KR100394940B1 - Method for measuring serum asialo-glycoprotein concentration for diagnosis of hepatic disease and a kit therefor - Google Patents

Abstract

The present invention provides a method for measuring the concentration of acaiallo glycoproteins by a sandwich assay using lectin as at least one of a capture protein and a probe protein, and a kit for measuring the same. The method and kit of the present invention can be usefully used for the early diagnosis and treatment of liver diseases such as cirrhosis and liver cancer, and have the advantages of high safety and reproducibility and accurate measurement of large quantities of samples at the same time. .

Description

Method for measuring serum aialallo glycoprotein levels for liver disease and measuring kit {Method for measuring serum asialo-glycoprotein concentration for diagnosis of hepatic disease and a kit therefor}

The present invention relates to a method for measuring and measuring kits of acaiallo glycoproteins by a sandwich type measuring method, and more particularly, to acaiallo α 1 -acid glycoprotein (AS-AGP) among acaiallo glycoproteins. In order to measure asialo haptoglobin (AS-HG) or asialo α 2-macroglobulin (AS-MG), antibodies and lectins to their glycoproteins may be used, or lectins that recognize acaiallo glycoproteins may be used. The present invention relates to a method for measuring the amount of asialoglycoprotein (ASGP, asialoglycoprotein) estimated to increase in blood at the onset of liver disease, and a kit for measurement therefor.

Liver diseases, including hepatitis, cirrhosis, and liver cancer, are the most common single disease in Korea, Japan, Taiwan, China, and most Southeast Asian countries. Currently, bilirubin or urobilinogen in urine is present. ), Or measure changes in biochemical components by measuring the amount of GOT (glutamic-oxaloacetic transaminase), GPT (glutamic pyruvic transaminase), total bilirubin, albumin, protein, lactic acid dehydrogenase, or type B Liver disease is diagnosed by detecting antigens or antibodies of hepatitis virus (HBV) or hepatitis C virus (HCV). In addition, alpha-feto protein (AFP) and carcinoembryonic antigen (CEA) tests are used to diagnose liver cancer. However, the liver has a number of complex functions, and there is a biological specificity that does not easily detect liver abnormalities, and liver disease is often diagnosed after aggravation due to the lack of early diagnosis technology for liver disease. There is difficulty in treatment. If liver disease progresses from acute hepatitis to chronic hepatitis, cirrhosis, liver tumor or from hepatitis to liver cancer, an effective diagnostic system is needed to track and diagnose the process, for effective treatment of the disease, and for fatal cirrhosis or liver cancer. Early diagnosis of liver disease is essential to prevent progression. Therefore, there is a need for an early clinical diagnosis of liver disease and a diagnostic method capable of sensitive and specific analysis of markers of liver disease that accurately reflects the progress of the disease in patients with liver disease.

The study of acai allo glycoprotein and acai allo glycoprotein receptors began mainly in animal models in the early 1970s, and from the early 1990s, studies on human acai allo glycoprotein and asai allo glycoprotein receptors began in earnest. . Asaiallo glycoproteins are serum markers that reflect the progression of liver disease and have been reported to increase rapidly during onset of acute hepatitis and to decrease again during the recovery phase (T. Sawamura, et al ., Gastroenterology, 1981; 81: 527). ~ 533). Concentrations of acai allo glycoprotein receptors and hepatic tissue asia alloglucoprotein receptors are symmetrical when hepatitis is induced in animals. It has been reported as a clinical marker capable of judging the condition (T. Sawamura, et al. , Gastroenterology, 1984; 87: 1217-1221). In addition, if the liver concentration and glycoproteins Asai allo shown that is directly proportional to the size of the liver tissue of the glycoprotein in blood Asai egg was found to be a marker to aid in assessing the status of liver cancer (T. Sawamura, et al., Gastrologia Japonica , 1985; 20: 201-208). As such, acaiallo glycoproteins and their receptors have specific relationships with normal liver function and their concentrations in blood or liver tissue have been reported to reflect normal liver function or the clinical condition of patients.

Currently, liver cancer markers such as AFP and CEA have low specificity, making it difficult to determine the therapeutic effect of patients. However, since asiaallo glycoprotein and its receptor have high specificity for liver disease, it may be used as a useful marker for early diagnosis of liver disease and determination of treatment outcome of patients. Attempts to use substances directly related to the function of the liver as markers for the development or progress of liver disease, rather than the existing markers for liver disease, suggest a new direction of liver disease diagnosis technology, as well as the early treatment by early diagnosis of liver disease. Will open the way.

