JPH08201383A - Method for determining sugar chain structure - Google Patents

Abstract

PURPOSE: To simply and efficiently determine a sugar chain structure by fixing the sugar chain or sugar peptide derived from composite carbohydrate to a solid phase, binding a marker material to it, and detecting the marked solid phase. CONSTITUTION: The composite carbohydrate such as sugar protein or glycolipid isolated from cells or a living sample derived from the cells is digested by enzyme to obtain the sugar chain or sugar peptide, and it is reacted with a solid phase (resins, celluloses, glass or the like) coated with a polymer having the reaction group (epoxy group, halogen atom or the like) capable of being reacted with the molecular terminal group of them and forming a covalent bond to be fixed. A marker material (e.g. marked lectin, marked antibody) peculiarly bonded with them is bonded, the marked solid phase is detected, or a bonding material (unmarked lectin and antibody or the like) peculiarly bonded with the sugar chain or sugar peptide is reacted, a material peculiarly bonded with the bonding material is reacted, and the generated marked solid phase is detected.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for determining a sugar chain structure, and more particularly to a method for simply and efficiently determining a sugar chain structure of a glycoconjugate such as glycoprotein, glycolipid and proteoglycan.

[0002]

BACKGROUND ART It is becoming clear that sugar chains of glycoconjugates widely distributed in nature are important constituents in the living body and are deeply involved in interactions between cells. Along with this, various techniques for microanalysis of sugar chain structures have been developed.These technologies are a combination of steps such as excision of sugar chains, separation and purification of sugar chains, and labeling of sugar chains. Ultimately, identification by NMR or MS is necessary to obtain reliable information on the obtained sugar, which is extremely troublesome. A method has also been proposed in which the sugar chain structure can be easily determined by omitting these steps.

The simple sugar chain structure analysis methods proposed so far include, for example, high performance liquid chromatography (HP).
As an application of the method of LC), an analytical method using a column on which a lectin having a sugar discrimination ability is immobilized (An
al. Biochem., 164, 374 (1987) Harada et al.) Two-dimensional HPLC combining two modes of HPLC (Anal.
Biochem., 171, 73 (1988) Tomiya et al.) And a method of pretreating a sample with a mixed enzyme series such as exoglycosidase and analyzing the treated sample by HPLC to determine the sugar chain structure (chemistry And creature 32 (10) 661
(1994) Konishi et al.). Each of these methods is a method for determining a sugar chain structure using the retention time of an HPLC chromatogram as an index.

However, the analysis method using the HPLC method is (1) only one sample can be analyzed at a time, and a large number of samples cannot be analyzed simultaneously. (2) HP
LC condition setting is delicate and retention time shifts, which is likely to be inaccurate. (3) Combination of HPLC with post-column reactor or pre-column reactor consumes large amount of reagent, (4) Lectin immobilization The use of a column has a problem that a glycopeptide adsorbed may change due to a change in the affinity of lectin for sugar.

[0005] Further, a method of immobilizing a sugar chain on a solid phase for analysis has been attempted, but immobilizing a highly hydrophilic sugar chain is technically difficult. Therefore, techniques for immobilizing sugar chains have been proposed. For example, a method of immobilizing an acid amide bond on an amino plate by using a reducing end of a sugar chain (Anal. Biochem., 182, 200 (1989) Oobayashi et al.), A method of imparting a high hydrophobicity to the sugar chain itself is used. A method of molecularizing and adsorbing onto a plate (Japanese Patent Laid-Open No. 62-21256).
8), a method in which a sugar chain is biotinylated, and the sugar chain is immobilized on a solid phase to which avidin is bound by utilizing the strong affinity of avidin and biotin. However,
In the method of immobilizing on a solid phase such as a plate, a reduction in immobilization yield due to pretreatment of sugar chains and immobilization using a condensing agent, complicated operation, and long time are required. Non-specific adsorption is likely to occur due to coexistence,
There were problems such as. Recently, it has been proposed to analyze sugar chains by an analysis method using surface plasmon resonance, but it is not a versatile method because an expensive special device is used.

