Soluble growth stimulating expression gene 2 protein antibody, detection kit and application thereof

Abstract

The present invention relates to the field of biomedicine technology, and provides soluble ST2 protein antibodies, detection kits, and applications thereof. The present invention provides soluble ST2 protein antibodies, intermediates, and detection kits containing the same, which can be used for the prediction and diagnosis of myocardial fibrosis, heart failure, and the like. The present invention obtains multiple antibodies with high sensitivity and good specificity by constructing recombinant soluble ST2 protein antigens, immunization, and cell line screening. Through appropriate reagent configuration, the good performance of the ST2 detection kit, especially the chemiluminescence detection kit, is achieved. The ST2 detection kit of the present invention has high accuracy, high sensitivity, a wide linear range, and a high degree of correlation; the reagents have high precision, good repeatability, and strong applicability.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a soluble growth stimulating expression gene 2 protein antibody, a detection kit and applications thereof. Background Art

[0002] Growth Stimulating Express Gene 2 (ST2), a member of the interleukin-1 (IL-1) receptor family, was first described in 1989. In 2005, ST2's specific ligand, interleukin-33 (IL-33), was discovered and reported. In 2013, the American College of Cardiology Foundation / Heart Association (ACCF / AHA) included IL-33 in its list of markers of myocardial fibrosis, suggesting its potential use in predicting disease severity and prognosis in patients with heart failure (HF). There are two main subtypes of ST2. One subtype lacks a transmembrane structure and is secreted outside the cell and exists in the circulatory system. It mainly exists in peripheral blood and is called soluble ST2 (sST2). The other subtype has a transmembrane structure and contains transmembrane fragments and the intracellular domain of the Toll / IL-1TR receptor. It mainly exists in cardiomyocytes and is called trans-membrane ST2 (ST2L).

[0003] ST2L and IL-33 form a signaling pathway that counteracts myocardial fibrosis and hypertrophy, slowing ventricular remodeling and thus exerting a protective effect on the myocardium. sST2, on the other hand, is secreted into the peripheral blood and competes with ST2L for IL-33 binding. This, in turn, blocks the beneficial effects of IL-33 by limiting the activation of the cascade triggered by the IL-33 / ST2L interaction. In healthy cardiovascular conditions, sST2 expression is low, allowing for full IL-33 / ST2L interaction, triggering a cascade of reactions that protect the heart through the activation of multiple intracellular kinases. In heart failure, however, sST2 expression is upregulated, acting as a "decoy" for binding to IL-33. Therefore, sST2 can be used as a biomarker for myocardial fibrosis. Higher levels of sST2 are associated with adverse cardiac remodeling, increased myocardial fibrosis, and worsened cardiovascular outcomes, making it a biomarker for both acute and chronic heart failure.

[0004] A follow-up study of over 500 patients found that the sST2 level in the blood can predict mortality within 1 year. Patients with sST2 concentrations higher than the average have a mortality rate 10 times higher within 1 year than patients with concentrations below the average. Moreover, sST2 concentrations higher than 700 ng / L can independently predict mortality within 1 year in patients with acute HF. In addition to baseline sST2 levels, dynamic concentration changes can also more effectively stratify the risk and assess the prognosis of patients with acute / chronic HF, and are basically unaffected by age, sex, body mass index, renal function, atrial fibrillation, anemia or HF etiology, and have small intra-individual variability. For patients with acute HF, a rapid short-term decrease in sST2 levels means that they have a good response to treatment and may have a better prognosis, while an increase in sST2 levels is more likely to reflect an increased risk of death.

[0005] Currently available immunoassay reagents and methods for soluble ST2 use the more traditional enzyme-linked immunosorbent assay (ELISA), which suffers from long detection cycles, low sensitivity, cumbersome procedures, and a lengthy process. Furthermore, the linear range is generally limited to 3-200 ng / ml, making it difficult for doctors to accurately assess a patient's condition. Subsequent methods developed include colloidal gold and immunofluorescence chromatography. While both offer rapid detection capabilities, they are limited to qualitative or semi-quantitative detection and suffer from low accuracy, making them incapable of providing precise clinical judgment. Furthermore, limitations inherent to the methods include the use of a heterogeneous reaction system, resulting in poor precision, and the inability to remove interfering substances from the operational procedures, compromising test accuracy. The immunoturbidimetric method for ST2 detection based on latex microspheres is also subject to significant interference from turbidity, such as lipids, that may be present in the sample.

