JPH10221344A - Assay for lentil lectin-binding cholinesterase - Google Patents

Abstract

(57) [Summary] [PROBLEMS] To provide a method for performing in vitro serum diagnosis of cirrhosis and hepatocellular carcinoma by a method similar to a simple enzyme immunoassay without using histological examination by biopsy or the like. Kind Code: A1 A method for measuring lentil lectin-binding cholinesterase using an immobilized monoclonal antibody and labeled lentil lectin, and a method for diagnosing cirrhosis and hepatocellular carcinoma from the results.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for detecting lentil lectin-binding cholinesterase in a sample and a method for diagnosing cirrhosis and hepatocellular carcinoma using the same.

[0002]

2. Description of the Related Art There are two types of cholinesterases which differ in enzymatic properties, physiological functions and biodistribution in living organisms. That is, one is acetylcholinesterase (EC 3.1.1.7), which specifically degrades acetylcholine and is abundantly present in erythrocytes, nervous tissues, muscles, etc., and is involved in its physiological functions and distributed. doing. Another is cholinesterase (pseudocholinesterase)
(EC 3.1.1.8), which acts on acylcholines such as benzoylcholine and butylcholine, is abundant in serum and liver, is produced in the liver, and its physiological effects are probably in the nervous-muscular system. It is believed that they are involved.

[0003] Currently, cholinesterase frequently measured in clinical tests is cholinesterase (seudocholinesterase) in serum. This enzyme has a molecular weight of about 34
A glycoprotein consisting of four identical subunits, each of which consists of 574 amino acids and has nine asparagine-linked sugar chains. Clinically, the measurement of this enzyme has a significant significance in reducing the activity of liver enzymes, especially in the determination of the degree of liver parenchymal dysfunction in chronic liver parenchyma, ie, cirrhosis and chronic hepatitis. Because cholinesterase in serum is produced in hepatocytes,
The decline is indicative of a chronic decline in hepatocyte function. In addition, an acute decrease in cholinesterase activity is seen in the case of organophosphate pesticide poisoning, in which case the activity of the enzyme must be measured and related to treatment. An increase in the activity of this enzyme is remarkably observed in nephrotic syndrome.

[0004] In the study of the sugar chain of cholinesterase, serum is treated by chromatography using an affinity column on which various lectins are immobilized, and the lectin-binding cholinesterase retained on the column is measured.
It has been reported that the sugar chain of cholinesterase is changed by disease (T. Okura, T. Hada et al., CAN).
SER RESEARCH 54, 55-61, Jan
uary 1, 1994). There is also a report that cirrhosis can be identified by measuring the serum cholinesterase-binding cholinesterase in the serum by a sandwich method using a monoclonal antibody against immobilized cholinesterase and labeled Hilochawantake lectin (M Kondo, T. Hada et al., Cli
nicaChimica Acta 243, 1-9,
1995, JP-A-8-285853).

On the other hand, in 1975 G. Caller and C.I. Milstein (Nature, 256, 495, 1
975), a monoclonal antibody production technique has been developed, and a large number of monoclonal antibodies against various antigens have been produced. Monoclonal antibodies are being used for in vitro diagnostics, in vivo diagnostics, therapeutics, and reagents for affinity purification, some of which have already been put into practical use, and some of which have been actively researched and developed. is there.

[0006] Lentil lectin is a protein containing metal and is relatively easy to prepare because it is prepared from lentils. Molecular weight 4
With a molecular weight of 6,000, a molecular weight of 5,710 (α) and 17,572
It is composed of a tetramer of α ₂ β ₂ consisting of the subunit (β). Lentil lectin includes two types of isolectins A and B having different isoelectric points. Lentil lectin has an affinity for α-D-mannose and α-D-glucose, and in particular, L-α fucose is located at the C-6 position of the N-acetylglucosamine residue closest to asparagine in the protein. Shows strong binding to the structure to which is bonded. On the other hand, it is known that the presence of bisecting N-acetylglucosamine does not affect the binding to lentil lectin. In addition, it has been reported that lentil lectin-binding sugar chains appear in α-fetoprotein in the serum of hepatocellular carcinoma patients (K. Taketa, Y. Pea et al., CANSER RESEARCH 53, 5419).
-5423,1993), it has become a vitro serum diagnostic agent as the AFP-L ₃ fraction ratio measured in the serum.

[0007] Diagnosis of liver disease includes biochemical examination, ultrasonic diagnosis, X-ray tomography, and the like. Currently, diagnosis of hepatocellular carcinoma is relatively effective in image diagnosis such as ultrasonic diagnosis and X-ray tomography, but α-fetoprotein and PIVKA-I, which are performed as extracorporeal serum diagnosis items,
The measurement of I has not been effectively diagnosed because the former has low specificity and the latter has low sensitivity. For cirrhosis, type IV collagen 7S, hyaluronic acid, and procollagen III peptide are used as in vitro serum diagnosis items. However, even chronic hepatitis often exhibits high values similar to those of cirrhosis, and diagnosis is difficult. After all, the diagnosis of cirrhosis and hepatocellular carcinoma, especially the diagnosis of cirrhosis, can only be made by histological examination of a liver tissue sample collected by biopsy or surgery, and this places a heavy burden on patients.

[0008]

In liver diseases, chronic hepatitis, liver cirrhosis and hepatocellular carcinoma have greatly different treatment methods, and their identification is important. Conventional diagnosis of liver cirrhosis and hepatocellular carcinoma by histological examination of liver tissue specimens by biopsy or surgery is a heavy burden on patients, and it would be very significant if this could be done by in vitro serum diagnosis. Becomes However, the method of treating serum by column chromatography using an affinity column immobilized lectin reported in the paper and measuring lectin-binding cholinesterase retained on the column is a method called column chromatography, Complicated operations such as adsorption, elution, volume measurement, and activity measurement must be repeated, and reproducibility and cost due to column deterioration are problems. AFP-L ₃ fractions ratio measured in serum, but the use of electrophoresis and antibody affinity transfer method, the operation is complicated and cirrhosis can not identify.

Therefore, the present inventors have developed a method for simultaneously measuring lentil lectin-binding cholinesterase in a large number of samples by a simple operation without using column chromatography, electrophoresis or the like. The purpose was to diagnose hepatocellular carcinoma.

[0010]

Means for Solving the Problems As a result of diligent studies, the present inventors have found that cholinesterase in serum of healthy subjects and patients with chronic hepatitis has little lentil lectin-binding sugar chain, but it is difficult to treat cirrhosis patients and hepatocytes. It has been found that cholinesterase in the serum of cancer patients has a lot of lentil lectin-binding sugar chains, and the present invention has been completed.

That is, the present invention is characterized by reacting an immobilized monoclonal antibody recognizing cholinesterase, a labeled lentil lectin, and a sample, and detecting a reacted or unreacted lentil lectin label. This is a method for measuring lentil lectin-binding cholinesterase. The present invention is also a method for diagnosing cirrhosis and hepatocellular carcinoma, which comprises measuring lentil lectin-binding cholinesterase by this method. Hereinafter, the present invention will be described in more detail.

In the method for measuring lentil lectin-binding cholinesterase according to the present invention, a monoclonal antibody recognizing cholinesterase is immobilized. A monoclonal antibody prepared by a known method may be used.
For example, by fusing a mouse antibody-producing lymphocyte immunized with cholinesterase prepared from human serum and myeloma cells, a hybridoma producing a monoclonal antibody recognizing the cholinesterase is obtained, and then the hybridoma or a derivative thereof. The cell line to be cultured can be cultured and collected from the culture. A hybridoma that produces a monoclonal antibody that recognizes cholinesterase can be produced by a cell fusion method known per se.

In general, when a monoclonal antibody is obtained from a hybridoma or a culture of animal cells derived from the hybridoma, it is necessary to eliminate the sugar chain because the antibody has a lentil lectin-binding sugar chain in the Fc portion. is there.
For example, a monoclonal antibody is obtained by a culture method in which a sugar chain synthesis inhibitor is added to a medium of a hybridoma or an animal cell derived from the hybridoma, which is subjected to limited hydrolysis with a protease such as pepsin or papain to eliminate the Fc portion. And treating the monoclonal antibody with a glycolytic enzyme, or treating the monoclonal antibody with periodate to eliminate the sugar chains.

As a method for immobilizing a monoclonal antibody having no sugar chain, a known method can be employed. Examples of immobilizing antibodies include glass, polystyrene, polyethylene, polyvinyl chloride, latex, agarose,
Beads and microplates using cellulose, methacrylate and the like are preferably used.

[0015] The labeling method and means for labeling lentil lectin, and the detection method and means therefor are not particularly limited, and can be carried out by known methods and means. As a labeling agent, an enzyme such as alkaline phosphatase, peroxidase or β-D-galactosidase can be used. In a method using a radioactive substance, ¹²⁵ I, ³
In the method in which H or the like uses a fluorescent substance, fluorescein / isothiocyanate or the like is usually used, but other substances may be used. In the case of labeling with an enzyme, if the enzyme itself has a lentil lectin-binding sugar chain, it is necessary to eliminate the sugar chain by eliminating the sugar chain.

When the labeling agent is an enzyme, a substrate is used to measure its activity. For example, alkaline
As a substrate for phosphatase, p-nitrophenyl phosphate, 4-methylumbelliferyl phosphate or the like is used. As a substrate for horseradish peroxidase, 2,2′-azinodi- [3-ethylbenzthiazolinesulfonic acid] diammonium is used. salts -H ₂ O _2, 5- aminosalicylic acid -H ₂ O _2, o- phenylenediamine -H ₂ O ₂
And the like, as a substrate for β-D-galactosidase, o-
Nitrophenol-β-D-galactopyranoside and the like can be mentioned. For the measurement, known reagents such as a solubilizer, a detergent, and a reaction terminator are used in addition to these substrates.

In the present invention, the reaction order of the monoclonal antibody, lentil lectin, and the sample is not particularly limited, and they may be reacted simultaneously or in any order. However, first react the sample with the monoclonal antibody,
If lentil lectin is reacted after removing the unreacted sample, the influence of a lentil lectin binding substance other than cholinesterase in the sample can be eliminated, which is a preferable method.

In the measurement method according to the present invention, rather than measuring the amount of lentil lectin-binding cholinesterase as a protein, the C content of the N-acetylglucosamine residue closest to asparagine in the protein is measured.
The lentil lectin-binding cholinesterase is measured by measuring the amount of the structure in which L-α fucose is bound to the −6 position in the sugar chain of cholinesterase.

In the method of measuring lentil lectin-binding cholinesterase according to the present invention, the sample is used for the reaction as it is or after appropriately diluting it. If the total cholinesterase concentration in the sample is measured after being diluted to a specific value, when comparing a plurality of samples, the N-acetyl at the position closest to asparagine in the protein relative to the total cholinesterase amount is compared with the total cholinesterase amount. This is a preferable method because the structure in which L-α fucose is bonded to the C-6 position of the glucosamine residue shows a numerical value reflecting the amount present in the sugar chain of cholinesterase.

In the method of the present invention, it is difficult to measure as an absolute amount of lentil lectin-binding cholinesterase. Therefore, for example, it may be expressed as a relative value based on a standard sample from which a certain numerical value is obtained.

By measuring lentil lectin-binding cholinesterase by the above methods, cirrhosis and hepatocellular carcinoma can be diagnosed. As a sample, a bodily fluid such as serum may be used as it is or appropriately diluted for measurement. In the case of serum, the dilution is preferably up to 1/20. On the basis of the lentil lectin-binding cholinesterase value in the serum of healthy volunteers, the values for chronic hepatitis patients are similar, but the values for cirrhosis patients and hepatocellular carcinoma patients are clearly higher, and cirrhosis patients and hepatocellular carcinoma Can be identified.

By comparing the measured value of lentil lectin-binding cholinesterase obtained as described above to the amount relative to the total cholinesterase, cirrhosis and hepatocellular carcinoma can be identified. In this case, the total cholinesterase concentration can be separately measured by a known method and calculated. Alternatively, the total cholinesterase concentration in the sample is measured in advance, and the sample is diluted so that the total cholinesterase concentration in the sample becomes a specific value, preferably 0.1 to 0.5 μg / ml. You may. In this case as well, the value of chronic hepatitis patients is similar to that of lentil lectin-binding cholinesterase in the serum of healthy subjects, but the values of cirrhosis patients and hepatocellular carcinoma patients are clearly higher, and cirrhosis And hepatocellular carcinoma.

[0023]

The method for detecting lentil lectin-binding cholinesterase of the present invention is a simple method similar to the so-called enzyme-linked immunosorbent assay without using complicated methods such as affinity chromatography. It is effective for measuring a sample.

Furthermore, the method for identifying cirrhosis and hepatocellular carcinoma of the present invention provides a serodiagnosis with less burden on a patient without the conventional histological examination of liver tissue specimens by biopsy or surgery. It is very significant.

[0025]

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

Example 1 Detection of lentil lectin-binding cholinesterase using a sample obtained by diluting serum so that the total cholinesterase concentration becomes the same Preparation of immobilized antibody Untreated 96-well microplate (Maxisorp,
To each well of Nunc Corporation, add phosphate buffered saline (P
100 μl of an F (ab ′) ₂ fragment of a monoclonal antibody against cholinesterase (BS) dissolved at 1 μg / ml (see JP-A-8-285853) and incubated at 37 ° C. for 3 hours. Next, the solution in each well was removed, washed twice with PBS, 300 μl of PBS containing 1% bovine serum albumin (BSA) was added, and blocking treatment was performed at 4 ° C. for 16 hours (with antigen or antibody present on the carrier). BSA was adsorbed to the non-specific binding site) and stored at 4 ° C as it was.

Detection of lentil lectin-binding cholinesterase The total cholinesterase concentration in the serum was measured in advance by an immunoassay using an antibody against cholinesterase (see Japanese Patent Application Laid-Open No. 8-285853). It was diluted with PBS containing 2% BSA and 0.05% Tween-20 to 5 μg / ml. The microplate on which the antibody was immobilized in this example was returned to room temperature and washed twice with PBS. The cholinesterase concentration was 0.5 μg / ml in each well.
Was added to each of the samples. 25 ° C
After 1 hour of incubation, the solution in the wells was removed, and the wells were washed three times with PBS containing 0.05% Tween-20 (hereinafter, PBS-T).

Next, 4 μg / ml biotin-labeled lentil lectin (manufactured by Honen) and 0.5 μg / ml were added to each well.
100 μl of PBS containing 2 ml of streptavidin-labeled alkaline phosphatase (manufactured by Jackson Immuno Research Laboratory) and 2% BSA and 0.05% Tween-20 were added, and the mixture was incubated at 25 ° C. for 1 hour. After removing the solution in the wells and washing three times with PBS-T, each well was added with 10 mM p-nitrophenyl phosphate, 2 mM MgCl ₂ , 0.3 M 2-
100 μl of amino-2-methyl-1-propanol was added and incubated at 25 ° C. for 30 minutes. 1N ・ Na
100 μl of OH was added to stop the reaction, 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 microplate reader. The results were classified into healthy subjects, chronic hepatitis patients, cirrhosis patients, and hepatocellular carcinoma patients, and the results shown in FIG.
hE). The vertical axis indicates the absorbance at 405 nm.

Example 2 Detection of lentil lectin-binding cholinesterase using a sample obtained by diluting serum at the same magnification. Serum was uniformly reduced to 1/10 with PBS containing 2% BSA and 0.05% Tween-20. Lentil lectin-binding cholinesterase was detected in the same manner as in Example 1 except that the sample diluted to the concentration was used as a sample. The results were classified into healthy subjects, chronic hepatitis patients, cirrhosis patients, and hepatocellular carcinoma patients, and the results in FIG. 2 were obtained. The vertical axis indicates the absorbance at 405 nm.

The detection results of lentil lectin-binding cholinesterase obtained by using the same cholinesterase concentration as a sample (FIG. 1) and the detection results of lentil lectin-binding cholinesterase of a sample obtained by diluting serum at the same magnification (FIG. 2) In both cases, the value is low in healthy subjects and chronic hepatitis patients, and high in cirrhosis patients and hepatocellular carcinoma patients. Therefore, both methods can be used as a serum diagnosis for cirrhosis and hepatocellular carcinoma.

[Brief description of the drawings]

FIG. 1 is a graph showing detected values of lentil lectin-binding cholinesterase by disease in Example 1.

FIG. 2 is a graph showing detected values of lentil lectin-binding cholinesterase for each disease in Example 2.

Claims (2)

[Claims] 1. An immobilized monoclonal antibody recognizing cholinesterase, a labeled lentil lectin,
And reacting the sample, and detecting the label of the reacted or unreacted lentil lectin, the method for measuring lentil lectin-binding cholinesterase. 2. A method for diagnosing cirrhosis and hepatocellular carcinoma, which comprises measuring lentil lectin-binding cholinesterase by the method according to claim 1.