WO2014136301A1 - Method for detecting glycoproteins - Google Patents

Abstract

Provided is a method for detecting multi-fucose glycoproteins. This method for detecting multi-fucose glycoproteins included in a body fluid comprises: (B) a step for bringing, into contact with lectin, a product obtained by eliminating sialic acids linked to sugar chains in the total glycoproteins included in said body fluid; and (D) a step for measuring the amount of presence of multi-fucose glycoproteins that have linked to said lectin. Note that a multi-fucose glycoprotein refers to a glycoprotein that includes, per glycoprotein molecule, at least one N-linked sugar chain including at least two fucoses per sugar chain.

Description

Glycoprotein detection method

The present invention relates to a method for detecting a glycoprotein. Specifically, the present invention relates to a method for detecting a glycoprotein having at least one N-linked sugar chain having at least two fucose per sugar chain per molecule of glycoprotein.

Currently, the diagnosis of cancer is centered on image diagnosis such as endoscope, PET, and MRI, but these are not necessarily diagnoses that a healthy person regularly receives because of the great pain and cost burden to patients. Not popular. A blood test that is simpler and less costly than image diagnosis is desirable to detect early-stage cancer without subjective symptoms. However, because current cancer markers are not accurate enough, healthy individuals should regularly It is not implemented in the health check you receive. If this is carried out, healthy persons who are determined to be false positives will continue, and depending on their additional examination (such as image diagnosis), the diagnostic function of the hospital will be paralyzed. Although a cancer marker with high discrimination accuracy is desired, there is actually no cancer marker having performance equivalent to that of image diagnosis.

Glycoproteins contained in serum have been known to change in sugar chain structure with canceration since ancient times, and in particular, it is known that fucosylation of sugar chains proceeds with the progression of cancer (Non-Patent Document 1, Non-patent document 2). For example, it is disclosed that sialyl Lewis X-type fucose in a three-chain or four-chain N-linked sugar chain is significantly increased in the serum of a liver cancer patient (Non-patent Document 1, Non-patent Document 2).

As a method for detecting fucosylation of an N-linked sugar chain as described above, a method using a lectin (a generic term for sugar chain-binding proteins) is disclosed. In particular, AAL lectin (Hirochawantake lectin) has excellent binding power and selectivity for fucose recognition, and has been widely used to search for cancer markers.
Patent Document 1 discloses a method comprising contacting a sample with a lectin to form a complex between the glycosylated protein and the lectin in the sample, and then detecting the glycosylated protein-lectin complex. Is disclosed. It is also described that the method may include a step of first contacting the sample with an antibody specific to the glycoprotein to be detected to capture the glycosylated protein to be detected in the sample. Has been.

Patent Document 2 discloses that the α1-acid glycoprotein erodes cancer by determining the abundance ratio of N-type sugar chains having 3- and 4-chain structures and the modification rate of fucose added to the N-type sugar chains. Disclosed is a method for determining the prognosis of a patient after organ organ resection surgery, and describes a method for determining the modification rate of fucose by cross-affinity immunoelectrophoresis using an AAL lectin.

Further, Non-Patent Document 3 discloses that a serum sample is first treated with sialidase for the purpose of detecting a γGTP protein having bisecting type GlcNAc, and this is treated with red kidney bean E ₄ lectin (E-PHA). An experimental example on an agarose column is disclosed.

Special table 2008-541060 Japanese Patent Laying-Open No. 2005-069846

Biochem Biophys Res Commun. Vol.374, No.2, p219-225 Hepatology, vol. 46, No.5, p1426-1435 (2007) Noguchi Laboratory Time Report, No. 49, p. 4-20 (2006)

As described in Non-Patent Documents 1 and 2, the relationship between fucosylation of N-linked sugar chains and cancer has been widely studied so far, and fucosylation of glycoproteins in serum progresses as cancer progresses. It is known. However, when only the presence or absence of fucosylation of sugar chains was detected, specificity and sensitivity were insufficient.
Accordingly, the present inventors have searched and analyzed the structure of a sugar chain that can be a cancer marker. As a result, the N-linked sugar chain has at least two fucose per sugar chain instead of one fucose per molecule. Glycoprotein having at least one N-linked sugar chain per molecule of glycoprotein (hereinafter sometimes referred to as “multifucose glycoprotein”) is significantly increased in body fluids collected from cancer patients. I found it. The present inventors also found that the multifucose glycoprotein is excellent in specificity and sensitivity as a cancer marker.