[0006] Chemiluminescence assays offer the advantages of high sensitivity, strong precision, a wide linear range, and resistance to interference, making them widely used in clinical disease marker detection. The performance of current ST2 detection reagents is primarily determined by the functional properties of ST2-binding antibodies. Optimal antibody pairing can effectively improve disease diagnostic efficiency. Considering the low concentration of soluble ST2 in some clinical samples (1-200 ng / mL) and the presence of structurally homologous proteins, screening for highly sensitive and specific antibody pairs for ST2 diagnosis is crucial. Summary of the Invention

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

[0008] In a first aspect, the present invention provides a soluble growth-stimulating gene expression 2 protein antibody or an antigen-binding fragment thereof, comprising: VHCDR1, VHCDR2, and VHCDR3 as shown in the amino acid sequences of SEQ ID NOs: 1-3, and VLCDR1 as shown in SEQ ID NO: 4, VLCDR2 with the sequence NAK, and VLCDR3 as shown in SEQ ID NO: 5; or

[0009] The amino acid sequences of VHCDR1, VHCDR2 and VHCDR3 are shown in SEQ ID NOs: 6-8, as well as VLCDR1 shown in SEQ ID NO: 9, VLCDR2 with the sequence TAS, and VLCDR3 shown in SEQ ID NO: 10.

[0010] As an optional embodiment, the soluble growth stimulating expression gene 2 protein antibody or its antigen-binding fragment 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; or 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.

[0011] Alternatively, the antibody or antigen-binding fragment thereof can specifically recognize and / or bind to an epitope of a soluble growth stimulating gene expression 2 protein antigen as shown in SEQ ID NO: 15 or SEQ ID NO: 16.

[0012] Alternatively, the antibody comprises a monoclonal antibody; the antigen-binding fragment of the antibody is selected from F(ab')2, F(ab)2, Fab', Fab, Fv or scFv.

[0013] In a second aspect, the present invention provides a nucleic acid encoding the above-mentioned soluble growth stimulating gene expression 2 protein antibody or an antigen-binding fragment thereof.

[0014] The third aspect of the present invention provides an expression vector comprising the above nucleic acid.

[0015] The fourth aspect of the present invention provides a transgenic cell line or recombinant bacteria comprising the above-mentioned nucleic acid or the above-mentioned expression vector.

[0016] In a fifth aspect, the present invention provides an immunoconjugate comprising an antibody portion and a coupling portion coupled to the antibody portion, wherein the antibody portion comprises the above-mentioned soluble growth stimulating expression gene 2 protein antibody or an antigen-binding fragment thereof, and the coupling portion is selected from a fluorescent substance, a chemiluminescent substance, a colored substance, an enzyme or a combination thereof.

[0017] In a sixth aspect, the present invention provides a detection kit for soluble growth stimulating expression gene 2 protein, which comprises the above-mentioned soluble growth stimulating expression gene 2 protein antibody or antigen-binding fragment thereof.

[0018] Alternatively, the assay is designed for immunochemiluminescence detection of soluble GREX2 protein.

[0019] As an optional embodiment, the detection kit 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; 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 NOs: 1-3, and VLCDR1 as shown in SEQ ID NO: 4, VLCDR2 with a sequence of NAK, and VLCDR3 as shown in SEQ ID NO: 5; the second antibody comprises VHCDR1, VHCDR2 and VHCDR3 with amino acid sequences as shown in SEQ ID NOs: 6-8, and VLCDR1 as shown in SEQ ID NO: 9, VLCDR2 with a sequence of TAS, and VLCDR3 as shown in SEQ ID NO: 10.

