JPH10311832A - Sandwich determination method and differentiation method of liver disease - Google Patents

Abstract

PROBLEM TO BE SOLVED: To execute the diagnosis of a liver disease by external serum diagnosis by setting the quantity of used monoclonal antibody to the equivalent or less of cholinesterase quantity in a sample. SOLUTION: The quantity of a solidification monoclonal antibody for identifying cholinesterase is set to the equivalent or less of cholinesterase quantity in a sample. The antigen connecting position of the monoclonal antibody is thus perfectly occupied by the cholinesterase in the sample. On the other hand, the quantity of labeled aleuria aurantia lectin is set to more than a quantity capable of connecting the sugar chains of all connective cholinesterase in the cholinesterase captured by the solidification monoclonal antibody. Thus, the quantity of aleuria aurantia lectin connective sugar chain of aleuria aurantia lectin connective cholinesterase per fixed quantity of cholinesterase can be determined with high sensitivity by one determining operation without directly determining the total quantity of cholinesterase. Therefore, liver cirrhosis and chronic liver disease can be differentiated by the serum of a patient.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for determining the amount of sugarcane-binding glycan of Hilochawantake lectin-binding cholinesterase per fixed amount of cholinesterase in serum, and a method for distinguishing liver diseases by the method. It is about.

[0002]

2. Description of the Related Art Cholinesterase (also called pseudocholinesterase, butylcholinesterase) (E.
C. 3.1.1.8) is a glycoprotein having a molecular weight of about 340,000 and consisting of four identical subunits, each subunit consisting of 574 amino acids and having nine asparagine-linked sugar chains. It acts on cholines such as benzoylcholine and butylcholine, is abundant in serum and liver, is produced in the liver, and its physiological actions are thought to be possibly involved in the nervous-muscle system.

[0003] Clinically, the measurement of this enzyme is carried out to determine the degree of liver disease, particularly chronic liver parenchymal dysfunction, ie, hepatic parenchymal dysfunction in cirrhosis and chronic hepatitis. Have. That is, since serum cholinesterase is produced in hepatocytes, the decrease indicates a decrease in hepatocyte function.

[0004] Hilochawantake ( Aleuria a)
urantia ) lectin is a sugar-free protein prepared from the Japanese radish mushroom, rich in serine and glycine, with a molecular weight of 72,000 and a molecular weight of 31,000.
And has one sugar binding site per subunit. Monosaccharides have an affinity for L-fucose, L-fucose α1 → 2, L-fucose α1 → 3
It has a binding property to residues, but L-fucose α1 → 6
It binds particularly strongly to sugar chains having residues.

[0005] Okura Takashi, Hada Toshikazu et al.
Serum was processed by affinity column chromatography in which Hilo chawantake lectin was immobilized, the enzymatic activity of Hilochawantake lectin-binding cholinesterase retained in the column was measured, and the ratio to the total cholinesterase enzyme activity in serum was determined. The proportion of patients with hepatocellular carcinoma and cirrhosis was significantly higher than those with chronic hepatitis and healthy subjects (CAN).
SER RESEARCH, 54, 55, 199
4 years). However, since this method is a column chromatography method, complicated operations such as adsorption, elution, volume measurement, and activity measurement must be repeated, and it is difficult to measure a large number of samples in a short time. Was.

Further, there is a report that cirrhosis can be differentiated by measuring the serum cholinesterase-binding cholinesterase in serum by a sandwich method using an immobilized monoclonal antibody against cholinesterase and labeled Hilochawantake lectin ( M. Kondo, T. Hada et al., Cini
ca Chimica Acta 243, 1-9, 1
995, JP-A-8-285853).

This method is very effective, but is disclosed in
-285853, that is, the total cholinesterase concentration in the sample is measured in advance, and then diluted so that the total cholinesterase concentration in the sample becomes a constant value. In the method of measuring, the total cholinesterase concentration varies greatly depending on the sample, so it is necessary to change the dilution ratio for each sample, which is troublesome, and in a sample with a low dilution ratio, it is caused by substances other than cholinesterase. There is a problem that non-specific adsorption increases. When measuring the total cholinesterase concentration in the sample in advance, when trying to determine the amount of cholinesterase from the enzyme activity of cholinesterase,
In some cases, the enzyme was inactivated or had a mutant with a genetically low enzyme activity, and the cholinesterase level could not be measured correctly.

On the other hand, in the second method described in JP-A-8-285853, that is, the method of measuring the cholinesterase-binding cholinesterase in a sample diluted at the same magnification, the concentration of total cholinesterase in each sample is determined. Therefore, the amount of the sugar chain of Hilochawantake lectin-binding cholinesterase-bound cholinesterase per fixed amount of cholinesterase was not always measured.