Conventionally, in order to measure the concentration of acaiallo glycoprotein, acaiallo glycoprotein receptor is isolated and purified from humans or other animals (rabbits, rats, etc.) and used as a capture protein, and a competitive radioactive receptor assay method using a radiolabeled substrate ( competitive radioreceptor assay or electroimmunodiffusion (JS Marshall, et al ., J. Lab. Clin. Med., 1978; 92: 30-37 and N. Serbource-Goguel, et al ., Hepatology, 1983; 3: 356-359. However, it is difficult to secure sufficient amounts of acaiallo glycoprotein receptors necessary for the development of acaiallo glycoprotein measurement kits, and in the case of competitive radioactive receptor assays, there is a risk of radiation exposure because of the use of radioactive materials and special treatment for waste treatment. There are various difficulties, such as the need for facilities, and there are problems in that the quantitative analysis is difficult in the case of the electroimmunity diffusion method. In particular, competitive assays have low precision and reproducibility, making them unsuitable for commonly used diagnostic kits. The present inventors have developed a reproducible and accurate measurement method and kit that can be used for diagnosing liver function or treating liver disease by measuring blood asiaallo glycoproteins.

There are many methods for qualitative or quantitative analysis of specific substances, but among these methods, particle agglutination assays, radioimmunoassays (RIA), and enzyme immunoassays (EIA). And fluoroimmunoassays (FIA) are the most widely used. Although radioimmunoassays have high sensitivity, they have various problems due to the use of radiolabels. Therefore, high sensitivity, safe and simple enzyme immunoassay methods and fluorescence immunoassay methods are more commonly used. Enzyme immunoassay methods generally use high-safety enzymes such as alkaline phosphatase, horseradish peroxidase, and glucose oxidase. Color substrates with high over-over rate are selected and used. This method has been widely used because it can solve the shortcomings of radioimmunoassays with sensitivity and specificity.

The present inventors have high safety and reproducibility compared to the conventional methods for measuring the concentration of acyalo glycoproteins, and as a measuring method and kit for measuring a plurality of samples simultaneously, glycoproteins ( α 1 -acid glycoprotein, haptoggle By developing a sandwich-type measuring method using an antibody against robin or α 2-macroglobulin) and a lectin that recognizes acaiallo glycoprotein, and a measurement kit therefor, the blood concentration of acaiallo glycoprotein can be effectively measured. It became.

SUMMARY OF THE INVENTION An object of the present invention is to provide a sandwich-type measuring method capable of measuring reproducibly and accurately the blood concentration of acaiallo glycoproteins that is increased in blood during the development of liver disease.

Another object of the invention is to provide a kit for measurement for this purpose.

Figure 1 is a graph showing a result of separation of proteins (α 1-acid glycoprotein, AGP ) per 1- α acids from human plasma by gel filtration (gel filtration) using a DEAE- cellulose column chromatography.

Figure 2 is a graph showing the results of purifying sialic acid desialylated α 1 -acid glycoprotein by gel filtration using Sephadex G-200 column chromatography.

Figures 3a and 3b shows the results of PAGE analysis of purified AGP and sialic acid-free AGP (Asialo α 1-acid glycoprotein, AS-AGP) and Western blotting using anti-AGP antibody, respectively. 3C and 3D show the results of SDS-PAGE analysis of purified AGP and AS-AGP and Western blotting using anti-AGP antibody, respectively. Picture.

Figures 4a, 4b and 4c is a graph showing the reactivity according to the concentration of acaiallo glycoproteins in the solid-phase sandwich assay using the antibody and lectin to the glycoprotein, Figure 4a is a GP and AGP, Figure 4b between the alsan is removed Happ toggle Robin (haptoglobin, HG) and between the alsan is removed HG, Figure 4c α 2- macro globulin (α 2-macroglobulin, MG) and between the removal alsan MG Graph showing the reactivity according to the concentration of.

5 is a graph showing the reactivity according to the concentration of acai allo glycoprotein in the lectin-lectin solid sandwich type measurement method using the lectin to recognize the asai allo glycoprotein, AGP, HG and sialic acid from which AGP and sialic acid are removed Graph showing the reactivity according to the concentration of the removed HG and MG and MG removed sialic acid.

