WO2023109384A1 - 去唾液酸糖蛋白受体片段sH2a作为标志物的应用 - Google Patents

去唾液酸糖蛋白受体片段sH2a作为标志物的应用 Download PDF

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WO2023109384A1
WO2023109384A1 PCT/CN2022/131117 CN2022131117W WO2023109384A1 WO 2023109384 A1 WO2023109384 A1 WO 2023109384A1 CN 2022131117 W CN2022131117 W CN 2022131117W WO 2023109384 A1 WO2023109384 A1 WO 2023109384A1
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sh2a
liver disease
active
asialoglycoprotein receptor
receptor fragment
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PCT/CN2022/131117
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English (en)
French (fr)
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姜芳
曹丽娟
王晨颖
张鹤耀
孙玉龙
王弢
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江苏为真生物医药技术股份有限公司
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere

Definitions

  • the present application relates to the field of liver diseases, in particular to the application of an asialoglycoprotein receptor fragment sH2a as a marker in the preparation of products for diagnosing active liver diseases.
  • the liver is the largest digestive gland in the human body and the central station of material and energy metabolism in the body. It is estimated that there are more than 500 chemical reactions taking place in the liver. First of all, it secretes bile to help digest food; it synthesizes protein from absorbed amino acids to provide energy for the body; it can store and burn fat in the body and control body shape; it is a storage organ for fat-soluble vitamins; it can also oxidize, reduce, and decompose in the body It is the largest detoxification organ of the human body. Experiments have shown that animals can only survive for more than 50 hours after the liver is completely removed, even if given corresponding treatment, which shows that the liver is an essential organ for maintaining life activities. Liver disease is a common disease and frequently-occurring disease. Usually, liver disease can be divided into several categories such as viral hepatitis, fatty liver, alcoholic liver disease, drug-induced liver injury, autoimmune hepatitis, liver cirrhosis and liver cancer.
  • Hepatitis B is a hepatitis B virus infection that damages the liver and can cause acute or chronic diseases.
  • the main manifestations are loss of appetite, nausea, upper abdominal discomfort, pain in the liver area, and fatigue. Some patients may have jaundice, fever and hepatomegaly with liver function damage. Some patients can become chronic and even develop into liver cirrhosis, and a few can develop into liver cancer.
  • hepatitis B cannot be distinguished from hepatitis caused by other viral agents. Therefore, it must be confirmed by laboratory testing. Several blood testing methods can be used to diagnose and monitor hepatitis B patients.
  • hepatitis B surface antigen HBsAg
  • HBsAb hepatitis B surface antibody
  • HeAg hepatitis B e antigen
  • HBeAb hepatitis B e antibody
  • HBcAb hepatitis B core antibody
  • Liver cirrhosis is a common clinical chronic progressive liver disease, which is diffuse liver damage caused by long-term or repeated effects of one or more causes. Most of them are post-hepatitic cirrhosis, and a small part are alcoholic cirrhosis and schistosome cirrhosis. Histopathologically, extensive liver cell necrosis, nodular regeneration of residual liver cells, connective tissue hyperplasia, and fibrous septum formation lead to structural destruction of hepatic lobules and formation of pseudolobules. The liver gradually deforms and hardens and develops into cirrhosis. In the early stage, there are no obvious symptoms due to the strong compensatory function of the liver.
  • liver function damage and portal hypertension are the main manifestations, and multiple systems are involved.
  • Autoimmune liver disease is a group of chronic injury inflammatory diseases of liver cells or bile duct epithelium caused by the imbalance of liver immune tolerance mechanism. According to its clinical manifestations, biochemical, immunological, imaging and histopathological characteristics, it can be divided into Autoimmune hepatitis (AIH) with progressive liver parenchymal cell damage, primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) with biliary system involvement.
  • AIH Autoimmune hepatitis
  • PBC primary biliary cirrhosis
  • PSC primary sclerosing cholangitis
  • Autoimmune liver disease can occur alone, or AIH and PBC, or AIH and PSC can occur simultaneously, which is called "overlap syndrome", in which all types of active autoimmune liver diseases have alkaline phosphate Enzymes, transaminases, etc. increased. Different types of autoimmune liver diseases have different demographic characteristics, clinical manifestations, and liver pathological changes. The specific pathogenesis of the disease is still unclear, and most patients are accompanied by other autoimmune diseases, such as diabetes and Hashimoto's thyroiditis.
  • Active liver disease specifically refers to chronic active hepatitis (CAH), a progressive inflammatory liver disease.
  • CAH chronic active hepatitis
  • HBV hepatitis B virus
  • NANB non-A, non-B hepatitis virus
  • some drugs can also cause pathological changes similar to CAH.
  • HBV hepatitis B virus
  • NANB non-A, non-B hepatitis virus
  • the cause of liver cell injury in CAH patients is not directly caused by the hepatitis virus itself, but by the host's immune response triggered by it, including cellular immunity and humoral immunity.
  • LSP specific lipoprotein
  • CAH includes two very characteristic situations, one is the common feature of organ-specific autoimmune diseases, such as the active autoimmune liver disease disclosed in this application, and the other is related to chronic viral infection (caused immune reaction), such as the active hepatitis B viral hepatitis, active hepatitis B cirrhosis, etc. disclosed in this application.
  • Active liver disease that is, chronic active hepatitis, is characterized by continuous inflammatory activities, and the pathogenesis of different liver diseases is basically the same, all due to immune responses, including host immunity and autoimmunity, mainly cellular immunity and humoral immunity, causing liver disease. cell damage.
  • a marker with high specificity and sensitivity that can be used for diagnosing active liver disease is provided.
  • an application of an asialoglycoprotein receptor fragment sH2a as a marker in the preparation of a product for diagnosing active liver disease is provided.
  • the product is a kit, a detection test strip or a detection chip.
  • the kit includes a capture antibody capable of specifically binding to the asialoglycoprotein receptor fragment sH2a.
  • the kit includes a detection antibody capable of specifically binding to the asialoglycoprotein receptor fragment sH2a, and the detection antibody is labeled with a detection marker.
  • the detection label is selected from the group consisting of fluorescent labels, chemiluminescent labels, electron-dense labels, metal particles, radioactive labels, chromophore labels or enzyme labels.
  • the detection label is selected from rhodamine, fluorescein, quantum dots, digoxin-labeled probes, radioactive isotopes, fluorescent microspheres, colloidal gold, acridinium esters, luciferase, horseradish peroxidized
  • the kit further includes asialoglycoprotein receptor fragment sH2a.
  • the kit further includes one or more of a solid phase carrier, a coating buffer and a washing buffer.
  • the solid phase carrier is magnetic particles, latex particles, microtiter plate or microfluidic chip.
  • the active liver disease includes active hepatitis B virus hepatitis, active hepatitis B cirrhosis and active autoimmune liver disease.
  • the product is used for diagnosing whether a subject suffers from active liver disease by detecting the content of the asialoglycoprotein receptor fragment sH2a in a sample.
  • the sample is selected from one or more of plasma, whole blood, lymph, urine, sweat, mucus, sputum and saliva.
  • a method for diagnosing active liver disease is also provided, wherein whether a subject suffers from active liver disease is diagnosed by detecting the content of asialoglycoprotein receptor fragment sH2a in a sample.
