JPH08134100A - Production of saccharide chain-specific antibody receptor - Google Patents

Abstract

PURPOSE: To obtain a saccharide chain-specific antibody which can simply detect a glycoprotein having a cancer-specific glycoside chain and is useful in the diagnosis and treatment of cancers by immunizing an animal which becomes immunologically tolerant to a soluble glycoprotein having no saccharide chain bonded with a soluble glycoprotein bound with a saccharide chain. CONSTITUTION: In a gene coding a soluble glycoprotein such as α-fetoprotein, a codon responding to amino acid (Asp) to which the saccharide chain bonds is exchanged with a codon responding to another amino acid by the mutation transduction technique to prepare a recombinant plasmid. Then, the plasmid is expressed in a COS7 cell originated from monkey kidney to prepare a soluble protein having no saccharide chain bonded. This protein is dosed to a newborn of an animal such as mouse to obtain an animal immunologically tolerant to the soluble glycoprotein having no saccharide chain bonded. Then, this animal is immunized with a soluble protein bound with a saccharide chain to obtain this saccharide chain-specific antibody which acts on the saccharide chain moiety of the soluble glycoprotein, thus can simply determine the soluble glycoprotein having a saccharide chain structure specific to some diseases, for example, cancer or the like.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a sugar chain-specific antibody that specifically acts on the sugar chain portion of a soluble glycoprotein.

[0002]

2. Description of the Related Art Alpha-fetoprotein (AFP)
Carcinoembryonic antigen (CEA), chorionic gonadotropin,
It is known that in soluble glycoproteins such as gamma-glutamine transferase protein, the structure of the sugar chain changes due to canceration of cells.

For example, alpha-fetoprotein is a kind of soluble glycoprotein known to be released into the blood in liver diseases such as liver cancer and is an important measurement item in the diagnosis of liver cancer. . In recent years, it has been reported that there are molecular species with different sugar chain structures in alpha-fetoprotein, and fractional quantification based on the difference in sugar chain structures, for the discrimination between benign diseases such as jaundice and hepatocellular carcinoma, It has become known to be useful. For this purpose, so far, 1) a method of immobilizing an anti-alphafetoprotein antibody on a solid phase to capture alphafetoprotein, and detecting only alphafetoprotein that binds to a specific lectin with an enzyme-labeled lectin, 2) A method of separating alpha-fetoprotein based on the difference in affinity for sugar chains by agarose gel electrophoresis containing a lectin, transferring it to a nitrocellulose membrane, etc., and then detecting it by immunostaining using an anti-alpha-fetoprotein antibody [Cancer Lett.]
1986; 31: 325-331] has been developed. However, 1)
This method has a low affinity for the sugar chain of lectin, and the detection sensitivity was not practically applicable to clinical laboratory. In addition, the method 2) can efficiently separate alpha-fetoprotein based on the difference in sugar chains, and has the ability to be sufficiently used in clinical tests, but it is complicated and time-consuming to separate proteins. In addition, there is a problem that a special device for measuring the intensity of color development after immunostaining is required.

[0004]

Therefore, as a method for solving such a problem, an antibody that specifically recognizes a soluble glycoprotein containing a sugar chain that appears specifically in a disease such as cancer is used. Development of diagnostic agents and therapeutic agents has been desired.

As a method for producing a tumor-specific antibody, for example, Japanese Patent Application Laid-Open No. 60-190721 discloses administration of human normal cells or normal tissues or membrane components thereof to animals for immunological tolerance (immunological tolerance). A method of inducing a tumor-specific monoclonal antibody by inducing an unresponsive state) and then immunizing with human tumor cells or tumor tissues or their membrane components is described. But according to this method,
Although it is possible to prepare an antibody against an antigen that is present on the surface of the membrane of tumor tissue or tumor cells and whose expression level increases when a normal cell turns into a tumor, it is secreted from the cell and turned into a tumor. It was difficult to obtain an antibody that specifically acts on a soluble glycoprotein containing a sugar chain whose structure is changed by.

An object of the present invention is to provide a method for producing a sugar chain-specific antibody that specifically acts on the sugar chain portion of a soluble glycoprotein having a sugar chain structure specific for diseases such as cancer. Is.

[0007]

Means for Solving the Problems As a result of extensive studies to solve such problems, the present inventor has determined that animals that have become immunologically tolerant to soluble glycoproteins to which sugar chains are not bound are The inventors have found that immunization with a bound soluble glycoprotein gives an antibody that specifically recognizes the sugar chain portion of the soluble glycoprotein, and arrived at the present invention.

