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WO2001035105A1 - Procede de mesure de la concentration serique d'asialoglycoproteine utile pour diagnostiquer les maladies hepatiques et trousse associee - Google Patents

Procede de mesure de la concentration serique d'asialoglycoproteine utile pour diagnostiquer les maladies hepatiques et trousse associee Download PDF

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Publication number
WO2001035105A1
WO2001035105A1 PCT/KR2000/000840 KR0000840W WO0135105A1 WO 2001035105 A1 WO2001035105 A1 WO 2001035105A1 KR 0000840 W KR0000840 W KR 0000840W WO 0135105 A1 WO0135105 A1 WO 0135105A1
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Prior art keywords
glycoprotein
asialo
lectin
coupled
concentration
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Inventor
Tai Wha Chung
Eun Young Song
Kyoung A. Kim
Dur Han Kwon
Hong Soo Lee
Yung Dai Kim
Yong Kung Choe
In Seong Choe
Hee Jung Kim
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KOBIAS CO Ltd
Korea Research Institute of Bioscience and Biotechnology KRIBB
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KOBIAS CO Ltd
Korea Research Institute of Bioscience and Biotechnology KRIBB
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Priority to AU61873/00A priority Critical patent/AU6187300A/en
Publication of WO2001035105A1 publication Critical patent/WO2001035105A1/fr
Anticipated expiration legal-status Critical
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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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/415—Assays involving biological materials from specific organisms or of a specific nature from plants
    • G01N2333/42—Lectins, e.g. concanavalin, phytohaemagglutinin

Definitions

  • the present invention relates to a method for measuring asialo-glycoprotein concentration through a sandwich assay and a kit therefor, more particularly to a method and a kit for measuring the amount of asialo-glycoprotein(ASGP) such as asialo ⁇ 1- acid glycoprotein(AS-AGP), asialo haptoglobin(AS-HG), or asialo ⁇ 2- macroglobulin(AS-MG) suspected of being increased in the blood in case of an attack of a hepatic disease by using an antibody against glycoprotein and a lectin recognizing asialo-glycoprotein, or using lectins.
  • ASGP as asialo ⁇ 1- acid glycoprotein
  • AS-HG asialo haptoglobin
  • AS-MG macroglobulin
  • Hepatic diseases such as hepatitis, liver cirrhosis, hepatocellular carcinoma are the ones that have the largest number of patients as a single disease in Korea, Japan, Taiwan, China, and the most part of Southeast Asian countries, and presently, a hepatic disease is diagnosed through the methods such as of checking the level of bilirubin or urobilinogen in urea, of measuring the total amount of GOT(glutamic-oxaloacetic transaminase), GPT(glutamic pyruvic transaminase).
  • hepatitis B virus(HBV) hepatitis B virus
  • HCV hepatitis C virus
  • CEA carcinoembryonic antigen
  • a hepatic disease developed from acute hepatitis into chronic hepatitis, liver cirrhosis and a tumor of the liver, or from hepatitis into hepatocellular carcinoma it is necessary to establish an effective diagnostic system for tracing and/or diagnosing their processes, and is also necessary to make an early diagnosis for treating these diseases effectively and preventing the disease from developing into fatal liver cirrhosis or hepatocellular carcinoma. Therefore, a method for diagnosis that can keenly and specifically analyze the marker of a hepatic disease which reflects the development of the hepatic disease in the patient accurately is required.
  • asialo-glycoprotein and asialo-glycoprotein receptor had begun in the early 1970's mainly for animal models and has been in progress from the early 1990's for the human bodies. It has been reported that asialo-glycoprotein is a marker in the serum for reflecting the progressive state of a hepatic disease because it is increasing rapidly in a development stage of an acute hepatitis and decreasing in a convalescent stage (T. Sawamura, et al., Gastroenterology, 1981 ; 81 :527-533).
  • asialo-glycoprotein is a marker indicating the stage of the hepatocellular carcinoma (T. Sawamura, et al., Gastrologia Japonica, 1985; 20:201-208). It has been reported, as described above, asialo-glycoprotein and its receptor have relationship specific with the function of the liver, and the concentration in blood and that in the liver tissue reflect the function state of the liver or the clinical state of a patient.
