Soluble growth stimulation expression gene 2 protein antibody, detection kit and application of soluble growth stimulation expression gene 2 protein antibody

Abstract

The invention relates to the technical field of biological medicine, and provides a soluble growth stimulation expression gene 2 protein antibody, a detection kit and application thereof. The invention provides a soluble ST2 protein antibody, an intermediate and a detection kit comprising the same. The soluble ST2 protein antibody, the intermediate and the detection kit can be used for prediction and diagnosis of myocardial fibrosis, heart failure and the like. A plurality of strains of antibodies with high sensitivity and good specificity are obtained by constructing and recombining soluble ST2 protein antigens, immunizing and screening cell strains. Through proper reagent configuration, good performance of an ST2 detection kit, especially a chemiluminescence method detection kit, is realized. The ST2 detection kit disclosed by the invention is high in accuracy, high in sensitivity, wide in linear range and high in correlation degree; the reagent is high in precision, good in repeatability and high in applicability.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a soluble growth stimulating express gene 2 protein antibody, a detection kit and its application. Background Art

[0002] Growth Stimulating Express Gene 2 protein (ST2) is a member of the interleukin-1 (IL-1) receptor family and was first reported in 1989. In 2005, the specific ligand of ST2, interleukin-33 (IL-33), was discovered and reported. In 2013, the American College of Cardiology Foundation / Heart Association (ACCF / AHA) included it in the list of markers for myocardial fibrosis and can be used to predict the disease severity and prognosis level of patients with heart failure (HF). ST2 has two main subtypes. One lacks a transmembrane structure and is the subtype secreted extracellularly and present in the circulatory system, mainly present in peripheral blood, called soluble ST2 (sST2). The other has a transmembrane structure, contains a transmembrane fragment and an intracellular domain of the Toll / IL-1TR receptor, and is mainly present in cardiomyocytes, called transmembrane ST2 (ST2L).

[0003] ST2L and IL-33 form a signaling pathway that counteracts myocardial fibrosis and myocardial hypertrophy, slows down ventricular remodeling, and thus plays a role in protecting the myocardium; while sST2 is secreted into peripheral blood and competitively binds to IL-33 with ST2L, blocking the beneficial effects of IL-33 by restricting the cascade activation triggered by the interaction of IL-33 / ST2L. Under cardiovascular healthy conditions, the expression level of sST2 is low, which allows IL-33 / ST2L to fully interact, causing a cascade reaction through the activation of multiple intracellular kinases, thereby protecting the heart. In the state of heart failure, the expression of sST2 is up-regulated and binds to IL-33 as a "decoy". Therefore, sST2 can be used as a detection marker for myocardial fibrosis. Higher levels of sST2 are associated with adverse cardiac remodeling, aggravated myocardial fibrosis, and deterioration of cardiovascular outcomes, and are biomarkers for acute and chronic heart failure.

[0004] A follow-up study of more than 500 cases found that the sST2 content in the blood can predict the mortality within 1 year. For patients with sST2 concentration higher than the average value, the 1-year case fatality rate is more than 10 times higher than that of patients with concentration lower than the average value. Moreover, when the sST2 concentration is higher than 700 ng / L, it can independently predict the 1-year case fatality rate of acute HF patients. In addition to the baseline sST2 level, the dynamic concentration change can also more effectively stratify the risks and evaluate the prognosis of acute / chronic HF patients, and is basically not affected by age, gender, body mass index, renal function, atrial fibrillation, anemia or the cause of HF, and the intra-individual variation is small. For acute HF patients, a rapid short-term decrease in the sST2 level means a good treatment response and may have a better prognosis, while an increase in the sST2 level is more likely to reflect an increase in the death risk.

[0005] The currently commercially available immunoassay reagents and methods for soluble ST2 apply the relatively traditional enzyme-linked immunosorbent assay (ELISA), which have the disadvantages of long detection cycle, low sensitivity, cumbersome operation and long time consumption. At the same time, the linear range is generally only 3 - 200 ng / ml, and it cannot help doctors judge the patient's condition in a timely and accurate manner. The subsequent developed methods include colloidal gold method and immunofluorescence chromatography method. Although the two can detect quickly, they can only perform qualitative or semi-quantitative detection, and the accuracy is low, and they cannot provide accurate clinical judgment basis. At the same time, limited by the method itself, due to the use of a heterogeneous reaction system, the detection precision is poor, and the interference substances cannot be removed from the operation procedure, so that the accuracy of the test cannot be guaranteed. The immunoturbidimetric method based on latex microspheres for detecting ST2 is also greatly interfered by turbidity substances such as lipids that may exist in the sample.

