JPS6217711B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for quantifying cancer-related glycoproteins in body fluids, and more particularly to a method for quantifying cancer-associated glycoproteins in body fluids, and more specifically, the present invention relates to a method for quantifying cancer-related glycoproteins in body fluids, and more specifically, to quantifying cancer-related glycoproteins (tumor associated glycoproteins) that increase with the proliferation of undifferentiated cells, especially cancer cells.
This article relates to a method for quantifying glycoprotein (hereinafter abbreviated as TAG).

Conventionally, as a method for diagnosing cancer, a method has been used to measure a specific glycoprotein that is specifically produced in a cancer patient. This method mainly utilizes the antigenicity of the protein portion, and is known to be used, for example, to diagnose primary liver cancer by measuring α ₁ -fetoprotein, or to diagnose gastrointestinal cancer, especially rectal cancer, by measuring CEA. ing. However, a method for diagnosing cancer that utilizes the sugar residue binding specificity of cancer-related glycoproteins is not yet known.

The present inventor discovered that TAG is produced by undifferentiated cells (mainly cancer cells) and released into the body fluid of cancer patients, and that this is produced by differentiated cells (mainly normal cells). We focused on the fact that the structures, lengths, and constituent sugar residues of the sugar chains of the glycoproteins that are produced and released into body fluids differ considerably, and as a result of extensive research, we found that
TAG is galactose-(β1→3 or β1→4)
- It has an acetylglucosamine terminal and binds specifically to lectin. Therefore, by reacting TAG in body fluids with lectin and measuring this, it is possible to know the presence or absence of cancer cells, the degree of proliferation, and the fate of cancer cells. discovered that it is possible to diagnose cancer using
The invention has been completed.

That is, in the present invention, a labeled lectin is added to a body fluid and reacted, the resulting TAG-labeled lectin conjugate or unreacted labeled lectin is separated, and the TAG-labeled lectin is separated.
This is a method for measuring TAG in body fluids, which is characterized by measuring the amount of labeled lectin conjugate or the amount of unreacted labeled lectin.

Various body fluids can be used as the body fluids used in the present invention, such as blood, cell tissue fluid, lymph fluid,
Examples include pleural fluid, ascites fluid, amniotic fluid, gastric fluid, urine, pancreatic fluid, spinal fluid, saliva, etc. Among them, it is particularly preferable to use blood as serum or plasma. Approximately 2 to 5 ml of body fluid may be collected for quantitative determination.

Among the collected body fluids, those other than blood can be used as test samples (hereinafter abbreviated as "sample") as they are, but it is desirable to add a protective protein such as bovine serum albumin to this. In the case of blood, serum obtained by a known serum collection method or plasma obtained by a plasma collection method using an anticoagulant such as heparin, EDTA, citric acid, etc. can be used as a sample. In particular, it is preferable to use plasma collected and prepared using heparin as an anticoagulant as a sample. The above sample may also be diluted if necessary.

Furthermore, the lectin used in the present invention is one that specifically binds to galactose-(β1→3 or β1→4)-acetylglucosamine [JBC, 250,
8518-8523 (1975); Biochem. Biophys Res.
Comm. 62 , 144 (1975): Z. Immunitaetsforch,
138 , 423-433 (1969); Br.J.Exp. Pathol. 27 ,
228-236 (1946); Proc. Natl. Acad. Sci USA,
75 , No. 5, 2215-2219 (1978)], for example, peanut lectin.

Examples of lectin labeling substances include various enzymes, various fluorescent substances, and various radioactive substances. Examples of enzymes include active fragments of enzymes such as glucoamylase, glucose oxidase, peroxidase, alkaline phosphatase, β-galactooxidase or heme octapeptide; examples of fluorescent substances include fluorescein, fluorescein isothiocyanate, Rhodamine, dansyl chloride, etc.; examples of radioactive substances include radioactive iodine, radioactive tritium, etc.

In the method of the present invention, the labeled lectin is preferably used in addition to a suitable solvent. Any solvent can be used as long as it does not denature the lectin, such as physiological saline, water, 0.1M Tris-HCl buffer (PH≒7.5), 0.1M phosphate buffer (PH≒7.4), etc. can be mentioned. The amount of lectin contained can be selected appropriately depending on the labeling substance or measuring substance, but it is usually 0.01 to 100 μg/
ml, especially 0.03 to 40 μg/ml is preferred. Furthermore, the labeled lectin solution described above can also be used after being diluted appropriately.

To carry out the method of the present invention, first, a labeled lectin is added and mixed to a certain amount of body fluid, and this is heated below 40°C.
Preferably, the reaction is carried out at 20 to 40°C. generated
There are no particular limitations on the method for separating the TAG-labeled lectin conjugate or unreacted labeled lectin, and conventional separation methods can be used, such as chromatography, electrophoresis, salting out, fractionation, and dialysis. law,
Gel filtration, adsorption, etc., a combination of these methods, or a separation method using agar gel, agarose gel, or polyacrylamide gel (Japanese Patent Application No. 59388/1983 by the present inventor) may also be used.