6a, 6b and 6c is a solid sandwich-type measuring method using an antibody and lectin to the glycoprotein, acai allo α 1-acid glycoprotein (AS-AGP), acai according to the dilution of the serum of patients with liver disease Graphs showing the results of concentration measurement of allo haptoglobin (AS-HG) and asialo α 2-macroglobulin (AS-MG), respectively.

Figure 7 is an example of measuring the amount of acyallo glycoproteins in the serum of patients with liver disease in a sandwich-type measuring method using the antibody and lectin for glycoprotein, the solid-type sandwich method using the antibody and lectin for AGP Chart measuring concentration of AS-AGP in blood of liver disease patients.

8 is a chart measuring the amount of acai allo glycoproteins in serum of patients with liver disease in a lectin-lectin sandwich type measurement method using a lectin to recognize acai allo glycoprotein.

The present invention recognizes acaiallo glycoprotein as one of the capture protein and probe protein in a sandwich-type measuring method for measuring the concentration of acaiallo glycoproteins in excess in the blood when the liver transitions from normal to hepatitis, cirrhosis or liver cancer. The present invention relates to a method for measuring the concentration of acaiallo glycoproteins using a lectin and a kit for measurement therefor.

Therefore, the method of the present invention specifically binds the antibody to glycoprotein to a solid body such as a microtiter plate, and (b) a serum sample is added to the solid body to bind the blood acyallo glycoprotein to the antibody. (C) adding a lectin to which the label is bound to bind to the aceallo glycoprotein bound to the antibody, and (d) detecting the label to measure the concentration of the aciallo glycoprotein.

In addition, the method of the present invention, (a) a lectin that recognizes acyalo glycoproteins is adsorbed to a solid body such as a microtiter plate, (b) a serum sample is added to the solid body to the blood asaiallo glycoproteins to the lectin (C) adding a lectin bound to a label to bind to the acyalo glycoprotein bound to the lectin, and (d) detecting the label to measure the concentration of acaiallo glycoprotein. .

In addition, the method of the present invention, (a) adsorbed lectins that recognize acaiallo glycoproteins to a solid body such as a microtiter plate, (b) a serum sample is added to the solid body to the blood asaiallo glycoproteins lectin (C) adding an antibody to the glycoprotein to which the label is bound to bind to the aceallo glycoprotein bound to the lectin, and (d) detecting the label to measure the concentration of the aceallo glycoprotein. Steps.

The present invention glycoprotein acid Asai allo The α 1- removed between alsan include glycoproteins from the (α 1-acid glycoprotein, AGP ), between the alsan is removed Happ toggle Robin (haptoglobin, HG), α 2 between the alsan is removed -Macroglobulin ( α 2-macroglobulin, MG) and the like.

In addition, lectins that specifically recognize acaiallo glycoproteins include Arachius hypogaea agglutinin (PNA), Ricinus communis agglutinin (RCA), and Agarius bisporus . Agarius bisporus agglutinin (ABA) and Viscum album agglutinin (VAA) and the like are exemplified, and it is preferable to use double PNA or RCA, and most preferably RCA. .

As a label to be bound to the lectin, enzymes or fluorescent materials can be used. When the enzyme is selected and used as a labeling substance, it is preferable to use an enzyme having high stability such as alkaline phosphatase, horseradish peroxidase, and glucose oxidase. The amount of acyalo glycoproteins can be measured by coloring by adding a coloration substrate having a high turn-over rate, for example, ortho-phenylene diamine (OPD). In the case of using a fluorescent material as a labeling material, it is preferable to use fluorescein, rhodamine, etc., and the amount of acyallo glycoprotein can be measured by the fluorescence intensity measurement method.

More specifically, the method of the present invention can be carried out as follows, for example. The antibody against the glycoprotein is added to a solid body such as a well of a microtiter plate, and left to stand for at least 1 hour to adsorb the antibody to the microtiter plate, and then a bovine serum albumin solution is added to adsorb albumin to the space on the surface of the solid body. Let's do it. After washing the wells with a rinsing solution, for example, a phosphate buffer containing a Tween-based surfactant, a serum sample dilution solution is added to each well and allowed to react at room temperature. After washing the wells with the wash solution, lectins labeled with enzymes or fluorescent materials, such as RCA-horseradish peroxidase conjugates, are added to each well and allowed to react at room temperature. After washing the wells with the above washing solution, a color developing substrate of an enzyme, for example, an ortho-phenylenediamine substrate solution, is added to each well to develop color. After a certain period of time, the reaction is stopped, the absorbance is measured at the appropriate wavelength, and the concentration of acaiallo glycoprotein in the blood is calculated by comparing the absorbance of the acaiallo glycoprotein standard solution. If the labeling substance is a fluorescent substance, a lectin labeled with a fluorescent substance is added to each well, reacted at room temperature, washed with a washing solution, and fluorescence intensity is measured.

In addition, in the above method, the antibody against the glycoprotein is replaced with a lectin that recognizes an acyallo glycoprotein, and the modified lectin is adsorbed on a microtiter plate and the antibody against the glycoprotein is labeled with a labeling substance. And modified methods in which lectins that recognize acaiallo glycoproteins are adsorbed onto a microtiter plate and the lectins are labeled and used with a label.

According to the measuring method of the present invention, in the case of using acyalo alpha 1-acid glycoprotein as a reference substance, blood acyalo glycoprotein can be detected in a concentration range of 0.03 µg / ml to 4 µg / ml.

The present invention also provides a kit for measuring aceallo glycoprotein, comprising a lectin conjugated with a solid substance to which an antibody or lectin is adsorbed to a glycoprotein and a label. In the measurement kit of the present invention, preferably, an antibody against α 1-acid glycoprotein, haptoglobin or α 2-macroglobulin adsorbed on a solid body, or a lectin or labeling substance that recognizes asiaallo glycoprotein is bound. Lectin solution, detection solution for labeling substance, serum dilution solution, washing solution and aceallo glycoprotein standard solution. When the labeling substance is an enzyme, the substrate solution of the enzyme is used as a detection solution of the labeling substance. When the labeling substance is a fluorescent substance, the fluorescence intensity emitted from the labeling substance is directly measured.

Specifically, for example, the measurement kit of the present invention is a microtiter plate, horseradish peroxidase, to which an antibody or lectin to α 1-acid glycoprotein, haptoglobin, α 2-macroglobulin is adsorbed, and RCA binder solution, ortho-phenylenediamine substrate solution, phosphate buffer containing tween as wash solution, serum dilution solution and asiaallo glycoprotein standard solution.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are merely provided to illustrate the present invention, but the protection scope of the present invention is not limited by these examples.

Example 1 Isolation and Purification of Acyalo glycoproteins from Serum

Among the glycoproteins contained in human plasma, α- acid glycoprotein (AGP) was isolated and purified as follows.

Thrombin 2 NIH unit was added to 200 mL of human plasma, and left at 37 ° C. for 2 hours and 4 ° C. overnight, and then centrifuged to remove blood clots. The prepared serum was dialyzed with 0.05 M sodium acetate buffer (pH 4.3), then loaded into a DEAE-cellulose column equilibrated with the same buffer and 0.05 M sodium acetate buffer (pH 4.3) and 0.1 M sodium acetate buffer (pH 4.3) As a result of measuring the absorbance at 280 nm by mixing and eluting with a linear concentration gradient (linear concentration gradient) increases as shown in FIG. Fractions containing AGP and other proteins were collected and ammonium sulfate was added to 0.5 g / ml to precipitate the protein. After centrifugation, ammonium sulfate was added to the supernatant again in an amount of 0.18 g / ml to precipitate the protein. The precipitate was dissolved in a small amount of distilled water and then dialyzed sufficiently with distilled water and freeze-dried.

40 mg of AGP thus separated was hydrolyzed with 6 ml of 0.1 N sulfuric acid solution at 80 ° C. for 2 hours, neutralized with 1 N sodium hydroxide, and dialyzed with 0.01 M phosphate buffer (pH 7.4). 28 mg of α 1-acid glycoprotein (AS-AGP) from which sialic acid was removed was loaded on a Sephadex G-200 column, gel-filtered with 0.01 M phosphate buffer (pH 7.4), and absorbance was measured at 280 nm. Fractions containing protein were collected. The result is shown in FIG.

To confirm the properties of the isolated AGP and AS-AGP, they were electrophoresed on polyacrylamide gels and SDS-polyacrylamide gels, and then stained with Coomassie blue, and the proteins separated on the gels. Western blotting analysis was performed using an anti-AGP antibody (Sigma, USA), and the results are shown in FIGS. 3A, 3B, 3C and 3D. 3A and 3B are photographs showing PAGE results of purified AGP and AS-AGP and Western blotting analysis results using anti-AGP antibody, wherein the first column is AGP and the second column is AS- AGP. 3C and 3D show SDS-PAGE results for purified AGP and AS-AGP and Western blotting analysis using anti-AGP antibodies, respectively, wherein column 1 is a standard molecular weight label, and Column 2 is AGP, and column 3 is AS-AGP.