[0006] Chemiluminescence method has the advantages of high sensitivity, strong precision, wide linear range and not being easily interfered, and is widely used in the detection of clinical disease indicators. The main determinant of the performance of current ST2 detection reagents lies in the functional characteristics of ST2-binding antibodies. A good antibody pair can effectively improve the disease diagnosis efficiency. Considering that the soluble ST2 concentration in some clinical samples is low (1 - 200 ng / mL), and there are structurally homologous similar proteins, therefore, screening high-sensitivity and specific antibody pairs for use in the diagnosis of ST2 is of great significance. Summary of the Invention

[0007] The present invention provides antibody raw materials for soluble growth stimulation expression gene 2 protein (sST2), ST2 antigen detection products and application schemes, etc., to solve the problems of low sensitivity, narrow linear range and poor precision of existing ST2 detection antibodies.

[0008] In the first aspect of the present invention, there is provided a soluble growth stimulating expressed gene 2 protein antibody or an antigen-binding fragment thereof, comprising: VHCDR1, VHCDR2 and VHCDR3 with amino acid sequences shown in SEQ ID NO: 1-3, and VLCDR1 with the amino acid sequence shown in SEQ ID NO: 4, VLCDR2 with the sequence NAK, and VLCDR3 with the amino acid sequence shown in SEQ ID NO: 5; or, VHCDR1, VHCDR2 and VHCDR3 with amino acid sequences shown in SEQ ID NO: 6-8, and VLCDR1 with the amino acid sequence shown in SEQ ID NO: 9, VLCDR2 with the sequence TAS, and VLCDR3 with the amino acid sequence shown in SEQ ID NO: 10.

[0009] As an alternative, the soluble growth stimulating expressed gene 2 protein antibody or an antigen-binding fragment thereof comprises a heavy chain variable region with an amino acid sequence shown in SEQ ID NO: 11 and a light chain variable region with an amino acid sequence shown in SEQ ID NO: 12; or a heavy chain variable region with an amino acid sequence shown in SEQ ID NO: 13 and a light chain variable region with an amino acid sequence shown in SEQ ID NO: 14.

[0010] As an alternative, the antibody or an antigen-binding fragment thereof can specifically recognize and / or bind to an epitope of the soluble growth stimulating expressed gene 2 protein antigen shown in SEQ ID NO: 15 or SEQ ID NO: 16.

[0011] As an alternative, the antibody includes a monoclonal antibody; the antigen-binding fragment of the antibody is selected from F(ab’)2, F(ab)2, Fab’, Fab, Fv or scFv.

[0012] In the second aspect of the present invention, there is provided a nucleic acid encoding the above-mentioned soluble growth stimulating expressed gene 2 protein antibody or an antigen-binding fragment thereof.

[0013] In the third aspect of the present invention, there is provided an expression vector containing the above nucleic acid.

[0014] In the fourth aspect of the present invention, there is provided a transgenic cell line or recombinant bacterium containing the above nucleic acid or the above expression vector.

[0015] In the fifth aspect of the present invention, there is provided an immunoconjugate, comprising an antibody part and a conjugate part conjugated to the antibody part, wherein the antibody part comprises the above-mentioned soluble growth stimulating expressed gene 2 protein antibody or an antigen-binding fragment thereof, and the conjugate part is selected from a fluorescent substance, a chemiluminescent substance, a colored substance, an enzyme or a combination thereof.

[0016] In a sixth aspect of the present invention, there is provided a detection kit for soluble growth-stimulating expressed gene 2 protein, which detection kit comprises the above-mentioned soluble growth-stimulating expressed gene 2 protein antibody or its antigen-binding fragment.

[0017] As an alternative, the detection kit is used for immunochemiluminescence detection of soluble growth-stimulating expressed gene 2 protein.

[0018] As an alternative, the detection kit comprises a first antibody and a second antibody used in combination; the first antibody serves as a capture antibody and the second antibody serves as a detection antibody; or, the first antibody serves as a detection antibody and the second antibody serves as a capture antibody; the first antibody comprises VHCDR1, VHCDR2 and VHCDR3 with amino acid sequences as shown in SEQ ID NO: 1-3, and VLCDR1 with an amino acid sequence as shown in SEQ ID NO: 4, VLCDR2 with the sequence NAK, and VLCDR3 with an amino acid sequence as shown in SEQ ID NO: 5; the second antibody comprises VHCDR1, VHCDR2 and VHCDR3 with amino acid sequences as shown in SEQ ID NO: 6-8, and VLCDR1 with an amino acid sequence as shown in SEQ ID NO: 9, VLCDR2 with the sequence TAS, and VLCDR3 with an amino acid sequence as shown in SEQ ID NO: 10.