  • the up-regulation of the content of the asialoglycoprotein receptor fragment sH2a in the subject sample is an indication that the subject suffers from active liver disease.
  • the sample is selected from one or more of plasma, whole blood, serum, lymph, urine, sweat, mucus, sputum and saliva.
  • the active liver disease includes one or more of active hepatitis B virus hepatitis, active hepatitis B cirrhosis and active autoimmune liver disease.
  • Fig. 1 is the SDS-PAGE electrophoresis purity identification diagram of the sH2a recombinant protein of one or more embodiments of the present application, the molecular weight is 35-40kDa, and the gray scale analysis shows that the protein purity reaches more than 95%;
  • Fig. 2 is the calibration curve of the sH2a detection kit of one or more embodiments of the present application, the linear range is 6.25ng/mL ⁇ 200ng/mL;
  • Fig. 3 is a sample concentration scatter diagram of the sH2a detection kit in one or more embodiments of the present application to detect the concentration of sH2a in plasma of active liver disease and healthy people;
  • Fig. 4 is the ROC curve of the sH2a detection kit of one or more embodiments of the present application to detect the concentration of sH2a in plasma of active liver disease and healthy people;
  • Figure 5 is a sample concentration scatter diagram of the sH2a detection kit in one or more embodiments of the present application detecting the sH2a concentration in plasma of inactive liver disease and healthy people;
  • Fig. 6 is a sample concentration scatter diagram of the sH2a detection kit in one or more embodiments of the present application to detect active liver disease, inactive liver disease, other types of liver disease, and sH2a concentration in plasma of healthy people;
  • Fig. 7 is a sample concentration scatter diagram of sH2a concentration in the serum of active liver disease, inactive liver disease and healthy people detected by the sH2a detection kit of one or more embodiments of the present application;
  • Fig. 8 is a sample concentration scatter diagram of the sH2a detection kit in one or more embodiments of the present application to detect the sH2a concentration in whole blood of active liver disease, inactive liver disease and healthy people.
  • the present application provides an application of an asialoglycoprotein receptor fragment sH2a as a marker in the preparation of a product for diagnosing active liver disease.
  • ASGPR asialoglycoprotein receptor, asialoglycoprotein receptor
  • Ashwell and his companion Morell also known as Ashwell-Morell receptor
  • mainly expressed in hepatic parenchymal cells in the sinusoidal space of the liver Surface due to the calcium ion dependence of its binding ligands, belongs to the C-type lectins. It can mediate desialylated glycoproteins to be endocytosed and degraded by hepatocytes, and plays an important role in many physiological links such as glycoprotein metabolism, lipid metabolism, degradation of apoptotic cells, regulation of coagulation, mediation of virus entry into cells, and autoimmune inflammatory response. play an important role.
  • the sH2a is derived from the H2 subunit of the liver ASGPR, and H2 has three alternative splicing forms.
  • H2a is the longest alternatively spliced form, containing an intracellular region of 57 nucleotides and an immediate transmembrane region of 15 nucleotides compared to H2b and H2c. Since the 5 amino acids encoded by these 15 nucleotides can serve as a proteolytic signal, H2a is cleaved into a 35kDa fragment immediately adjacent to the pentapeptide, and is secreted out of the cell in a soluble form, namely sH2a.
  • the present application found that by measuring the content of the asialoglycoprotein receptor fragment sH2a in biological samples, the differential diagnosis of active liver disease can be very good, so that it can be applied to the early diagnosis and differential diagnosis of active liver disease, and Provide a strong basis for disease treatment.
  • the experimental data shows that when sH2a is used as the detection index to detect active liver disease samples, the statistical results of the ROC curve show that the area under the curve is 0.9583, and 65.77ng/mL is used as the detection reference value, the specificity of the sH2a detection kit is 94.50%, and the sensitivity is 85.33 %.
  • the value of diagnostic markers for active liver disease, and the diagnostic value is high. The discovery of this marker provides a new experimental theoretical basis and a new direction for further research on the mechanism of active liver disease and the treatment of active liver disease, and enriches the diagnostic and detection methods of active liver disease.
  • the above-mentioned products are kits, detection test strips or detection chips. It can be understood that the specific types are not limited thereto, and various protein detection products are acceptable.
  • the above product detects the asialoglycoprotein receptor fragment sH2a by a double-antibody sandwich method. It can be understood that the method for detecting the target protein is not limited thereto, and can be adjusted as needed, such as competition method and the like.
  • the above kit includes a capture antibody capable of specifically binding to the above-mentioned asialoglycoprotein receptor fragment sH2a.
  • the above kit includes a detection antibody that can specifically bind to the asialoglycoprotein receptor fragment sH2a, and the detection antibody is labeled with a detection marker.
  • a detection marker that can specifically bind to the asialoglycoprotein receptor fragment sH2a
  • corresponding detection methods and detection reagents can be selected according to needs, for example, fluorescent markers are detected by fluorescent detection methods and corresponding reagents, enzyme labels are detected by substrate reactions and related reagents, etc.
  • the detection label is selected from the group consisting of fluorescent labels, chemiluminescent labels, electron-dense labels, metal particles, radioactive labels, chromophore labels or enzyme labels.
  • the above-mentioned detection label is selected from rhodamine, fluorescein, quantum dots, digoxin-labeled probes, radioactive isotopes, fluorescent microspheres, colloidal gold, acridinium esters, luciferase, horseradish peroxidized
  • the above kit detects asialoglycoprotein receptor fragment sH2a by enzyme-linked immunosorbent assay.
  • the basic principle is that the enzyme molecule is covalently bound to the antibody or anti-antibody molecule, and this combination will not change the immunology of the antibody. characteristics, and does not affect the biological activity of the enzyme.
  • This enzyme-labeled antibody can specifically bind to the antigen or antibody adsorbed on the solid-phase carrier. After the substrate solution is added dropwise, the hydrogen donor contained in the substrate can change from the colorless reduced form to the colored oxidized form under the action of the enzyme, resulting in a color reaction.
  • This chromogenic reaction can be quantitatively determined by an ELISA detector, which combines the sensitivity of the enzyme chemical reaction with the specificity of the antigen-antibody reaction, making the ELISA method a specific and sensitive detection method.
  • the detection marker is horseradish peroxidase
  • the kit also includes a TMB substrate.
  • the above kit further includes asialoglycoprotein receptor fragment sH2a, which can be used as a standard for drawing a standard curve and the like.
  • the above kit further includes one or more of a solid phase carrier, a coating buffer and a washing buffer.
  • a solid phase carrier is selected from the group consisting of magnetic particles, latex particles, microtiter plate or microfluidic chip, but is not limited thereto, and other conventional solid-phase carriers can be selected as required.
  • the aforementioned active liver disease includes active hepatitis B virus hepatitis, active hepatitis B cirrhosis, and active autoimmune liver disease.
  • the product is used to diagnose whether a subject suffers from active liver disease by detecting the content of the asialoglycoprotein receptor fragment sH2a in a sample.
  • the sample is selected from one or more of plasma, whole blood, lymph, urine, sweat, mucus, sputum and saliva.
