirements. The correlation between the theoretical value and the measured value is: r = 0.9997.

[0159] Table 5 Linear table

[0160]

[0161] 4. Accuracy

[0162] Different concentrations of NUP85 antigen were added to blank distilled water samples, and the recovery rates were measured according to the operating procedures of the NUP85 antigen double antibody sandwich kit. The results are shown in Table 6. As can be seen from the table, the recovery rate of the NUP85 antigen double antibody sandwich kit is between 89.89% and 103.20%, indicating that the kit has high accuracy.

[0163] Table 6 NUP85 antigen double antibody sandwich kit recovery test results

[0164]

[0165] 5. Repeatability

[0166] 5.1 Intra-batch reproducibility

[0167] The same batch of NUP85 detection kits trial-produced in the laboratory were used to test 30 sera with known background, including 10 negative sera, 10 strongly positive sera and 10 weakly positive sera, on the 1st, 2nd, 3rd, 5th and 7th days respectively. Each serum was repeated 3 times, and the standard deviation and coefficient of variation were calculated. The intra-batch repeatability was analyzed. The test results are shown in Tables 7, 8 and 9. It can be seen from the results in the table that the coefficient of variation of the 30 sera was less than 15%, which indicates that the intra-batch repeatability of the kits from the same batch is good.

[0168] Table 7 Intra-batch repeatability test results

[0169]

[0170]

[0171] Table 8 Intra-batch repeatability test results

[0172]

[0173] Table 9 Intra-batch repeatability test results

[0174]

[0175] 5.2 Batch-to-batch reproducibility

[0176] Three different batches of NUP85 detection kits (batch numbers 20221201, 20221215, and 20230104) produced in the laboratory were used to test 30 sera with known background, including 10 negative sera, 10 strongly positive sera, and 10 weakly positive sera. The test was repeated three times for each serum. The results are shown in Tables 10, 11, and 12.

[0177] From the results in the table, it can be seen that the coefficients of variation of the 30 sera are all less than 15%, which means that the batch reproducibility of the three batches of test kits is good.

[0178] Table 10 Results of inter-batch repeatability test

[0179]

[0180] Table 11 Results of inter-batch repeatability test

[0181]

[0182] Table 12 Results of inter-batch repeatability test

[0183]

[0184] 6. Stability test

[0185] The NUP85 detection kits of three different batches (batch numbers were 20221201, 20221215, and 20230104) were stored at 2-8°C and taken out at 0 months, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, 10 months, and 12 months, respectively, and tested with the same 50 sera. The results are shown in Table 13. It can be seen from the results that the sensitivity and specificity of the kit are relatively stable, indicating that the kit can be stored for 8 months or more.

[0186] Table 13 Kit stability test results

[0187]

[0188] By testing the kit's minimum detection line, relative sensitivity, relative specificity, compliance rate, linear range, accuracy, repeatability, batch difference, and stability, it can be seen that all performance characteristics of the product meet the requirements, and therefore the quality of the kit is qualified.

[0189] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A human NUP85 antigen epitope polypeptide I, II, characterized in that: The amino acid sequences of the polypeptides I and II are shown in SEQ ID No. 1 and SEQ ID No. 2, respectively.

2. A method for preparing the human NUP85 antigen epitope polypeptides I and II according to claim 1 by solid phase synthesis.

3. A hapten of human NUP85, characterized in that: The hapten is the human NUP85 antigen epitope polypeptides I and II as described in claim 1.

4. A complete antigen of human NUP85, characterized in that: The complete antigen is a hapten-carrier complex formed by chemically coupling NUP85 polypeptides I and II with a carrier protein.

5. An anti-human NUP85 monoclonal antibody I and II or an antigen-binding fragment thereof, characterized in that: The antigen epitopes recognized and bound by the monoclonal antibody or its antigen-binding fragment are shown in SEQ ID No. 1 and SEQ ID No. 2, respectively.

6. A human NUP85 in vitro diagnostic kit, characterized in that: The kit comprises a coating antibody and a biotin-labeled binding antibody, wherein when the coating antibody is derived from one of the monoclonal antibodies I and II described in claim 5, the binding antibody is derived from the other of the monoclonal antibodies I and II described in claim 5.

7. A method for preparing the diagnostic kit for quantitatively detecting human NUP85 in a sample as described in claim 6, characterized in that: The following steps are involved: (1) providing a first NUP85 antibody as a capture antibody (coating antibody); (2) Providing a second NUP85 antibody as a binding antibody.

8. The kit according to claim 6 or 7, characterized in that: The kit also contains horseradish peroxidase labeled with streptavidin, substrate solution (developer), stop solution, calibration substance and / or quality control substance.

9. Use of the anti-human NUP85 monoclonal antibodies I and II or their antigen-binding fragments according to claim 5 in the preparation of liver cancer diagnostic reagents or kits.

10. The use according to claim 9, characterized in that The kit can detect NUP85 in human tissues, cells or body fluids based on an immunoassay method, and optionally detect NUP85 in blood sample serum or plasma.

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