[0019] As an alternative, the detection kit includes magnetic particles coated with a capture antibody, a detection antibody labeled with a luminescent label, a substrate and an excitation solution suitable for the luminescent label, and a series of quality control products and calibration products of soluble growth-stimulating expressed gene 2 protein antigen.

[0020] As an alternative, the capture antibody comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 11 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 12; the detection antibody comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 13 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 14.

[0021] In a seventh aspect of the present invention, there is provided the use of the above-mentioned soluble growth-stimulating expressed gene 2 protein antibody or its antigen-binding fragment, the above-mentioned nucleic acid, the above-mentioned expression vector, the above-mentioned transgenic cell line or recombinant bacterium or the above-mentioned immunoconjugate in the preparation of a detection reagent or kit for diagnosing or predicting myocardial fibrosis or heart failure.

[0022] The present invention provides a soluble growth stimulation expressed gene 2 protein antibody, an intermediate, and a detection kit containing the same, which can be used for the prediction and diagnosis of myocardial fibrosis, heart failure, etc. By constructing a recombinant soluble ST2 protein antigen, immunizing and screening cell lines, the present invention obtains two antibodies with high sensitivity and good specificity. Further, through appropriate reagent configuration, good performance of the sST2 detection kit, especially the chemiluminescence detection kit, is achieved. The sST2 detection kit of the present invention has high accuracy, high sensitivity, a wide linear range, and a high degree of correlation; the reagent has high precision, good repeatability, and strong applicability. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is an SDS-PAGE electrophoresis diagram of recombinant expressed sST2 protein.

[0024] Figure 2 It is a color gradient heat map of the paired detection results of the sST2 antibody. In the figure, the depth of the color block reflects the detection signal intensity (OD value) of the paired combination; the value in each cell represents the level of the OD value; for the color, from small to large in value, the color changes from blue to light white, from white to light red, and to dark red; among them, the darker the blue, the weaker the signal generated by the antibody pairing; the darker the red, the stronger the signal generated by the antibody pairing.

[0025] Figure 3 It is an analysis curve of the detection results of the linear range of the sST2 kit in Example 6. DETAILED DESCRIPTION OF THE INVENTION

[0026] The present invention provides an antibody or its antigen-binding fragment that can specifically bind to soluble growth stimulation expressed gene 2 protein, namely an antibody raw material, an sST2 antigen detection product, and an application solution.

[0027] The present invention constructs a human soluble growth stimulation expressed gene 2 protein expression gene, and obtains a recombinant human soluble growth stimulation expressed gene 2 protein antigen with biological activity through recombinant expression; through independent animal immunization and monoclonal cell line screening, 9 monoclonal antibodies that can efficiently bind to recombinant human growth stimulation expressed gene 2 protein are obtained; further, a group of monoclonal antibody pairs ST2-7# and ST2-15# are preferably selected, which can bind to human soluble growth stimulation expressed gene 2 protein with high sensitivity and strong specificity by a double antibody sandwich method. That is, the present invention provides the application of these two antibodies in detecting soluble growth stimulation expressed gene 2 protein, detecting heart failure and myocardial fibrosis, and in the preparation of reagents or kits for detecting myocardial fibrosis and heart failure.

[0028] For antibodies, in some embodiments, the antibody comprises all or a portion of the constant region of the antibody. In some embodiments, the constant region is selected from the following various types, including: IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3 or IgG4), and IgM, etc. The present invention also discloses antigen-binding fragments capable of specifically binding to anti-ST2 protein antibodies. Examples of antigen-binding fragments include F(ab’)2, F(ab)2, Fab’, Fab, Fv, scFv, etc.

[0029] For the monoclonal antibodies of the present invention, they can be obtained from a single clone by any method available or known in the art, including expression in any eukaryotic, prokaryotic or phage system. The monoclonal antibodies for use in the present invention can be prepared using a wide variety of techniques known in the art, including hybridoma technology, recombinant technology or phage display technology, etc.

[0030] Products in other aspects of the present invention also include intermediates in the process of preparing antibodies, including expression vectors, transgenic cell lines or recombinant bacteria, or compositions containing the above substances. Specifically, for the expression vector, it can be a recombinant expression vector such as a eukaryotic expression vector. For the transgenic cell line or recombinant bacteria, it also includes eukaryotic cells or prokaryotic cells, such as Escherichia coli, yeast or animal cells (e.g., mammalian cells, such as mouse cells, human cells, etc.) and cell lines.

