Diagnostic kits for liver fibrosis

Abstract

This invention relates to a diagnostic kit for liver fibrosis. The kit comprises a solid-phase conjugate containing anti-MASP2 antibody and an anti-MASP2 antibody labeled with a chemiluminescent marker. This invention has found that using MASP2 as a marker of liver fibrosis, combined with chemiluminescent detection, can improve the sensitivity and specificity of liver fibrosis diagnosis, thereby increasing its accuracy.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a diagnostic kit for liver fibrosis. Background Technology

[0002] Viral hepatitis, fatty liver, and alcoholic liver disease are among the most common and prevalent liver diseases. Liver fibrosis is a pathological change present in most chronic liver diseases. Essentially, it is a reversible, excessive repair response to liver tissue damage during the course of chronic liver disease. Further progression of liver fibrosis can lead to cirrhosis, severely impacting the patient's health and life.

[0003] According to the "Guidelines for the Diagnosis and Treatment of Liver Fibrosis Using Integrated Traditional Chinese and Western Medicine (2019 Edition)," the commonly used diagnostic methods for liver fibrosis in clinical practice are divided into three categories: pathology, imaging, and serological examinations. Among these, liver biopsy pathology is the irreplaceable "gold standard," but it is an invasive procedure and has limitations due to specimen and interpreter errors, which greatly restricts its application. Imaging examinations vary considerably in their specificity and sensitivity for diagnosing liver fibrosis and are easily affected by factors such as equipment, diagnostic site, and the operator's skill and experience. Commonly used serological markers for liver fibrosis include hyaluronic acid, type III procollagen and type III procollagen peptides or N-terminal peptides, type IV collagen, and laminin. However, the results of these serological marker tests only indicate abnormalities in the metabolism of the extracellular matrix (ECM) within liver tissue. Elevated fibrosis markers can also occur in some internal medicine diseases, easily leading to misdiagnosis and affecting the accuracy of liver fibrosis diagnosis. Summary of the Invention

[0004] This invention reveals that the MASP2 gene is specifically expressed in the liver, exhibiting tissue and organ specificity. The level of MASP2 protein in blood samples is associated with liver fibrosis. In liver fibrosis samples, the MASP2 protein level is lower than in normal samples. Diagnosing liver fibrosis by detecting the MASP2 protein level in blood samples can improve the current problem of low diagnostic accuracy in liver fibrosis diagnosis.

[0005] Therefore, the primary objective of this invention is to provide an application of MASP2 as a marker of liver fibrosis in the preparation of diagnostic products for liver fibrosis.

[0006] In one embodiment, the diagnostic product is a chemiluminescence diagnostic product.

[0007] Another object of the present invention is to provide a diagnostic kit for liver fibrosis.

[0008] A diagnostic kit for liver fibrosis, comprising:

[0009] A solid-phase conjugate, the solid-phase conjugate comprising a solid-phase support and an anti-MASP2 antibody coated on the solid-phase support; and

[0010] The labeled antibody is an anti-MASP2 antibody labeled with a chemiluminescent marker.

[0011] In one embodiment, the anti-MASP2 antibody of the labeled antibody binds to a different antigenic epitope of MASP2 than the anti-MASP2 antibody of the solid-phase conjugate binds to a different antigenic epitope of MASP2.

[0012] In one embodiment, the chemiluminescent label is selected from one of acridine-based chemiluminescent substances, ruthenium terpyridine, adamantane, luminol, luminol derivatives, isoluminol, isoluminol derivatives, horseradish peroxidase, and alkaline phosphatase.

[0013] In one embodiment, the acridine-based chemiluminescent material is selected from at least one of AE-NHS, DMAE-NHS, Me-DMAE-NHS, NSP-DMAE-NHS, NSP-SA, NSP-SA-NHS, and NSP-SA-ADH.

[0014] In one embodiment, the diagnostic kit further includes an antigen diluent comprising: 10 mM to 50 mM phosphate, 500 mM to 1 M sodium chloride, 0.1 w / v% to 2 w / v% EDTA-2Na, 1 w / v% to 10 w / v% trehalose, 1 w / v% to 10 w / v% mannitol, 1 w / v% to 10 w / v% lysine, and 0.1 v / v% to 2 v / v% surfactant.

[0015] In one embodiment, the surfactant is selected from at least one of Tween20, Tween80, Triton X-100, Triton X-405 and Tetronic 1307.