That is, according to the present invention, an animal tolerated with a soluble glycoprotein to which a sugar chain is not bound is immunized with a soluble glycoprotein to which a sugar chain is bound to specifically immobilize the sugar chain portion of the soluble glycoprotein. The gist is a method for producing a sugar chain-specific antibody, which comprises producing a sugar chain-specific antibody that acts.

The present invention will be described in detail below. Examples of the soluble glycoprotein used in the present invention include alpha fetoprotein, carcinoembryonic antigen (CEA), chorionic gonadotropin, gamma glutamyl transferase protein, and the like.

In the present invention, first, immunological tolerance is carried out with soluble glycoproteins to which sugar chains of these soluble glycoproteins have been removed but which have no sugar chains attached. Examples of soluble glycoproteins to which these sugar chains are not bound include, for example, soluble glycoproteins purified from human serum or cells producing these soluble glycoproteins, are treated with glycopeptidase F (Takara Shuzo) or the like. And can be obtained by removing the sugar chain portion. In addition, these soluble glycoproteins to which sugar chains are not bound can be produced by Escherichia coli using a gene recombination technique. In this case, in order to maintain an intramolecular disulfide bond and obtain a soluble glycoprotein without a sugar chain having a structure similar to a natural substance, C
It is desirable to use eukaryotic cells such as OS cells, CHO cells, L cells and the like. In order to produce soluble glycoproteins to which these sugar chains are not bound, a method of culturing the cells in the presence of a sugar chain synthesis inhibitor such as tunicamycin, or the amino acid residue to which the sugar chain is bound, can be used. , For example, asparagine residue of alphafetoprotein [Asn232, Proceedings of the National Academy
Of Science USA (Proc., Natl., Aca
d., Sci., USA.), 80: 4604-4608, 1983].
Examples thereof include a method of substituting with another amino acid by a technique such as NA amplification method (Polymerase chain reaction: PCR) and expressing it in the cells as described above.

[0011] The thus obtained sugar chain-unbound soluble glycoprotein or cells producing a sugar chain-unbound soluble glycoprotein is used, for example, in neonates such as mice, rats, rabbits and goats, preferably Can be injected into newborns within 24 hours of age to induce immune tolerance in animals. In addition, as described above, a gene for a soluble glycoprotein in which a sugar chain binding site is mutated by introducing an amino acid substitution is introduced into an animal embryo by a conventional method together with a marker gene such as a neomycin resistance gene, and transgenic. Immune tolerance can also be induced by producing animals. In addition, oral tolerance can be induced by orally administering a soluble glycoprotein to which no sugar chain is bound. In this way, an animal that has become immunotolerant to a soluble glycoprotein without a sugar chain attached thereto has the ability to produce an antibody against the immunized soluble glycoprotein without a sugar chain attached thereto. There is no.

Next, the animal tolerated to the soluble glycoprotein to which the sugar chain is not bound may be immunized with the soluble glycoprotein to which the sugar chain is bound. These soluble glycoproteins can be purified, for example, from the serum of choriocarcinoma patients or hepatocellular carcinoma patients or from the culture medium of cells that produce these soluble glycoproteins. For example, in the case of alpha fetoprotein, , Taketa et al. [Cancer Lett., 31: 325-331, 1
986], the L of alpha-fetoprotein fractionated by
It is preferable to use 3 fractions.

The amount of the soluble glycoprotein to which these sugar chains are bound, the administration site, the use of an adjuvant and the like may be immunized according to the conventional method for obtaining antiserum. For example,
When using a mouse, 0.001 to 10 mg per mouse, preferably 0.01 to 1 mg per mouse
The soluble glycoprotein having a sugar chain bound thereto is mixed well with an adjuvant (for example, Freund's complete adjuvant) for the first time and administered subcutaneously or intraperitoneally. Furthermore, after 2 weeks or more, only the soluble glycoprotein to which the sugar chain is bound may be administered intravenously, subcutaneously or intraperitoneally.