[0031] The present invention also provides an sST2 detection kit or detection reagent, which kit or reagent contains the antibody or its antigen-binding fragment of the present invention. In some embodiments, the kit further includes a solid-phase substrate for the attachment of the antibody or its antigen-binding fragment, and the solid-phase substrate includes but is not limited to microtiter plates, magnetic particles, filter paper for immunochromatography, polymers such as polystyrene, glass filters and other insoluble carriers, etc. In some embodiments, the kit further contains some other components, including but not limited to enzymes for labeling, corresponding substrates, radioisotopes, fluorescent substances, colored substances, buffers, etc.

[0032] When using the above kit or detection reagent to detect sST2, in some embodiments, the detection method includes: binding the antibody or its antigen-binding fragment of the present invention to the sST2 antigen in the sample to be tested to form an antibody-antigen or antibody fragment-antigen complex; subsequently, detecting the sample complex to determine whether there is a target antigen in the sample.

[0033] Specifically, in some embodiments, the detection reagent or kit comprises an antibody of the present invention or its antigen-binding fragment (as a capture antibody, a first antibody), and a detectable, labeled second antibody (detection antibody). Specific detection methods include: adsorbing the first antibody onto a solid support; subsequently adding a test sample that may contain sST2 to the above support; adding a second antibody with a label to the above support; and detecting the presence of the label to determine whether sST2 is present. Additionally, specific detection methods may also be known detection methods such as enzyme immunoassay, immunochemiluminescence assay, radioimmunoassay, fluorescence immunoassay, immunochromatography, competitive method, or sandwich method, etc.

[0034] In a preferred embodiment, the present invention provides an immunochemiluminescence assay kit prepared using anti-soluble growth stimulation expressed gene 2 protein antibodies (ST2-7# and ST2-15#). Through the independent screening of the best monoclonal antibody pairings with the highest sensitivity and specificity, and appropriate reagent configuration, this kit achieves good performance of the sST2 detection kit, and has characteristics such as high accuracy and sensitivity, a wide linear range, and considerable precision.

[0035] More specifically, the detection kit of the present invention has the following advantages: 1. The reagent has high accuracy, and the relative deviation of the detection results of samples at each concentration does not exceed 5%; 2. The reagent has high sensitivity, a wide linear range, and a high degree of correlation. The lower limit of the linear range (2 ng / mL) is much lower than the reference value (35 ng / mL), which can effectively avoid false positive misjudgments; the actual linear range is much higher than the upper limit of the expected linear range (300 ng / mL), and can even reach the level of 500 ng / mL, covering the vast majority of sample concentrations, enabling high-concentration samples to maintain accurate results without dilution during detection. At the same time, within this range, the linear correlation coefficient r of the reagent is as high as 0.9996, ensuring the accuracy of the full-range detection results and being able to accurately monitor the fluctuations of the patient's indicators; 3. The reagent has high precision and good repeatability. Whether it is within-laboratory or between-laboratories precision, the CV is less than 5%, which can provide a reliable traceability basis for the detection data; 4. The reagent has good applicability. It is applicable to both high-speed fully automatic chemiluminescence workstations and small chemiluminescence analyzers, can meet various hospital application scenarios including outpatient clinics, emergency departments, and community hospitals, and at the same time maintain stable detection performance.

[0036] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0037] In the embodiments of the present invention, the experimental materials used can be obtained from commercial channels without special instructions. If there are no special instructions for the experimental operation methods, the well-known means in the art are adopted.

[0038] Example 1: Recombinant expression of sST2 antigen

[0039] According to the growth stimulation expressed gene 2 protein sequence information (NP_003847.2) published by the National Center for Biotechnology Information (NCBI) in the United States, its full-length sequence was selected and cloned into the pCDNA3.4 eukaryotic expression vector. The obtained plasmid was transfected into HEK293 cells (human embryonic kidney cells 293) through a PEI transfection reagent (polyethylenimine). After culturing in a constant temperature shaker at 37 °C, 8% CO2, and an appropriate rotation speed for 7 days, the cell supernatant was collected by centrifugation, and the recombinantly expressed sST2 protein was obtained through nickel medium affinity chromatography purification, with a size of approximately 37.8 kDa ( Figure 1 )

[0040] Example 2: Immunization of animals with recombinant sST2 protein

[0041] The recombinant sST2 antigen prepared in Example 1 was diluted to 1 mg / mL with a 20 mM PBS pH7.4 solution (phosphate buffer) and emulsified with Freund's adjuvant in a 1:1 ratio. The emulsified recombinant sST2 antigen was intraperitoneally injected to immunize Balb / c mice aged 6 to 8 weeks, with 100 μg for each mouse. Freund's complete adjuvant was used for the primary immunization, and Freund's incomplete adjuvant was used for the booster immunization. After three immunizations, tail vein blood was collected to measure the titer, and those with a titer exceeding 1 million were selected for cell fusion. Direct immunization of the spleen was performed 72 hours before fusion, and 30 μg of sST2 antigen was injected.