[0016] In one embodiment, the antigen diluent further includes a preservative; the concentration of the preservative in the antigen diluent is 0.1 w / v% to 0.5 w / v%.

[0017] In one embodiment, the antigen diluent comprises: 15 mM to 40 mM phosphate, 500 mM to 800 M sodium chloride, 1 w / v% to 2 w / v% EDTA-2Na, 5 w / v% to 10 w / v% trehalose, 1 w / v% to 5 w / v% mannitol, 1 w / v% to 5 w / v% lysine, 0.5 w / v% to 1.5 w / v% surfactant, and 0.1 w / v% to 0.5 w / v% preservative. Attached Figure Description

[0018] Figure 1 The ROC curve of the kit in Example 1;

[0019] Figure 2 This is the ROC curve for reagent A. Detailed Implementation

[0020] To facilitate understanding of the present invention, a more comprehensive description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Each example is provided as an explanation and not as a limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment can be used in another embodiment to produce further embodiments.

[0021] The term "and / or" includes any and all combinations of one or more of the associated listed items. The terms "contains," "includes," and "comprises" as used herein are synonyms and are inclusive or open-ended, not excluding additional, uncited members, elements, or method steps. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0023] Mannose-associated serine protease 2 (MASP2) is a member of the MASPs family. The human MASP2 gene is located at 1p36.23-31, is approximately 20 kb in length, and encodes a 686-residue peptide chain. It is specifically expressed in the liver. This gene has two alternative splice variants, MASP2 and MAP19, both synthesized and secreted into the bloodstream by the liver and exist as zymogens. MASP2 is associated with human immunodeficiency diseases and plays an important role in the body's innate immune defense. It is the central protease molecule in the complement lectin pathway. In the complement-activated lectin pathway, mannose-binding lectin (MBL) binds to MASP2 through its collagen-like region (CLR) to form a complex. Then, its C-terminal sugar recognition domain recognizes and binds to mannose, fucose, and other structures on the surface of pathogenic microorganisms, leading to a conformational change. MASP2 is then activated, cleaving C4 and C2 into C4b and C4a, and C2b and C2a. Subsequently, C4b and C2b participate in the formation of C3 convertase, further activating downstream complement components. This study found that MASP2, as a marker of liver fibrosis, combined with chemiluminescence detection, can improve the sensitivity and specificity of liver fibrosis diagnosis, thus enhancing diagnostic accuracy.

[0024] Based on the above, one embodiment of this application provides the application of MASP2 as a marker of liver fibrosis in the preparation of diagnostic products for liver fibrosis.

[0025] Based on the above, one embodiment of this application also provides the application of a reagent for detecting the biomarker MASP2 in the preparation of diagnostic products for liver fibrosis.

[0026] In some embodiments, the diagnostic product is a diagnostic kit. Further, the diagnostic product is a chemiluminescence diagnostic kit.

[0027] Furthermore, one embodiment of this application also provides a diagnostic kit for liver fibrosis, the diagnostic kit comprising a solid-phase conjugate and a labeled antibody.

[0028] Specifically, the solid-phase conjugate includes a solid-phase carrier and an anti-MASP2 antibody coated on the solid-phase carrier.

[0029] Optionally, the solid support can be a test tube, EP tube, multi-well plate, micro-reaction plate with recesses, microspheres, or discs.

[0030] In some embodiments, the multiwell plate is an enzyme-linked immunosorbent assay (ELISA) plate. In one optional specific example, the multiwell plate has 16, 32, 48, 64, 96 or more wells.

[0031] In this invention, the term "microsphere" can refer to a sphere, near-sphere, cube, polyhedron, or irregular shape. In some embodiments, the diameter of the microsphere is 10 nm to 1 mm. In an optional specific example, the diameter of the microsphere is 100 nm, 500 nm, 1 μm, 10 μm, 100 μm, or 500 μm. Further, the diameter of the microsphere is 400 nm to 10 μm.

[0032] In some embodiments, the microspheres are magnetic beads containing magnetic materials. The magnetic materials can be metals (metallic elements or alloys), nonmetals, or composites of metals and nonmetals. Examples of metals include iron, aluminum, nickel, and cobalt; examples of nonmetals include ferrite nonmetals (e.g., Fe₂O₃ or Fe₃O₄ magnetic nanoparticles); and examples of composites of metals and nonmetals include neodymium iron boron rubber magnetic composite materials.