  • the present application also provides a method for diagnosing active liver disease, by detecting the content of the asialoglycoprotein receptor fragment sH2a in the sample of the subject to diagnose whether the subject has active liver disease. Specifically, there is a significant difference in sH2a content between patients with active liver disease and healthy people, and the sH2a content is significantly up-regulated in patients with active liver disease, while there is no significant difference in sH2a content between patients with inactive liver disease and healthy people. Patients with active liver disease and patients with inactive liver disease. Therefore, the up-regulation of the content of the asialoglycoprotein receptor fragment sH2a in the subject sample is an indication that the subject suffers from active liver disease.
  • the subject's sample when the sH2a content in the subject's sample is significantly up-regulated, according to the type of liver disease the subject suffers from, it can be diagnosed that the subject has active HBV hepatitis, active HBV cirrhosis and active autoimmune liver disease at least one of the
  • the subject sample is selected from one or more of plasma, whole blood, lymph, urine, sweat, mucus, sputum, and saliva, and the subject sample includes but is not limited to the above samples, as long as it can be achieved
  • the samples of patients with active liver disease and patients with inactive liver disease can be used as subject samples by the sH2a content of the samples.
  • the method for diagnosing active liver disease of the present application has high specificity and good sensitivity.
  • the sH2a detection kit used in the diagnostic method has a specificity of 94.50% and a sensitivity of 85.33%.
  • the present application also provides a diagnostic system for active liver disease, including:
  • Information acquisition module used to acquire the content information of the asialoglycoprotein receptor fragment sH2a in the subject sample;
  • Diagnosis module used for diagnosing the active liver disease of the subject according to the content information of the asialoglycoprotein receptor fragment sH2a in the subject sample.
  • the diagnosis module specifically includes:
  • Judgment unit used to judge whether the content of asialoglycoprotein receptor fragment sH2a in the subject sample is up-regulated according to the content information of the asialoglycoprotein receptor fragment sH2a in the subject sample;
  • Output unit used to output the diagnosis result that the subject has active liver disease when the content of the asialoglycoprotein receptor fragment sH2a in the subject sample is up-regulated.
  • the method for preparing the asialoglycoprotein receptor fragment sH2a includes the following steps: loading the sH2a gene fragment into an expression vector to obtain a recombinant plasmid, transferring the recombinant plasmid into eukaryotic cells, and transfecting for 18-22 hours Add a transfection enhancer, collect the supernatant after 5-7 days, filter the supernatant with a filter membrane, and then purify to obtain the sH2a recombinant protein.
  • the above-mentioned purification comprises the following steps: performing Ni-NTA affinity chromatography on the filtrate obtained by filter membrane filtration under non-denaturing conditions, the equilibration buffer is 50mM PBS, 10mM imidazole, 150mM NaCl, pH 7.2-7.4, After sample loading, wash 10mL; elute with 50mM PBS, 250mM imidazole, 150mM NaCl, pH 7.2-7.4, collect the eluate; use a 10kDa ultrafiltration tube to concentrate the protein solution, and store it in 50mM PBS (pH 7.2-7.4) buffer, store at -80°C.
  • asialoglycoprotein receptor fragment sH2a in the diagnosis of active liver disease will be described in further detail below mainly in conjunction with the specific embodiments and accompanying drawings.
  • Active liver disease that is, chronic active hepatitis, is mainly diagnosed based on the following aspects: (1) Abnormal liver function, manifested as an increase in ALT (alanine aminotransferase), AST (aspartate aminotransferase) or an increase in jaundice.
  • liver inflammation When liver inflammation is active, transaminases are released from liver cells into the blood, and transaminases are elevated in peripheral blood, so elevated transaminases are one of the more sensitive indicators for diagnosing hepatitis activity; (2) liver function in some patients The performance is normal, and the liver biopsy shows interlobular inflammation or interface hepatitis, lymphocyte, neutrophil infiltration and other manifestations, indicating that the liver cells have inflammatory activities, which is also one of the diagnostic basis for active hepatitis.
  • sample inclusion criteria for active liver disease in this example include:
  • Hepatitis B viral hepatitis HBsAg (hepatitis B surface antigen) positive, ALT>2 times the upper limit of normal value, AST/ALT>1.5, HBV DNA>20000IU/ml;
  • HBV hepatitis, autoimmune liver disease, and active cirrhosis that do not meet the above diagnostic criteria are correspondingly diagnosed as inactive HBV hepatitis, inactive autoimmune liver disease, and inactive active cirrhosis.
  • Embodiment 1 sH2a recombinant protein expression and purification identification
  • Protein expression NCBI searched the human ASGPR2 sequence, selected the extracellular segment, namely sH2a amino acid sequence, to synthesize the gene and optimized the codons for mammalian expression, and loaded it into the pcDNA3.1 vector at the same time. Transform the obtained sH2a recombinant plasmid (pcDNA3.1-sH2a-His) into eukaryotic mammalian cells, and use the pcDNA3.1 empty vector as a negative control, add a transfection enhancer for 18-22 hours after transfection, 5-7 days Collect the supernatant and filter the supernatant with a 0.22 ⁇ m filter membrane.
  • Protein purification The resulting filtrate was subjected to Ni-NTA affinity chromatography under non-denaturing conditions, and the equilibration buffer was 50mM PBS, 10mM imidazole, 150mM NaCl, pH 7.2-7.4. After sample loading, wash 10 mL; elute with 50 mM PBS, 250 mM imidazole, 150 mM NaCl, pH 7.2-7.4, and collect the eluate. Concentrate the protein solution using a 10kD ultrafiltration tube, store the protein in 50mM PBS buffer at pH 7.2-7.4, and store at -80°C. Purified protein was identified by SDS-PAGE electrophoresis, the molecular weight was 35-40kDa, and the gray scale analysis showed that the protein purity was above 95%, as shown in Figure 1 .
  • sH2a recombinant protein activity Dilute sH2a recombinant protein to 1 ⁇ g/mL with coating buffer (pH9.6) and add to a microwell plate, 100 ⁇ L per well, coat overnight at 4°C, and coat the antibody on the microwell plate that was patted dry And add 150-200 ⁇ L ELISA plate stabilizer, and incubate at 37°C for 1-2 hours.
  • the capture antibody (manufacturer: SinoBiological, article number: 13908-R002) and the detection antibody (manufacturer: Thermo, article number: MA5-29046) are well matched and can be used for the construction of a double-antibody sandwich system, as shown in Table 2.
  • Embodiment 3 sH2a kit and sH2a calibration curve drawing
  • the sH2a kit includes the aforementioned capture antibody, detection antibody and sH2a calibrator, etc.
  • dilute the capture antibody to 3 ⁇ g/mL with coating buffer (pH 9.6), add 100 ⁇ L per well to the microtiter plate, and incubate at 4°C overnight; dilute the sH2a calibrator protein to 0 ng/mL with protein stabilizer mL, 6.25ng/mL, 12.5ng/mL, 25ng/mL, 50ng/mL, 100ng/mL, 200ng/mL, and add the calibrator into the pre-coated microtiter plate in order, 100 ⁇ L per well, incubate at 37°C 1 hour; wash 4 times with washing solution, add horseradish peroxidase-labeled detection antibody at a concentration of 10 ng/mL, 100 ⁇ L per well, incubate at 37°C for 1 hour; wash 4 times with washing solution, add TMB substrate to detect Color solution and measure the OD value of
  • the regression equation is obtained by fitting the logistic four-parameter method, and the OD value of the sample to be tested is substituted into the regression equation, Obtain the corresponding sH2a antigen content.