[0042] Example 3: Screening of monoclonal antibodies

[0043] (1)Fusion cells: The mice to be fused were sacrificed by cervical dislocation. Their spleens were ground and filtered to prepare a spleen cell suspension, which was transferred to a 50 mL centrifuge tube. It was resuspended with serum-free RPMI1640 medium and the cell suspension was washed 3 times, centrifuged at 15000 rpm for 5 min each time. The myeloma cells were resuspended with serum-free RPMI1640 medium and the cell suspension was washed 3 times, centrifuged at 15000 rpm for 5 min each time. The spleen cell suspension and the myeloma cell suspension were mixed, centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and the bottom of the tube was gently flicked to disperse the cells. At the same time, 1 mL of PEG1450 pre-warmed to 37 °C was slowly added, and after adding it within 60 s, it was pipetted and mixed evenly for 30 s. Subsequently, 40 mL of RPMI 1640 medium pre-warmed to 37 °C was immediately added to terminate the fusion reaction, and it was transferred to RPMI 1640 HAT cell screening medium, mixed evenly and plated on a 96-well cell culture plate. After 7 days, the RPMI 1640 cell medium was changed, and after another 3 days, a part of the supernatant was aspirated for subsequent operations.

[0044] (2)Supernatant detection: The recombinantly expressed sST2 protein was coated on a polystyrene 96-well enzyme-linked immunosorbent assay (ELISA) plate at a total amount of 50 ng / well. The cell supernatant aspirated in Example 3(1) was added to the coated 96-well plate. After incubation and washing, HRP-labeled goat anti-mouse secondary antibody, reaction substrate and termination solution were incubated in sequence. The cell wells corresponding to the high-value positive wells were selected for subcloning. After 5 subclonings, finally 9 hybridoma monoclonal cells with good reactivity (OD value ≥ 3.0) to the recombinant sST2 protein were selected.

[0045] Example 4: Pairing and screening of monoclonal antibodies

[0046] (1)HRP-labeled antibody: 30 mg of HRP dry powder was dissolved in 1.5 mL of pure water, 1.2 mL of 25 mg / mL NaIO4 aqueous solution was added and mixed evenly, and the reaction was carried out at 4 °C for 30 min. After taking it out, 0.2 mL of ethylene glycol solution was added, and it was left at room temperature for 30 min to complete the activation of HRP, and it was stored at 4 °C for later use. The antibody was diluted to 2 mg / mL with 20 mM carbonate buffer pH 9.6. 2 mL of the antibody to be labeled was mixed evenly with the activated HRP labeling solution and dialyzed overnight in 20 mM carbonate buffer pH 9.6. After taking it out, 0.2 mL of 2 mg / mL NaBH4 solution was added, and the reaction was carried out at 4 °C for 2 hours. An equal volume of saturated ammonium sulfate solution was added, and the reaction was carried out at 4 °C for 30 min and then centrifuged. The precipitate was resuspended with 1 mL of 20 mM PBS pH 7.4 solution, and an equal volume of glycerol was added and mixed evenly for storage.

[0047] (2)Pairing detection: Dilute the anti-ST2 antibody to 1 μg / mL with 20 mM PB pH 7.4 solution, coat 100 μL per well on a polystyrene 96-well ELISA plate, and incubate at 37 °C for 2 hours. Wash the 96-well plate 5 times with PBST, add the blocking solution for blocking, and discard the blocking solution after blocking. Add 100 μL per well of the recombinant ST2 protein diluted to 10 ng / mL to the 96-well plate, incubate at 37 °C for 1 hour, wash the 96-well plate 5 times with PBST, add the HRP-labeled anti-ST2 antibody, incubate at 37 °C for 30 min, wash the 96-well plate 5 times with PBST, then incubate the reaction substrate and the termination solution in sequence, and place it in an ELISA reader for reading. Pair all 9 antibodies in pairs as the coated antibody and the labeled antibody, and after detection, select the monoclonal antibody pair with the highest sensitivity and the best specificity, namely monoclonal antibodies ST2-7# and ST2-15# ( Figure 2 ).