[0033] In some embodiments, the surface of the microspheres is modified with one or more active functional groups. Optionally, the active functional groups include one or more of -OH, -COOH, -NH2, -CHO, and -SO3H. In some embodiments, the coated antibody is conjugated or bound to the microspheres by physical adsorption or direct chemical conjugation (e.g., bridging via a bridging agent).

[0034] In other embodiments, the solid support is made of one or more of the following materials: polystyrene, plastic, cellulose, polyacrylamide, polyethylene polypropylene, cross-linked dextran, glass, silicone rubber, and agarose gel.

[0035] Specifically, the labeled antibody is an anti-MASP2 antibody labeled with a chemiluminescent marker. In some embodiments, the labeled anti-MASP2 antibody binds to a different MASP2 epitope than the anti-MASP2 antibody of the solid-phase conjugate binds to. In this case, the diagnostic kit utilizes the solid-phase conjugate to bind to MASP2 in the test sample, forming a complex with the labeled antibody to detect the amount of MASP2 in the test sample; that is, it uses the principle of the immunosandwich assay to detect the amount of MASP2 in the test sample. It is understood that in other embodiments, the labeled anti-MASP2 antibody binds to the same MASP2 epitope as the anti-MASP2 antibody of the solid-phase conjugate, in which case the competitive assay is used to detect the amount of MASP2 in the test sample.

[0036] In some embodiments, the anti-MASP2 antibody coated on the solid-phase support and the chemiluminescently labeled anti-MASP2 antibody are each independently selected from all domesticated (e.g., livestock and pets) and wild animals and birds, including, but not limited to, cattle, horses, dairy cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, mink, chickens, ducks, geese, turkeys, fighting cocks, etc. In some embodiments, the anti-MASP2 antibody coated on the solid-phase support and the chemiluminescently labeled anti-MASP2 antibody are from the same source. Antibodies from the same species can effectively reduce cross-reactivity and improve accuracy.

[0037] In some embodiments, the anti-MASP2 antibody coated on the solid-phase support is a polyclonal antibody. This configuration can increase the sensitivity of the detection.

[0038] It should be noted that the term "antibody" in this article includes both naturally occurring antibodies and non-naturally occurring antibodies, including, for example, chimeric, bifunctional, humanized antibodies and human antibodies, as well as related synthetic isoforms. Furthermore, antibodies also include functional fragments capable of binding to desired antigens, such as Fab, F(ab')2, Fd, Fv, scFv, bispecific antibodies, and the smallest antibody recognition unit, as well as single-chain derivatives of these antibodies and fragments, such as scFv-Fc. Antibody types can be IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD.

[0039] In some embodiments, the chemiluminescent label is selected from one of acridine chemiluminescent substances, ruthenium terpyridine, adamantane, luminol, luminol derivatives, isoluminol, isoluminol derivatives, horseradish peroxidase, and alkaline phosphatase.

[0040] In one embodiment, the acridine-based chemiluminescent material is selected from at least one of acridine esters and acridine sulfonamides. Optionally, the acridine-based chemiluminescent material is selected from at least one of AE-NHS, DMAE-NHS, Me-DMAE-NHS, NSP-DMAE-NHS, NSP-SA, NSP-SA-NHS, and NSP-SA-ADH.

[0041] It should be noted that in this article, "AE-NHS" is the abbreviation for "acridine succinimide"; "DMAE-NHS" is the abbreviation for "(6'-Dimethyl-4'-(N-succinimidyloxycarbonyl)phenyl-10-methyl-acridinium-9-carboxylate methosulfate)"; "Me-DMAE-NHS" is the abbreviation for "2',6'-Dimethylcarbonylphenyl10-Methyl-9-acridinecarboxylate 4'-NHS Ester Triflate"; and "NSP-DMAE-NHS" is the abbreviation for "2',6'-DiMethylcarbonylphenyl-10-sulfopropylacridinium-9-carboxylate". "NSP-SA" is an abbreviation for "4'-NHSEster"; "NSP-SA" is an abbreviation for "3-[9-(((3-(carboxypropyl)[4-methxylphenyl]sulfonyl)amine)carboxyl]-10-acridiniumyl)-1-propanesulfonateinner salt"; "NSP-SA-NHS" is an abbreviation for "3-[9-(((3-(N-succinimidyloxycarboxypropyl)[4-methxylphenyl]sulfonyl)amine)carboxyl]-10-acridiniumyl)-1-propanesulfonateinner salt"; "NSP-SA-ADH" is an abbreviation for "2',6'-Dimethylcarbonylphenyl10-Methyl-9-acridinecarboxylate 4'-NHSEster Triflate". AE-NHS, DMAE-NHS, Me-DMAE-NHS, and NSP-DMAE-NHS belong to acridine esters; NSP-SA and NSP-SA-NHS belong to acridine salts; and NSP-SA-ADH is an acridine hydrazide.