  • the linear range of the calibration curve is 6.25-200 ng/mL, as shown in Figure 2 is the calibration curve of the sH2a detection kit.
  • the above-mentioned sH2a detection kit adopts the double-antibody sandwich method. The method is simple and easy to operate, and the detection is fast and sensitive. The microplate reader used is simple, popular, and low in price. 5ng/mL.
  • inactive liver diseases 80 cases of hepatitis B virus hepatitis, 60 cases of hepatitis B cirrhosis, and 60 cases of autoimmune liver disease
  • plasma samples of 200 healthy blood donors were collected from blood banks.
  • the aforementioned sH2a detection kit was used to detect the sH2a concentration in the plasma of inactive liver disease and healthy subjects.
  • the sample concentration scatter plot showed that there was no significant difference in sH2a content between inactive liver disease and healthy people, as shown in Figure 5.
  • 300 plasma samples of active liver disease (100 cases of HBV, 100 cases of HBV cirrhosis, 100 cases of autoimmune liver disease), 200 plasma samples of inactive liver disease (80 cases of HBV, 80 cases of HBV) were collected from the hospital.
  • 60 cases of cirrhosis, 60 cases of autoimmune liver disease) 200 plasma samples of other types of liver disease (100 cases of drug-induced hepatitis, 100 cases of fatty liver); at the same time, plasma samples of 200 healthy blood donors were collected from the blood bank.
  • Use the aforementioned sH2a detection kit to detect the sH2a concentration in plasma of active liver disease, inactive liver disease, other types of liver disease and healthy people.
  • the sample concentration scatter plot showed that there was a significant difference in sH2a content between active liver disease and healthy people, and there was no significant difference in sH2a content between inactive liver disease and other types of liver disease and healthy people, as shown in Figure 6.
  • Serum samples of 100 cases of active liver disease 50 cases of HBV hepatitis, 30 cases of HBV cirrhosis, 20 cases of autoimmune liver disease
  • 100 serum samples of inactive liver disease 50 cases of HBV hepatitis, 50 cases of HBV
  • 30 cases of cirrhosis and 20 cases of autoimmune liver disease 100 cases of serum from healthy blood donors were collected from the blood bank.
  • Use the aforementioned sH2a detection kit to detect the concentration of sH2a in the serum of active liver disease, inactive liver disease, other types of liver disease and healthy people.
  • the sample concentration scatter plot shows that there is a significant difference in sH2a content between active liver disease and healthy people, and there is no significant difference in sH2a content between inactive liver disease and healthy people, as shown in Figure 7.
  • the present application provides a marker sH2a with high sensitivity and good specificity that can be used to diagnose active liver disease.
  • a marker sH2a with high sensitivity and good specificity that can be used to diagnose active liver disease.