As can be seen in the PAGE results of FIG. 3A, AS-AGP is more positively charged than AGP due to the removal of the sialyl group, thereby lowering the mobility on the polyacrylamide gel, and removing the sialyl group from the SDS-PAGE of FIG. 3C. It can be seen that the molecular weight of AGP is lower than that of AGP. In addition, the results of Western blotting analysis of FIGS. 3B and 3D showed that the monoclonal antibody against AGP specifically reacted with both AGP and AS-AGP. Therefore, in the present invention, the antibody against the glycoprotein was used for the measurement of acaiallo glycoprotein in the blood.

Example 2 Lectin-Lectin Sandwich Type Test Method

Sandwich type measurement method using lectin as both the capture protein and the probe protein to confirm the reactivity according to the sample concentration, Arachius hypoaza aglutinin (PNA), Ricinus communis aglutinin (RCA), Agarius Lectins such as bisporus aglutinin (ABA) and biscum alum aglutinin (VAA) were used as capture proteins and probe proteins, and sandwiched measurement methods were applied as follows.

100 μl of a solution of the diluted lectin at a concentration of 0.13 μg / ml to 4 μg / ml was added to each well of the microtiter plate and left overnight at 4 ° C. to adsorb the lectin to the microtiter plate, followed by 3% Bovine serum albumin solution was added to adsorb albumin to the space on the solid surface. After washing the wells with phosphate buffer containing 0.05% Tween (“washing solution”), add 100 μl of the diluted solution of AS-AGP obtained in Example 1 in the range of 0 to 4 μg / ml to each well and at room temperature. The reaction was carried out for 2 hours. After washing the wells three times with the washing solution, 100 μl of a solution diluted with horseradish peroxidase-coupled probe lectin (EY Laboratories, USA) to a concentration of 0.05 μg / ml to 0.5 μg / ml was added to each well. The reaction was carried out at room temperature for 1 hour. The wells were washed three times with the washing solution, and 100 µl of ortho-phenylenediamine (OPD) substrate solution (Sigma, USA) was added to each well and developed. After 15 minutes, 2.5N sulfuric acid solution was added to stop the reaction and the absorbance was measured at 490 nm. As a result, the reactivity according to the sample concentration was as shown in Table 1 below.

In the results of Table 1, a positive response (low background, high reactivity) according to the sample concentration was shown when PNA or RCA was used as the capture protein and RCA was used as the probe protein. When the OD ₄₉₀ value of the background is 0.05 or less, it is set as "low", and when the OD ₄₉₀ value of the reactivity according to the concentration is less than 0.2, it is set as "low". If the value is "0.8" or more, the expression is referred to as "high".

Example 3 Lectin-Antibody Sandwich Method Test

In order to confirm the reactivity according to the sample concentration by using a lectin as a capture protein and an antibody as a probe protein, horseradish peroxidase is used instead of lectin combined with horseradish peroxidase as a probe protein. Except for using the anti-AGP antibody (EY Laboratories, USA) to which I bound was carried out in the same manner as in Example 2 to obtain the results shown in Table 2.

In the results of Table 2, a positive response (low background, high reactivity) according to the sample concentration was shown when PNA was used as the capture protein and anti-AGP antibody was used as the probe protein. The criteria for evaluating background and reactivity are the same as described in connection with Table 1 above.

Example 4 Antibody-Lectin Sandwich Measurement Test

The reactivity according to the concentration of acaiallo glycoproteins in the sample was examined by a sandwich-type measurement method using an antibody against AGP, HG or MG as a capture protein and a lectin-peroxidase conjugate as a probe protein.

100 microliters of an appropriately diluted solution of anti-AGP antibody, anti-HG antibody and anti-MG antibody (Sigma, USA), respectively, were placed in a well of a microtiter plate and allowed to stand overnight at 4 ° C. Then, 3% bovine serum albumin solution was added to adsorb albumin to the space on the solid surface. After washing the wells with phosphate buffer containing 0.05% Tween (“washing solution”), add 100 μl of each solution diluted in multiples of AGP, HG, and MG in the range of 0.03 to 4 μg / mL to each well. It was added and reacted at room temperature for 2 hours. After washing the wells three times with the washing solution, 100 μl of a solution diluted with horseradish peroxidase-coupled probe lectin used in Example 2 to an appropriate concentration was added to each well and allowed to react at room temperature for 1 hour. The wells were washed three times with the washing solution, and 100 µl of ortho-phenylenediamine substrate solution was added to each well to develop color. After 15 minutes, 2.5N sulfuric acid solution was added to stop the reaction and the absorbance was measured at 490 nm. As a result, the reactivity according to the sample concentration was as shown in Table 3 below.