  • the marker of hepatocellular carcinoma such as AFP and CEA has difficulty in judging the curative effect of a patient because of its low specificity.
  • asialo-glycoprotein and its receptor has high specificity for a hepatic disease, they are expected as an effective marker for an early diagnosis and a judgment on the curative effect of a patient.
  • An attempt to use a substance directly related to the functions of a liver as a marker instead of the existing marker for diagnosis of a hepatic disease will not only present a new direction for a diagnostic technique of a hepatic disease but give a way for an early treatment of the disease by an early diagnosis.
  • asialo- glycoprotein receptor is isolated from a human bodies or other animals such as rabbit and rat, purified, and used as a capture protein, and a competitive radioreceptor assay using radioactive labeling substance or an eletroimmunodiffusion is employed (J. S. Marshall, et al., J. Lab. Clin. Med., 1978; 92:30-37 and N. Serbource-Goguel, et al., Hepatology, 1983; 3:356-359).
  • the asialo-glycoprotein receptor is difficult to obtain as large an amount as necessary for the development of the kit for measuring asialo-glycoprotein.
  • the competitive radioreceptor assay has many difficulties such as a risk of suffering from radioactive rays and necessity of a particular facility for radioactive waste disposal, due to the use of radioactive substance and the eletroimmunodiffusion has a problem in difficulty of quantitative analysis. Particularly, in the competitive assay, accuracy and reproducibility are so low that the assay is not suitable for the diagnostic kit.
  • the present inventors have developed a method and a kit for measuring serum asialo- glycoprotein concentration reproducibly and accurately to diagnose liver functions and treat hepatic diseases.
  • a radioimmunoassay RIA
  • an enzyme immunoassay EIA
  • a fluoroimmunoassay FIA
  • the radioimmunoassay has high sensibility but has many problems due to the use of radioactive labeling substance, so the enzyme immunoassay and the fluoroimmunoassay are more widely used because they are safe and simple as well as high in sensibility.
  • enzyme immunoassay stable enzymes such as alkaline phosphatase, horseradish peroxidase, or glucose oxidase are used generally, and color developer of high turn-over rate is selected and used as a substrate of enzyme.
  • This assay has similar sensibility and specificity to the radioimmunoassay and can solve the problems of the radioimmunoassay, thus bringing on a wide use.
  • the present inventors have developed a sandwich assay method and a kit for measuring the serum concentration of asialo-glycoprotein effectively by using an antibody against glycoprotein( ⁇ 1-acid glycoprotein, haptoglobin, or ⁇ 2- macroglobulin) and a lectin recognizing asialo-glycoprotein, said method being able to measure many samples simultaneously as well as are higher in safety and reproducibility than that of the prior art for measuring the concentration of asialo- glycoprotein.
  • the present invention provides a method and a kit for measuring the concentration of asialo-glycoprotein by using lectin recognizing asialo-glycoprotein as at least one of a capture protein or a probe protein through a sandwich assay to measure the concentration of asialo-glycoprotein being present excessively in the blood when developing from normal into hepatitis, liver cirrhosis, or hepatocellular carcinoma.
  • the method of the present invention comprises: (a) adsorbing an antibody against glycoprotein onto a solid phase such as a microtiter plate, (b) adding serum sample to the solid phase to bind an asialo-glycoprotein in serum to the antibody. (c) adding a lectin coupled with labeling substance to bind it to the asialo-glycoprotein bound with antibody, and (d) detecting the labeling substance to measure the concentration of asialo-glycoprotein.
  • the method of the present invention comprises: (a) adsorbing a lectin recognizing asialo-glycoprotein onto a solid phase such as a microtiter plate, (b) adding serum sample to the solid phase to bind an asialo-glycoprotein in serum to the lectin, (c) adding a lectin coupled with labeling substance to bind it to the asialo-glycoprotein bound with lectin, and (d) detecting the labeling substance to measure the concentration of asialo-glycoprotein.