[0006]

An object of the present invention is to efficiently immobilize a sugar chain or glycopeptide derived from a glycoconjugate on a solid phase in a single step, and further immobilize a labeling substance thereon, An object of the present invention is to provide a method capable of simply and efficiently determining a sugar chain structure by detecting the resulting labeled solid phase. According to the method of the present invention, a series of reactions required for analysis can be efficiently performed using a conventional detection device, and a large number of trace samples can be analyzed at one time.

[0007]

According to the present invention, (1)
Immobilization by reacting a sugar chain or glycopeptide derived from a glycoconjugate with a solid phase coated with a polymer having a reactive group capable of reacting with a molecular end group of the sugar chain or the glycopeptide to form a covalent bond Producing a sugar chain or an immobilized glycopeptide, (2) reacting the immobilized sugar chain or the immobilized glycopeptide with a labeled substance that specifically binds to these, and (3) producing a labeling A method for determining a sugar chain structure is provided, which comprises detecting the solid phase.

Further, according to the present invention, (1) it has a sugar chain or glycopeptide derived from a glycoconjugate and a reactive group capable of reacting with the molecular end group of the sugar chain or the glycopeptide to form a covalent bond. Reacting with a solid phase coated with a polymer to produce immobilized sugar chains or immobilized glycopeptides,
(2) The immobilized sugar chain or the immobilized glycopeptide is reacted with a binding substance that specifically binds to them, and (3) the resulting product and a label that specifically binds to the binding substance. There is provided a method for determining a sugar chain structure, which comprises reacting with a substance and (4) detecting the resulting labeled solid phase.

The method for determining the sugar chain structure of the present invention will be described in detail below. The measurement target of the present invention is a glycoconjugate, that is, a sugar chain structure of a sugar containing non-sugar components such as glycoprotein, glycolipid, and proteoglycan, and the sugar itself is composed of monosaccharides, oligosaccharides and polysaccharides. Both are not particularly limited.

The sample to be used is not particularly limited, and various sugar chains contained in the sample, for example, N-glycoside type, O-glycoside type, collagen type glycoprotein and ganglio type, globo type, Examples thereof include sugar chains such as lacto-based glycolipids. As a method for separating a sugar chain from a glycoconjugate such as glycoprotein, glycolipid, and proteoglycan, a commonly used method, for example, hydrazinolysis method, thorough digestion with pronase, N-glycanase treatment, O-glycanase treatment, trifluoro A decomposition method with acetic acid and the like can be mentioned, and a treatment operation and the like with them can be performed in the same manner as that usually adopted in this kind of method. (Methods Enzymol., Vol 83, 263 (198
2))

In the method of the present invention, first, a solid phase on which a sugar chain or sugar peptide derived from a glycoconjugate is immobilized is prepared. For that purpose, first, glycoconjugates such as glycoproteins, glycolipids and proteoglycans isolated from cells or biological preparations derived therefrom are digested with an enzyme or the like to obtain sugar chains or fragments of sugar peptides. Each fragment is separated and purified by HPLC and the like, and each peak fraction is collected in an appropriate amount to obtain a sugar chain or sugar peptide fragment for immobilization. Various chromatographies can be used for the fractionation of sugar chains, but especially for the separation of low molecular weight substances such as sugar peptides, reverse-phase HPL using C18 etc.
C is suitable. 210-230 for the elution fraction monitor
It is convenient to detect the absorption at nm. The obtained fraction is fractionally dried for each peak and used for the reaction with the solid phase for immobilization.

The solid phase on which the sugar chain or the sugar peptide fragment is to be immobilized is not particularly limited, and its material is fluorinated such as polyolefin, polystyrene, substituted polystyrene, polytetrafluoroethylene, and polyvinylidene fluoride. Polymers, polysulfones, polyesters, polycarbonates, polyacrylonitriles, nylons, polyacrylamides, polymethacrylates, polyvinyl alcohols, polyurethanes and copolymers thereof such as water-insoluble or insoluble synthetic polymers, celluloses, glass and the like. . The form of the solid phase is not particularly limited, and typical forms include microplates, membranes, fibers, papers, etc., with microtiter plates being most preferred. Further, its size and thickness are not particularly limited.

When the sugar chain or sugar peptide is immobilized on the solid phase, the surface of the solid phase is previously fixed to the molecular end group of the sugar chain or sugar peptide, ie, the sugar chain, in order to enhance the binding force. It is coated with a polymer having a reducing group or a reactive group capable of reacting with an amino group or a carboxyl group at the molecular end of a sugar peptide to form a covalent bond (for example, an acid amide bond). Examples of the reactive group capable of forming such a covalent bond include an epoxy group, a halogen atom, an amino group, a hydrazino group, a carboxyl group, an acid anhydride group and a formyl group.

The polymer having a reactive group as described above is
It is prepared by polymerizing a monomer having a reactive group as described above and chemically modifying a polymer having another reactive group. Preferred polymers include polymers of acid anhydride monomers such as maleic anhydride, or vinyl monomers copolymerizable therewith, such as methyl vinyl ether,
Examples thereof include maleic anhydride copolymers with ethyl vinyl ether, butyl vinyl ether, isobutyl vinyl ether, styrene and the like. Further, polymers and copolymers such as glycidyl methacrylate or glycidyl acrylate, which is a monomer having an epoxy group, or acrylic acid or methacrylic acid, which is a monomer having a carboxyl group, can be mentioned. It is also possible to chemically modify the functional group of the obtained polymer to obtain a polymer having a different functional group, for example, a polymer having an epoxy group or an acid anhydride may be a compound such as ammonia, hydrazine or propanediamine. A polymer having an amino group can be obtained by reacting with various diaminoalkanes, basic amino acids, polyamines and the like. Also,
A polymer having an amino group can be converted to a polymer having a carboxyl group by reacting with an acid anhydride such as succinic anhydride. In the case of a polymer having a hydroxyl group, for example, it is reacted with diglycidyl ethers such as 1,4-butanediol diglycidyl ether and diepoxides such as 1,7-octadiene diepoxide under alkaline conditions. It may be a polymer having an epoxy group.

The molecular weight of these coating polymers is not particularly limited, but in order to have hydrophobicity and viscosity suitable for coating on a solid surface, the molecular weight is 40.
It may be 0 or more, and particularly preferably 20,000 or more and 1 million or less. The method of coating the polymer having the above-mentioned reactive group on the solid phase is not particularly limited, and a dipping method, a coating method or other coating method of an organic solvent solution of the polymer can be adopted.

The reaction conditions between the sugar chain or sugar peptide fraction and the immobilized solid phase are not particularly limited, and may be generally the same as in the case of this type of detection method. That is, generally under a temperature condition of 45 ° C or lower, preferably about 4 to 40 ° C,
The reaction is performed for about 1 to 40 hours. At that time, as the reaction medium, an ordinary solvent that does not adversely influence the reaction, for example, a buffer solution having a pH of about 4 to 8 such as a citrate buffer solution, a phosphate buffer solution, a Tris-hydrochloric acid buffer solution, and an acetate buffer solution is preferable. Can be used as a thing.

In the method of the present invention, after immobilizing on the solid phase of the sugar chain or sugar peptide as described above, (1) the sugar chain or sugar peptide and a label that specifically binds to these Or a bound substance that specifically binds to the sugar chain or sugar peptide is detected. After the reaction, the binding substance and the labeling substance that specifically binds are reacted and bound, and the resulting labeled solid phase is detected.