[0020] As an optional option, the detection kit includes magnetic particles coated with capture antibodies, detection antibodies labeled with luminescent markers, substrates and excitation fluids suitable for the luminescent markers, and a series of quality control products and calibrators of soluble growth stimulating expression gene 2 protein antigens.

[0021] As an optional embodiment, 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.

[0022] In a seventh aspect, the present invention provides the use of the above-mentioned soluble growth-stimulating gene expression 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 bacteria, or the above-mentioned immunoconjugate in the preparation of a detection reagent or kit for diagnosing or predicting myocardial fibrosis or heart failure.

[0023] The present invention provides soluble ST2 protein antibodies, intermediates, and detection kits containing the same, which can be used for the prediction and diagnosis of myocardial fibrosis, heart failure, and the like. By constructing a recombinant soluble ST2 protein antigen and conducting immunization and cell line screening, the present invention obtains two antibodies with high sensitivity and excellent specificity. Furthermore, through appropriate reagent configuration, the excellent 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 reagents are highly precise, reproducible, and highly applicable. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the SDS-PAGE electrophoresis diagram of the recombinantly expressed sST2 protein.

[0025] Figure 2 This is a color gradient heatmap of the sST2 antibody pairing test results. In the figure, the color shades reflect the detection signal intensity (OD value) of the pairing combination; the value of each cell represents the high and low OD value. The color gradient changes from light blue to white, from white to light red, and finally to dark red, as the value increases. Darker blue indicates a weaker signal generated by the antibody pairing, while darker red indicates a stronger signal.

[0026] Figure 3 This is the test result analysis curve of the linear range of the sST2 kit in Example 6. DETAILED DESCRIPTION

[0027] The present invention provides an antibody capable of specifically binding to a soluble growth stimulating expression gene 2 protein or an antigen binding fragment thereof, namely, an antibody raw material, an sST2 antigen detection product and an application scheme.

[0028] The present invention constructs a human soluble growth-stimulating gene 2 protein expression gene and obtains a biologically active recombinant human soluble growth-stimulating gene 2 protein antigen through recombinant expression. Through independent animal immunization and monoclonal cell line screening, nine monoclonal antibodies capable of efficiently binding to the recombinant human growth-stimulating gene 2 protein are obtained. Furthermore, a pair of monoclonal antibodies, ST2-7# and ST2-15#, is preferably selected, capable of binding to the human soluble growth-stimulating gene 2 protein with high sensitivity and strong specificity using a double antibody sandwich method. That is, the present invention provides the use of these two antibodies in detecting soluble growth-stimulating gene 2 protein, detecting heart failure and myocardial fibrosis, and in preparing reagents or kits for detecting myocardial fibrosis and heart failure.

[0029] Regarding antibodies, in some embodiments, the antibody comprises all or a portion of an antibody constant region. In some embodiments, the constant region is selected from various types including IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3, or IgG4), and IgM. The present invention also discloses antigen-binding fragments that specifically bind to anti-ST2 protein antibodies. Examples of antigen-binding fragments include F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, and the like.

[0030] For monoclonal antibodies of the present invention, they can be obtained from a single clone by any means available or known in the art, including expression in any eukaryotic, prokaryotic or phage system. Monoclonal antibodies for 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.

[0031] Other products of the present invention also include intermediates used in the preparation of antibodies, including expression vectors, transgenic cell lines, or recombinant bacteria, or compositions comprising the foregoing. Specifically, expression vectors may be recombinant expression vectors, such as eukaryotic expression vectors, while transgenic cell lines or recombinant bacteria may also include eukaryotic or prokaryotic cells, such as Escherichia coli, yeast, or animal cells (e.g., mammalian cells, such as mouse cells or human cells), as well as cell lines.