[0048] Example 5: Sequence determination and binding epitope analysis of monoclonal antibodies

[0049] (1)Antibody sequence determination: Entrust Nanjing Genscript Biotech Co., Ltd. to perform de-novo protein de novo sequencing on two monoclonal antibodies, ST2-7# and ST2-15#. After deconvolution analysis, the amino acid sequences of the heavy and light chain variable regions of the two monoclonal antibodies, ST2-7# and ST2-15#, are obtained respectively. The specific amino acid sequences are shown in Table 1. The isotypes of both antibodies are mouse IgG1.

[0050] Table 1 Amino acid sequences of the variable regions of monoclonal antibodies

[0051] (2)Antibody binding epitope analysis: Entrust Nanjing Genscript Biotech Co., Ltd. to analyze the binding epitopes of two monoclonal antibodies, ST2-7# and ST2-15#. The binding epitopes of both antibodies are linear epitopes, and the relevant information is shown in Table 2. Among them, the binding epitope of monoclonal antibody ST2-7# is the amino acids QYDCLALNLHG (Gln-Tyr-Asp-Cys-Leu-Ala-Leu-Asn-Leu-His-Gly) at positions 300 to 310 of the full-length ST2 protein, and the binding epitope of monoclonal antibody ST2-15# is the amino acid sequence RRHTVRLS (Arg-Arg-His-Thr-Val-Arg-Leu-Ser) at positions 312 to 319 of the full-length ST2 protein.

[0052] Table 2 Binding epitope information of monoclonal antibodies

[0053] Example 6: Preparation of sST2 Detection Reagent and Kit (1) Preparation of antibody buffer: Add 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) with pH 7.3, 0.5% polyoxyethylene lauryl ether, 50 mM tris(hydroxymethyl)aminomethane (Tris), 2% sucrose, 4% arginine, 2.5% casein, and 0.1% sodium azide to prepare the antibody buffer, and adjust the pH to 7.30 ± 0.05 (25°C). Filter through a 0.8 μm filter membrane to remove large particle impurities.

[0054] (2) Preparation of magnetic bead storage buffer: Add 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) with pH 7.3, 0.5% polyoxyethylene lauryl ether, 50 mM potassium dihydrogen phosphate, 20 mM sodium chloride, 2% sucrose, 4% arginine, 1% bovine serum albumin, and 0.1% sodium azide to prepare the magnetic bead storage buffer, and adjust the pH to 7.40 ± 0.05 (25°C). Filter through a 0.8 μm filter membrane to remove large particle impurities.

[0055] (3) Biotinylation of antibody: Dissolve biotin in dimethyl sulfoxide solution to 1 mg / ml, add 2 mg / mL sST2 capture antibody, i.e., ST2-7#, mix at a mass ratio of 1:10, then add an equal volume of carbonate buffer pH 9.6 for dilution, and incubate at 37°C for 2 hours to complete the labeling. Then ultrafilter using a 50 kDa ultrafiltration tube to replace the buffer with antibody buffer, and the final concentration of biotinylated sST2 capture antibody is 10 μg / mL.

[0056] (4) Coupling of magnetic bead reagent: Magnetically separate 20 mg of concentrated streptavidin-labeled magnetic beads, wash 3 times with antibody buffer, add the above biotinylated sST2 capture antibody, mix at a mass ratio of 50:1, react at 30°C for 15 min in the environment of antibody buffer to complete the coupling of antibody and magnetic beads, magnetically separate and discard the supernatant, resuspend with magnetic bead storage buffer, and dilute and volume-fix to 200 mL to obtain the magnetic bead reagent with completed antibody coupling.

[0057] (5) Coupling of acridinium ester-labeled reagent: Dissolve acridinium ester in dimethyl sulfoxide solution to 5 mg / mL, add 2 mg / mL sST2 detection antibody, i.e., ST2-15#, mix at a mass ratio of 1:10, then add an equal volume of carbonate buffer pH 9.6 for dilution, and incubate at 37°C for 2 hours to complete the labeling. Then ultrafilter using a 50 kDa ultrafiltration tube to replace the buffer with antibody buffer, and dilute and volume-fix to 2 L to obtain the acridinium ester-labeled reagent with completed antibody coupling.

[0058] (6)Preparation of calibrators and quality control materials: Recombinant ST2 antigen was formulated into a series of calibrators with a calibrator diluent at concentrations of 0 ng / mL, 5 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, and 300 ng / mL. The composition of the calibrator diluent was 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) at pH 7.3, 0.5% polyoxyethylene lauryl ether, 50 mM potassium chloride, 2% sucrose, 4% arginine, and 0.1% sodium azide. A high-concentration ST2 sample (100 ng / mL) was diluted with the calibrator diluent to 8 ng / mL and 50 ng / mL, serving as the low-value quality control material and the high-value quality control material, respectively.