  • a good differential diagnosis of active liver disease can be applied to the early diagnosis and differential diagnosis of active liver disease, and provide a strong basis for disease treatment.

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Abstract

去唾液酸糖蛋白受体片段sH2a作为诊断活动期肝病的标志物，以及去唾液酸糖蛋白受体片段sH2a作为标志物在制备用于诊断活动期肝病的产品中的应用。

Description

去唾液酸糖蛋白受体片段sH2a作为标志物的应用
相关申请的交叉引用
本申请要求于2021年12月14日申请的，申请号为2021115268060，名称为“去唾液酸糖蛋白受体片段sH2a作为标志物的应用”的中国专利申请的优先权，在此将其全文引入作为参考。
技术领域
本申请涉及肝病领域，特别是涉及一种去唾液酸糖蛋白受体片段sH2a作为标志物在制备用于诊断活动期肝病的产品中的应用。
背景技术
肝脏是人体内最大的消化腺，是体内物质能量代谢的中心站。据估计，在肝脏中发生的化学反应有500种以上。首先它分泌胆汁，帮助消化饮食；它把吸收的氨基酸合成蛋白质供给机体能量；它能贮藏和燃烧体内的脂肪，控制体形；它是脂溶性维生素的贮存器官；它还能够氧化、还原、分解体内的毒素，吞噬不小心吃入体内的细菌，是人体最大的解毒器官。实验证明，动物在完全摘除肝脏后即使给予相应的治疗，最多也只能生存50多个小时，这说明肝脏是维持生命活动的一个必不可少的重要器官。而肝病是常见病和多发病，通常可将肝病分为病毒性肝炎、脂肪肝、酒精性肝病、药物性肝损伤、自身免疫性肝炎、肝硬化及肝癌等几大类。
乙型肝炎是一种损害肝脏的乙型肝炎病毒感染，可引起急性或慢性疾病，临床上以食欲减退、恶心、上腹部不适、肝区痛、乏力为主要表现。部分患者可有黄疸发热和肝大伴有肝功能损害。有些患者可慢性化，甚至发展成肝硬化，少数可发展为肝癌。临床上还不能将乙型肝炎同由其它病毒因子引起的肝炎区分开来，因此，必须通过实验室检测来确诊，可用若干血液检测方法对乙肝患者进行诊断和监测。目前主要依靠乙型肝炎表面抗原(HBsAg)、乙肝表面抗体(HBsAb)、乙肝e抗原(HBeAg)、乙肝e抗体(HBeAb)及乙肝核心抗体(HBcAb)等指标和乙肝病毒感染病程是否超过6个月来区分急性和慢性乙型肝炎，结合碱性磷酸酶、转氨酶(如ALT、AST等)、HBV DNA等指标区分肝病活动期及非活动期。肝硬化是临床常见的慢性进行性肝病，由一种或多种病因长期或反复作用 形成的弥漫性肝损害。大多数为肝炎后肝硬化，少部分为酒精性肝硬化和血吸虫性肝硬化。病理组织学上有广泛的肝细胞坏死、残存肝细胞结节性再生、结缔组织增生与纤维隔形成，导致肝小叶结构破坏和假小叶形成，肝脏逐渐变形、变硬而发展为肝硬化。早期由于肝脏代偿功能较强可无明显症状，后期则以肝功能损害和门脉高压为主要表现，并有多系统受累，晚期常出现上消化道出血、肝性脑病、继发感染、脾功能亢进、腹水、癌变等并发症。自身免疫性肝病(AILD)是一组肝脏免疫耐受机制失衡引起的肝细胞或胆管上皮慢性损伤性炎症疾病，根据其临床表现、生化、免疫学、影像学和组织病理学特点，可分为以肝实质细胞进行性损伤为主的自身免疫性肝炎(AIH)，以胆道系统受累为主的原发性胆汁性肝硬化(PBC)和原发性硬化型胆管炎(PSC)。自身免疫性肝病可单独发病，也可出现AIH与PBC、或AIH与PSC同时发病的情况，被称为“重叠综合征”(overlap syndrome)，其中各类型活动期自身免疫肝病均出现碱性磷酸酶、转氨酶等含量升高。不同类型的自身免疫性肝病，其人口学特征、临床表现、肝脏的病理改变各有不同。该病具体的发病机制尚不明了，患者多伴有其他自身免疫性疾病，如糖尿病、桥本甲状腺炎等。
活动期肝病特指慢性活动性肝炎(CAH)这一类进展性炎性肝脏疾病。慢性活动性肝炎(CAH)这一术语由saint在1953年首次提出。CAH的病因90％左右为乙型肝炎病毒(HBV)，其次非甲非乙型肝炎病毒(NANB)及某些药物也可引起类似CAH的病变。然而，已报导CAH患者肝细胞损伤的原因并不是肝炎病毒本身直接引起的，而是由其引发的宿主的免疫反应，包括细胞免疫和体液免疫，来发挥作用。此外，在遗传、环境因素作用下，如药物等，也会使得肝细胞表面特异性脂蛋白(LSP)特性发生改变，被自身抗体识别为“抗原”，导致自身抗体错误攻击自身正常组织和细胞导致组织、器官损伤，形成自身免疫性疾病。
研究发现CAH包括两类非常特征性的情况，一类是器官特异性自身免疫性疾病共同的特征，如本申请公开的活动期自身免疫性肝病，另一类则与慢性病毒感染(引起的免疫反应)有关，如本申请公开的活动期乙肝病毒型肝炎、活动期乙肝肝硬化等。
活动期肝病，即慢性活动性肝炎，均以持续发生的炎症活动为特征，且不同 肝病发病原理基本相同，均因免疫反应，包括细胞免疫和体液免疫为主的宿主免疫和自身免疫，引起肝细胞损伤。
目前，如何将活动期肝病与非活动期肝病、以及其他类型肝病(药物性肝炎、脂肪肝等)快速便捷地区分鉴别开来仍然是不清楚的。
发明内容
基于此，根据本申请的各实施例，提供了一种特异性和灵敏度较高的可用于诊断活动期肝病的标志物。
根据本申请的各实施例，提供了一种去唾液酸糖蛋白受体片段sH2a作为标志物在制备用于诊断活动期肝病的产品中的应用。
在其中一个实施例中，产品为试剂盒、检测试纸条或检测芯片。
在其中一个实施例中，试剂盒包括捕获抗体，捕获抗体能够特异性结合去唾液酸糖蛋白受体片段sH2a。
在其中一个实施例中，试剂盒包括检测抗体，检测抗体能够特异性结合去唾液酸糖蛋白受体片段sH2a，且检测抗体上标记有检测标记物。
在其中一个实施例中，检测标记物选自荧光标记、化学发光标记、电子致密标记、金属粒子、放射性标记、发色团标记或酶标记构成的组。
在其中一个实施例中，检测标记物选自罗丹明、荧光素、量子点、地高辛标记探针、放射性同位素、荧光微球、胶体金、吖啶酯、荧光素酶、辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶或葡萄糖-6-磷酸脱氢酶构成的组。
在其中一个实施例中，试剂盒还包括去唾液酸糖蛋白受体片段sH2a。
在其中一个实施例中，试剂盒还包括固相载体、包被缓冲液和洗涤缓冲液中的一种或多种。
在其中一个实施例中，固相载体为磁性颗粒、胶乳粒子、酶标板或微流控芯片。
在其中一个实施例中，活动期肝病包括活动期乙肝病毒型肝炎、活动期乙肝肝硬化和活动期自身免疫性肝病。
在其中一个实施例中，产品用于通过检测样本中去唾液酸糖蛋白受体片段sH2a的含量诊断受试者是否患有活动期肝病。
在其中一个实施例中，样本选自血浆、全血、淋巴液、尿液、汗液、粘液、痰液和唾液中的一种或多种。
根据本申请的各实施例，还提供了一种诊断活动期肝病的方法，通过检测样本中去唾液酸糖蛋白受体片段sH2a的含量诊断受试者是否患有活动期肝病。
在其中一个实施例中，受试者样本中去唾液酸糖蛋白受体片段sH2a的含量上调是受试者患有活动期肝病的指征。
在其中一个实施例中，所述样本选自血浆、全血、血清、淋巴液、尿液、汗液、粘液、痰液和唾液中的一种或多种。
在其中一个实施例中，所述活动期肝病包括活动期乙肝病毒型肝炎、活动期乙肝肝硬化和活动期自身免疫性肝病中的一种或多种。