The results in Table 3 showed a positive response (low background, high reactivity) when anti-AGP antibody, anti-HG antibody, and anti-MG antibody were used as capture proteins and RCA as probe protein, respectively. The criteria for evaluating background and reactivity are the same as described in connection with Table 1 above.

Example 5 Concentration of Acyalo glycoprotein Concentration by the Antibody-Lectin Sandwich Method

Reactivity Test

In order to establish a method for measuring acaiallo glycoproteins using antibodies and lectins, a microtiter plate was used as a solid body, and a sandwich type measurement using lectins bound to horseradish peroxidase enzyme was made by adsorbing monoclonal antibody to the solid body. Method was used.

That is, 100 μl of a properly diluted solution of monoclonal antibody or polyclonal antibody specific for AGP and HG, respectively, was added to the well of a microtiter plate and left overnight at 4 ° C. to adsorb the antibody to the microtiter plate. Then, 3% bovine serum albumin solution was added to adsorb albumin to the space on the solid surface. After washing the wells with phosphate buffer containing 0.05% Tween (“washing liquid”), dilute AGP, HG, MG and sialic acid-free AGP, HG, MG in 2-fold dilutions in the range 0.03-4 μg / ml. 100 μl each was added to each well and allowed to react at room temperature for 2 hours. After washing the wells three times with the washing solution, 100 μl of a properly diluted solution of the RCA-horseradish peroxidase conjugate was added to each well and allowed to react at room temperature for 1 hour. The wells were washed three times with the washing solution, and 100 µl of ortho-phenylenediamine substrate solution was added to each well to develop color. After 15 minutes, 2.5N sulfuric acid solution was added to stop the reaction, and the absorbance was measured at 490 nm to determine the reactivity according to the concentration of acyalo glycoprotein.

As shown in Figures 4a, 4b and 4c, by using the measuring method of the present invention, the α 1-acid glycoprotein, haptoglobin, and α 2-macroglobulin, each of which the sialic acid is removed from the asiaallo glycoprotein, was 0.03. Measurements can be made at concentrations between μg / ml and 1.0 μg / ml.

Example 6 Concentration of Acyalo glycoproteins by Lectin-Lectin Sandwich-type Measuring Method

Reactivity Test

Another method for measuring acaiallo glycoproteins is to use a sandwich-type measurement method in which a lectin that recognizes acaiallo glycoprotein is adsorbed onto a solid body and a lectin bound to horseradish peroxidase enzyme is used as a probe.

That is, 100 μl of a solution diluted with 4 μg / ml of lectin (RCA, EY Labs.) Was added to the well of the microtiter plate and left overnight at 4 ° C. for adsorption on the microtiter plate. Serum albumin solution was added to adsorb the space on the solid surface with albumin. After washing the wells with phosphate buffer containing 0.05% Tween (“washing liquid”), dilute AGP, HG, MG and sialic acid-free AGP, HG, MG in 2-fold at 0.03 to 5.0 μg / ml. 100 μl each was added to each well and allowed to react at room temperature for 2 hours. After washing the wells three times with the washing solution, 100 μl of a properly diluted solution of the RCA-horseradish peroxidase conjugate was added to each well and allowed to react at room temperature for 1 hour. The wells were washed three times with the washing solution, and 100 µl of ortho-phenylenediamine substrate solution was added to each well to develop color. After 15 minutes, 2.5N sulfuric acid solution was added to stop the reaction, and the absorbance was measured at 490 nm to determine the reactivity according to the concentration of acyalo glycoprotein.

As shown in Figure 5, using the measuring method of the present invention, each of the α 1-acid glycoprotein, haptoglobin and α 2-macroglobulin from which the sialic acid is removed from the asiaallo glycoprotein is 0.03 µg / ml to 5.0 µg, respectively. Measurements can be made at concentrations between / ml.