More specifically, when separating unreacted labeled lectin, for example, add an appropriate amount of a glycoprotein-lectin conjugate precipitant, such as polyethylene glycol, saturated ammonium sulfate, ribanol, etc., to the above reaction solution and separate by centrifugation. Remove the conjugate.

In addition, when separating a TAG-labeled lectin conjugate, for example, the TAG-labeled lectin conjugate produced by the above reaction and unreacted labeled lectin can be separated using the difference in diffusion rate between an agar gel, an agarose gel, or a polyacrylamide gel. The conjugate can be easily separated. The method for preparing the gel is not particularly limited, and any conventional method can be used. For example, add an appropriate amount of agar, agarose, or polyacrylamide to a diluted solution such as distilled water, citric acid or Tris-HCl buffer with a pH of about 7.5, and gently stir for 60 minutes.
The mixture is heated to ~80°C to dissolve, placed in a suitable container such as a test tube, and allowed to cool to solidify into a jelly. A preservative may be added to this gel if necessary.
The surface of the gel thus prepared may be flat, but it is preferable to make it concave because the resulting composite will not adhere to the tube wall.

The amount of TAG in the body fluid can be calculated by measuring the amount of the TAG-labeled lectin conjugate separated as described above or the amount of unreacted labeled lectin using a conventional method.

The measurement method is appropriately selected depending on the lectin labeling substance. For example, if a lectin is labeled with an enzyme, by selecting an appropriate enzyme substrate for a colorimetric or fluorometric assay and measuring its enzyme activity, if the labeled substance is a fluorescent substance, the fluorescence can be detected. The amount of TAG-labeled lectin conjugate or the amount of unreacted labeled lectin can be measured by measuring the intensity, or in the case of a radioactive substance, its radiation.

As described above, according to the present invention, TAG in body fluids can be advantageously quantified. By knowing the amount of TAG, it is possible to diagnose any cancer from the early stage to the terminal stage, and it is particularly useful for early detection of cancer. Furthermore, the method of the present invention can be applied to any cancer, such as gastric cancer, breast cancer, colon cancer, rectal cancer, ovarian cancer, oral cavity cancer, tongue cancer, laryngeal cancer, prostate cancer, liposarcoma, malignant melanoma, malignant lymphoma, primary gastric sarcoma, etc. It can also be used as a diagnostic method for cancer.

Next, reference examples and examples will be given and explained.

Reference example (i) Activation of peroxidase: 5 mg of peroxidase (horseradish)
It was dissolved in 1 ml of 0.3M aqueous sodium bicarbonate solution. Add 0.1 ml of 0.1M fluorodinitrobenzene ethanol solution to this, stir gently at room temperature for 1 hour, then add 1 ml of 0.06M NaIo ₄ solution at room temperature
Stir gently for a minute. Furthermore, 1 ml of 0.16M ethylene glycol was added, and the mixture was gently stirred at room temperature for 1 hour. Next, the mixture was dialyzed overnight at 4°C using 0.01M carbonate-sodium hydrogen carbonate buffer (PH9.5).

(ii) Lectin-peroxidase labeling method: 5 mg of lectin was dissolved in 3 ml of activated peroxidase obtained in (i), and reacted at room temperature for 2 to 3 hours with gentle stirring. 5 mg of NaBH ₄ was added to this, and the mixture was reacted at 4°C for 3 hours. Next, this solution was dialyzed overnight against 0.1M Tris-HCl buffer (PH7.4), and gel filtration was performed using Sephadex G150 gel column chromatography (eluent: 0.1M Tris-HCl buffer PH7.4). Ivy. Each fraction was measured at OD ₂₈₀ and OD ₄₀₃ , and OD ₂₈₀ and OD ₄₀₃
Collected overlapping peaks.

(iii) Preparation of agarose gel: Agarose (manufactured by Iwai Chemical Co., Ltd.) was mixed with 0.01M Tris-HCl buffer (PH
7.5), heat and dissolve at 70-80°C, and add 0.01 V/V% thimerosal.
Dispense 1 ml of the obtained solution into test tubes and leave to stand at room temperature to prepare a 1 W/W % agarose gel.

Example 1 (i) Preparation of test samples 25 patients with cancer, 2 patients with other diseases, and healthy subjects
From 23 cases, 5 ml of blood was collected using a syringe treated with heparin (500 units), centrifuged at 2000 rpm for 10 minutes, and the supernatant was taken as a test sample.