本申请的一个或多个实施例的细节在下面的附图和描述中提出。本申请的其他特征、目的和优点将从说明书、附图以及权利要求书中变得明显。
附图说明
为了更好地描述和说明这里公开的那些发明的实施例和/或展示例，可以参考一幅或多幅附图。用于描述附图的附加细节或示例不应当被认为是对所公开的发明、目前描述的实施例和/或实施例以及目前理解的这些发明的最佳模式中的任何一者的范围的限制。
图1为本申请一个或多个实施例的sH2a重组蛋白的SDS-PAGE电泳纯度鉴定图，分子量大小在35-40kDa，灰度分析表明蛋白纯度达到95％以上；
图2为本申请一个或多个实施例的sH2a检测试剂盒的校准曲线，线性范围6.25ng/mL～200ng/mL；
图3为本申请一个或多个实施例的sH2a检测试剂盒检测活动期肝病及健康人血浆中sH2a浓度的样本浓度散点图；
图4为本申请一个或多个实施例的sH2a检测试剂盒检测活动期肝病及健康人血浆中sH2a浓度的ROC曲线；
图5为本申请一个或多个实施例的sH2a检测试剂盒检测非活动期肝病及健康人血浆中sH2a浓度的样本浓度散点图；
图6为本申请一个或多个实施例的sH2a检测试剂盒检测活动期肝病、非活动期肝病、其他类型肝病及健康人血浆中sH2a浓度的样本浓度散点图；
图7为本申请一个或多个实施例的sH2a检测试剂盒检测活动期肝病、非活动期肝病及健康人血清中sH2a浓度的样本浓度散点图；
图8为本申请一个或多个实施例的sH2a检测试剂盒检测活动期肝病、非活动期肝病及健康人全血中sH2a浓度的样本浓度散点图。
具体实施方式
为了便于理解本申请，下面将对本申请进行更全面的描述，并给出了本申请的较佳实施例。但是，本申请可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使对本申请的公开内容的理解更加透彻全面。
除非另有定义，本文所使用的所有的技术和科学术语与属于本申请的技术领域的技术人员通常理解的含义相同。本文中在本申请的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本申请。本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
本申请提供了一种去唾液酸糖蛋白受体片段sH2a作为标志物在制备用于诊断活动期肝病的产品中的应用。
ASGPR(asialoglycoprotein receptor，去唾液酸糖蛋白受体)是第一个被发现的凝集素，由Ashwell及其同伴Morell发现，也称Ashwell-Morell受体，主要表达于肝窦状间隙的肝实质细胞表面，由于其结合配体的钙离子依赖性，因此属于C型凝集素。其能够介导去唾液酸化的糖蛋白被肝细胞内吞降解，在糖蛋白代谢、脂代谢、凋亡细胞降解、调节凝血、介导病毒进入细胞和自身免疫性炎症反应等多个生理环节均发挥着重要的作用。而sH2a则是源于肝脏ASGPR的H2亚基，H2有3种可变剪切形式。H2a为最长的可变剪切形式，相比H2b和H2c，它包含有57个核苷酸的胞内区域，以及15个核苷酸的紧邻跨膜区。由于这15个核苷酸编码的5个氨基酸可以作为蛋白水解信号，H2a在紧邻五肽处裂解为35kDa的片段，以可溶形式分泌至细胞外，即sH2a。
本申请通过研究发现，通过对生物样品中的去唾液酸糖蛋白受体片段sH2a 的含量进行测定，能够很好地鉴别诊断活动期肝病，从而应用于活动期肝病的早期诊断、鉴别诊断，并为疾病治疗提供有力依据。实验数据表明，以sH2a作为检测指标检测活动期肝病样本时，ROC曲线统计结果显示曲线下面积为0.9583，以65.77ng/mL作为检测参考值，sH2a检测试剂盒特异性为94.50％，灵敏度为85.33％。ROC曲线下面积越接近1越具有诊断价值，这表明通过检测sH2a可以高效地将活动期肝病与非活动期肝病、其他类型肝病(药物性肝炎、脂肪肝等)区分鉴别开来，sH2a具备作为活动期肝病诊断标志物的价值，且诊断价值较高。该标志物的发现，为进一步研究活动期肝病的机理，探索活动期肝病的治疗提供了新的实验理论基础和新的方向，丰富了活动期肝病的诊断和检测手段。
在一个具体示例中，上述产品为试剂盒、检测试纸条或检测芯片，可以理解，具体类型不限于此，各种蛋白检测产品均可。
在一个具体示例中，上述产品通过双抗体夹心法检测去唾液酸糖蛋白受体片段sH2a。可以理解，检测目的蛋白的方法不限于此，可以根据需要进行调整，例如竞争法等。
在一个具体示例中，上述试剂盒包括捕获抗体，捕获抗体能够特异性结合上述去唾液酸糖蛋白受体片段sH2a。
在一个具体示例中，上述试剂盒包括检测抗体，检测抗体能够特异性结合去唾液酸糖蛋白受体片段sH2a，且检测抗体上标记有检测标记物。可以理解，根据检测标记物的不同，相应的检测方法和检测试剂可根据需要选择，例如荧光标记通过荧光检测方法和相应试剂进行测定，酶标记通过底物反应和相关试剂进行测定等。
在一个具体示例中，上述检测标记物选自荧光标记、化学发光标记、电子致密标记、金属粒子、放射性标记、发色团标记或酶标记构成的组。
在一个具体示例中，上述检测标记物选自罗丹明、荧光素、量子点、地高辛标记探针、放射性同位素、荧光微球、胶体金、吖啶酯、荧光素酶、辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶或葡萄糖-6-磷酸脱氢酶构成的组。可以理解，检测标记物的具体种类不限于此，可根据需要进行选择。
可选地，上述试剂盒通过酶联免疫吸附法检测去唾液酸糖蛋白受体片段 sH2a，其基本原理是酶分子与抗体或抗抗体分子共价结合，此种结合不会改变抗体的免疫学特性，也不影响酶的生物学活性。此种酶标记抗体可与吸附在固相载体上的抗原或抗体发生特异性结合。滴加底物溶液后，底物可在酶作用下使其所含的供氢体由无色的还原型变成有色的氧化型，出现颜色反应。因此，可通过底物的颜色反应来判定有无相应的免疫反应，颜色反应的深浅与标本中相应抗体或抗原的量呈正比。此种显色反应可通过ELISA检测仪进行定量测定，这样就将酶化学反应的敏感性和抗原抗体反应的特异性结合起来，使ELISA方法成为一种既特异又敏感的检测方法。
在一个具体示例中，上述检测标记物为辣根过氧化物酶，试剂盒还包括TMB底物。
在一个具体示例中，上述试剂盒还包括去唾液酸糖蛋白受体片段sH2a，其可作为标准品，用于标准曲线的绘制等。
在一个具体示例中，上述试剂盒还包括固相载体、包被缓冲液和洗涤缓冲液中的一种或多种。可选地，上述固相载体选自磁性颗粒、胶乳粒子、酶标板或微流控芯片构成的组，但不限于此，可根据需要选择其他常规的固相载体。
在一个具体示例中，上述活动期肝病包括活动期乙肝病毒型肝炎、活动期乙肝肝硬化和活动期自身免疫性肝病等。
在一个具体示例中，产品用于通过检测样本中去唾液酸糖蛋白受体片段sH2a的含量诊断受试者是否患有活动期肝病。
在一个具体示例中，样本选自血浆、全血、淋巴液、尿液、汗液、粘液、痰液和唾液中的一种或多种。
本申请还提供了一种诊断活动期肝病的方法，通过检测受试者样本中去唾液酸糖蛋白受体片段sH2a的含量诊断受试者是否患有活动期肝病。具体地，活动期肝病患者和健康人之间sH2a含量存在显著性差异，活动期肝病患者sH2a含量显著上调，而非活动期肝病患者和健康人之间sH2a含量无显著性差异，以此可以区分活动期肝病患者和非活动期肝病患者。因此，受试者样本中去唾液酸糖蛋白受体片段sH2a的含量上调是受试者患有活动期肝病的指征。