  • the method of the present invention comprises: (a) adsorbing a lectin recognizing asialo-glycoprotein onto a solid phase such as a microtiter plate, (b) adding serum sample to the solid phase to bind an asialo-glycoprotein in serum to the lectin, (c) adding an antibody against glycoprotein coupled with labeling substance to bind it to the asialo-glycoprotein bound with lectin, and (d) detecting the labeling subtance to measure the concentration of asialo-glycoprotein.
  • an asialo-glycoprotein may be desialylated ⁇ 1-acid glycoprotein(AGP). desialylated haptoglobin(HG), or desialylated ⁇ 2- macroglobulin(MG).
  • a lectin recognizing specifically asialo-glycoprotein may be Arachius hypogaea agglutinin(PNA), Ricinus communis agglutinin(RCA), Agarius bisporus agglutinin(ABA), or Viscum album agglutinin(VAA), and it is more preferable to use PNA or RCA, and most preferable to use RCA.
  • PNA Arachius hypogaea agglutinin
  • RCA Ricinus communis agglutinin
  • ABA Agarius bisporus agglutinin
  • VAA Viscum album agglutinin
  • a labeling substance to be coupled with lectin or antibody may be enzyme or fluorescent material.
  • enzyme it is preferable to use the enzyme of high stability such as alkaline phosphatase, horseradish peroxidase, or glucose oxidase, and the amount of asialo-glycoprotein can be measured by adding a color developer of high turn-over rate, for example ortho-phenylene diamine(OPD).
  • fluorescent material it is preferable to use fluorescein, or rhodamine, and the amount of asialo-glycoprotein can be measured by analyzing fluorescent strength.
  • the method of the present invention comprises as follows: adding an antibody against glycoprotein to a solid phase such as a microtiter plate well, allowing it to stand more than 1 hour to adsorb the antibody onto the microtiter plate well, adding a bovine serum albumin solution to adsorb the albumin onto remaining spaces of well, washing the well with a detergent such as a phosphate buffer containing a Tween-type surfactant, adding a dilution of the serum sample to each well to react them at room temperature, washing the well with above detergent again to react them at room temperature, adding lectin labeled with an enzyme or fluorescent material such as a RCA-horseradish peroxidase complex to each well to react them at room temperature, washing the well with above detergent, adding color- developer of enzyme such as ortho-phenylene diamine solution, stopping the reaction after a predetermined period, measuring absorbance at an appropriate wave length, and determining the concentration of serum asialo-glycoprotein by comparing
  • the present invention includes a modified method that replacing above antibody against glycoprotein with lectin recognizing asialo-glycoprotein, the lectin is adsorbed onto a microtiter plate well and the antibody against glycoprotein labeled with a labeling substance is used, and a modified method that the lectin is adsorbed onto a microtiter plate well and the lectin labeled with a labeling substance is used.
  • the serum asialo-glycoprotein can be detected at the range of 0.03 ⁇ g/ml ⁇ 4 ⁇ g/ml.
  • the present invention provides a kit for measuring the level of asialo- glycoprotein that comprises a solid phase adsorbed with an antibody against glycoprotein or a lectin and a lectin coupled with labeling substance.
  • the kit of the present invention comprises a solid phase adsorbed with an antibody against ⁇ 1-acid glycoprotein, haptoglobin or ⁇ 2-macroglobulin, or a lectin recognizing asialo- glycoprotein, a lectin solution coupled with labeling substance, a detecting solution of labeling substance, a serum dilution, detergent, and a standard solution for asialo- glycoprotein.
  • the labeling substance is an enzyme
  • a substrate solution of enzyme is used as the detecting solution.
  • the labeling substance is a fluorescent material, fluorescent strength emitted from the fluorescent material is measured directly.
  • the kit of the present invention comprises a microtiter plate adsorbed with an antibody against ⁇ 1-acid glycoprotein, haptoglobin or ⁇ 2- macroglobulin, or with a lectin, a RCA-horseradish peroxidase complex solution, an ortho-phenylene diamine solution, a phosphate buffer containing Tween as a detergent, a serum dilution, and a standard solution for asialo-glycoprotein.
  • Fig. 1 is a graph illustrating the result on isolation of ⁇ 1 -acid glycoprotein( AGP) from the blood plasma of a human body by gel filtration using DEAE-cellulose column chromatography.