When this multifucose glycoprotein is detected, in the methods described in Patent Documents 1 and 2 above, since the lectin binds regardless of the number of fucose per sugar chain, only the multifucose glycoprotein is selectively used. There is a problem that it cannot be detected. At present, the only method for detecting multifucose glycoprotein is to use a mass spectrometer. However, this method using a mass spectrometer requires complicated processing and is expensive. Therefore, there is a problem that it is not practical as a diagnostic test method.

In Non-Patent Document 3, bisecting type GlcNAc is detected, and there is no description or suggestion of a method for detecting multifucose glycoprotein.
An object of the present invention is to provide a method for detecting multifucose glycoprotein more efficiently.

In order to solve the above-mentioned problems, the present inventors include lectins and N-linked sugar chains having at least two fucose per sugar chain (hereinafter referred to as “multifucose sugar chains”) included in the multifucose glycoprotein. ). As a result, in an N-linked sugar chain containing fucose from which sialic acid has been eliminated, a sugar chain having one fucose bond does not bind to an AAL lectin, whereas a multi-chain having two or more fucose bonds. It has been found that the fucose sugar chain strongly binds to the AAL lectin even after sialic acid is eliminated. Furthermore, if the sialic acid of the glycoprotein contained in the body fluid of the test animal is desorbed before contacting the AAL lectin, the AAL lectin can selectively recognize only the multifucose glycoprotein, Completed the invention.

That is, the gist of the present invention resides in the following (1) to (12).
(1) A method for detecting multifucose glycoprotein contained in a body fluid,
(B) contacting the lectin with a product obtained by eliminating sialic acid that binds to the sugar chains of all glycoproteins contained in the body fluid; and
(D) measuring the abundance of multifucose glycoprotein bound to the lectin,
A detection method characterized by comprising:
However, the multifucose glycoprotein is a glycoprotein having at least one N-linked sugar chain having at least two fucose per sugar chain per molecule of glycoprotein.

(2) The detection method according to (1), wherein the lectin is an AAL lectin (Hirochawantake lectin).
(3) The detection method according to (1) or (2), wherein in the step (B), sialic acid is eliminated by bringing the total glycoprotein into contact with a sialidase enzyme.
(4) Prior to the step (B),
(A) contacting the body fluid with a carrier on which an antibody that recognizes a protein portion of a glycoprotein is immobilized,
And said (B) process,
(B ′) a step of contacting a lectin with a product from which sialic acid bound to the sugar chain of the glycoprotein bound to the carrier in step (A) has been eliminated,
The detection method according to any one of (1) to (3), wherein
(5) Between the step (B) or the step (B ′) and the step (D),
(C) eluting multifucose glycoprotein bound to the lectin from the lectin;
The detection method according to any one of (1) to (4), comprising:
(6) In the step (A), the step (B ′) is performed on the glycoprotein bound to the antibody without detaching the glycoprotein from the antibody. (4) ) Or the detection method according to (5).
(7) In the step (A), the glycoprotein bound to the antibody is desorbed from the antibody, and the step (B ′) is performed on the desorbed glycoprotein, The detection method according to (4) or (5).
(8) In the step (A), the number of moles of the multifucose glycoprotein is the same as the number of moles of the multifucose glycoprotein with respect to the number of moles of the antibody; Characterized in that the concentration of the antibody and / or the glycoprotein is adjusted so that the total number of non-glycoproteins (hereinafter referred to as “non-multifucose glycoproteins”) is equal to or greater than the same number of moles. The detection method according to any one of (4) to (7).
(9) In the step (D), the ratio of the abundance of the multifucose glycoprotein to the abundance of the total glycoprotein contained in the body fluid is further obtained. (1) to (8) ).
(10) In the step (D), the ratio of the abundance of the multifucose glycoprotein to the abundance of the non-multifucose glycoprotein bound to the antibody in the step (A) is further obtained. (4) The detection method according to any one of (9).
(11) The detection method according to any one of (1) to (10), wherein the multifucose glycoprotein is significantly increased or decreased by the onset or progression of a specific disease.
(12) The detection method according to (11), wherein the specific disease is cancer, and cancer is detected using the abundance of the multifucose glycoprotein contained in a body fluid as an index.

According to the present invention, a method for efficiently detecting multifucose glycoprotein can be provided. Thereby, in particular, it is possible to provide a method for detecting cancer with higher sensitivity and specificity than conventional methods.

The measurement result obtained in Reference Example 1 is shown. The correlation of the measurement result by a two-step extraction method and the measurement result by a mass spectrometer about multifucose glycoprotein in Example 2 is shown. The correlation of the measurement result by the two-step extraction method about the single fucose glycoprotein in Example 2 and the measurement result by a mass spectrometer is shown.

The detection method of the present invention detects a glycoprotein having at least one N-linked sugar chain having at least two fucose per sugar chain per glycoprotein molecule (ie, “multifucose glycoprotein”). This is a detection method of interest, (B) a step in which sialic acid bound to the sugar chains of all glycoproteins contained in the body fluid has been removed is brought into contact with lectin, and then (D) bound to the lectin. A step of measuring the abundance of multifucose glycoprotein.

Moreover, the detection method of the present invention can be provided with the following steps in addition to the step (B) and the step (D). Prior to the step (B), the detection method of the present invention preferably has a step of (A) bringing the body fluid into contact with a carrier immobilizing an antibody that recognizes the protein portion of the multifucose glycoprotein. That is, prior to the step (B), (A) the step of contacting the body fluid with a carrier on which an antibody that recognizes the protein portion of the glycoprotein is immobilized, and the step (B) comprises (B ′ It is preferable that the step is a step of contacting the lectin with the sialic acid bound to the sugar chain of the glycoprotein bound to the carrier in step (A).

The detection method of the present invention can be a method having, for example, the following steps (A), (B ′), and (D).
(A) A step of bringing the body fluid into contact with a carrier on which an antibody that recognizes a protein portion of a glycoprotein is immobilized.
(B ′) a step of bringing a lectin into contact with sialic acid bound to a sugar chain of a glycoprotein bound to the carrier.
(D) A step of measuring the abundance of multifucose glycoprotein bound to the lectin.
In the detection method of the present invention, (C) the multifucose glycoprotein bound to the lectin is eluted from the lectin between the step (B) or (B ′) and the step (D). Steps can also be added.

When the detection method of the present invention includes all the steps (A) to (D), the detection method of the present invention is as follows. However, in this case, it is preferable that after the step (A), the glycoprotein bound to the antibody is desorbed from the antibody before proceeding to the step (B ′).
(A) A step of bringing the body fluid into contact with a carrier on which an antibody that recognizes a protein portion of a glycoprotein is immobilized.
(B ′) a step of bringing a lectin into contact with sialic acid bound to a sugar chain of a glycoprotein bound to the carrier.
(C) A step of eluting the multifucose glycoprotein bound to the lectin from the lectin.
(D) A step of measuring the abundance of multifucose glycoprotein bound to the lectin.

As a sample for the detection method of the present invention, a body fluid collected from a test animal is used. As the body fluid, blood, lymph fluid, cerebrospinal fluid, urine and processed products thereof are used, preferably blood, and more preferably serum or plasma obtained by separating the blood. The test animal is preferably a human, but the detection method of the present invention can also be used for animal experiments other than humans.
Hereinafter, after explaining the multifucose glycoprotein to be detected by the detection method of the present invention, the detection method of the present invention (each step (A) to (D)) will be described in detail.

[Multifucose glycoprotein]
In the present invention, the multifucose glycoprotein refers to a glycoprotein having at least one N-linked sugar chain having at least two fucose per sugar chain per molecule of glycoprotein. Here, the N-linked sugar chain refers to a sugar chain that binds to asparagine of a protein.