Example 7 Patient Serum Dilution with Antibody-Lectin Sandwich Kit

Kits for measuring acaiallo glycoprotein concentration were prepared using the following components:

A. Solid antibody: A microtiter plate to which the antibody is adsorbed, 100 μl of the antibody against AGP, HG or MG was added to the microtiter plate and allowed to stand at 4 ° C. overnight, and albumin was added to the space on the solid surface. Prepared by adsorption.

B. Enzyme-linked lectins: RCA solution (RCA-peroxidase conjugate) with horseradish peroxidase

C. Serum Dilutions

D. Ortho-phenylenediamine Substrate Solution

E. Wash: Phosphate Buffer with 0.05% Tween

F. Standard Solution: Acyalosaccharide Protein Standard Solution

Using this kit, the serum dilution response of asaiallo glycoproteins in serum of normal, liver cirrhosis patients, liver cancer patients and chronic hepatitis patients was examined as follows.

Each serum sample was properly diluted with a serum dilution solution (C) to a solid-type antibody of A, that is, an antibody against a glycoprotein, and diluted to 100 μl per well, followed by the constitution of B, D, and E. Asia alloglucoprotein concentration was measured by the same sandwich method as Example 5 using urea.

Figure 6a is an anti-AGP antibody and RCA-HRP, Figure 6b is an anti-HG antibody and RCA-HRP, and Figure 6c is a serum showing the results of experiments performed with a sandwich-type kit using anti-MG antibody and RCA-HRP, respectively Dilution response curve.

Example 8 Determination of Blood Acyalo glycoprotein Concentrations in Patients with an Antibody-Lectin Sandwich Kit

Anti-AGP antibodies were used to measure blood acyallo glycoprotein concentrations in patients with the kit used in Example 7. After diluting the serum of normal persons (50), hepatitis patients (26), cirrhosis patients (45), and liver cancer patients (37) by 10-fold, the acai allotrope in acai allo glycoprotein was prepared by the method of Example 5. As a result of measuring the concentration of AGP, cirrhosis and liver cancer showed AGP concentrations higher than normal, and hepatitis showed a similar concentration to normal (FIG. 7, Table 4). These results indicate that elevated blood levels of acaiallo glycoproteins are associated with the development of cirrhosis or liver cancer.

The cut-off value in FIG. 7 was determined as the average of 50 normal serum samples plus 2 times the standard deviation (arithmetic mean + 2SD).

Example 9 Measurement of Blood Asia Alloprotein Levels in Patients by Lectin-Lectin Sandwich Kit

The following components were used to prepare kits for the determination of aceallo glycoprotein concentration.

A. Solid antibody: Microtiter plate adsorbed with lectin (RCA), 100 μl of appropriate concentration of RCA was added to the microtiter plate and allowed to stand overnight at 4 ° C., followed by solid body surface with 3% bovine serum albumin. It was prepared by adsorption in the space of.

B. Enzyme-linked lectins: RCA solution (RCA-peroxidase conjugate) with horseradish peroxidase

C. Serum Dilutions

D. Ortho-phenylenediamine Substrate Solution

E. Wash: Phosphate Buffer with 0.05% Tween

The kit measured the amount of acyallo glycoproteins in the blood of the patient. After diluting the serum of normal people (41), hepatitis patients (59), cirrhosis patients (98), liver cancers (81), and patients with other diseases other than liver disease (53) by 10-fold, respectively, the method of Example 6 As a result of measuring the concentration of allo glycoproteins, the concentration of acai allo glycoproteins in blood of liver cirrhosis and liver cancer patients was found to be higher than the normal value as in Example 8. In FIG. 8, the cut-off value was defined as the average absorbance of 41 normal serum samples plus 2 times the standard deviation value (arithmetic mean + 2SD).

On the other hand, the concentrations of acai alloglucoprotein in blood of patients with diseases other than liver disease were similar to those of normal people. Therefore, acai alloglucoprotein is specific for liver disease, and the diagnosis of liver disease such as cirrhosis and liver cancer is judged. It shows that it can be used as a marker.

The antibody-lectin or lectin-lectin sandwich type method and measuring kit of the present invention for measuring the concentration of acyallo glycoprotein in blood can be usefully used for the early diagnosis and treatment of liver disease such as cirrhosis and liver cancer. A large amount of samples can be accurately measured with high reproducibility at the same time, which will be very useful for medical diagnosis and evaluation of treatment status.