[0032] The present invention also provides an sST2 detection kit or reagent comprising an antibody or antigen-binding fragment thereof of the present invention. In some embodiments, the kit further comprises a solid substrate for attachment of the antibody or antigen-binding fragment thereof, including but not limited to microplates, magnetic particles, immunochromatographic filter paper, polymers such as polystyrene, glass filters, and other insoluble supports. In some embodiments, the kit further comprises other components, including but not limited to a labeling enzyme, a corresponding substrate, a radioisotope, a fluorescent substance, a colored substance, a buffer, and the like.

[0033] sST2 is detected using the above-mentioned kit or detection reagent. In some embodiments, the detection method includes: binding the antibody or antigen-binding fragment thereof of the present invention to the sST2 antigen in the sample to be tested to form an antibody-antigen or antibody fragment-antigen complex; then, detecting the sample complex to determine whether the target antigen is present in the sample.

[0034] Specifically, in some embodiments, the detection reagent or kit comprises an antibody or antigen-binding fragment thereof of the present invention (serving as a capture antibody, or primary antibody) and a detectably labeled secondary antibody (detection antibody). The specific detection method comprises: adsorbing the primary antibody onto a solid support; subsequently adding a test sample potentially containing sST2 to the support; adding a labeled secondary antibody to the support; and detecting the presence of the labeled secondary antibody to determine the presence of sST2. Alternatively, the specific detection method may include known methods such as enzyme immunoassay, immunochemiluminescence, radioimmunoassay, fluorescence immunoassay, immunochromatography, competitive assay, or sandwich assay.

[0035] In a preferred embodiment, the present invention provides an immunochemiluminescent detection kit prepared using anti-soluble growth-stimulating gene 2 protein antibodies (ST2-7# and ST2-15#). Through independent screening of monoclonal antibody pairs with the highest sensitivity and specificity, and through appropriate reagent configuration, this kit achieves excellent performance for sST2 detection, exhibiting high accuracy and sensitivity, a wide linear range, and considerable precision.

[0036] More specifically, the detection kit of the present invention has the following advantages:

[0037] 1. The reagent has high accuracy, and the relative deviation of the test results of samples of various concentrations does not exceed 5%;

[0038] 2. The reagent has high sensitivity, a wide linear range, and a high degree of correlation. The lower limit of the linear range (2ng / mL) is much lower than the reference value (35ng / mL), which can effectively avoid false positives. The actual linear range is much higher than the upper limit of the expected linear range (300ng / mL), even reaching 500ng / mL, covering the vast majority of sample concentrations. This allows high-concentration samples to be tested without dilution and maintain accurate results. 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 test results across the entire range and enabling precise monitoring of changes and fluctuations in patient indicators.

[0039] 3. The reagents have high precision and good repeatability. Both the indoor and inter-laboratory precision CV are less than 5%, which can provide a reliable traceability basis for the test data;

[0040] 4. The reagent has good applicability, suitable for both high-speed fully automatic chemiluminescence workstations and small chemiluminescence analyzers. It can meet a variety of hospital application scenarios including outpatient clinics, emergency departments and community hospitals, while maintaining stable detection performance.

[0041] The technical solution of the present invention is described in detail below with reference to the embodiments.

[0042] Unless otherwise specified, the experimental materials used in the examples of the present invention can be obtained from commercial channels, and the experimental operation methods, unless otherwise specified, are performed using means known in the art.

[0043] Example 1: Recombinant expression of sST2 antigen

[0044] Based on the sequence information of the growth-stimulating expressed gene 2 protein (NP_003847.2) published by the National Center for Biotechnology Information (NCBI), the full-length sequence was selected and cloned into the pCDNA3.4 eukaryotic expression vector. The resulting plasmid was transfected into HEK293 cells (human embryonic kidney cells 293) using PEI transfection reagent (polyethylenimine). After culturing for 7 days in a constant temperature shaker at 37°C, 8% CO2, and an appropriate speed, the cell supernatant was collected by centrifugation and purified by nickel-based affinity chromatography to obtain the recombinant expressed sST2 protein, which was approximately 37.8 kDa ( Figure 1 ).