  • Fig. 2 is a graph illustrating the result on purification of desialylated ⁇ 1-acid glycoprotein by gel filtration using Sepadex G-200 column chromatography.
  • Fig. 3a and Fig. 3b are photographs illustrating results on analysis of purified AGP and desialylated AGP(asialo ⁇ 1-acid glycoprotein, AS- AGP) by PAGE and by
  • FIG. 3c and Fig. 3d are the photographs illustrating results on analysis of purified AGP and AS- AGP by SDS-
  • Fig. 4a, Fig. 4b and Fig. 4c are graphs illustrating dose-response depending on the concentration of asialo-glycoprotein by the solid phase sandwich assay using antibody against glycoprotein and lectin.
  • Fig. 4a is of AGP and desialylated AGP
  • Fig. 4b is of haptoglobin(HG) and desialylated HG
  • Fig. 4c is of ⁇ 2-macroglobulin(MG) and desialylated MG, respectively.
  • Fig. 5 is a graph illustrating dose-response depending on the concentration of asialo-glycoprotein i.e., AGP. desialylated AGP, HG, desialylated HG, MG, and desialylated MG by the lectin-lectin solid-phase sandwich assay using a lectin recognizing asialo-glycoprotein, respectively.
  • 6c are graphs illustrating results on measurement of the concentrations of asialo ⁇ 1-acid glycoprotein(AS-AGP), asialo haptoglobin(AS-HG), and asialo ⁇ 2-macroglobulin(AS-MG) depending on the dilution of serum of a hepatic disease patients by the solid-phase sandwich assay using antibody against glycoprotein and lectin, respectively.
  • Fig. 7 is a diagram illustrating the measured concentration of the serum AS-AGP of hepatic disease patients by the solid-phase sandwich assay using antibody against AGP and lectin, as an example of measuring the concentrationl of the serum asialo- glycoprotein of hepatic disease patients by sandwich assay using antibody and lectin.
  • Fig. 8 is a diagram illustrating the measured level of the serum asialo-glycoprotein of hepatic disease patients by the lectin-lectin sandwich assay using lectin recognizing asialo-glycoprotein.
  • Example 1 Isolation and Purification of Asialo-glycoprotein from the Serum ⁇ 1-acid glycoprotein(AGP) was isolated and purified from human blood plasma containing glycoprotein by the following method.
  • FIG. 3a is that of AGP
  • column 2 is that of AS- AGP.
  • FIG. 3d are photographs illustrating results on analysis of purified AGP and AS-AGP by SDS-PAGE and by Western blotting using anti-AGP antibody, respectively.
  • column 1 is the mark of the standard molecular weight
  • column 2 is that of AGP
  • column 3 is that of AS-AGP.
  • AS-AGP is lower in migrating rate on the polyacrylamide gel than that of AGP because AS-AGP lacking of sialyl group is more positive than AGP.
  • PAGE of Fig. 3a As shown the result of SDS-PAGE in Fig. 3c, the molecular weight of AS-AGP due to desialylation is lower than that of AGP.
  • a monoclonal antibody against AGP is shown to react specifically with both of AGP and AS-AGP in the result of Western blotting analysis, as shown in Fig. 3b and Fig. 3d. Therefore, in the present invention, the antibody against glycoprotein was used for measuring serum asialo-glycoprotein.
  • the following sandwich assay was carried out by using lectin such as Arachius hypogaea agglutinin(PNA), Ricinus communis agglutinin(RCA), Agarius bisporus agglutinin(ABA), and Viscum album agglutinin(VAA) as capture protein and probe protein.
  • lectin such as Arachius hypogaea agglutinin(PNA), Ricinus communis agglutinin(RCA), Agarius bisporus agglutinin(ABA), and Viscum album agglutinin(VAA) as capture protein and probe protein.
  • each lOOu 1 of the dilution of 0.13 ⁇ g/ml ⁇ 4 ⁇ g/ml of the capture lectin was added to each well of a microtiter plate, and allowed to stand overnight at 4 ° C to adsorb the lectin onto the microtiter plate. Thereafter, 3% the bovine serum albumin solution was added to adsorb the albumin on the remaining spaces of the solid phase surface. After the wells were washed with the phosphate buffer containing 0.05% Tween(a detergent), each 100 ⁇ 1 of the double dilution of 0-4 ⁇ g/ml of the AS-AGP obtained in the above example 1 was added to each well to react for 2 hours at room temperature.
  • each 100 ⁇ 1 of the dilution of 0.05 ⁇ g/ml ⁇ 0.5 ⁇ g/ml of the probe lectin coupled with horseradish peroxidase(E-Y Laboratories, USA) was added to each well to react for lhour at room temperature.
  • each 100 ⁇ 1 of the ortho-phenylene diamine(OPD) solution(Sigma, USA) was added to each well to be color-developed. After 15minutes, the reaction was stopped by adding 2.5N sulfuric acid solution and measured its absorbance at 490nm. As a result, dose-response depending on the sample concentration is shown in Table 1.
  • Example 3 Lectin- Antibody Sandwich Assay Examination To confirm dose-response depending on the sample concentration by sandwich assay using lectin as a capture protein and antibody as a probe protein, the assay was carried out in the same manner as the Example 2 above described except using an anti- AGP antibody coupled with horseradish peroxidase(E-Y Laboratories, USA) instead of a lectin coupled with horseradish peroxidase as a probe protein. The result is shown in Table 2.
  • Example 4 Antibody-Lectin Sandwich Assay Examination By sandwich assay using antibody against AGP, HG or MG as a capture protein and lectin-peroxidase complex as a probe protein, dose-response depending on asialo- glycoprotein concentration in the sample was examined as follows.
  • Each lOO ⁇ 1 of the appropriately diluted solution of anti-AGP antibody, anti-HG antibody or anti-MG antibody(Sigma, USA) was added to each well of a microtiter plate, and allowed to stand overnight at 4 ° C to adsorb the antibody onto the microtiter plate. Thereafter, 3% the bovine serum albumin solution was added to adsorb the albumin on the remaining spaces of the solid phase surface. After the wells were washed with the phosphate buffer containing 0.05% Tween(a detergent), each 100 ⁇ 1 of the double dilution of 0.03-4 ⁇ g/ml of the desialylated AGP, HG and MG was added to each well to react for 2 hours at room temperature.
  • each 100 ⁇ 1 of appropriately diluted solution of the probe lectin coupled with horseradish peroxidase as used in Example 2 was added to each well to react for 1 hour at room temperature.
  • each 100 ⁇ 1 of the ortho-phenylene diamine solution was added to each well to be color-developed. After 15minutes, the reaction was stopped by adding 2.5N sulfuric acid solution and measured its absorbance at 490nm. As a result, dose-response depending on the sample concentration is shown in Table 3.
  • each lOO ⁇ 1 of the appropriately diluted solution of monoclonal antibodies specific for AGP and HG, and polyclonal antibody specific for MG was added to each well of a microtiter plate, and allowed to stand overnight at 4 ° C to adsorb the antibody onto the microtiter plate. Thereafter, 3% the bovine serum albumin solution was added to adsorb the albumin on the remaining spaces of the solid phase surface.
  • each 100 ⁇ 1 of the double dilution of 0.03-4 ⁇ g/ml of the AGP, HG, MG, desialylated AGP, desialylated HG and desialylated MG was added to each well to react for 2 hours at room temperature.
  • each 100 ⁇ 1 of appropriately diluted solution of the lectin- horseradish peroxidase complex was added to each well to react for 1 hour at room temperature.
  • each 100 ⁇ 1 of the ortho-phenylene diamine solution was added to each well to be color-developed.
  • Another method for measuring asialo-glycoprotein is sandwich assay using a lectin recognizing asialo-glycoprotein adsorbed onto a solid phase as a capture protein, and a lectin coupled with horseradish peroxidase as a probe protein. That is, each lOO ⁇ 1 of the dilution of 4 ⁇ g/ml of lectin(RC A, EY Labs.) was added to each well of a microtiter plate, and allowed to stand overnight at 4 ° C to adsorb the lectin onto the microtiter plate. Thereafter, 1% the bovine serum albumin solution was added to adsorb the albumin on the remaining spaces of the solid phase surface.
  • each 100 ⁇ 1 of the double dilution of 0.03-5.0 ⁇ g/ml of the AGP, HG, MG, desialylated AGP, desialylated HG and desialylated MG was added to each well to react for 2 hours at room temperature.
  • each 100 ⁇ 1 of appropriately diluted solution of the RCA- horseradish peroxidase complex was added to each well to react for 1 hour at room temperature.
  • each 100 ⁇ 1 of the ortho- phenylene diamine solution was added to each well to be color-developed. After 15minutes, the reaction was stopped by adding 2.5N sulfuric acid solution and measured its absorbance at 490nm.
  • desialylated ⁇ 1-acid glycoprotein, haptoglobin, and ⁇ 2-macroglobulin of the asialo-glycoprotein could be measured in the range of 0.03 ⁇ g/ml ⁇ 5.0 ⁇ g/ml, respectively.
  • Example 7 Examination of Serum Dilution of Patient by the Anti-Lectin Sandwich Assay Kit A kit for measuring the concentration of asialo-glycoprotein comprising the following components was prepared.
  • A. solid antibody an antibody adsorbed onto a microtiter plate. It was prepared by adding each 100 ⁇ 1 of an antibody against AGP, HG or MG to each well of the microtiter plate, allowing to stand overnight at 4 ° C, and then adsorbing an albumin onto the spaces of solid phase surface.
  • a reaction on dilution of the asialo-glycoprotein in the serum of the healthy subject and patients of liver cirrhosis, hepatocellular carcinoma and chronic hepatitis was examined as follows.
  • the concentration of asialo-glycoprotein was measured by adding each 100 ⁇ 1 of the appropriately diluted solution of serum to the A component of solid antibody i.e., an antibody adsorbed onto a microtiter plate well and using components of B, D and E through the sandwich assay as the above described in Example 5.
  • Fig. 6a, Fig. 6b and Fig. 6c are the serum dilution reaction curves illustrating results obtained by the sandwich kit using anti-AGP antibody and RCA-HRP(in Fig. 6a), anti-HG antibody and RCA-HRP(in Fig. 6b), and anti-MG antibody and RCA-HRP(in Fig. 6c).
  • Example 8 Measurement of the Serum Asialo-glycoprotein Concentration of Patients by the Antibody-Lectin Sandwich Kit
  • the serum asialo-glycoprotein concetrations of patients were measured by the kit used in the Example 7 using an anti-AGP antibody.
  • the concentrations of asialo AGP of the asialo-glycoprotein were measured by the method as described in the Example 5 using dilution in ten times of the serums of the healthy subjects(50 person s), the hepatitis patients(26 persons), liver cirrhosis patients(45 persons), hepatocell ular carcinoma patients(37 persons), and non-hepatic disease patients(39 persons).
  • the cut-off value in Fig. 7 was calculated by the sum of the arithmatic mean for serum samples from 50 healthy subjects and double of SD(mean+2SD).
  • Example 9 Measurement of the Serum Asialo-glycoprotein Concentration of Patients by the Lectin-Lectin Sandwich Kit A kit for measuring the concentration of asialo-glycoprotein comprising the following components was prepared.
  • A. solid lectin a lectin adsorbed onto a microtiter plate. It was prepared by adding each 100 ⁇ 1 of RCA to each well of the microtiter plate, allowing to stand overnight at 4 ° C , and then adsorbing 3% bovine serum albumin onto the spaces of solid phase surface.
  • E. detergent a phosphate buffer containing 0.05% Tween
  • the serum asialo-glycoprotein level of patients was measured by the kit above described.
  • the level of asialo AGP of the asialo-glycoprotein was measured by the method as described in the Example 6 using dilution in ten times of the serums of the healthy subjects(41 persons), the hepatitis patients(59 persons), liver cirrhosis patients(98 persons), hepatocellular carcinoma patients(81 persons), and non-hepatic disease patients(53 persons).
  • the results showed that, the serum asialo-glycoprotein concentrations in liver cirrhosis and hepatocellular carcinoma patients were higher than the normal value as that in Example 8.
  • the cut-off value in Fig. 8 was calculated by the sum of the arithmatic mean of absorbance for the serum samples from 41 healthy subjects and double of SD(mean+2SD).