Claims (23)

delete delete (a) adsorbing an antibody against a glycoprotein selected from the group of glycoproteins consisting of α 1-acid glycoprotein, heptoglobin, α 2-macroglobulin and mixtures thereof, to a solid body such as a microtiter plate, (b) adding a sample containing asiaallo glycoprotein from which the sialic acid has been removed from the glycoprotein to the solid body to bind the asiaallo glycoprotein to the antibody; (c) Ricinus communis agglutinin (RCA) bound with horseradish peroxidase is added to bind to the acyallo glycoprotein bound to the antibody, (d) detecting the label and measuring the concentration of acaiallo glycoproteins, the method comprising measuring the concentration of acaiallo glycoproteins in the sample. delete The method of claim 3, The antibody to the glycoprotein is a monoclonal antibody or a polyclonal antibody. delete delete delete (a) adsorbing the lectin to a solid, such as a microtiter plate, (b) adding a sample containing acaiallo glycoprotein to the solid to bind the acaiallo glycoprotein to the lectin, (c) adding a lectin bound to the labeling substance to bind to the acyallo glycoprotein bound to the lectin, (d) detecting the label and measuring the concentration of acaiallo glycoproteins. The method of claim 9, The lectin of step (a) is PNA, and the lectin is bound to the labeling material of step (c) is characterized in that the RCA combined with horseradish peroxidase. The method of claim 9, The lectin of step (a) is RCA, characterized in that the lectin bound to the labeling material of step (c) is RCA combined with horseradish peroxidase. The method of claim 1, (a) adsorbing the lectin to a solid, such as a microtiter plate, (b) adding a sample containing acaiallo glycoprotein to the solid to bind the acaiallo glycoprotein to the lectin, (c) adding an antibody to the glycoprotein to which the labeling substance is bound to bind to the asiaallo glycoprotein bound to the lectin, (d) detecting the label and measuring the concentration of acaiallo glycoproteins. The method of claim 12, The method of claim 1, wherein the lectin of step (a) is PNA, and the antibody to the glycoprotein to which the labeling material of step (c) is bound is an anti-AGP antibody to which horseradish peroxidase is bound. A kit for measuring the concentration of acaiallo glycoproteins by a sandwich type measurement method, characterized in that it comprises a lectin bound to an antibody and a labeling substance to a glycoprotein adsorbed on a solid body. The method of claim 14, Kit for measuring the concentration of acyallo glycoproteins in serum samples. The method of claim 14, A kit comprising an antibody against a glycoprotein adsorbed on a solid body, a lectin solution bound to an enzyme, an enzyme substrate solution, a serum sample dilution solution, a wash solution and an aceallo glycoprotein standard solution. The method of claim 16, Antibodies against glycoproteins adsorbed on microtiter plates, horseradish peroxidase-RCA conjugate solution, ortho-phenylenediamine substrate solution, serum sample dilution solution, phosphate buffer containing tween as a washing solution, and acai Kits comprising allo glycoprotein standard solution. As a kit for measuring the concentration of acyallo glycoproteins in serum samples, anti-AGP antibody adsorbed on microtiter plate, horseradish peroxidase-RCA conjugate solution, ortho-phenylenediamine substrate solution, A kit comprising a serum sample dilution solution, a phosphate buffer solution containing tween as a wash solution, and an aceallo glycoprotein standard solution. A kit for measuring the concentration of acaiallo glycoproteins by a sandwich-type measuring method, characterized in that it comprises a lectin and a labeling substance bound to the lectin that recognizes the acaiallo glycoprotein adsorbed on the solid body. The method of claim 19, Kit for measuring the concentration of acyallo glycoproteins in serum samples. The method of claim 19, A kit comprising a lectin that recognizes an aceallo glycoprotein adsorbed on a solid body, a lectin solution bound to an enzyme, an enzyme substrate solution, a serum sample dilution solution, a wash solution, and a aceallo glycoprotein standard solution. The method of claim 21, Lectin, horseradish peroxidase-lectin conjugate solution, ortho-phenylenediamine substrate solution, serum sample dilution solution, phosphate buffer containing tween as wash solution to recognize acyallo glycoprotein adsorbed on microtiter plate And acyalo glycoprotein standard solution. As a kit for measuring the concentration of acyallo glycoproteins in serum samples, RCA adsorbed on microtiter plates, horseradish peroxidase-RCA conjugate solution, ortho-phenylenediamine substrate solution, serum sample dilution Kit comprising a solution, a phosphate buffer containing tween as a wash solution, and an asiaallo glycoprotein standard solution.