[0045] Example 2: Immunization of animals with recombinant sST2 protein

[0046] The recombinant sST2 antigen prepared in Example 1 was diluted to 1 mg / mL with 20 mM PBS (phosphate buffered saline) at pH 7.4 and emulsified with Freund's adjuvant at a 1:1 ratio. Six- to eight-week-old Balb / c mice were immunized intraperitoneally with 100 μg of the emulsified recombinant sST2 antigen per mouse. Freund's complete adjuvant was used for the primary immunization, and incomplete adjuvant was used for the booster immunization. After three immunizations, tail vein blood was collected and titered. Cells with titers exceeding 1 million were selected for cell fusion. 72 hours prior to fusion, the spleen was directly immunized with 30 μg of sST2 antigen.

[0047] Example 3: Screening of monoclonal antibodies

[0048] (1) Fusion cells: The mice to be fused were killed by cervical dislocation, and their spleens were removed, ground and filtered to prepare a spleen cell suspension, which was then transferred to a 50 mL centrifuge tube. The cells were resuspended in serum-free RPMI1640 medium and washed three times, each time by centrifugation at 15,000 rpm for 5 min. The myeloma cells were resuspended in serum-free RPMI1640 medium and washed three times, each time by centrifugation at 15,000 rpm for 5 min. The spleen cell suspension and myeloma cell suspension were mixed, centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and the bottom of the tube was flicked to disperse the cells. At the same time, 1 mL of PEG1450 pre-warmed to 37°C was slowly added within 60 s. After the addition was completed, the mixture was pipetted and mixed for 30 s. Then, 40 mL of RPMI 1640 medium pre-warmed to 37°C was immediately added to terminate the fusion reaction. The cells were transferred to RPMI 1640 HAT cell screening medium, mixed, and plated on a 96-well cell culture plate. After 7 days, the RPMI 1640 cell culture medium was replaced, and after another 3 days, part of the supernatant was aspirated for subsequent operations.

[0049] (2) Supernatant detection: The recombinantly expressed sST2 protein was coated in a polystyrene 96-well ELISA plate at a total amount of 50 ng / well. The cell supernatant obtained in Example 3 (1) was added to the coated 96-well plate. After incubation and washing, the plate was incubated with HRP-labeled goat anti-mouse secondary antibody, reaction substrate, and stop solution. The cell wells corresponding to the high-value positive wells were selected for subcloning. After five subclones, nine hybridoma monoclonal cells with good reactivity to the recombinant sST2 protein (OD value ≥ 3.0) were finally selected.

[0050] Example 4: Paired screening of monoclonal antibodies

[0051] (1) HRP labeled antibody: Dissolve 30 mg of HRP dry powder in 1.5 mL of pure water, add 1.2 mL of 25 mg / mL NaIO4 aqueous solution, mix well, and react at 4°C for 30 min. Remove and add 0.2 mL of ethylene glycol solution, let it sit at room temperature for 30 min to complete HRP activation, and store at 4°C for use. Dilute the antibody to 2 mg / mL with 20 mM carbonate buffer pH 9.6, mix 2 mL of the antibody to be labeled with the activated HRP labeling solution, and dialyze overnight in 20 mM carbonate buffer pH 9.6. Remove and add 0.2 mL of 2 mg / mL NaBH4 solution, let it react at 4°C for 2 hours. Add an equal volume of saturated ammonium sulfate solution, let it react at 4°C for 30 min, then centrifuge. Resuspend the precipitate with 1 mL of 20 mM PBS pH 7.4 solution, add an equal volume of glycerol, mix well, and store.

[0052] (2) Pairing test: Use 20mM PB pH7.4 solution to dilute the anti-ST2 antibody to 1μg / mL, 100μL / well is coated on a polystyrene 96-well enzyme-labeled plate, and incubated at 37℃ for 2 hours. Use PBST to wash the 96-well plate 5 times, add blocking solution for blocking, and discard the blocking solution after blocking. Add 100μL / well of recombinant ST2 protein diluted to 10ng / mL to the 96-well plate, incubate at 37℃ for 1 hour, wash the 96-well plate 5 times with PBST, add HRP-labeled anti-ST2 antibody, incubate at 37℃ for 30min, wash the 96-well plate 5 times with PBST, and then incubate the reaction substrate and stop solution in sequence, and place it in an enzyme-labeled instrument for reading. All 9 antibodies are paired in pairs as coating antibodies and labeled antibodies. After testing, the monoclonal antibody pair with the highest sensitivity and best specificity is selected, namely monoclonal antibodies ST2-7#, ST2-15# ( Figure 2 ).