进一步，当受试者样本中sH2a含量显著上调，根据受试者所患肝病种类的 不同，可以诊断受试者患有活动期乙肝病毒型肝炎、活动期乙肝肝硬化和活动期自身免疫性肝病中的至少一种。
具体地，受试者样本选自血浆、全血、淋巴液、尿液、汗液、粘液、痰液和唾液中的一种或多种，受试者样本包括但不限于上述样本，只要能实现通过样本的sH2a含量区分活动期肝病患者和非活动期肝病患者的样本都可以作为受试者样本。
本申请的诊断活动期肝病的方法特异性高，灵敏度好，该诊断方法使用的sH2a检测试剂盒特异性为94.50％，灵敏度为85.33％。
相应地，本申请还提供了一种活动期肝病的诊断系统，包括：
信息获取模块：用于获取受试者样本中去唾液酸糖蛋白受体片段sH2a的含量信息；
诊断模块：用于根据受试者样本中去唾液酸糖蛋白受体片段sH2a的含量信息对受试者进行活动期肝病诊断。
一些实施例中，诊断模块具体包括：
判断单元：用于根据受试者样本中去唾液酸糖蛋白受体片段sH2a的含量信息判断受试者样本中去唾液酸糖蛋白受体片段sH2a的含量是否上调；
输出单元：用于在受试者样本中去唾液酸糖蛋白受体片段sH2a的含量上调时输出受试者患有活动期肝病的诊断结果。
在一个具体示例中，去唾液酸糖蛋白受体片段sH2a的制备方法包括以下步骤：将sH2a基因片段装载至表达载体上得到重组质粒，将重组质粒转入真核细胞中，转染18～22h添加转染增强剂，5～7天收集上清，利用滤膜过滤上清液，然后进行纯化得到sH2a重组蛋白。
在一个具体示例中，上述纯化包括以下步骤：将滤膜过滤所得滤液进行非变性条件下的Ni-NTA亲和层析，平衡缓冲液为50mM PBS、10mM咪唑、150mM NaCl，pH 7.2～7.4，上样完毕后，清洗10mL；用50mM PBS、250mM咪唑、150mM NaCl，pH7.2～7.4洗脱，收集洗脱液；利用10kDa超滤管浓缩蛋白溶液，保存于50mM PBS(pH 7.2～7.4)缓冲液中，-80℃保存。
下面主要结合具体实施方式和附图对去唾液酸糖蛋白受体片段sH2a在诊断 活动期肝病中的应用作进一步详细的说明。
活动期肝病，也即慢性活动性肝炎，主要诊断依据包括以下方面：(1)肝脏功能出现异常，表现为ALT(谷丙转氨酶)、AST(谷草转氨酶)上升或伴有黄疸指标升高。当肝脏炎症活动时，转氨酶从肝细胞内释放到血液中，在外周血中就表现为转氨酶升高，因此转氨酶升高是诊断肝炎活动比较敏感的指标之一；(2)有的患者肝功能表现正常，在肝脏活检中表现肝小叶间炎症或者界面性肝炎、淋巴细胞、中性细胞浸润等表现，说明肝细胞有炎症活动，也是活动性肝炎的诊断依据之一。
因此，本实施例活动期肝病的样本入组标准包括：
(1)乙肝病毒性肝炎：HBsAg(乙肝表面抗原)阳性，ALT＞2倍正常值上限，AST/ALT＞1.5，HBV DNA>20000IU/ml；
(2)自身免疫性肝病：排除病毒性肝炎，转氨酶＞2倍正常值上限，自身抗体阳性(ANA、SMA或LMKl抗体)滴度≥1：80，γ-球蛋白或IgG>正常上限1.5倍。
(3)活动性肝硬化(代偿期)：乙肝“二对半”检查为“大三阳”或“小三阳”，HBV-DNA为阳性；轻微的门脉高压症(食管静脉曲张、腹壁静脉显现等)；AST/ALT>1.5，球蛋白升高(>正常值)。
(4)活动性肝硬化(失代偿期)：乙肝“二对半”检查为“大三阳”或“小三阳”，HBv-DNA为阳性；血浆白蛋白降低(低于正常值)，球蛋白增高(大于正常值)，白/球蛋白<1.5，AST/ALT>1.5。
不满足上述诊断标准的乙肝病毒性肝炎、自身免疫性肝病和活动性肝硬化则相应地诊断为非活动期乙肝病毒性肝炎、非活动期自身免疫性肝病和非活动期活动性肝硬化。
实施例1 sH2a重组蛋白表达及纯化鉴定
蛋白表达：NCBI搜索人源ASGPR2序列，选择胞外段即sH2a氨基酸序列合成基因并优化为哺乳动物表达密码子，同时装载至pcDNA3.1载体上。将获取的sH2a重组质粒(pcDNA3.1-sH2a-His)转入真核哺乳动物细胞中，并以pcDNA3.1空载体作为阴性对照，转染18～22h添加转染增强剂，5～7天收集上 清，利用0.22μm滤膜过滤上清液。
蛋白纯化：将所得滤液进行非变性条件下的Ni-NTA亲和层析，平衡缓冲液为50mM PBS、10mM咪唑、150mM NaCl，pH7.2～7.4。上样完毕后，清洗10mL；用50mM PBS、250mM咪唑、150mM NaCl，pH7.2～7.4洗脱，收集洗脱液。利用10kD超滤管浓缩蛋白溶液，将蛋白保存于pH7.2～7.4 50mM PBS缓冲液中，-80℃保存。纯化的蛋白进行SDS-PAGE电泳纯度鉴定，分子量大小在35～40kDa，灰度分析表明蛋白纯度达到95％以上，如图1所示。
sH2a重组蛋白活性鉴定：将sH2a重组蛋白用包被缓冲液(pH9.6)稀释至1μg/mL加入微孔板，每孔100μL，4℃包被过夜，拍干的微孔板中包被抗体并加入150～200μL酶标板稳定剂，37℃孵育1～2小时。弃去封闭液并拍干，37℃干燥20～40min；梯度稀释捕获抗体和酶标抗体(浓度为0～200ng/mL)，37℃孵育0.5～1小时，洗涤后加入羊抗兔IgG-HRP(100ng/mL)，37℃孵育0.5～1小时，洗涤后显色。检测发现捕获抗体和检测抗体在100ng/mL时的OD值大于1.0，蛋白与抗体的反应曲线R2>0.99，蛋白的反应活性满足要求，如表1所示。
表1 sH2a真核蛋白活性鉴定
Figure PCTCN2022131117-appb-000001
实施例2 sH2a抗体配对
采用3μg/mL捕获抗体包被酶标板，加入不同浓度(6.25～200ng/mL)的sH2a校准品，37℃孵育1小时，洗涤后加入100μL浓度为10ng/mL的HPR标记的检测抗体，并于37℃孵育1小时，洗涤后加入TMB底物显色液并测定各孔OD值。从结果上看，捕获抗体(厂家：SinoBiological，货号：13908-R002)和检测抗体(厂家：Thermo，货号：MA5-29046)配对良好，可用于双抗夹心体系构建，如表2所示。
表2
Figure PCTCN2022131117-appb-000002
实施例3 sH2a试剂盒及sH2a校准曲线绘制
sH2a试剂盒包括前述捕获抗体、检测抗体和sH2a校准品等。首先将捕获抗体用包被缓冲液(pH9.6)稀释至3μg/mL，按每孔100μL加入酶标板中，4℃孵育过夜；将sH2a校准品蛋白用蛋白质稳定剂稀释分别稀释至0ng/mL、6.25ng/mL、12.5ng/mL、25ng/mL、50ng/mL、100ng/mL、200ng/mL，并按照顺序将校准品加入预包被酶标板中，每孔100μL，37℃孵育1小时；用洗涤液清洗4次，加入辣根过氧化物酶标记的检测抗体，浓度10ng/mL，每孔100μL，37℃孵育1小时；再用洗涤液清洗4次，加入TMB底物显色液并测定各孔OD值。以标准品含量自变量(X)，以其相对应的吸光值(A)作因变量(Y)，以logistic四参数法拟合得到回归方程将待测样本测得的OD值代入回归方程，求得相对应的sH2a抗原含量。校准曲线线性范围6.25～200ng/mL，如图2所示为sH2a检测试剂盒校准曲线。上述sH2a检测试剂盒采用双抗夹心法，方法操作简便易行，检测快速灵敏，所用酶标仪简单、普及，价格低廉，该检测试剂盒线性范围达到6.25～200ng/mL，最低检测限可达5ng/mL。