[0009] On the other hand, since treatment strategies are completely different between chronic hepatitis and cirrhosis, it is very important to diagnose the transition from chronic hepatitis to cirrhosis clinically. At present, the discrimination is largely based on the empirical judgment of a physician based on the history of chronic hepatitis patients and the results of biochemical tests. In the serodiagnosis, measurement of hyaluronic acid, type IV collagen 7S, and procollagen type III-N-terminal peptide has been performed. Is not enough. In addition, image diagnosis such as ultrasonography and CT examination is also performed. Diagnosis of tumorous lesions such as hepatocellular carcinoma can be performed relatively early, but for diffuse lesions such as cirrhosis. Is difficult to diagnose. After all, the diagnosis of chronic hepatitis and cirrhosis can only be made by histological examination of a liver tissue sample obtained by biopsy or surgery.
It is a heavy burden on the patient.

[0010]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for diagnosing a liver disease which presently imposes a heavy burden on a patient by in vitro serum diagnosis. More specifically, instead of using a complicated column method, a method similar to enzyme immunoassay was used to develop a method that is not affected by the dilution ratio of the sample or the difference in the concentration of total cholinesterase in the sample. It is intended to be used for discrimination.

[0011]

Means for Solving the Problems The present inventors have made intensive studies on the above problems, and as a result, have reached the present invention. That is, the present invention is characterized in that in a sandwich assay using an immobilized monoclonal antibody recognizing cholinesterase and labeled Hilochawantake lectin, the amount of the monoclonal antibody used is equal to or less than the amount of cholinesterase in the sample. Measurement method.
Further, the present invention is a method for identifying a liver disease, characterized by using the above-mentioned measurement method. Hereinafter, the present invention will be described in more detail.

In the present invention, an immobilized monoclonal antibody recognizing cholinesterase and a labeled Chinese cabbage lectin are used. There are no particular limitations on the immobilized monoclonal antibody and the labeled Hilochawantake lectin used herein.
No. 5853 can be used.

The amount of the monoclonal antibody used is not more than the equivalent of the amount of cholinesterase present in the sample. As a result, the monoclonal antibody completely occupies the antigen-binding site by the cholinesterase contained in the sample in the measurement system. On the other hand, there is no particular limitation on the amount of labeled G. lucidum lectin used, but the excess amount, that is, of the cholinesterase captured by the immobilized monoclonal antibody, binds to the sugar chain of all G. lucidum-binding cholinesterase. By using more than the possible amount, it is possible to measure with high sensitivity.

By using the measuring system as in the present invention, it is possible to directly measure the total amount of cholinesterase without directly measuring it.
In one measurement operation, a certain amount of cholinesterase
Amount of sugar chain, or L, of the Chinese lantern lectin-binding cholinesterase
-The amount of fucose α1 → 2 and L-fucose α1 → 3 and L-fucose α1 → 6 residues can be measured. This value is not affected by the total cholinesterase concentration contained in the sample.

In the present invention, as described above, Hilocha wantake lectin labeled with a labeling agent is used. If the sensitivity of the labeling agent is too high, the sensitivity of the labeling agent itself may be reduced or an unlabeled Hilocha wantake lectin may be used. Sensitivity can be reduced by adding an amount simultaneously.

The value determined by the method of the present invention, that is, the amount of the sugar chain of the Japanese terrestrial lectin-binding cholinesterase, ie, L-fucose α, per unit amount of the cholinesterase.
1 → 2 and L-fucose α1 → 3 and L-fucose α1 → 6 residues) show higher values in the sera of patients with cirrhosis and hepatocellular carcinoma than those of chronic hepatitis patients and healthy persons. . Therefore, by the measurement method of the present invention, cirrhosis / hepatocellular carcinoma can be distinguished from chronic hepatitis / healthy person. In particular, as described above, it is very important clinically to distinguish between cirrhosis and chronic hepatitis, and this distinction can be made by the method of the present invention.

[0017]

The measuring method according to the present invention is a simple method which does not require a complicated method such as affinity chromatography. In addition, it is not necessary to change the dilution ratio for each serum when measuring Hilocha wantake lectin-binding cholinesterase, and the measurement can be performed at the same dilution ratio. Therefore, the measurement is simple, and the cholinesterase, which tends to occur when the dilution ratio is changed, is also easy. The effect of non-specific adsorption caused by substances other than zeolites can be avoided.