[0053] Example 5: Sequence determination and binding epitope analysis of monoclonal antibodies

[0054] (1) Antibody sequence determination: Nanjing GenScript Biotech Co., Ltd. was commissioned to perform de-novo protein sequencing on two monoclonal antibodies, ST2-7# and ST2-15#. After spectral analysis, the amino acid sequences of the heavy chain and light chain variable regions of the two monoclonal antibodies, ST2-7# and ST2-15#, were obtained. The specific amino acid sequences are shown in Table 1. Both antibodies are mouse IgG1 subtypes.

[0055] Table 1 Amino acid sequences of the variable regions of monoclonal antibodies

[0056]

[0057] (2) Antibody binding epitope analysis: Nanjing GenScript Biotech Co., Ltd. was commissioned to analyze the binding epitopes of two monoclonal antibodies, ST2-7# and ST2-15#. The binding epitopes of both antibodies were linear epitopes, and the relevant information is shown in Table 2. Among them, the binding epitope of monoclonal antibody ST2-7# is the amino acid sequence QYDCLALNLHG (Gln-Tyr-Asp-Cys-Leu-Ala-Leu-Asn-Leu-His-Gly) from position 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) from position 312 to 319 of the full-length ST2 protein.

[0058] Table 2 Binding epitope information of monoclonal antibodies

[0059]

[0060] Example 6: Configuration of sST2 detection reagents and kits

[0061] (1) Preparation of antibody buffer: Add 50 mM pH 7.3 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), 0.5% lauryl polyoxyethylene ether, 50 mM tris(hydroxymethylaminomethane) (Tris), 2% sucrose, 4% arginine, 2.5% casein, and 0.1% sodium azide to prepare antibody buffer. Adjust the pH to 7.30 ± 0.05 (25°C). Filter through a 0.8 μm filter to remove large particles.

[0062] (2) Preparation of magnetic bead storage buffer: Add 50 mM pH 7.3 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), 0.5% lauryl polyoxyethylene 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 magnetic bead storage buffer. Adjust the pH to 7.40 ± 0.05 (25°C). Filter through a 0.8 μm filter to remove large particles.

[0063] (3) Biotin labeling of antibodies: Dissolve biotin to 1 mg / ml using dimethyl sulfoxide solution, add 2 mg / mL of sST2 capture antibody, i.e., ST2-7#, and mix at a mass ratio of 1:10. Then, dilute with an equal volume of carbonate buffer, pH 9.6, and incubate at 37°C for 2 hours to complete the labeling. Then, ultrafiltration was performed using a 50 kDa ultrafiltration tube to replace the buffer with antibody buffer. The final concentration of the biotin-labeled sST2 capture antibody was 10 μg / mL.

[0064] (4) Coupling of magnetic bead reagent: 20 mg of concentrated streptavidin-labeled magnetic beads were magnetically separated and washed three times with antibody buffer. The biotin-labeled sST2 capture antibody was added and mixed at a mass ratio of 50:1. The mixture was reacted at 30°C in an antibody buffer environment for 15 minutes to complete the coupling of the antibody and magnetic beads. The supernatant was discarded after magnetic separation and resuspended with magnetic bead storage buffer. The volume was diluted to 200 mL to obtain the magnetic bead reagent with completed antibody coupling.