(ii) Measurement method 200ml of each sample was divided into two test tubes, and peroxidase-labeled lectin obtained in the reference example (hereinafter abbreviated as "enzyme-labeled lectin") [3.5μg/ml as lectin, 0.1M Tris-HCl buffer (PH≈7.5)] was added in an amount of 50μ each. After mixing with gentle stirring, the mixture was left to react at 20 to 30°C for 1 hour. Then, 250μ of a polyethylene glycol (mw6000) solution prepared at 8% (W/V) with 0.1M Tris-HCl buffer was added to one of the two test tubes (sample A), and 0.1μ to the other (sample B). M Tris-HCl buffer 250μ
were added to each and mixed gently. Specimen A, B
After standing to react for 30 to 60 minutes at 20 to 30°C, incubate at 1000 x g using a swing rotor at 40°C.
Centrifuged for ~60 min. Gently separate 50μ of the supernatant liquid, add it to 2ml of physiological saline prepared in advance, stir thoroughly, and add 50μ of the peroxidase substrate solution (hereinafter abbreviated as “substrate solution”).
μ was added and reacted for 30 minutes in the dark at 20-30°C. The substrate solution used was a 0.1M citric acid buffer solution to which orthophenylenediamine and hydrogen peroxide were added to final concentrations of 6% and 0.1%, respectively. Further, it is desirable that the substrate solution be prepared before use and kept at 4°C until use. After the reaction, 1 ml of 2N hydrochloric acid was added to stop the reaction, and the color development was measured by absorbance at a wavelength of 492 nm using a spectrophotometer.

The value c obtained by subtracting the absorbance a of specimen A from the absorbance b of specimen B was taken as the amount of TAG-binding lectin and plotted. The results are as shown in FIG. In addition, ○ in the figure indicates healthy people, 1 to 27
means the following patients.

1,18 Gastric cancer, 2,6,9,16,17 Lung cancer, 3,5,7,8,13 Early gastric cancer, 4,14
Advanced gastric cancer, 10 cardiac dysfunction, 11 lymphadenitis, 12 metastasis from colon cancer to liver, 15 metastasis from uterine cancer to lung, 19 hydatidiform mole, 20 colon cancer, 21
Uterine cancer, 22, 26 Ovarian cancer, 23 Rectal cancer, 24
Prostate cancer, 25 urethral cancers, 27 liver cancers.

As shown in FIG. 1, a sample showing a c value higher than that of a healthy person means that the amount of TAG in the plasma is large, and this suggests that cancer cells are present in the subject.

Example 2 (i) Preparation of test samples Example 1 from 14 cancer-bearing patients and 8 healthy individuals
The sample collected and prepared in the same manner as above was used as the test sample.

(ii) Measurement method H solution (in distilled water, sodium chloride 8, potassium chloride 0.4, sodium phosphate-hydrogen 0.05,
Potassium dihydrogen phosphate 0.06, magnesium sulfate
0.05, magnesium chloride 0.05, calcium chloride
50μ of the sample diluted ¹⁰⁴ times with 0.1M phosphate buffer (containing 1W/W% glucose) was incubated in a water bath for 15 minutes at 23°C, and then diluted with 0.1M phosphate buffer (PH
Mix 50μ of peroxidase-labeled lectin diluted 100 times with 7.4) and further incubate for 23 minutes in a water bath.
℃ for 15 minutes. After incubation, 20μ of the mixture was gently layered on the surface of the agarose gel prepared in Reference Example (iii). This tube was further incubated in a water bath at 23℃ for 1 hour, and then transferred to an agarose gel tube with 0.1M
2 ml of phosphate buffer (PH≈7.4) was added and stirred, and the supernatant was immediately transferred to a clean test tube. To this test tube, 400μ of the peroxidase substrate solution prepared in the same manner as in Example 1 was added, stirred, and allowed to react at room temperature for 15 minutes. After the reaction, 1 ml of 1N hydrochloric acid was added to stop the enzyme reaction, and the mixture was sufficiently stirred and mixed using a thermomixer or similar device.

The absorbance of the reaction solution thus obtained was measured at a wavelength of 492 nm using a spectrophotometer. As a blank, a mixture of 400μ of substrate solution and 1ml of 1N hydrochloric acid was used. The results are as shown in FIG. In the figure, ● means a healthy person, and numbers 1 to 10 mean the following patients.

1 gastric cancer, 2 hydatidiform mole, 3 colon cancer, 4 uterine cancer,
5 ovarian cancer, 6 rectal cancer, 7 prostate cancer, 8 urethral cancer,
9 liver cancer, 10 breast cancer.

[Brief explanation of the drawing]

Figure 1 shows the amount of cancer-related glycoproteins in Example 1 of the present invention.
FIG. 2 shows the results measured by the method of Example 2.

Claims (1)

[Claims] 1. Add labeled lectin to body fluid and react, separate the resulting cancer-related glycoprotein-labeled lectin conjugate or unreacted labeled lectin, and measure the amount of the glycoprotein-labeled lectin conjugate or unreacted labeled lectin. A method for quantifying cancer-related glycoproteins in body fluids, characterized by: