Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1996-04-17
2001-12-25
Celsa, Bennett (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S004000, C435S007920, C435S007400
Reexamination Certificate
active
06333162
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for measuring total cholinesterase in, for example, serum; a method for measuring
Aleuria aurantia
lectin-reactive cholinesterase in, for example, serum; and an in vitro diagnostic method for distinguishing liver cirrhosis, liver carcinoma and hepatitis using those methods.
2. Related Art
Two types of cholinesterase exist in the body that differ in terms of enzymological properties, physiological function and distribution in the body. Namely, the first is acetylcholinesterase (E.C.3.1.1.7), which specifically breaks down acetylcholine, exists in a large amount in erythrocytes, neural tissue, muscle and so forth, and is distributed in relation to these physiological functions. The other is cholinesterase (also referred to as pseudocholinesterase or butylcholinesterase) (E.C.3.1.1.8), which acts on cholines, such as benzoylcholine and butylcholine, exists in a large amount in the serum and liver, is produced in the liver, and the physiological action of which is considered to most likely be involved with the neuromuscular system.
At present, the cholinesterase that is frequently measured in clinical laboratory examinations is cholinesterase in serum (pseudocholinesterase or butylcholinesterase). This enzyme is a glycoprotein having a molecular weight of approximately 340,000 and is composed of four identical subunits. Each subunit is composed of 574 amino acids and has nine asparagine-coupled carbohydrate chains. Clinically, a decrease in its activity, as determined by measuring this enzyme, has significance in terms of determining the degree of functional impairment the liver parenchyma in liver disease, and particularly chronic liver parenchymal disorders such as liver cirrhosis and chronic hepatitis. Since serum cholinesterase is produced in liver parenchymal cells, its decrease indicates a chronic functional decrease of liver cells. In addition, acute decreases in cholinesterase activity are observed in cases of poisoning by organic phosphorous-based agricultural chemicals, measurement of the activity of this enzyme is indispensable in these cases. In addition, increases in the activity of this enzyme are observed prominently in nephrotic syndromes.
In the past, measurement of cholinesterase was performed by various methods including a thiocholine method, wherein thiocholine released by cholinesterase is measured by coloring it with an SH group assay reagent using the synthetic substrates of acetylthiocholine, propionylthiocholine and butylthiocholine; a UV method wherein a direct decrease in substrate is measured in the form of the reduction in absorbance of the ultraviolet using benzylcholine as the substrate; a pH colorimetric method wherein an organic acid produced by cholinesterase is measured using a pH indicator; and an enzyme method (cholinoxidase method) wherein the hydrogen peroxide produced during specific decomposition of choline by cholinesterase is measured with a coloration system using benzoylcholine as the substrate and cholinoxidase and peroxidase as cooperative enzymes.
In addition, ever since the development of technology for producing monoclonal antibodies by G. Kohler and C. Milstein in 1975 (Nature, Vol. 256, p. 495, 1975), numerous monoclonal antibodies have been prepared for various antigens. The use of monoclonal antibodies has proceeded in the fields of in vitro diagnostic drugs, in vivo diagnostic drugs, therapeutic drugs and affinity purification reagents. Some of these have already reached the level of practical application, while research and development are being actively conducted, on others, to reach practical application.
Monoclonal antibodies to cholinesterase have also been produced. Examples of methods that have already been reported include a method wherein cholinesterase is measured by an enzyme immunoassay method using immobilized polyclonal antibody to cholinesterase and monoclonal antibody to cholinesterase (A. Broch, et al., J. Clin. Chem. Clin. Biochem., Vol. 28, p. 222, 1990), and a method wherein cholinesterase is measured by an enzyme immunoassay method using immobilized monoclonal antibody to cholinesterase and polyclonal antibody to cholinesterase (M. Whittaker, et al., Hum. Hered, Vol. 40, p. 153, 1990).
Aleuria aurantia
lectin is a protein that is free of carbohydrate chains prepared from
Aleuria aurantia
. It contains large amounts of serine and glycine, has a molecular weight of 72,000 and has subunits having a molecular weight of 31,000. It has one carbohydrate binding site per subunit. It has affinity for L-fucose as a -monosaccharide. The lectin binds with &agr;1→2 L-fucose and &agr;1→3 L-fucose residues, and binds strongly with a carbohydrate chain having an &agr;1→6 L-fucose residue.
Recently, a serum was treated by chromatography using an affinity column on which
Aleuria aurantia
was immobilized (T. Ohkura and T. Hada et al.) When this column was used to measure the enzyme activity of
Aleuria aurantia
lectin-reactive cholinesterase retained on the column and to determine the ratio of that cholinesterase to the total enzyme activity of cholinesterase originally present in the serum, that ratio was indicated as being significantly higher in patients with hepatocellular carcinoma and liver cirrhosis than in patients with chronic hepatitis and normal controls (Cancer Research, Vol. 54, p. 55, 1994). In addition, there is no other method at present for diagnosing liver cirrhosis and chronic hepatitis than by performing histological examinations of liver tissue specimens obtained by biopsy or operation, thus causing a considerable burden on patients.
SUMMARY OF THE INVENTION
Methods for measuring cholinesterase of the prior art, including the thiocholine method, UV method, pH colorimetric method and enzyme method (cholinoxidase method), all measure the enzyme-activity of cholinesterase, and do not measure the amount of cholinesterase as protein.
In addition, in the case of methods that measure enzyme activity, depending on the selection of the substrate, there is the possibility of also measuring the activity of substances (enzymes) other than cholinesterase that have esterase activity. Since that which appears as a result of damage to liver parenchymal cells is inherently the synthesis of cholinesterase itself, it is necessary to measure the amount of cholinesterase as protein. In addition, even in the case of a decrease in cholinesterase activity caused by poisoning by organic phosphorous-based agricultural chemicals, measuring the amount of cholinesterase as protein along with enzyme activity allows a more accurate judgment to be made.
Moreover, in the case of the reported enzyme immunoassay methods, since polyclonal antibody is used either for the immobilized antibody or enzyme-labeled antibody, it is difficult to supply a uniform antibody semi-permanently.
Therefore, as a first object of the present invention, the inventors of the present invention attempted to measure the amount of cholinesterase as protein, without being affected by substances (enzymes) other than cholinesterase that have esterase activity and without being affected by cholinesterase activity inhibitors, by using an enzyme immunoassay method that uses monoclonal antibodies that can be supplied semi-permanently in uniform quality without using polyclonal antibodies.
Moreover, since diagnosis of liver cirrhosis and chronic hepatitis by histological examinations using liver tissue specimens obtained by biopsy or operation as performed in the past causes a considerable burden on patients, a method that enables this to be performed by serodiagnosis would be extremely significant. However, the method for determining the ratio of
Aleuria aurantia
lectin-reactive cholinesterase to total enzyme activity of cholinesterase originally present in serum by treating serum with column chromatography using an affinity column on which
Aleuria aurantia
lectin is immobilized and measuring the enzyme activity of
Aleuria aurantia
lectin-reactive
Hada Toshikazu
Kondo Masahide
Yasukawa Kiyoshi
Celsa Bennett
Sughrue & Mion, PLLC
Tosoh Corporation
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