In the above method (1), a labeled lectin and a labeled antibody are preferably used as the labeled substance that specifically binds to the immobilized sugar chain or sugar peptide. Here, as the labeled lectin and the labeled antibody, various lectins and antibodies labeled with various labeling agents such as ordinary radioactive substances, enzyme labeling substances and fluorescent substances are used. Examples of radioactive substances as labeling agents include 125I and other radioactive iodine agents,
Examples of the fluorescent substance include fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), substituted rhodamine isothiocyanate (XRITC), rhodamine B isothiocyanate, dichlorotriazine fluorescein (DTA).
F) and the like, and as enzyme labeling substances, peroxidase, microperoxidase, chymotrypsinogen, procarboxypeptidase, glyceroaldehyde-3-phosphate dehydrogenase, amylase, phosphorylase, DNA-nase, β-galactosidase, etc. Can be mentioned respectively.

Various known lectin to be labeled, for example, ConA, RCA60, RCA12.
0, LCA, PSA, VFA, L-PHA, E-PH
A, WGA, PNA, etc. can be illustrated. The above various lectins can be obtained as commercial products, or can be produced according to a conventional method (J. Biol. Chem., 254, 9349-.
9351 (1979): Nature, 194, 495 (1962)); fluorescent antibody method,
Laboratory of Medical Chemistry 4,263-270; Acta. Endocrinol. Suppl.,
168, 206 (1972) etc.). The antibody to be labeled is also not particularly limited as long as it is an antibody that specifically recognizes a sugar chain or a sugar peptide. By proper selection of the antibody, a labeled antibody having high specificity and affinity for the immobilized carbohydrate or glycopeptide can be obtained.

In the above method (2), unlabeled lectin and antibody are preferably used as the binding substance that specifically binds to the immobilized sugar chain or sugar peptide. The lectin and antibody are not particularly limited as long as they can specifically recognize a sugar chain or a sugar peptide, like the lectin and antibody to be labeled used in the method (1) above. You can choose between lectins and antibodies.

In the above-mentioned method (2), after binding an unlabeled binding substance, a labeling substance which specifically binds to the binding substance is reacted to bind. Examples of the labeling substance include labeled antibodies having an affinity for lectins or antibodies, or substances such as red blood cells and polymer latex having a sugar having an affinity for lectins.

The above methods (1) and (2) have merits and demerits when comparing the two, but the method (2) is superior in the following points. That is, in the above method (1),
In general, the labeled lectins or labeled antibodies that can be used are limited and are difficult to obtain, and when attempting to prepare the labeled lectins or labeled antibodies, it takes time to purify the obtained preparations. In addition, it may cause denaturation in which the specificity and affinity of lectins and antibodies are changed. In contrast to this, in the method of (2) above, a wide range of labeled antibodies can be utilized by interposing a binding substance, and there is no difficulty thereof.

The binding reaction of the labeled lectin or labeled antibody in the above method (1) and the binding reaction of the unlabeled binding substance and the binding reaction of the labeling substance in the above method (2) are both As in the case of immobilizing the sugar chain or sugar peptide, it can be carried out in the same manner as a usual reaction of this type. At this time, the above-mentioned various solvents that do not adversely affect the reaction can be used, and if desired, metal ions such as calcium and manganese can be added to the reaction system. The above reaction is generally carried out under a temperature condition of 45 ° C. or lower, preferably about 4 to 40 ° C., for about 1 to 40 hours.

In both the above methods (1) and (2),
The finally obtained immobilized labeled substance is appropriately detected by measuring the change in the absorption spectrum or measuring the radioactivity depending on the shape and optical properties of the solid phase. For example, if the solid phase is a microplate,
It can be easily detected with a plate reader or the like used in a general enzyme immunoassay (ELISA).

[0025]

EXAMPLES The present invention will be specifically described below by showing Examples of the present invention, but the present invention is not limited to these Examples. Example 1 MMAC (methyl vinyl ether / maleic anhydride copolymer, / n-263, molecular weight 41000) was dissolved in hexane at a concentration of 5 mg / ml, and this solution was added to a 96-well microtiter plate (Immulon 200, CA Greiner and Sohn
e, GmbH & CoKG, Germany), 100 μl was added to each well, and the plate was allowed to stand at room temperature for 30 minutes. After discarding the supernatant,
A 2-fold diluted aqueous solution of p-aminophenylgalactopyranoside (used as a simple model of glycopeptide) dissolved in 50 mM Tris-maleic acid buffer (pH 7.0) (1.56
-400 μg / ml) was added to each well in an amount of 50 μl to perform immobilization. After discarding the supernatant and washing 3 times with TBS, 5% BS
The cells were blocked with A (bovine serum albumin) / TBS (Tris-hydrochloric acid buffer containing 0.15M NaCl) for 1 hour. After discarding the supernatant, 100 labeled HRP (horse radish peroxidase) -labeled RCA (castor seed lectin), which is a labeled lectin capable of specifically recognizing galactose, was added.
Each μl was added and left to stand. Furthermore, discard the supernatant and discard TBS.
The plate was washed with, and color was developed using O-phenylenediamine, which is a substrate for HRP, and the absorbance at 490 nm was measured.

An experiment similar to the above was performed using B instead of MMAC.
Dissolve S3 (bis-sulfosacrene imidyl suberate) in 5 mM phosphate buffer (pH 5.0) to a concentration of 0.1 mg / ml and add 100 μl to each well,
After standing at room temperature for 2 hours, the supernatant was discarded and the plate was used for comparison. The results are shown in Fig. 1. As shown in Fig. 1, the concentration of the ligand is 5 μg / m.
It was found that at a concentration of 1 or less, almost no difference was observed between BS3 and MMAC, but at 5 μg / ml or more, MMAC was much more efficiently immobilized in a concentration-dependent manner.

Example 2 The same experiment as in Example 1 was conducted using MMAC while changing the time for immobilizing sugar to 1, 3, 6, 18 hours, and the influence of the immobilization time was investigated. . The result is shown in FIG. As shown in FIG. 2, concentration-dependent results were obtained at all immobilization times, but almost equilibrium was reached in about 6 hours.

Example 3 The reaction time of HRP-labeled lectin was 15 minutes, 30 minutes,
Example 1 using MMAC by changing the time from 1 hour to 2 hours
The same experiment was performed. The results are shown in Fig. 3. The reaction yield reached equilibrium in 1 hour.

Example 4 In the same manner as in Example 1, a 2-fold dilution series solution (1.56-400 μl / ml) of p-aminophenylgalactopyranoside was added to a microtiter plate coated with MMAC for immobilization. RCA after blocking with 5% BSA / TBS
50 μl each was added and the mixture was allowed to stand at room temperature for 1 hour. After further washing and blocking, 100% of 4% human type A red blood cells
μl was added and left still for 1 hour to lyse the red blood cells. For detection, the absorbance at 415 nm was measured with a micro reader. As a comparison, the same experiment was performed on a plate not coated with MMAC. The results are shown in Fig. 4. It was clarified that the one immobilized with aminated sugar via MMAC was easily detected subsequently with lectin and erythrocyte, as compared with the one immobilized without MMAC.

Example 5 In the same manner as in Example 1, p-aminophenylgalactohydraside was immobilized on a microtiter plate coated with MMAC. At that time, the concentration of the immobilized sugar was 6.2.
After changing to 5-400 μg / ml and blocking with 5% BSA / TBS, RCA 0.5 mg / ml was added by 100 μl and left at room temperature for 1 hour. Furthermore, washing and blocking are performed, and an antibody against the plant-type N-linked sugar chain (anti-B
-SJA-II antibody, diluted 1000 times) was added in 100 μl portions and reacted at room temperature for 1 hour. After washing and blocking, 100 μl of HRP-labeled anti-rabbit IgG antibody (goat) was added as a secondary antibody, and the mixture was allowed to stand at room temperature for 30 minutes. Color development was performed by a method using O-phenylenediamine, and the absorbance at 490 nm was measured. The results are shown in FIG. As a result, it was revealed that aminated sugars can be detected with high sensitivity by an antibody against sugar and its secondary antibody.