Serum is collected from the animals 1 to 4 weeks after the final immunization as described above. This serum, for example,
By adsorbing to a column packed with a protein A column, preferably a soluble glycoprotein to which a sugar chain used for immunization is bound, for example, the L3 fraction of alphafetoprotein (eg, sepharose), and eluted, An antibody that specifically acts on the sugar chain portion of the soluble glycoprotein can be obtained. In addition, animals tolerized as described above, preferably mice or rats, are immunized with a sugar chain-bound soluble protein, and production of antibodies is confirmed, and then spleen cells or iliac lymph nodes are isolated from the animals. It is also possible to prepare cells and obtain a hybridoma producing a monoclonal antibody by a known method.

Since the antibody thus obtained acts specifically on the sugar chain portion of the soluble glycoprotein, for example, when alpha-fetoprotein having a sugar chain structure peculiar to cancer patients is used as the soluble glycoprotein. This antibody can be used to selectively detect alpha-fetoprotein found in patients with benign liver diseases such as jaundice and alpha-fetoprotein found specifically in cancer patients such as liver cancer.

[0016]

EXAMPLES Next, the present invention will be specifically described with reference to examples.

Reference Example 1 (Preparation of alpha-fetoprotein to which sugar chain is not bound) Morinaga et al. [Proceeding of the National Academy of Science USA (Proc. N
Atl. Acad. Sci. USA) 80, 4606-4608, 1983] based on the nucleotide sequence of human-derived alphafetoprotein described in Hepat.
oma cDNA library (Toyobo, Code No. CLHL101
From 5b), a gene fragment encoding alpha-fetoprotein was amplified by the PCR method, and a DNA fragment of a desired size was separated and purified by agarose gel electrophoresis. After blunting the ends of the obtained DNA fragment using a blunting kit (Takara Shuzo), EcoRI linkers (Takara Shuzo) were ligated to both ends (Takara Shuzo).
Was used to bind. The obtained alpha-fetoprotein DNA fragment was digested with EcoRI (Takara Shuzo Co., Ltd.) overnight, and then separated and purified by agarose gel electrophoresis. This fragment was designated as pCDM8 vector (Funakoshi, IV-3082).
-01) was ligated to the HindIII site using a ligation kit (Takara Shuzo) to transform E. coli HB101 strain (Takara Shuzo). A plasmid in which the alpha-fetoprotein gene was inserted in the forward direction was selected from the obtained colonies and named pCDM 8-AFP.

Next, a plasmid in which the codon AAT corresponding to the amino acid residue Asp232 of the alphafetoprotein encoded by pCDM 8-AFP was replaced with AGT was prepared by the mutagenesis method using the PCR method, and pCDM 8- It was named AFP (NS). The obtained pCDM 8-AFP (NS) was added to monkey kidney-derived COS7.
It introduced into the cell (ATCC CRL 1651) using the DEAE dextran method. After the introduction, the cells were cultured in a CO ₂ incubator to produce alpha fetoprotein in the medium. When this culture supernatant was analyzed by electrophoresis, the cultured cells produced alpha-fetoprotein to which sugar chains were not bound. The culture supernatant was recovered and the anti-alpha fetoprotein antibody NO. 5107 [manufactured by Medix Biochemica] was used as the antibody / antigen immobilization kit No. Purification was performed using an anti-alpha-fetoprotein antibody column immobilized with 44895 (manufactured by Pierce), and as a result, alpha-fetoprotein (1) in which almost no single sugar chain was electrophoretically bound
I got About 1250 μg of alpha-fetoprotein (1) having no sugar chain bound thereto was recovered from the culture supernatant of 50 petri dishes having a diameter of 10 cm.

Reference Example 2 (Preparation of alpha-fetoprotein to which sugar chain is bound) Human liver cancer-derived HepG2 cells (ATCC HB 8065) are cultured [medium: minimum essential medium (Minimum Essen)
tial medium) 41500-034, manufactured by Lifetec Oriental Co., Ltd.], alpha-fetoprotein to which a sugar chain is bound is accumulated in a culture solution, and purified by using column chromatography in the same manner as in Reference Example 1 to obtain SDS. Electrophoretically almost uniform sugar chain-bound alpha-fetoprotein was obtained. About 1400 μg was collected per culture supernatant of 10 plates with a diameter of 10 cm. This is further taken by Taketa et al. [Cancer Lett.] 1986, 3
1,325-331], that is, separated by agarose gel electrophoresis containing lectin, and from the fraction corresponding to L3, 300 μg of alpha-fetoprotein (2) having a sugar chain attached thereto
Was recovered.

Example 1 COS7 cells producing alpha-fetoprotein to which sugar chains were not bound, prepared in Reference Example 1, were used as newborn rabbit 3
The cells were intraperitoneally administered to animals (within 24 hours after birth) at 1 × 10 ⁵ cells / animal. One month later, serum was collected from the ears of the rabbit, and the antibody titer to the sugar chain-unbound alphafetoprotein (1) was measured by enzyme-linked immunosorbent assay (ELISA). That is, a 96-well microtiter plate was physically adsorbed with the alpha-fetoprotein (1) having no sugar chain prepared in Reference Example 1, and the serum collected by the above method was added to the phosphate-buffered sarin (PBS). ) In 50, 500, 50
10 times diluted to 100 times for each well
After adding 0 microliter each, the mixture was allowed to stand at room temperature (25 ° C) for 2 hours, washed with PBS, further added with horseradish-derived peroxidase-labeled goat anti-rabbit IgG antibody (manufactured by Bio-Rad) and allowed to stand for 2 hours. PB this plate
After washing with S, a color reaction was carried out by adding a substrate solution containing 3,3 ', 5,5', tetramethylbenzidine and hydrogen peroxide, and alpha-fetoprotein (1) with no sugar chain attached Antibodies against were not contained in any of the sera.

Next, 50 μg of alpha-fetoprotein (1) having no sugar chain prepared in Reference Example 1 was mixed with an equal volume of Freund's complete adjuvant (manufactured by Nacalai TeX Co., Ltd.) to prepare an emulsion. The above rabbit was subcutaneously administered and immunized. Two weeks later, 100 μg of alpha-fetoprotein (1) having no sugar chain prepared in Reference Example 1 was emulsified with an equal volume of Freund's complete adjuvant (manufactured by Nacalai Techs) and additionally administered. After further 3 weeks, the final immunization was performed with the alpha-fetoprotein (1) to which the sugar chain was not bound, which was prepared in Reference Example 1, and 2 weeks after that, blood was collected from the ear and serum was separated and collected. The antibody titer to the alpha-fetoprotein (1) to which the sugar chain of this serum was not bound was measured in the same manner as above.

The results are shown in Table 1. The antibody titer is
It was represented by the average value of the color development intensity (OD ₄₀₅ ) in the ELISA obtained using the sera of 3 rabbits.

[0023]

[Table 1]

As shown in Table 1, no antibody against alpha-fetoprotein (1) to which no sugar chain was bound was detected in the serum of any of the rabbits. From this, it can be seen that administration of alpha-fetoprotein (1) to which a sugar chain is not bound to newborns induces immune tolerance to alpha-fetoprotein (1) to which a sugar chain is not bound.

[0025] Next, in three newborn rabbits in which immunotolerance was induced in this manner, one month later, the sugar chain-conjugated alpha-fetoprotein (2) 50 prepared in Reference Example 2 was used.
μg was mixed with an equal volume of Freund's complete adjuvant (manufactured by Nacalai Techs) to prepare an emulsion, which was subcutaneously administered to immunize. Two more weeks later, reference example 2
100 μg of the alpha-fetoprotein (2) having a sugar chain bound thereto prepared in step 1 was emulsified with an equal volume of Freund's incomplete adjuvant (manufactured by Nacalai TeX Co., Ltd.) for additional administration. After further 3 weeks, the final immunization was performed with the α-fetoprotein (2) to which the sugar chain prepared in Reference Example 2 was alone, and 2 weeks after that, 49 ml of serum was separated and collected.

Next, the antibody titer to the alpha-fetoprotein (1) in which the sugar chain of this serum was not bound was measured by the above method. Further, a 96-well microtiter plate on which the alpha-fetoprotein (2) to which the sugar chain was bound was immobilized was prepared, and the antibody titer to the alpha-fetoprotein (2) to which the sugar chain was bound was measured in the same manner. Furthermore, alpha-fetoprotein with a sugar chain attached (2)
Was treated with glycopeptidase F (manufactured by Takara Shuzo Co., Ltd.) to remove the sugar chain portion, to prepare alpha fetoprotein (3) having no sugar chain bound, which was fixed on a 96-well microtiter plate, and similarly. The antibody titer to alpha-fetoprotein (3) to which sugar chain was not bound was measured.

The results are shown in Table 2.

[0028]

[Table 2]

As shown in Table 2, no antibodies against alpha-fetoprotein (1) with no sugar chain attached and alpha-fetoprotein (3) with no sugar chain attached were detected in any of the sera. Thus, an antibody against the alpha-fetoprotein (2) having a sugar chain attached was detected in all the sera. From this, it can be seen that the obtained antibody recognizes the sugar chain portion of the alpha-fetoprotein (2) to which the sugar chain is bound.

Example 2 The COS7 cells producing the alpha-fetoprotein (1) to which no sugar chain was bound prepared in Reference Example 1 were ablated into the abdominal cavity of newborn BALB / c mice (within 24 hours after birth) at 1 × 10 ^5. Cells / mouse were administered. After 6 weeks, the sugar chain-bound alpha fetoprotein (2) prepared in Reference Example 2
Was emulsified with an equal volume of Freund's complete adjuvant (manufactured by Nakarai Techs Co., Ltd.), and this was subcutaneously administered. After two more weeks, the alpha-fetoprotein (2) to which the sugar chain was bound prepared in Reference Example 2 was emulsified with Freund's incomplete adjuvant (manufactured by Nacalai Techs), and this was additionally administered. Two more weeks later, reference example 2
The final immunization was carried out with the alpha-fetoprotein (2) having a sugar chain bound thereto prepared in (1), and 3 days later, blood was collected from the tail to obtain serum. The antibody titers of the alpha-fetoprotein (1) to which the sugar chain was not bound, the alpha-fetoprotein (2) to which the sugar chain was bound, and the alpha-fetoprotein (3) to which the sugar chain was not bound were determined in Example 1 of this serum.
When measured in the same manner as in 1., only an antibody that binds to the alpha-fetoprotein (2) having a sugar chain bound thereto was detected.

Therefore, the spleen of this mouse was removed, and the cells were suspended in RPMI1640 medium (manufactured by Gikob) and cultured in advance for myeloma cells NS-1 (ATCC TIB 1
8) was mixed with 5 of splenocytes obtained from 1 mouse in 1 of myeloma cells, and the mixture was fused with 50% by weight of polyethylene glycol 4000 (manufactured by Sigma). After the cell fusion, the cells were mixed with HAT medium (GIT medium (manufactured by Nippon Pharmaceutical Co., Ltd.) in HAT-Media Suplemen).
t (made by Boehringer Mannheim) was added thereto, and the mixture was dispensed into a 96-well microtiter plate and cultured at 37 ° C. for 10 days. The antibody titer against the sugar chain-bound alphafetoprotein (2) in the culture supernatant was measured in the same manner as in Example 1, and a hole showing antibody production positive for the sugar chain-bound alphafetoprotein was selected.
Cloning was performed by repeating the limiting dilution method twice. As a result, hybridoma strains MAH1 to MAH5 that produce an antibody that recognizes the alpha-fetoprotein (2) having a sugar chain bound thereto were obtained. The obtained hybridoma strain was cultured in 10 ml of GIT medium (manufactured by Nippon Pharmaceutical Co., Ltd.), and the culture supernatant was used as a crude monoclonal antibody solution. This crude monoclonal antibody solution was tested for antibody titers against alpha-fetoprotein (1) with no sugar chain attached, alpha-fetoprotein (2) with an attached sugar chain and alpha-fetoprotein (3) without an attached sugar chain in Example 1.
It measured similarly to. As a control, the antibody titer against carcinoembryonic antigen (C0214, manufactured by Script Laboratories) was measured in the same manner.

The results are shown in Table 3.

[0033]

[Table 3]

As shown in Table 3, each of the obtained monoclonal antibodies specifically reacted with alpha-fetoprotein (2) having a sugar chain bound thereto.

Reference Example 3 Antibody / antigen immobilization kit No. 44895 (made by Pierce)
Anti-alpha fetoprotein antibody No. 51
07 [Medix Biochemic
a) manufactured by the company] was immobilized to prepare an anti-alpha-fetoprotein antibody column. Next, 500 ml of serum of a liver cancer patient showing an alphafetoprotein value of 494.2 ng / ml [measured using an Ergia-AFP kit (Midori Cross)] was diluted 5 times with PBS and then equilibrated with PBS. The sample was passed through an anti-alpha fetoprotein antibody column that had been denatured, and the non-adsorbed fraction was collected and used as sample A. Moreover, after diluting 500 ml of serum of the same liver cancer patient 5 times with PBS, a column on which anti-alphafetoprotein antibody was not immobilized [Antibody / antigen immobilization kit No. Attached to 44895 (manufactured by Pierce)
Then, the non-adsorbed fraction was collected in the same manner as above, and this was designated as Sample B. The alpha fetoprotein value of sample B was 83.1 ng / ml. Furthermore, a serum sample of a chronic hepatitis patient showing an alpha fetoprotein value of 92.3 ng / ml was used as sample C.

