JPH07104350B2 - Kit for detecting tumor, cancer, and precancerous symptoms - Google Patents

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for specifically analyzing and detecting sugar chains of glycoproteins in biological samples such as serum, urine, feces and mucus. It is a method for simultaneously detecting T-antigen and H-antigen in a sample to be produced simultaneously by using one reagent. By this method, a large number of healthy people can be easily detected and screened for tumors, cancers, precancerous symptoms, and the like.

[0002]

2. Description of the Related Art Glycoprotein exists in a living body such as serum or mucus, and is a protein having many sugar chains around a long protein molecule. There are many types of glycoproteins, and the structures of sugar chains are complicated and there are many types. In healthy individuals, mucin-type serine / threonine-type glycoproteins produced by mucous membranes are present in the mucus (including serum, milk, etc.) of living organisms. As shown schematically in Fig. 1, this glycoprotein has a β structure in which the tip is closed by sialic acid (N-Acetylneuraminic acid) to the long molecular skeleton (protein skeleton) 1 of the protein.
-D-Gal (1 → 3) -D-GalNAc (β-galactose-N
It has a large number of acetylgalactosamine) sugar chains 2 (hereinafter referred to as A-type sugar chains).

According to a recent study, when a tumor or cancer, particularly a colon cancer or a precancerous tumor, occurs, the sialic acid at the tip of the A-type sugar chain of the side chain of the mucin-type glycoprotein is missing and released. Glycoprotein that has been modified into a B-shaped sugar chain (hereinafter referred to as T-antigen)
Has been found to produce.

On the other hand, the use of lectins for the analysis of these glycoproteins is widespread. Lectins are sugar-binding proteins isolated from plants, animals, and microorganisms, and there are many types, but various lectins specifically recognize and bind to a certain sugar chain structure. For example, mushroom-derived lectin (hereinafter referred to as ABA), wheat germ-derived lectin (hereinafter WGA).
) And the like specifically bind to a glycoprotein having an A-type sugar chain regardless of the presence or absence of sialic acid. On the other hand, peanut-derived lectin (hereinafter referred to as PNA) has been known to specifically bind to T-antigen (glycoprotein having a B-type sugar chain).

Utilizing each of the above facts, US Pat. No.
4,857,457, Aug. 15,1989, (S
screeni; g Test For Large I
ntestinal Cancer), various detection methods using PNA, for example, insolubilization (adhesion fixation) on the surface of a solid such as plastic, contact with intestinal mucus collected by fingers to adsorb mucin-type glycoprotein in the sample, Then enzyme-labeled PNA was added or biotinylated PNA
By adding lectin and using enzyme-labeled adibin to determine the degree of color development to detect the presence of T-antigen in a biological sample, or by the method of agglutination using PNA-sensitized latex on the same principle, A method of screening a healthy subject for the development of colorectal cancer by a method of detecting an antigen has been proposed.

[0006] In the article "A new screening method for colorectal cancer using T-antigen in rectal mucus" (Tada et al., Laboratory, Vol.34.No.10.October 1990), T-antigen was directly analyzed.
A method of detecting by chiff reaction is disclosed.

[0007]

However, as a result of further research, T-antigen is also generated in a biological sample due to the development of cancer, tumor, etc., for example, "mucous composition of colon goblet cells",
Kenjiro Taketake, History of Medicine, Vol.147, No.5, 1988.1
As suggested in 0.29, α-L-Fuc (1 → 2)
A glycoprotein having a β-D-Gal (1 → 4) β-D-GulNAc (α-L fucose β-galactose-N-acetylglucosamine) sugar chain (hereinafter referred to as C-type sugar chain) (hereinafter referred to as H-antigen) also appears. I understand. There are cases where T-antigen is produced and cases where H-antigen is produced depending on the site, type, degree of production of cancer or tumor. The above-described conventional known method can detect T-antigen but cannot detect H-antigen, and thus has a problem that the occurrence of cancer or tumor is often overlooked.

[0008]

The present invention is a detection method characterized by detecting T-antigen and H-antigen in a biological sample at the same time with a single reagent. Is a method of screening.

To simultaneously detect T-antigen and H-antigen from one biological sample, the following method is specifically used. 1. Lectin-lectin sandwich method A lectin that binds to a mucin-type glycoprotein having an A-type sugar chain such as ABA and WGA is insolubilized on a solid surface and brought into contact with a biological sample to bind and capture the mucin-type glycoprotein in the sample.
Next, an enzyme-labeled PN that specifically binds to T-antigen
Enzyme-labeled UAE ₁ (Horseinida-derived lectin) that specifically binds to A and H-antigens, preferably in approximately equal amounts,
This is a method in which a mixed reagent is added and then a substrate solution is added for color development. In this case, a lectin that specifically binds to the H-antigen, for example, an anti-H lectin such as ESH (eel serum lectin) can be used instead of UAE ₁ . Enzyme labeling (eg Yoshitake et al. (Immunity Experimental Method XI, P8
497-8519, 1982. POD (peroxidase) enzyme label may be used as the Japanese Society of Immunology (see Japanese). In addition, if a biotinylated lectin is used in place of the enzyme-labeled lectin and the color degree is determined by the enzyme-labeled adibin, the detection becomes more reliable. It is advisable to measure the absorbance for the determination of coloration.

Further, as a result of the research, the present inventor preferably uses a lectin such as ABA or WGA that specifically binds to an A-type sugar chain as an insolubilized lectin, but a mucin-type glycoprotein has many side chains. Some of them have B lacking sialic acid
It becomes a T-antigen by being transformed into a type G sugar chain, and therefore PN
It was found that even if A or UAE ₁ was used as the insolubilized lectin, the mucin-type glycoprotein having T-antigen and H-antigen could be captured and the same result could be obtained. In addition, since the castor-derived lectin (hereinafter referred to as RCA ₁ ) binds to both T-antigen and H-antigen relatively well, it can be used alone in the present invention as an enzyme-labeled lectin.

2. Lectin-antibody sandwich method 1. As described in 1, the lectin that binds to the mucin-type glycoprotein is insolubilized on the solid surface to capture the glycoprotein in the biological sample. Antibodies produced from each of T-antigen and H-antigen are mixed (preferably approximately equal amount) and then labeled with an enzyme, or each antibody is labeled with an enzyme and mixed (preferably approximately equal amount). Then, an enzyme-labeled antibody reagent is prepared, and this reagent is dropped to cause an antigen-antibody reaction. T-antigen, H
Any of the antigens is capable of producing an antibody, which is the usual method for producing an antibody, ie, immunizing animals such as rabbits and goats with T-antigen or H-antigen and collecting the serum to purify it. Good. Further, it is suitable to use a POD (peroxidase enzyme) label as the labeling enzyme. When the antigen-antibody reaction is completed, a substrate solution (color reaction solution) is added to this to develop color, and the absorbance is measured to determine and detect.

3. Antibody-antibody sandwich method Various antibodies have the property of binding to their respective antigens. Therefore, it was found that the antibody of each antigen may be insolubilized on the solid surface, a biological sample may be brought into contact with the surface to capture the antigen in the sample, and the captured antigen may be colored with the enzyme-labeled antibody. In the present invention, anti-T-antigen and anti-H-antigen antibodies are insolubilized on the surface of the same microplate, or a mixture of antibodies of both antigens, preferably in approximately equal amounts, is insolubilized on the surface of the microplate. A biological sample is contacted with the surface of the plate in which both antibodies have been insolubilized to capture both antigens in the sample, and separately described in 2. above. After the enzyme-labeled antibody reagent described in 1) is dropped, a substrate solution is added to develop color, and the absorbance is measured to determine.

4. Lectin-sensitized latex agglutination reaction method Sensitizing latex particles by adding a mixture of anti-T lectin such as RCA ₁ or PNA and anti-H lectin such as UEA ₁ to a suspension of latex particles, preferably approximately equal amounts To do. The sample solution of the sample is dropped on a slide glass for agglutination reaction, and the above-mentioned latex reagent is added thereto and shaken vertically and horizontally to observe the presence or absence of aggregation of latex particles after a certain time. The sensitized latex aggregates due to the presence of either T-antigen or H-antigen. The state of aggregation can be determined by classifying it into three stages according to the criteria shown in FIG.

[0014]

The method of carrying out the method of the present invention on a stool sample for the detection of colorectal cancer and its function will be described below.

1. As a sample, 50 μl of feces juice containing 10 to 20 mg of feces of the patient to be examined mixed with 1 ml of deionized water or one drop of a hemochaser container is used. On the other hand, ABA lectin (eg.
Honen, Seikagaku Corporation) 3 μg / ml solution (0.1 mol
/ L Tris buffer (pH 8.4) is dispensed into each well of a microplate (a plastic plate with many small-diameter wells) in an amount of 100 µl and left overnight at 4 ° C to immobilize (insolubilize) the surface. . 1% BSA (0.1 mol Tris buffer pH
8.0), add the above stool sample and react at 37 ° C for 1 hour to trap and capture the glycoprotein in the sample. After washing, 100 μl of a solution prepared by mixing POD (peroxidase) -labeled PNA lectin and POD-labeled UEA ₁ lectin in equal amounts is added, and the mixture is further reacted at 37 ° C. for 1 hour. Then, wash and add 50 μl of TMB solution (made by KPL) and add 50 μl of hydrogen peroxide buffer (made by KPL) for color development. After 2 minutes, add 50 μl of 2.7 g / dl NaF solution to stop color development. Then, the color development is measured by using a colorimeter (Sanko Junyaku Co., Ltd. auto reader) with light having two wavelengths of 630 nm and 492 nm as targets. Then, the T-antigen and H-antigen in the stool sample react with the POD-labeled PNA lectin and the POD-labeled UEA ₁ lectin, respectively, and the presence of either one or both can be detected simultaneously.

POD Labeling-PNA Lectin and POD Labeling-U
Even if the POD-labeled RCA ₁ lectin is used instead of the mixture of EA ₁ lectin, the same detection can be performed although the reliability is slightly inferior.

2. As a stool sample, 50 μl of stool juice prepared by mixing 10 to 20 mg of the stool of the patient to be examined with 1 ml of deionized water or a drop of a container for a hemochaser is used. On the other hand, ABA lectin (eg, Honen, Seikagaku Corporation) 3 μg / ml solution (0.
100 μl of 1 mol / l Tris buffer (pH 8.4) was dispensed into each well of the microplate and left overnight at 4 ° C. to immobilize on the surface. Each we of this microplate
1% BSA (0.1 mol Tris buffer pH 8.0)
Then, the sample is added and reacted at 37 ° C. for 1 hour to trap the glycoprotein in the sample. After washing, mix 1 volume of POD-labeled anti-T antibody and POD-labeled anti-H antibody.
Add 00 μl and incubate at 37 ℃ for 1 hour. Next, wash and add 50 μl of TMB solution (made by KPL) and 50 μl of hydrogen peroxide buffer (made by KPL) to develop color. After 2 minutes, add 50 μl of 2.7 g / dl NaF solution to develop color. Stop. Color development is measured using a colorimeter (Sanko Junyaku Auto Reader) with two wavelengths of light at 630 nm and 492 nm. Then, T-antigen and H-antigen in the stool sample react with POD-labeled anti-T antibody and POD-labeled anti-H antibody, respectively, and it is possible to detect the presence of either one or both at the same time, and the presence of tumor such as precancerous symptoms. Can be detected.

3. The same stool sample as above is used. On the other hand, a solution (0.1 mol / l Tris buffer pH 8.4) containing 3 μg / ml each of anti-T antibody and anti-H antibody was dispensed in 100 μl aliquots into each well of the microplate at 4 ° C. It was left overnight and solid-phased on the surface. Add 1% BSA (0.1 mol Tris buffer pH to each well of this microplate)
8.0), add the above sample and react at 37 ° C. for 1 hour to trap the glycoprotein in the sample. After washing, add 100 μl of a solution in which POD-labeled anti-T antibody and POD-labeled anti-H antibody were mixed in equal amounts, and further react at 37 ° C for 1 hour. Next, wash and add 50 μl of TMB solution (made by KPL) and 50 μl of hydrogen peroxide buffer (made by KPL) to develop color, and after 2 minutes add 50 μl of 2.7 g / dl NaF solution to develop color. After stopping, color development is performed using a colorimeter (Sanko Pure Chemical Industries, Ltd. auto reader) at two wavelengths with 630 nm and 492 nm as controls. Then, the T-antigen and H-antigen in the stool sample are captured by the insolubilized antibodies, and then POD-labeled anti-T antibody
Since it reacts with any of the POD-labeled anti-H antibodies and the presence of either one or both can be detected at the same time, the presence of a tumor such as a precancerous condition can be detected.

4. As a sample, 50 μl of feces juice containing 10 to 20 mg of feces of the patient to be examined mixed with 1 ml of deionized water or one drop of a hemochaser container is used. 1 mg of latex particles having a particle size of 0.1 to 1.0 μm was suspended in TBS so as to be 1%. To 4 volumes of this suspension, 1 volume of TBS containing approximately ₁ mg of RCA ₁ or PNA and UEA ₁ lectin per ml was added and sensitized at room temperature for 6 hours. The obtained sensitized latex was washed twice with TBS and suspended in TBS containing 0.1% bovine serum albumin at 0.5%. For preservation, an appropriate amount of preservative such as sodium azide is added.
0.05 ml of the sample solution was dropped on the slide glass for agglutination reaction, 0.05 ml of the above-mentioned sugar chain detection reagent was added on it, and the slide glass was shaken up, down, left and right, and after 3 minutes, the presence or absence of aggregation was determined. To do. The judgment is

FIG. 2 is performed according to the criteria shown in FIG. By this determination, the presence of one or both of T-antigen and H-antigen in the sample can be simultaneously detected.

[0020]

Example 1 A stool sample of 60 persons was collected and the operation of 1.
Detection was performed by the method described in. ABA lectin was used as the insolubilized lectin. The captured A-type glycoprotein was color-tested using POD-labeled PNA lectin alone, POD-labeled UEA ₁ lectin alone, and a detection reagent in which both were mixed in equal amounts. It was as 1.

[Table 1]

From these results, when the POD-labeled PNA lectin was used, the presence of T-antigen was (-) and the POD-labeled UE was
There are 6 samples (No. 19, 22, 30, 36, 43, 44) in which the presence of H-antigen by A ₁ lectin is (+), and 10% when only POD-labeled PNA lectin is used.
The presence of H-antigen is overlooked in the sample. In the case of only POD-labeled UEA ₁ lectin, there are 13 samples (No. 2, 5, 5) in which the POD-labeled PNA lectin is (+), that is, the T-antigen is present, even if the H-antigen is (−).
9, 11, 12, 16, 18, 20, 23, 26, 2
8, 32, 41), and similarly the detection is insufficient. On the contrary, it can be seen that when the mixed reagent of the present invention is used, (+) judgment can be obtained in all the above examples.

[0022]

Example 2 The same sample and method as in Example 1 were used for POD labeled RC.
The determination was carried out using the A ₁ lectin. As a result, the same judgment result as that obtained by mixing POD-labeled PNA lectin and POD-labeled UEA ₁ lectin in equal amounts was obtained.

[0023]

As described in detail above, when the method of the present invention is used to detect various biological samples, the production of glycoprotein T-antigen and / or H-antigen by cancer and tumor is suppressed. It can be judged at the same time, so all dangerous signs can be found. Further, the method of the present invention may be detected by an ordinary method using only one specific detection reagent, the detection work is simple, and it is most suitable for use in screening for cancer, tumor and the like.

[Brief description of drawings]

FIG. 1 is a schematic diagram of a type A glycoprotein molecule.

2 (a), (b) and (c) are diagrams showing the degree of aggregation by latex in the case of Example 2. FIG.

[Explanation of symbols]

 1 Protein skeleton 2 A-type sugar chain 3 Sialic acid

Claims (7)

[Claims] 1. Sa-β-DG contained in a biological sample
al (1 → 3) -D- GalNAc in (sialic acid -β galactose -N-acetylgalactosamine) sugar (A-type sugar chain) mucin-type serine / threonine type binding glycoprotein having typically the tip of Sa (sialic Acid, N-Acetyl neurom
inic acid) -deficient sugar chain (B-type sugar chain)
And T- antigen having, α-L-Fuc (1 → 2) β-D
-To simultaneously detect H-antigen having a Gal (1 → 4) β-D-GulNAc (α-L fucose β-galactose-N-acetylglucosamine) sugar chain (C-type sugar chain)
The kit of claim 1, wherein the mushroom-derived lectin (ABA) and wheat germ-derived lectin
Kuching (WGA), Lectin derived from peanut (PN
A), gorse-derived lectin (UAE ₁ ), etc.
Mucin-type serine / threoni having representatively A-type sugar chains
A lectin that specifically binds to a T- type binding glycoprotein, an enzyme-labeled PNA that specifically binds to the T-antigen, and
And an enzyme-labeled UAE ₁ that specifically binds to the H-antigen
And enzyme-labeled eel serum lectin
To the substrate solution that reacts with the labeling enzyme, and to this substrate solution.
And a corresponding coloring liquid, for detecting tumor, cancer, and precancerous condition.
And 2. T-antigen and H-antigen contained in a biological sample.
A kit for simultaneous detection of raw material and mucin-type serine / threonine typically having an A-type sugar chain
A lectin that specifically binds to a type-binding glycoprotein and a biotinylated lectin that specifically binds to a T-antigen
And a biotinylated lectic that specifically binds to H-antigen
Or T-antigen specific mixture.
And a lectin that specifically binds to H-antigen.
After mixing approximately equal amount of cutin, lectin or
And mixing the reagent obtained Te, to correspond to the enzyme-labeled avidin, and enzyme-labeled avidin
And a colored liquid for detecting a tumor, cancer, or precancerous condition.
And 3. T-antigen and H-antigen contained in a biological sample.
A kit for simultaneous detection of raw material and mucin-type serine / threonine typically having an A-type sugar chain
A lectin that specifically binds to a type-binding glycoprotein, an enzyme-labeled castor-derived lectin (RCA ₁ ), a substrate solution that reacts with the labeling enzyme, and a substrate solution
And a corresponding coloring liquid, for detecting tumor, cancer, and precancerous condition.
And 4. T-antigen and H-antigen contained in a biological sample.
A kit for simultaneous detection of raw material and mucin-type serine / threonine typically having an A-type sugar chain
A lectin that specifically binds to a type-binding glycoprotein, an anti-T antibody produced from the T-antigen and an H-antigen, and an anti-T antibody.
H-antibody is mixed in approximately equal amount and then enzyme-labeled, or
Is approximately equal after the enzyme labeling of the anti-T antibody and anti-H antibody.
Mixed with each other, or a mixed reagent obtained by the reaction, a substrate solution that reacts with the labeling enzyme, and the substrate solution
And a corresponding coloring liquid, for detecting tumor, cancer, and precancerous condition.
And 5. T-antigen and H-antigen contained in a biological sample.
A kit for simultaneous detection of raw material and anti-T antibody produced from the T-antigen and H-antigen.
And anti-H antibody, and the anti-T antibody and anti-H antibody were mixed in substantially equal amounts, and
Or labeled with anti-T antibody and anti-H antibody
After enzyme labeling, mix in approximately equal amounts, or
A combined reagent, a substrate solution that reacts with the labeling enzyme, and this substrate solution
And a corresponding coloring liquid, for detecting tumor, cancer, and precancerous condition.
And 6. Peroxidase (PO ) as a labeling enzyme
D) is used.
A kit for detecting a tumor, cancer, or precancerous condition according to any of the above. 7. An anti-T lectin such as RCA ₁ or PNA
And an anti-H lectin such as UEA ₁ were mixed in approximately equal amounts.
A mixed reagent is provided, and the mixed reagent is added to the suspension of latex particles to prepare a latex.
Particle sensitized and dropped on a slide glass for agglutination reaction
The sensitized latex suspension was added to the biological sample
T-antigen in a biological sample depending on the presence or absence of aggregation of tex particles
, Cancer, pre-detected to simultaneously detect and H-antigen
A kit for detecting cancer symptoms.