Example 6 1 mg of castor seed lectin (RCA) was added to 0.1 mM Ca.
_{Cl 2 -30mM NaCl-50mM Tris-} H
Trypsin (20 μg / 2) dissolved in 500 μl of Cl (pH 7.8), heated at 100 ° C. for 10 minutes, and dissolved in the same buffer solution.
μl) was added and the mixture was allowed to stand at 37 ° C. for 24 hours to digest the protein. In addition, 0.2M PMSF and 0.2M
The reaction was stopped by adding 5 μl of EDTA and heating at 100 ° C. for 10 minutes. A blank obtained by performing the same operation without adding protein was used. A part of this digest is C18
The peptide was separated by addition to a reverse phase HPLC column. For separation, a C18 reverse-phase column (4 × 150 mm) was used, and the flow rate was 1.
The detection was performed at 0 ml / min, a temperature of 40 ° C., and 220 nm, and a linear gradient from 0.05% trifluoroacetic acid to 60% 2-propanol: acetonitrile (7: 3) was used for 60 minutes. The obtained chromatogram is shown in FIG.
It was shown to.

100 μl of MMAC (1 mg / ml) dissolved in hexane was added to each well of the titer plate and left standing at room temperature for 30 minutes. The peptide fraction collected is
Dry with Speed Vac, add pure water and part of it to 50m
It was dissolved in M Tris-maleic acid buffer solution to make 50 μl, and the supernatant was added to the discarded well. The peptide was fixed by allowing to stand for 18 hours at 4 ° C. The same immobilization was performed using BS3 instead of MMAC. Dissolve BS3 in 5 mM phosphate buffer (pH 5.0) to a concentration of 0.1 mg / ml, and add 5 to the wells of a titer plate.
0 μl of each was added and coating was performed at 37 ° C. for 2 hours. Further, a part of the collected peptide fraction was dissolved in PBS to make 50 μl, added to the well and incubated with BS3. The peptide was immobilized by allowing it to stand at 4 ° C. for 18 hours.

The wells of the plate on which the peptide was immobilized were washed 3 times with TBS and blocked with 5% BSA / TBS for 1 hour at room temperature. The supernatant was discarded, 100 μl of a 1000-fold diluted solution of HRP-labeled (concanavalin A) Con A was added, and the mixture was reacted at room temperature for 1 hour. After washing, HRP-labeled ConA bound to the sugar-containing peptide in the well was subjected to color development by a method using O-phenylenediamine, and the absorbance at 490 nm was measured and detected.
The results are shown in Fig. 6. What is detected as positive is Fig. 6
Peak numbered at (29, 31, 44, 47, 47, 48, 5
1, 54, 58, 59, 60, 65, 67).

MM for these fractions
The result of comparison between the AC coating method and the BS3 method is shown in FIG. 7. As a result, the absorbance was higher when immobilized with MMAC as a whole, and the fractions (peaks 29, 44, 48, 51, 67) that were hardly detected by BS3 could be detected by MMAC. In particular, when the peptide without sugar chain was mixed in the fraction and the amount of sugar peptide was small (peaks 44 and 54), a difference in the immobilized amount of MMAC and BS3 was observed. Moreover, when the surface of the titer plate was not coated, no peptide was detected.

Example 7 As in Example 1, a certain amount of human milk oligosaccharide was added to a microtiter plate coated with MMAC, and 5% B was added.
Blocked with SA / TBS. 25 μg / ml monoclonal antibody (NS10C17) solution (0.15M Na
TB containing Cl, 10% BSA, 0.1% NaN ₃
S) was added and reacted at room temperature for 30 minutes. After reaction, TB
After washing 3 times with S, HRP-labeled anti-mouse IgM antibody solution was added as a secondary antibody, and the mixture was allowed to stand at room temperature for 30 minutes. Color was developed by a method using 0-phenylenediamine,
The absorbance at 0 nm was measured. As a result, LNDI (Le
Lacto-N-difucopentaose I [Fucαl → 2Galβl containing structure ^b [Fucαl → 2Galβl → 3 (Fucαl → 4) GlcNAc]]
→ 3 (Fucαl → 4) GlcNAcβl → 3Galβl → 4Glc]) was detected with high sensitivity.

Reference Example 1 A part of the tryptic digest of RCA in Example 6 was replaced with 0.1 M Ac0Na-1 mM CaCl ₂ -1 mMM.
C equilibrated with gCl ₂ -150 mM NaCl buffer
on Agarose column (3ml), buffer 20m
After washing with l, 0.1M methyl α-mannoside solution 10
Elute with ml. After concentrating this sugar peptide fraction, C
Figure 18 shows the chromatogram applied to an 18 reverse-phase HPLC column.
It was shown to. Similar to the peptide in Example 6, MMAC
As a result of detection by the method, 7 peaks that were positive were assigned numbers.

Reference Example 2 The amino acid sequence analysis of the seven peak fractions in Reference Example 1 and the 12 peak fractions obtained in Example 6 gave the results shown in Table 1. (The glycosylation site is underlined, the analyzed sequence is shown by a solid line, and the sequence known from a known report is shown by blank letters.) In Table 1, the peak number on the left vertical axis is the reverse phase of the RCA digest. The positive peak fraction number obtained by chromatography is shown, and the right vertical axis shows the Co of the RCA digest.
The positive peak fraction number obtained by affinity chromatography on a nA agarose column is shown.

In Table 1, peaks 1, 2, and 4 are RCA.
Of B-85 with a high mannose type sugar chain, and peaks 3, 5, 6 and 7 were peptides with a high mannose type sugar chain of B-135 of RCA. A-
The complex type sugar chain bound to 10 and B-73 could not be obtained on the ConA agarose column. On the other hand, according to the MMAC coating method, peaks 31,44,5
1, 66 and 67 are B-85, A-10 and B, respectively.
Glycopeptides corresponding to -135, A-10 and A-10, which could not be detected by the ConA agarose method, could be detected. Therefore, it was revealed that by detecting the sugar chain by the MMAC immobilization method, a sugar peptide which could not be obtained by the ConA agarose column can be detected. In addition,
Amino acid sequence analysis is performed by Applied Bionsystems in Protein se.
Quencher Model 476A was used for analysis.

[0039]

INDUSTRIAL APPLICABILITY According to the method of the present invention, by covalently binding a sugar chain to a solid phase, the interaction with a substance having a sugar recognizing ability such as a lectin, an antibody and a glycolytic enzyme is analyzed. Thus, the sugar chain structure can be determined easily and efficiently. Further, according to the method of the present invention, it is possible to detect a sugar chain which was not detected by the conventional method of analyzing by chromatography using immobilized lectin. In addition, it is possible to determine the sugar chain structures of various kinds of samples at one time. The method of the present invention is expected to be applied in fields where sugar chains are expected to be involved, such as the development of cancer diagnostic agents.

The method for determining the sugar chain structure of the present invention, that is, (1) a sugar chain or glycopeptide derived from a glycoconjugate is reacted with the sugar chain or a molecular end group of the glycopeptide to form a covalent bond. Reacting with a solid phase coated with a polymer having a reactive group capable of producing an immobilized sugar chain or immobilized glycopeptide, and (2-1) the immobilized sugar chain or immobilized glycopeptide and a specific thereof. Reacting with a labeled substance that specifically binds, or (2-2) reacting the immobilized sugar chain or immobilized glycopeptide with a binding substance that specifically binds to these, and further obtaining And a labeled substance that specifically binds to the binding substance, and then (3) detecting the labeled solid phase to be produced, for determining a sugar chain structure. The preferred embodiments of It is a cage.

(I) In the above (1), the sugar chain and sugar peptide to be immobilized on the solid phase are prepared by digesting a complex sugar with an enzyme. (Ii) In the above (1), the reactive group capable of forming a covalent bond is an epoxy group, a halogen atom, an amino group, a hydrazino group, a carboxyl group, an acid anhydride group or a formyl group. (Iii) In the above (1), the polymer having a reactive group has a molecular weight of 400 to 1,000,000.

(Iv) In (2-1) above, the labeled substance is a labeled lectin. (V) In (2-1) above, the labeled substance is a labeled antibody. (Vi) In the above (2-2), the binding substance is an unlabeled lectin or antibody, and the labeling substance that specifically binds to the binding substance is a labeled antibody, erythrocyte or polymer latex. is there.

[Brief description of drawings]

FIG. 1 shows the influence of a coating polymer and the concentration of the glycoside on immobilization of p-aminophenylglycoside on a plate, on the amount of RCA immobilization (expressed as absorbance).

FIG. 2 shows the immobilization time in immobilization of p-aminophenylglycoside and the effect of the glycoside concentration on the amount of RCA immobilization (expressed as absorbance).

FIG. 3 shows the reaction time of HRP-lectin (RCA) to immobilized p-aminophenylglycoside and the effect of the glycoside concentration on the amount of RCA immobilized (expressed as absorbance).

FIG. 4: MMAC in detection of lectin (RCA) bound to p-aminophenylglycoside by erythrocytes.
The effect of the presence or absence of a coat and the glycoside concentration on the amount of RCA immobilized (also expressed as absorbance) is shown.

FIG. 5 shows the influence of the same glycoside concentration on the amount of RCA immobilized (also expressed as absorbance) in the detection of p-aminophenyl glycoside using an antibody.

FIG. 6 shows the results of analysis of a tryptic digest of RCA by reverse phase high performance liquid chromatography.

FIG. 7: RCA-derived peptide fraction was analyzed using MMA
The results of immobilization on C coat and BS3 coat are shown for comparison.

FIG. 8 shows the results of analysis of RCA digests by affinity chromatography on a ConA agarose column.

[Table 1]

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Atsuko Ogawa 2-30, Nakameguro, Meguro-ku, Tokyo 111 (72) Inventor Isamu Matsumoto 4-3-13 Mihama, Urayasu, Chiba (72) Invention Person Tamami Nakayama 5-1 Ogimachi, Kawasaki-ku, Kawasaki-shi, Kanagawa Prefecture Chemical Research Laboratory, Showa Denko KK (72) Inventor Seiya Moriguchi 1-13-9 Shibadaimon, Minato-ku, Tokyo Inside Showa Denko KK

Claims (2)

[Claims] (1) A solid coated with a sugar chain or glycopeptide derived from a glycoconjugate and a polymer having a reactive group capable of reacting with a molecular end group of the sugar chain or the glycopeptide to form a covalent bond. To produce an immobilized sugar chain or immobilized glycopeptide, and (2) react the immobilized sugar chain or immobilized glycopeptide with a labeled substance that specifically binds to these. (3) A method for determining a sugar chain structure, which comprises detecting the produced labeled solid phase. 2. A solid coated with a sugar chain or glycopeptide derived from a glycoconjugate and a polymer having a reactive group capable of reacting with a molecular end group of the sugar chain or the glycopeptide to form a covalent bond. (2) reacting the immobilized sugar chain or immobilized glycopeptide with a binding substance that specifically binds to the immobilized sugar chain or immobilized glycopeptide, 3) Determine the sugar chain structure characterized by reacting the obtained product with a labeling substance that specifically binds to the binding substance, and (4) detecting the labeled solid phase produced. Method.