[0059] Example 7: Accuracy of the kit The accuracy of a reagent refers to the degree of agreement between the mean of the quantity values obtained from an infinite number of repeated measurements and a reference quantity value.

[0060] Using the detection kit in Example 6, one high-concentration (199.47 ± 3.56 ng / mL) and one low-concentration (19.87 ± 0.52 ng / mL) growth-stimulating expressed gene 2 protein enterprise reference product were detected respectively, and the detection was repeated 3 times. The detection results are shown in Table 3. According to the relative deviation of the detection results, the relative deviations of the 3 detection results of the two reference products were both within 5%, indicating that the kit had good accuracy.

[0061] Table 3 Detection results of the accuracy of the sST2 kit in Example 6

[0062] Example 8: Linear range of the kit The expected linear range of the sST2 kit in Example 6 was 2 - 500 ng / mL. One high-value sample (501.23 ng / mL) and one low-value sample (1.20 ng / mL) were selected near the upper and lower limits of this range. The high- and low-value samples were accurately diluted in different proportions with a zero-value sST2 calibrator to prepare the following 6 different concentration levels (1.20 ng / mL, 4.07 ng / mL, 29.91 ng / mL, 144.76 ng / mL, 288.31 ng / mL, 501.23 ng / mL) of test samples, and the sST2 kit in Example 6 was used to detect them.

[0063] The detection results are shown in Table 4. After fitting the curve and linear analysis, the linear regression equation of the detection kit was: y = 0.9972x + 0.0212, R 2 = 0.9996 > 0.995 ( Figure 3), initially judged to meet the requirements of the expected linear range; the relative deviation of each measured value from the theoretical value for each concentration sample is not more than 10%, and this expected linear range is acceptable.

[0064] Table 4 Detection results of the linear range of the sST2 kit in Example 6

[0065] Example 9: Precision of the kit Precision refers to the degree of agreement between the indicated values or measured values obtained by repeated measurement of the same or similar objects to be measured under specified conditions. The precision of the sST2 kit in Example 6 was evaluated through repeatability, intermediate precision (precision within the laboratory), and reproducibility (precision between laboratories). Three batches of samples to be tested, namely high-concentration quality control product 1 with a known concentration (target value 153.37 ng / mL), low-concentration quality control product 2 (target value 30.47 ng / mL), and a mixed quality control product with an equal ratio mixture (target value 91.92 ng / mL), were used to evaluate this performance.

[0066] In the same laboratory, using the same detection instrument, three batches of samples were detected separately. Two analytical batches were detected every day, and two parallel samples of each concentration in each batch were processed for detection to evaluate the precision within the laboratory of the kit.

[0067] In the same laboratory, using three different commercially available instruments (NRM411, NORMAN-CL 5, NRM411-S7) and a single batch of the detection kit, three batches of samples were detected separately. Each batch of samples was continuously detected for 5 days, and each batch of samples was repeatedly detected 5 times a day to evaluate the precision between laboratories of the kit.

[0068] The detection results of the precision within the laboratory and the precision between laboratories adopted a two-way analysis of variance model. The results of the analysis of variance are presented in Table 5 after being sorted out, the results of the standard deviation and CV analysis are presented in Table 6 after being sorted out, and the results of the confidence interval are presented in Table 7 after being sorted out. All of the above results meet the expectations and there are no abnormal values.

[0069] Table 5 Results of the analysis of variance of the precision of the sST2 kit in Example 6

[0070] Relevant explanations for Table 5: SS site: sum of squares - between laboratories (between instruments); MS site: mean square - between laboratories (between instruments); SS day: sum of squares - between days; MS day: mean square - between days; SS error: sum of squares - within batch; MS error: mean square - within batch; SS total: sum of squares - total.

[0071] Precision analysis results of the sST2 kit in Example 6, Table 6

[0072] Related explanations for Table 6: s: standard deviation; CV: coefficient of variation.

[0073] Precision confidence interval (95%, α = 0.05) of the sST2 kit in Example 6, Table 7

[0074] The present invention has been described in detail above in combination with specific embodiments and exemplary examples, but these descriptions should not be construed as limiting the present invention. Those skilled in the art understand that without departing from the spirit and scope of the present invention, various equivalent substitutions, modifications or improvements can be made to the technical solutions of the present invention and their implementation manners, and all of these fall within the scope of the present invention.