Next, from the serum separated and collected in Example 1,
The antibody was purified by protein A column. That is,
10 ml of the collected serum was diluted 50 times with a 50 mM sodium carbonate buffer (pH 9.6), and then passed through a protein A Sepharose column (manufactured by Pierce) equilibrated with the same buffer. After passing the solution, it was washed with 50 mM Tris-HCl buffer (pH 8.6) and eluted with glycine-HCl buffer (pH 2.3). Fractions containing the antibody were collected and dialyzed against PBS to obtain an antibody solution containing the anti-alphafetoprotein antibody (total protein amount 1.2 mg). The obtained antibody solution was diluted with 50 mM sodium carbonate buffer (pH 9.6), dispensed in 100-microliter aliquots into a 96-well microtiter plate, and left overnight at 4 ° C. After removing this liquid, add 1% to each hole.
(W / V) bovine serum albumin (BSA), PBS containing 0.15M sodium chloride is filled, and room temperature (25 ° C)
Left for 2 hours. Then, remove this liquid to 0.2%
(W / V) Tween 20 (Tween20), 0.2% (W / V)
BSA, 20 mM containing 0.15 M sodium chloride
Each hole was washed with a washing solution consisting of the sodium phosphate buffer solution (pH 7.2) of 1. to prepare an anti-alpha-fetoprotein-immobilized antibody plate.

Samples A, B and C were serially diluted and added to the anti-alpha-fetoprotein-immobilized antibody plate thus obtained in 50 microliter increments.
It was left at room temperature (25 ° C) for a minute. This plate was washed three times with the washing solution described above, and then the anti-alpha fetoprotein monoclonal antibody No. 5108 (manufactured by Medics) diluted to 3000 times with PBS was added to 30
It was left at room temperature (25 ° C) for a minute. Then, the cells were washed 3 times with PBS, and further, a goat anti-rabbit IgG antibody (manufactured by Bio-Rad) labeled with horseradish-derived peroxidase was added to
50 microliters of each diluted with 00 times were added to each well, and left at room temperature (25 ° C.) for 1 hour. After that, the plate was washed 5 times with the washing solution, 100 μl of the coloring substrate solution was added, and the mixture was left at room temperature (25 ° C.) for 30 minutes. 10%
The reaction was stopped by adding 50 microliters of (W / V) sodium dodecyl sulfate (SDS), and the absorbance at 415 nm was measured. The result is shown in FIG.

FIG. 1 is a diagram showing the results when alpha-fetoprotein was detected using the antibody produced according to the present invention, in which the vertical axis represents the absorbance (A460) and the horizontal axis represents the dilution ratio. As shown in FIG. 1, the antibody prepared in Example 1 did not react with the serum (sample A) of the liver cancer patient treated with the anti-alpha-fetoprotein antibody, whereas it was treated with the anti-alpha-fetoprotein antibody. This antibody is specific for alpha-fetoprotein, as it reacts with the serum of non-liver cancer patients (Sample B). Furthermore, it can be seen that the specificity for the serum of the liver cancer patient (Sample B) is much higher than that for the serum of the chronic hepatitis patient (Sample C). From this, the use of the assay reagent using the antibody obtained by the present invention enables specific detection of alpha-fetoprotein having a sugar chain structure associated with liver cancer.

[0039]

INDUSTRIAL APPLICABILITY According to the present invention, a sugar chain-specific antibody that specifically acts on the sugar chain portion of a soluble glycoprotein can be easily produced. Further, by using the sugar chain-specific antibody produced by the present invention, a soluble glycoprotein having a sugar chain structure unique to a disease such as cancer can be easily measured.

[Brief description of drawings]

FIG. 1 is a diagram showing the results when alpha-fetoprotein was detected using the antibody produced by the present invention.

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Claims (1)

[Claims] 1. A saccharide that specifically immunizes a sugar chain portion of a soluble glycoprotein by immunizing an animal tolerated with a soluble glycoprotein to which the sugar chain is not bound with a soluble glycoprotein to which the sugar chain is bound. A method for producing a sugar chain-specific antibody, which comprises producing a chain-specific antibody.