[0042] In some embodiments, the diagnostic kit described above also includes an antigen diluent. It is understood that the antigen diluent may be a solid (e.g., lyophilized powder or spray-dried powder) or a solution.

[0043] In some embodiments, the antigen diluent includes: 10 mM–50 mM phosphate, 500 mM–1 M sodium chloride, 0.1 w / v%–2 w / v% EDTA-2Na, 1 w / v%–10 w / v% trehalose, 1 w / v%–10 w / v% mannitol, 1 w / v%–10 w / v% lysine, and 0.1 v / v%–2 v / v% surfactant. Studies have confirmed that the MBL / MASP2 complex can only be separated in a high-salt solution containing EDTA (a high-salt solution refers to a salt solution with a concentration of 500 mM–1 M), thus improving the detection sensitivity of the reagent.

[0044] In some embodiments, the pH of the antigen diluent is 6.5 to 7.5. In one optional specific example, the pH of the antigen diluent is 6.8, 7, 7.2, or 7.5.

[0045] In some embodiments, the surfactant comprises at least one selected from Tween, Triton X-100, Triton X-405, and Tetronic 1307. Optionally, Tween is selected from at least one selected from Tween-20, Tween-80, and Tween-100. It is understood that in other embodiments, the surfactant is not limited to the above-described embodiments.

[0046] In some embodiments, the antigen diluent further includes a preservative. Optionally, the preservative is selected from at least one of sodium azide and proclin 300. In other embodiments, the preservative is not limited to those described above.

[0047] In some embodiments, the concentration of the preservative in the antigen diluent is 0.1 w / v% to 0.5 w / v%. Further, the concentration of the preservative in the antigen diluent is 0.1 w / v% to 0.2 w / v%.

[0048] Furthermore, the antigen diluent includes: 15mM–40mM phosphate, 500mM–800M sodium chloride, 1w / v%–2w / v% EDTA-2Na, 5w / v%–10w / v% trehalose, 1w / v%–5w / v% mannitol, 1w / v%–5w / v% lysine, 0.5w / v%–1.5w / v% surfactant, and 0.1w / v%–0.5w / v% preservative.

[0049] The aforementioned diagnostic kit for liver fibrosis uses MASP2 as a biomarker for liver fibrosis and combines it with chemiluminescence detection to improve the diagnostic sensitivity and specificity of liver fibrosis, thereby increasing the accuracy of diagnosis. Furthermore, when paired with a corresponding chemiluminescence analyzer, it can achieve automated (automatic sampling, automatic detection, and automatic reporting), high-throughput (300 samples / hour), and rapid detection of MASP2 (reporting in 30 minutes).

[0050] Furthermore, one embodiment of this application also provides a method for using the above-mentioned diagnostic kit for liver fibrosis, the method comprising the following steps: incubating the sample to be tested together with a solid-phase conjugate and a labeled antibody, conjugating the antibody and labeled antibody on the MASP2 solid-phase conjugate; washing away unbound non-specific antigens and labeled antibodies; and detecting the signal intensity of the labeled antibody.

[0051] In some embodiments, the antigen diluent, the sample to be tested, the solid-phase conjugate, and the labeled antibody are incubated together.

[0052] The term "detection" and similar terms are used in general to refer to the process or discovery or determination of the presence or absence of something, as well as its degree, quantity, or level, or the probability of its occurrence. For example, the term "detection" when used with respect to a target nucleic acid sequence can indicate the discovery or determination of the presence, absence, level, or quantity of said sequence, as well as the probability or likelihood of its presence or absence. It is to be understood that expressions such as "detection of presence or absence," "detection of presence or absence," and related expressions include both qualitative and quantitative detection. For example, quantitative detection includes determining the quantity or amount of MASP2 in a sample.