[0065] (5) Coupling of acridinium ester labeling reagent: Dissolve acridinium ester in dimethyl sulfoxide solution to 5 mg / mL, add 2 mg / mL of sST2 detection antibody, i.e., ST2-15#, and mix at a mass ratio of 1:10. Then, dilute with an equal volume of carbonate buffer, pH 9.6, and incubate at 37°C for 2 hours to complete the labeling. Then, ultrafiltration is performed using a 50 kDa ultrafiltration tube to replace the buffer with antibody buffer, and the volume is diluted to 2 L to obtain the acridinium ester labeling reagent for antibody coupling.

[0066] (6) Preparation of calibrators and quality control materials: Recombinant ST2 antigen was prepared into a series of calibrators with 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 pH 7.3 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), 0.5% lauryl polyoxyethylene ether, 50 mM potassium chloride, 2% sucrose, 4% arginine, and 0.1% sodium azide. The high-concentration ST2 sample (100 ng / mL) was diluted to 8 ng / mL and 50 ng / mL with the calibrator diluent, which were used as low-value quality control materials and high-value quality control materials, respectively.

[0067] Example 7: Accuracy of the kit

[0068] The accuracy of a reagent refers to the degree of agreement between the average value of the quantity obtained by repeated measurements an infinite number of times and a reference value.

[0069] The detection kit described in Example 6 was used to test one sample of the high-concentration (199.47±3.56 ng / mL) and one sample of the low-concentration (19.87±0.52 ng / mL) reference product for growth-stimulating expression gene 2 protein, respectively, three times. The test results are shown in Table 3. Based on the relative deviation of the test results, the relative deviation of the three test results for the two reference products was within 5%, indicating that the kit has good accuracy.

[0070] Table 3 Test results of the sST2 kit accuracy in Example 6

[0071]

[0072] Example 8: Linear range of the kit

[0073] The expected linear range of the sST2 kit in Example 6 is 2-500 ng / mL. A high-value sample (501.23 ng / mL) and a 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 using the zero-value sST2 calibrator to prepare the following six test samples at different concentration levels (1.20 ng / mL, 4.07 ng / mL, 29.91 ng / mL, 144.76 ng / mL, 288.31 ng / mL, and 501.23 ng / mL), which were then tested using the sST2 kit in Example 6.

[0074] The test results are shown in Table 4. After fitting curve and linear analysis, the linear regression equation of the test kit is: y=0.9972x+0.0212, R 2 =0.9996>0.995( Figure 3 ), it is preliminarily judged that it meets the expected linear range requirements; the relative deviation between each measured value and the theoretical value of each concentration sample is no more than 10%, and the expected linear range is acceptable.

[0075] Table 4 Detection results of the linear range of the sST2 kit in Example 6

[0076]

[0077] Example 9: Precision of the kit

[0078] Precision refers to the degree of agreement between labeled or measured values ​​obtained from repeated measurements of the same or similar analytes under specified conditions. The precision of the sST2 kit described in Example 6 was evaluated using repeatability, intermediate precision (within-laboratory precision), and reproducibility (between-laboratory precision). This performance evaluation was performed using three batches of test samples: a high-concentration quality control 1 (target value of 153.37 ng / mL), a low-concentration quality control 2 (target value of 30.47 ng / mL), and an equal-proportioned mixed quality control (target value of 91.92 ng / mL).

[0079] In the same laboratory, using the same detection instrument, three batches of samples were tested separately, with two analytical batches tested every day. Two samples of each concentration in each batch were processed in parallel for testing to evaluate the laboratory precision of the kit.

[0080] In the same laboratory, three different instruments (NRM411, NORMAN-CL 5, and NRM411-S7) and a single batch of the detection kit were used to test three batches of samples. Each batch of samples was tested continuously for five days, and each batch of samples was tested five times daily to evaluate the inter-laboratory precision of the kit.

[0081] The results of intra-laboratory precision and inter-laboratory precision tests were analyzed using a two-way analysis of variance model. The variance analysis results are shown in Table 5, the standard deviation and CV analysis results are shown in Table 6, and the confidence interval results are shown in Table 7. All of the above results are in line with expectations, with no abnormal values.

[0082] Table 5 Precision variance analysis results of the sST2 kit in Example 6

[0083]

[0084] Table 5 related notes: 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.