Claims

1. A soluble growth stimulating expressed gene 2 protein antibody or an antigen-binding fragment thereof, characterized in that, Comprising: VHCDR1, VHCDR2 and VHCDR3 with amino acid sequences as shown in SEQ ID NO: 1-3, and VLCDR1 with amino acid sequence as shown in SEQ ID NO: 4, VLCDR2 with sequence NAK, VLCDR3 with amino acid sequence as shown in SEQ ID NO: 5; or, VHCDR1, VHCDR2 and VHCDR3 with amino acid sequences as shown in SEQ ID NO: 6-8, and VLCDR1 with amino acid sequence as shown in SEQ ID NO: 9, VLCDR2 with sequence TAS, VLCDR3 with amino acid sequence as shown in SEQ ID NO:

10.

2. The soluble growth stimulation expressed gene 2 protein antibody or its antigen-binding fragment according to claim 1, characterized in that, Comprising a heavy chain variable region with amino acid sequence as shown in SEQ ID NO: 11 and a light chain variable region with amino acid sequence as shown in SEQ ID NO: 12; or a heavy chain variable region with amino acid sequence as shown in SEQ ID NO: 13 and a light chain variable region with amino acid sequence as shown in SEQ ID NO:

14.

3. The soluble growth stimulating expressed gene 2 protein antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment can specifically recognize and / or bind to an epitope of the soluble growth-stimulating expressed gene 2 protein antigen as shown in SEQ ID NO: 15 or SEQ ID NO:

16.

4. The soluble growth-stimulating expressed gene 2 protein antibody or antigen-binding fragment thereof according to claim 1, characterized in that The antibody includes a monoclonal antibody; the antigen-binding fragment of the antibody is selected from F(ab’)2, F(ab)2, Fab’, Fab, Fv or scFv.

5. A nucleic acid encoding the soluble growth-stimulating expressed gene 2 protein antibody or its antigen-binding fragment according to any one of claims 1-4.

6. An expression vector comprising the nucleic acid according to claim 5.

7. A transgenic cell line or recombinant bacterium comprising the nucleic acid according to claim 5 or the expression vector according to claim 6.

8. An immunoconjugate, characterized in that, Comprising an antibody portion and a conjugate portion conjugated to the antibody portion, the antibody portion comprising the soluble growth-stimulating expressed gene 2 protein antibody or its antigen-binding fragment according to any one of claims 1-4, and the conjugate portion being selected from a fluorescent substance, a chemiluminescent substance, a colored substance, an enzyme or a combination thereof.

9. A detection kit for soluble growth-stimulating expressed gene 2 protein, characterized in that, Comprising the soluble growth-stimulating expressed gene 2 protein antibody or its antigen-binding fragment according to any one of claims 1-4.

10. The detection kit according to claim 9, wherein A detection kit for immunochemiluminescence detection of soluble growth-stimulating expressed gene 2 protein.

11. The detection kit according to claim 9, wherein, It comprises a first antibody and a second antibody used in pair; the first antibody serves as a capture antibody and the second antibody serves as a detection antibody; alternatively, the first antibody serves as a detection antibody and the second antibody serves as a capture antibody; the first antibody comprises VHCDR1, VHCDR2 and VHCDR3 with amino acid sequences shown in SEQ ID NO: 1-3, and VLCDR1 with the amino acid sequence shown in SEQ ID NO: 4, VLCDR2 with the sequence of NAK, and VLCDR3 with the amino acid sequence shown in SEQ ID NO: 5; the second antibody comprises VHCDR1, VHCDR2 and VHCDR3 with amino acid sequences shown in SEQ ID NO: 6-8, and VLCDR1 with the amino acid sequence shown in SEQ ID NO: 9, VLCDR2 with the sequence of TAS, and VLCDR3 with the amino acid sequence shown in SEQ ID NO:

10.

12. The detection kit according to claim 9, wherein The detection kit includes magnetic particles coated with a capture antibody, a detection antibody labeled with a luminescent marker, a substrate and an excitation solution suitable for the luminescent marker, as well as a series of quality control products and calibration products of soluble growth stimulation expressed gene 2 protein antigen.

13. The detection kit according to claim 12, wherein The capture antibody comprises a heavy chain variable region with an amino acid sequence shown in SEQ ID NO: 11 and a light chain variable region with an amino acid sequence shown in SEQ ID NO: 12; the detection antibody comprises a heavy chain variable region with an amino acid sequence shown in SEQ ID NO: 13 and a light chain variable region with an amino acid sequence shown in SEQ ID NO:

14.

14. Use of the soluble growth stimulation expressed gene 2 protein antibody or its antigen-binding fragment according to any one of claims 1-4, the nucleic acid according to claim 5, the expression vector according to claim 6, the transgenic cell line or recombinant bacterium according to claim 7, or the immunoconjugate according to claim 8 in the preparation of a detection reagent or kit for diagnosing or predicting myocardial fibrosis or heart failure.

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