[0053] In some embodiments, the sample to be tested is blood containing or presumed to contain MASP2. In an alternative specific example, the sample to be tested is plasma or serum containing or presumed to contain MASP2. Specific Implementation

[0055] The following detailed description is provided with reference to specific embodiments. Unless otherwise specified, the embodiments do not include components other than unavoidable impurities. Unless otherwise specified, the reagents and instruments used in the embodiments are conventionally selected in the art. Experimental methods not specifying specific conditions in the embodiments are implemented according to conventional conditions, such as those described in literature, books, or methods recommended by the manufacturer.

[0056] Example 1

[0057] The kit in this embodiment mainly consists of calibrators and detection reagents. The detection reagents consist of three parts:

[0058] R1 reagent: Composed of paramagnetic magnetic beads and magnetic bead diluent. The magnetic beads are coated with anti-human MASP2 polyclonal antibody (Yahuilong). The magnetic bead concentration is 0.15 mg / mL. The magnetic bead diluent contains 50 mM Tris, 2 w / v% BSA, 5 w / v glycine, 0.5 w / v% surfactant (Triton X-405), 1 w / v% protein stabilizer (AEP-HBC), and 0.1 w / v% preservative (PC300). The pH of the magnetic bead diluent is 7.4.

[0059] Reagent R2 consists of mouse anti-human MASP2 monoclonal antibody (Yahirolong) labeled with acridine ester and acridine diluent. The concentration of mouse anti-human MASP2 monoclonal antibody in the acridine component is 200 ng / mL; the acridine diluent is a diluent containing 50 mM PBS, 2 w / v% BSA, 0.5 v / v% surfactant (Tetronic 1307), 0.05 w / v% mouse IgG, and 0.1 w / v% preservative (PC300), with a pH of 7.4.

[0060] Reagent R3 consists of 20 mM phosphate, 500 mM sodium chloride, 1.5 w / v% EDTA-2Na, 10 w / v% trehalose, 2 w / v% mannitol, 1 w / v% lysine, 1 v / v% Tween-20, 0.1 w / v% NaN3, and 0.1% PC300, pH 7.0.

[0061] Example 2

[0062] This embodiment describes the method of using the reagent kit, which includes, but is not limited to, the following steps:

[0063] The kit in Example 1 can be used with the iFlash-3000 series chemiluminescence analyzer manufactured by YHLO. The cleaning solution, pre-excitation solution, excitation solution, and the corresponding cleaning and luminescence reading steps are the default settings of the analyzer. The remaining steps are programmed manually and processed by the analyzer.

[0064] Step 1: Sample processing: Set the program, pipette 10 μL of sample into a reaction vessel, add 90 μL of reagent L3, and incubate at 37°C for 5 min to separate the MBL / MASP2 complex.

[0065] The second step involves adding magnetic beads / acridine: 50 μL of R1 reagent and 50 μL of R2 reagent are added to the reaction vessel and incubated at 37°C for 10 min. The MASP2 antigen in the sample binds to the anti-MASP2 antibody coated on the magnetic beads and the anti-MASP2 antibody on the acridine to form an antibody-antigen-antibody immune complex. Then, a washing procedure is performed to wash away the unbound and adsorbed sample.

[0066] The third step is to determine the concentration of the analyte: according to the pre-set luminescence program of the instrument, the chemiluminescent marker that has been bound to the magnetic beads through a series of reactions is excited, and the luminescence value is read by the chemiluminescence analyzer.

[0067] The fourth step is to generate a report: based on the calibration curve and reference range, the report shows the MASP2 content in the blood sample to assist in clinical judgment.

[0068] Example 3

[0069] Testing the MASP2 content in clinical samples

[0070] Serum samples were collected from 70 healthy individuals and 70 individuals clinically diagnosed with liver fibrosis. Following the steps in Example 2, a measurement program was set up to detect the MASP2 content in each serum sample. The MASP2 content in the serum samples from both healthy individuals and those clinically diagnosed with liver fibrosis was also detected according to the instructions of the purchased MASP2 ELISA kit (hereinafter referred to as Kit A). The results are shown in Table 1.

[0071] The MASP2 levels in patient and healthy samples obtained from the kit in Example 1 were analyzed for specificity and sensitivity using SPSS 22.0 statistical software. ROC curves were plotted, and the area under the curve (AUC) was calculated. The results are as follows: Figure 1 As shown. SPSS 22.0 statistical software was used to analyze the specificity and sensitivity of MASP2 levels in patient samples and healthy population samples measured by reagent A using ROC curves. ROC curves were plotted, and the area under the curve (AUC) was calculated. The results are shown below. Figure 2 As shown.

[0072] Table 1

[0073]

[0074]

[0075]

[0076]

[0077] As shown in Table 1, the content of MASP2 in healthy individuals was significantly higher than that in liver-damaged samples. When the cutoff value of the kit in Example 1 was set at 423.84 ng / mL, the area under the curve reached 0.953 (the lower limit of the asymptotic 95% confidence interval was 0.913, and the upper limit was 0.993; standard error...). a The value is 0.02, indicating asymptotic significance. b The area under the curve is 0, where a is under the nonparametric assumptions and b is the null hypothesis (real area = 0.5). The detection specificity can reach 95.7%, and the sensitivity is 92.9%. When the cutoff value of kit A is set at 345.58 ng / mL, its curve area reaches 0.865 (the lower limit of the asymptotic 95% confidence interval is 0.803, and the upper limit is 0.927; standard error). a The value is 0.032, indicating asymptotic significance. bThe value is 0, where a is under nonparametric assumptions and b is the null hypothesis (real area = 0.5). The detection specificity can reach 85.7% and the sensitivity is 78.6%. By comparison, it can be found that the detection sensitivity and specificity of the kit in Example 1 are similar to those of kit A.

[0078] Example 4

[0079] Test linearity

[0080] (1) Testing the linearity of the kit in Example 1: Select a high-value sample, dilute it with a zero-value buffer according to Table 2, set the measurement program according to the steps in Example 2, detect the MASP2 content in the diluted sample, calculate the deviation between the test concentration and the theoretical concentration, and the results are shown in Table 2.

[0081] Table 2

[0082]

[0083] As shown in Table 2, the dilution linearity deviation of the kit in Example 1 can be controlled within ±10% in the range of 0 ng / mL to 1200 ng / mL. The concentration of MASP2 in human serum is 70 ng / mL to 1200 ng / mL, which meets the requirements for clinical testing.

[0084] (2) Testing the linearity of kit A: A high-value sample was selected and diluted with a zero-value buffer according to Table 3. The concentration of MASP2 protein in the sample was detected using kit A, and the deviation between the test concentration and the theoretical concentration was calculated. The results are shown in Table 3.

[0085] Table 3

[0086]

[0087] As can be seen from Table 3, the detection linear range of kit A is in the range of 0 ng / mL to 900 ng / mL, which is much lower than the measurement linear range of kit A in Example 1.

[0088] Example 5

[0089] Test detection limit

[0090] The test method is as follows: the sample dilution solution is used as the zero sample for detection. The test is repeated 20 times, and the RLU value (relative luminescence value) of the 20 measurement results is obtained. The mean (M) and standard deviation (SD) are calculated. M+3SD is the detection limit. The results are shown in Table 4.

[0091] Table 4

[0092] Reagent test kit Detection limit The reagent kit of Example 1 0.83 ng / mL Reagent A 1.88 ng / mL

[0093] As shown in Table 4, the detection limit of the kit in Example 1 is much lower than that of the kit in Example A.

[0094] In summary, the kit in Example 1 has a low detection limit for MASP2, high sensitivity, a wide linear detection range, and high accuracy in the diagnosis of liver fibrosis.

[0095] Example 6

[0096] Compare the clinical concordance rates of different liver fibrosis markers.