实施例4 sH2a试剂盒临床性能验证1
从医院收集300例活动期肝病血浆样本(乙肝病毒型肝炎100例、乙肝肝硬化100例、自身免疫性肝病100例)，同时从血站收集200例健康献血人员血浆。使用前述sH2a检测试剂盒(捕获抗体、检测抗体、sH2a校准品等)检测活动期肝病及健康人血浆中的sH2a浓度。样本浓度散点图显示，sH2a对活动期肝病和健康人检测结果的区分具有统计学意义且具有显著性差异，如图3所示。ROC曲线统计结果显示，曲线下面积为0.9583，以65.77ng/mL作为检测参考值，sH2a检测试剂盒特异性为94.50％，灵敏度为85.33％，如图4所示。
实施例5 sH2a试剂盒临床性能验证2
从医院收集200例非活动期肝病血浆样本(乙肝病毒型肝炎80例、乙肝肝硬化60例、自身免疫性肝病60例)，同时从血站收集200例健康献血人员血浆。使用前述sH2a检测试剂盒检测非活动期肝病及健康人血浆中的sH2a浓度。样本浓度散点图显示，非活动期肝病和健康人之间sH2a含量无显著性差异，如图5所示。
实施例6 sH2a试剂盒临床性能验证3
从医院收集300例活动期肝病血浆样本(乙肝病毒型肝炎100例、乙肝肝硬化100例、自身免疫性肝病100例)、200例非活动期肝病血浆样本(乙肝病毒型肝炎80例、乙肝肝硬化60例、自身免疫性肝病60例)、200例其他类型肝病血浆样本(药物性肝炎100例、脂肪肝100例)；同时从血站收集200例健康献血人员血浆。使用前述sH2a检测试剂盒检测活动期肝病、非活动期肝病、其他类型肝病及健康人血浆中的sH2a浓度。样本浓度散点图显示，活动期肝病和健康人之间sH2a含量存在显著性差异，非活动期肝病及其他类型肝病和健康人之间sH2a含量无显著性差异，如图6所示。
实施例7 sH2a试剂盒临床性能验证4
从医院收集100例活动期肝病血清样本(乙肝病毒型肝炎50例、乙肝肝硬化30例、自身免疫性肝病20例)、100例非活动期肝病血清样本(乙肝病毒型肝炎50例、乙肝肝硬化30例、自身免疫性肝病20例)；同时从血站收集100例健康献血人员血清。使用前述sH2a检测试剂盒检测活动期肝病、非活动期肝病、其他类型肝病及健康人血清中的sH2a浓度。样本浓度散点图显示，活动期肝病和健康人之间sH2a含量存在显著性差异，非活动期肝病和健康人之间sH2a含量无显著性差异，如图7所示。
实施例8 sH2a试剂盒临床性能验证5
从医院收集100例活动期肝病全血样本(乙肝病毒型肝炎50例、乙肝肝硬化30例、自身免疫性肝病20例)、100例非活动期肝病全血样本(乙肝病毒型肝炎50例、乙肝肝硬化30例、自身免疫性肝病20例)；同时从血站收集100例健康献血人员全血。使用前述sH2a检测试剂盒检测活动期肝病、非活动期肝病、其他类型肝病及健康人全血中的sH2a浓度。样本浓度散点图显示，活动期肝病 和健康人之间sH2a含量存在显著性差异，非活动期肝病和健康人之间sH2a含量无显著性差异，如图8所示。
综上可知，本申请提供了一种灵敏度高、特异性好的可用于诊断活动期肝病的标志物sH2a，通过对生物样品中的去唾液酸糖蛋白受体片段sH2a的含量进行测定，能够很好地鉴别诊断活动期肝病，从而应用于活动期肝病的早期诊断、鉴别诊断，并为疾病治疗提供有力依据。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本申请的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本申请构思的前提下，还可以做出若干变形和改进，这些都属于本申请的保护范围。因此，本申请专利的保护范围应以所附权利要求为准。

Claims (18)

  1. 去唾液酸糖蛋白受体片段sH2a作为标志物在制备用于诊断活动期肝病的产品中的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述产品为试剂盒、检测试纸条或检测芯片。
  3. 根据权利要求2所述的应用，其特征在于，所述试剂盒包括捕获抗体，所述捕获抗体能够特异性结合所述去唾液酸糖蛋白受体片段sH2a。
  4. 根据权利要求2所述的应用，其特征在于，所述试剂盒包括检测抗体，所述检测抗体能够特异性结合所述去唾液酸糖蛋白受体片段sH2a，且所述检测抗体上标记有检测标记物。
  5. 根据权利要求4所述的应用，其特征在于，所述检测标记物选自荧光标记、化学发光标记、电子致密标记、金属粒子、放射性标记、发色团标记或酶标记构成的组。
  6. 根据权利要求4所述的应用，其特征在于，所述检测标记物选自罗丹明、荧光素、量子点、地高辛标记探针、放射性同位素、荧光微球、胶体金、吖啶酯、荧光素酶、辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶或葡萄糖-6-磷酸脱氢酶构成的组。
  7. 根据权利要求2～6任一项所述的应用，其特征在于，所述试剂盒还包括所述去唾液酸糖蛋白受体片段sH2a。
  8. 根据权利要求2～6任一项所述的应用，其特征在于，所述试剂盒还包括固相载体、包被缓冲液和洗涤缓冲液中的一种或多种。
  9. 根据权利要求8所述的应用，其特征在于，所述固相载体选自磁性颗粒、胶乳粒子、酶标板或微流控芯片构成的组。
  10. 根据权利要求1～6任一项所述的应用，其特征在于，所述活动期肝病包括活动期乙肝病毒型肝炎、活动期乙肝肝硬化和活动期自身免疫性肝病中的一种或多种。
  11. 根据权利要求1～6任一项所述的应用，其特征在于，所述产品用于通过检测样本中去唾液酸糖蛋白受体片段sH2a的含量诊断受试者是否患有活动期肝病。
  12. 根据权利要求11所述的应用，其特征在于，所述样本选自血浆、全血、血清、淋巴液、尿液、汗液、粘液、痰液和唾液中的一种或多种。
  13. 一种活动期肝病的诊断方法，其特征在于，根据受试者样本中去唾液酸糖蛋白受体片段sH2a的含量诊断受试者是否患有活动期肝病。
  14. 根据权利要求13所述的诊断方法，其特征在于，受试者样本中去唾液酸糖蛋白受体片段sH2a的含量上调是受试者患有活动期肝病的指征。
  15. 根据权利要求13或14所述的诊断方法，其特征在于，所述样本选自血浆、全血、血清、淋巴液、尿液、汗液、粘液、痰液和唾液中的一种或多种。
  16. 根据权利要求13或14所述的诊断方法，其特征在于，所述活动期肝病包括活动期乙肝病毒型肝炎、活动期乙肝肝硬化和活动期自身免疫性肝病中的一种或多种。
  17. 一种活动期肝病的诊断系统，其特征在于，包括：
    信息获取模块：用于获取受试者样本中去唾液酸糖蛋白受体片段sH2a的含量信息；
    诊断模块：用于根据受试者样本中去唾液酸糖蛋白受体片段sH2a的含量信息对受试者进行活动期肝病诊断。
  18. 根据权利要求17所述的诊断系统，其特征在于，诊断模块具体包括：
    判断单元：用于根据受试者样本中去唾液酸糖蛋白受体片段sH2a的含量信息判断受试者样本中去唾液酸糖蛋白受体片段sH2a的含量是否上调；
    输出单元：用于在受试者样本中去唾液酸糖蛋白受体片段sH2a的含量上调时输出受试者患有活动期肝病的诊断结果。
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