  • the serum asialoglycoprotein concentration of the non-hepatic disease patients was similar to the normal value. Therefore, it shows that the asialo-glycoprotein is specific for the hepatic diseases and can be used as a marker in a diagnosis and judgement of hepatic disease state.
  • the present sandwich assay method and the kit for measuring serum concentration asialo-glycoprotein can be used effectively in early diagnosis and judgement on treatment result of hepatic diseases including liver cirrhosis and hepatocellular carcinoma, said method being able to measure many samples simultaneously as well as are high in safety and reproducibility.

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Abstract

La présente invention concerne un procédé de mesure de la concentration d'asialoglycoprotéine au moyen d'un dosage de type sandwich dans lequel la lectine fait office de protéine de capture ou de protéine de sonde; et une trousse associée. Le procédé et la trousse selon la présente invention peuvent être utilisés efficacement dans le diagnostic précoce et le jugement des résultats du traitement des maladies hépatiques y compris la cirrhose du foie et l'hépatocarcinome, ledit procédé permettant de mesurer simultanément plusieurs échantillons et assurant une grande sécurité et une forte reproductibilité.
PCT/KR2000/000840 1999-11-10 2000-08-01 Procede de mesure de la concentration serique d'asialoglycoproteine utile pour diagnostiquer les maladies hepatiques et trousse associee Ceased WO2001035105A1 (fr)

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AU61873/00A AU6187300A (en) 1999-11-10 2000-08-01 Method for measuring serum asialo-glycoprotein concentration for diagnosis of hepatic disease and kit therefor

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Cited By (10)

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WO2004058823A1 (fr) * 2002-12-27 2004-07-15 Neobiodigm Co., Ltd Anticorps monoclonal dirige contre l'asialo alpha 1-acide glycoproteine, bandelette immunochromatographique comprenant l'anticorps monoclonal et methode de diagnostic de maladies hepatiques utilisant cette bandelette immunochromatographique
WO2006121892A3 (fr) * 2005-05-05 2007-02-22 Philadelphia Health & Educatio Diagnostic des maladies du foie au moyen de l'evaluation de la glycosylation des proteines
US20130323756A1 (en) * 2010-07-07 2013-12-05 Aethlon Medical, Inc. Methods and compositions for quantifying exosomes
CN104374919A (zh) * 2009-07-14 2015-02-25 独立行政法人产业技术综合研究所 糖蛋白的测定方法、试剂及糖链标记物
US9110078B2 (en) 2008-04-04 2015-08-18 Drexel University Diagnosis of liver pathology through assessment of anti-gal IgG glycosylation
US9796761B2 (en) 2009-07-14 2017-10-24 National Institute Of Advanced Industrial Science And Technology Glycan markers as measure of disease state of hepatic diseases
JP2019011978A (ja) * 2017-06-29 2019-01-24 国立大学法人群馬大学 糖タンパク質におけるフコシル糖鎖の量を測定する方法およびキット
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US9796761B2 (en) 2009-07-14 2017-10-24 National Institute Of Advanced Industrial Science And Technology Glycan markers as measure of disease state of hepatic diseases
EP2846162A1 (fr) * 2009-07-14 2015-03-11 National Institute of Advanced Industrial Science and Technology Procédé de mesure de la glycoprotéine, procédé d'examen d'une maladie hépatique, réactif pour la détermination quantitative d'une glycoprotéine et glycoprotéine en tant que marqueur de glycane comme index pour des états cliniques d'une maladie hépatique
CN104374919A (zh) * 2009-07-14 2015-02-25 独立行政法人产业技术综合研究所 糖蛋白的测定方法、试剂及糖链标记物
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EP3816629A1 (fr) * 2019-10-31 2021-05-05 Acebiomed, Inc. Kit de suivi et de diagnostic du degré de l'hépatite chronique progressive et de la fibrose hépatique par la mesure de l'asialo a1 glycoprotéine-acide en tant que marqueur de lésions hépatocellulaires et son utilisation
CN114280300A (zh) * 2021-12-28 2022-04-05 四川大学华西医院 尿液蛋白在诊断代谢性肝病中的应用
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