[0097] Serum samples were collected from 70 healthy individuals and 70 individuals clinically diagnosed with liver fibrosis. Following the steps outlined in Example 2, a measurement program was established to detect the MASP2 content in each serum sample. The contents of type III procollagen N-terminal peptide, type IV collagen, laminin, and serum hyaluronic acid in the serum samples from the 70 healthy individuals and 70 individuals clinically diagnosed with liver fibrosis were also measured according to the operating instructions of Company B's Type III procollagen N-terminal peptide assay kit (chemiluminescence method), Type IV collagen assay kit (chemiluminescence method), laminin assay kit (chemiluminescence method), and serum hyaluronic acid assay kit (chemiluminescence method). The clinical concordance rates of the five liver fibrosis markers were statistically analyzed, and the results are shown in Table 5. It should be noted that the normal value for MASP2 (the detection value in healthy individuals, the same below) is >423.84 ng / mL. If the detection value of MASP2 is less than or equal to 423.84 ng / mL, it is considered liver fibrosis. The normal value for type III procollagen N-terminal peptide is <30 ng / mL. If the detection value of type III procollagen N-terminal peptide is greater than or equal to 30 ng / mL, it is considered liver fibrosis. The normal value for type IV collagen is <30 ng / mL. If the detection value of type IV collagen is greater than or equal to 30 ng / mL, it is considered liver fibrosis. The normal value for laminin is <50 ng / mL. If the detection value of laminin is greater than or equal to 50 ng / mL, it is considered liver fibrosis. The normal value for serum hyaluronic acid is <100 ng / mL. If the detection value of serum hyaluronic acid is greater than or equal to 100 ng / mL, it is considered liver fibrosis. In Table 5, "compliant" means that the judgment result is consistent with the actual clinical diagnosis result; "inconsistent" means that the judgment result is inconsistent with the actual clinical diagnosis result.

[0098] Table 5: Clinical concordance rates of different biomarkers

[0099]

[0100]

[0101]

[0102]

[0103] As shown in Table 5, MASP2, as a marker of liver fibrosis, has a higher clinical concordance rate than traditional markers such as type III procollagen N-terminal peptide, type IV collagen, laminin, and serum hyaluronic acid.

[0104] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0105] The embodiments described above are merely illustrative of several implementations of the present invention, facilitating a detailed understanding of the technical solutions of the present invention, but should not be construed as limiting the scope of protection of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. It should be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided by the present invention through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this invention patent should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A diagnostic kit for liver fibrosis, characterized in that, include: A solid-phase conjugate, the solid-phase conjugate comprising a solid-phase support and an anti-MASP2 antibody coated on the solid-phase support; and The labeled antibody is an anti-MASP2 antibody labeled with a chemiluminescent marker; The diagnostic kit also includes an antigen diluent comprising: 10 mM to 50 mM phosphate, 500 mM to 1 M sodium chloride, 0.1 w / v% to 2 w / v% EDTA-2Na, 1 w / v% to 10 w / v% trehalose, 1 w / v% to 10 w / v% mannitol, 1 w / v% to 10 w / v% lysine, and 0.1 v / v% to 2 v / v% surfactant; The diagnostic kit has a detection specificity greater than 95% and a sensitivity greater than 92%. The detection linear range of the diagnostic kit is 0 ng / mL to 1200 ng / mL, and the limit of detection is 0.83 ng / mL.

2. The diagnostic kit according to claim 1, characterized in that, The anti-MASP2 antibody of the labeled antibody binds to a different antigenic epitope of MASP2 than the anti-MASP2 antibody of the solid-phase conjugate binds to a different antigenic epitope of MASP2.

3. The diagnostic kit according to claim 1, characterized in that, The chemiluminescent label is selected from one of the following: acridine-based chemiluminescent substances, ruthenium terpyridine, adamantane, luminol, luminol derivatives, isoluminol, isoluminol derivatives, horseradish peroxidase, and alkaline phosphatase.

4. The diagnostic kit according to claim 3, characterized in that, The acridine-based chemiluminescent material is selected from at least one of AE-NHS, DMAE-NHS, Me-DMAE-NHS, NSP-DMAE-NHS, NSP-SA, NSP-SA-NHS, and NSP-SA-ADH.

5. The diagnostic kit according to claim 1, characterized in that, The surfactant is selected from at least one of Tween20, Tween80, Triton X-100, Triton X-405 and Tetronic 1307.

6. The diagnostic kit according to claim 1, characterized in that, The antigen diluent also includes a preservative; in the antigen diluent, the concentration of the preservative is 0.1 w / v % ~ 0.5 w / v %.

7. The diagnostic kit according to claim 6, characterized in that, The antigen diluent comprises: 15 mM to 40 mM phosphate, 500 mM to 800 M sodium chloride, 1 w / v% to 2 w / v% EDTA-2Na, 5 w / v% to 10 w / v% trehalose, 1 w / v% to 5 w / v% mannitol, 1 w / v% to 5 w / v% lysine, 0.5 w / v% to 1.5 w / v% surfactant, and 0.1 w / v% to 0.5 w / v% preservative.

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