Furthermore, the sera of patients with liver diseases, particularly sera of patients with cirrhosis and patients with chronic hepatitis, are not affected by the difference in the concentration of total cholinesterase in serum, and are better than the known method (JP-A-8-285853). It can be distinguished from serum.

The present invention provides a measurement system that can be used to distinguish liver diseases, especially cirrhosis and chronic hepatitis, more effectively than known serum diagnostic items, and histological examination of liver tissue specimens by conventional biopsy or surgery. This can be provided as a serodiagnosis with little burden on the patient without any problem, and is very significant.

It is generally said that the cholinesterase concentration in serum greatly varies among individuals. For example, if cholinesterase is decreased in liver disease, the concentration may be as low as 1/50 of a healthy person. However, even if the cholinesterase concentration is such a low concentration, the amount of the antibody to be used must be adjusted so that the cholinesterase occupies the entire antigen-binding site of the immobilized antibody in the measurement method of the present application. As described above, even when the cholinesterase is at a low concentration, it is conventionally considered that it is extremely difficult to obtain the detection sensitivity without being buried in the background at the time of measurement, but in the present invention, the measurement is performed with a sufficient measurement sensitivity. I was able to.

[0021]

The present invention will be described below by way of examples. However, the present invention is not limited to only this embodiment.

Example 1 A method for measuring the amount of the sugar chain of Hilochawan lectin-binding cholinesterase in a single measurement of Cholinesterase lectin-binding cholinesterase An untreated 96-well microtiter plate (Maxisorp, Nunc) 0.1 μl in PBS
100 μl of an F (ab ′) ₂ fragment of a monoclonal antibody (named B-3) recognizing cholinesterase dissolved at g / ml was added and incubated at 37 ° C. for 2 hours. Next, the solution in each well was removed, and the well was washed twice with PBS, and the PBS containing 1% BSA (bovine serum albumin) was removed.
300 μl of BS was added thereto, and a blocking treatment (a treatment of adsorbing BSA to a non-specific binding site with an antigen or an antibody present on the carrier) at 4 ° C. for 16 hours was performed and stored at 4 ° C. as it was.

Microtiter plate with immobilized antibody
After returning to room temperature and washing twice with PBS, diluent T
(2% BSA, 0.05% Tween-20, 150m
50 m containing M-NaCl and 1 mM-MgCl ₂
100 μl of a 1/5 dilution of human serum with an MTris · HCl buffer solution (pH 8.1) was added.
Incubated for hours. 0.05 after removing the solution
The plate was washed three times with PBS containing% Tween-20. Then, 4 μg / ml of biotin-labeled Hilochawan lectin (manufactured by Honen) and 0.5 μg / ml of streptavidin-labeled alkaline phosphatase (manufactured by Jackson Immuno Research Laboratory) were added to each well.
Was added to the mixture, and the mixture was incubated at 25 ° C. for 1 hour. After removing the solution and washing three times with PBS, 100 μl of 10 mM p-nitrophenyl phosphate, 2 mM MgCl ₂ , 0.3 M 2-amino-2-methyl-1-propanol was added to each well, and 2 wells were added.
Incubate for 30 minutes at 5 ° C. The reaction was stopped by adding 100 μl of 1N NaOH, and the absorbance of each well was measured at a measurement wavelength of 405 nm and a control wavelength of 492 nm using an automatic microtiter plate reader. The value obtained by this one measurement indicates the amount of the sugar chain of the Japanese terrestrial lectin-binding cholinesterase per unit amount of cholinesterase. The absorbance at 405 nm was used as it was for the measured value.

The serum of a patient with hepatocellular carcinoma, the serum of a patient with cirrhosis, the serum of a patient with chronic hepatitis, and the serum of a healthy individual were measured to determine the amount of the sugar chain of Hilochawantake lectin-binding cholinesterase per fixed amount of cholinesterase. Was. The results are shown in FIG. As is clear from FIG. 1, the values of the serum of cirrhosis patients and the serum of hepatocellular carcinoma patients were higher than those of the sera of patients with chronic hepatitis and the sera of healthy individuals. Therefore, the method of the present invention can be used to distinguish between cirrhosis and hepatocellular carcinoma from chronic hepatitis and healthy subjects by serodiagnosis. These methods gave better results than the known methods.

[Brief description of the drawings]

FIG. 1 is a diagram showing the relationship between liver disease and absorbance measured in Example 1.

Claims (2)

[Claims] 1. A sandwich assay using an immobilized monoclonal antibody recognizing cholinesterase and labeled Hilochawantake lectin, wherein the amount of the monoclonal antibody used is equal to or less than the amount of cholinesterase in the sample. Measurement method. 2. A method for identifying a liver disease, comprising using the measurement method according to claim 1.