[0085] Table 6 Precision analysis results of the sST2 kit in Example 6

[0086]

[0087] Table 6 related notes: s: standard deviation; CV: coefficient of variation.

[0088] Table 7 Precision confidence intervals (95%, α=0.05) for the sST2 kit in Example 6

[0089]

[0090] The present invention has been described in detail above with reference to specific embodiments and exemplary examples. However, these descriptions are not to be construed as limiting the present invention. Those skilled in the art will appreciate that, without departing from the spirit and scope of the present invention, various equivalent substitutions, modifications, or improvements may be made to the technical solutions and implementations of the present invention, all of which fall within the scope of the present invention.

Claims

1. A soluble growth stimulating gene expression 2 protein antibody or an antigen-binding fragment thereof, characterized in that: comprising: VHCDR1 as shown in SEQ ID NO: 1, VHCDR2 as shown in SEQ ID NO: 2, and VHCDR3 as shown in SEQ ID NO: 3, as well as VLCDR1 as shown in SEQ ID NO: 4, VLCDR2 with the sequence of NAK, and VLCDR3 as shown in SEQ ID NO: 5; or, The amino acid sequence of VHCDR1 is shown in SEQ ID NO: 6, VHCDR2 is shown in SEQ ID NO: 7, and VHCDR3 is shown in SEQ ID NO: 8, as well as the VLCDR1 is shown in SEQ ID NO: 9, the VLCDR2 with the sequence of TAS, and the VLCDR3 is shown in SEQ ID NO:

10.

2. The soluble growth stimulating gene expression 2 protein antibody or antigen-binding fragment thereof according to claim 1, characterized in that: It 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; or 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.

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

16.

4. The soluble growth stimulating gene expression 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 expression gene 2 protein antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4.

6. An expression vector comprising the nucleic acid of claim 5.

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

8. An immunoconjugate, characterized in that The invention comprises an antibody portion and a coupling portion coupled to the antibody portion, wherein the antibody portion comprises the soluble growth stimulating expression gene 2 protein antibody or its antigen-binding fragment according to any one of claims 1 to 4, and the coupling portion is selected from a fluorescent substance, a chemiluminescent substance or a combination thereof.

9. A detection kit for soluble growth stimulating expression gene 2 protein, characterized in that: The invention comprises the soluble growth stimulating expression gene 2 protein antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

10. The detection kit according to claim 9, characterized in that The detection kit is used for the detection of soluble growth-stimulating gene 2 protein by immunochemiluminescence.

11. The detection kit according to claim 9, characterized in that The invention 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; or, the first antibody serves as a detection antibody and the second antibody serves as a capture antibody; the first antibody comprises an amino acid sequence of VHCDR1 as shown in SEQ ID NO: 1, a VHCDR2 as shown in SEQ ID NO: 2, and a VHCDR3 as shown in SEQ ID NO: 3, as well as a VLCDR1 as shown in SEQ ID NO: 4, a VLCDR2 with a sequence of NAK, and a VLCDR3 as shown in SEQ ID NO: 5; the second antibody comprises an amino acid sequence of VHCDR1 as shown in SEQ ID NO: 6, a VHCDR2 as shown in SEQ ID NO: 7, and a VHCDR3 as shown in SEQ ID NO: 8, as well as a VLCDR1 as shown in SEQ ID NO: 9, a VLCDR2 with a sequence of TAS, and a VLCDR3 as shown in SEQ ID NO:

10.

12. The detection kit according to claim 9, characterized in that The detection kit includes magnetic particles coated with capture antibodies, detection antibodies labeled with luminescent markers, substrates and excitation fluids suitable for the luminescent markers, and a series of quality control products and calibrators of soluble growth stimulating expression gene 2 protein antigens.

13. The detection kit according to claim 12, characterized in that 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.

14. Use of the soluble growth stimulating gene expression 2 protein antibody or its antigen-binding fragment according to any one of claims 1 to 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 myocardial fibrosis or heart failure.

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