The multifucose glycoprotein is a sugar chain part essentially comprising a protein part and an “N-linked sugar chain having at least two fucose per sugar chain” (hereinafter sometimes referred to as “multifucose sugar chain”). And have. The multifucose glycoprotein is not particularly limited as long as it has a multifucose sugar chain in its sugar chain part.
In the present invention, the entire multifucose glycoprotein may be detected, but the multifucose glycoprotein may be detected by peptide fragmentation.
Hereinafter, the multi-fucose glycoprotein to be detected according to the present invention will be described separately for multi-fucose sugar chains and glycoproteins.

(Multifucose sugar chain)
The multi-fucose sugar chain is an N-linked sugar chain and has at least two fucose, that is, multi-fucose per one N-linked sugar chain.

The basic skeleton of the multifucose sugar chain is not particularly limited, but is usually branched, preferably 3 branches or more, more preferably 3 branches or 4 branches, most preferably 4 branches. It is. This is because the multi-fucose is more likely to be generated in the four branched chains, and the ratio of the four branched chains in the multi-fucose sugar chain is large. The fucose that binds to this N-linked sugar chain is per sugar chain (here, per sugar chain means not one per branched chain but the entire sugar chain molecule that can bind to one asparagine molecule). It is counted as a book.), Preferably 2 or more, and preferably 5 or less, more preferably 4 or less. This is because it is extremely rare that 6 or more fucose bonds per N-linked sugar chain.

As long as at least two fucose possessed by the multifucose sugar chain are fucose, there are no particular restrictions on the mode of binding to the N-linked sugar chain, and α1-6 bonds to GlcNAc present at the reducing end of the N-linked sugar chain. Although it may be a core fucose or other outer fucose, it is preferably an outer fucose. A sugar chain that does not have sialic acid and has only one outer fucose bound thereto has a tendency to be unable to bind to the AAL lectin. Therefore, the outer fucose has the effect of the present invention. Because it is easy to be done.
As outer fucose, fucose (Lewis X-type fucose) that binds α1-3 to GlcNAc present at the reducing end of N-linked sugar chain and α1-4 bond to GlcNAc present at the reducing end of N-linked sugar chain Examples include fucose (Lewis A type fucose). The multifucose sugar chain has at least two fucose, and the binding mode of these two fucose may be the same or different.

In addition, the multifucose sugar chain before being subjected to the step (B) or (B ′) is not particularly limited in terms of the number of sialic acid residues bound to the N-linked sugar chain and the number thereof.
Specific structures of the multifucose sugar chain include, for example, A2G2FcFo, A2G2Fo2, A2G2FcFo2, A3G3FcFo2, A3G3FcFo3, A3G3Fo4, A3G3Fo4, A4G4F4F4, A4G4F4F4, A4G4F4F4 The thing which the acid couple | bonded arbitrary number is mentioned. Here, A is the number of branches, G is the number of galactose, S is the number of sialic acid (N-acetylneuraminic acid), Fo is the number of outer fucose, and Fc is the core fucose. As preferred examples of specific structures, the structures of A3G3S2Fo2, A3G3S3Fo2, A3G3S3Fo3, A4G4S3Fo2, A4G4S4Fo2 and A4G4S4Fo3 are shown below.
In the structural formulas below, “Gal” is galactose, “GlcNAc” is N-acetylglucosamine, “Man” is mannose, “Fuc” is fucose, and “NeuAc” is N-acetylneuraminic acid. Represents (sialic acid).

[A3G3S2Fo2]

[A3G3S3Fo2]

[A3G3S3Fo3]

[A4G4S3Fo2]

[A4G4S4Fo2]

[A4G4S4Fo3]

That is, the multifucose sugar chain is an N-linked sugar chain having 3 or 4 branched chains, has 3 or 4 galactoses, has 0 to 4 sialic acids, It is preferable that two or three fucose bonds per N-linked sugar chain which is a chain. In this case, there is no particular limitation on the bonding position between sialic acid and fucose.
The multifucose glycoprotein of the present invention is required to have at least one multifucose sugar chain per molecule of glycoprotein, but has two or more multifucose sugar chains per molecule of glycoprotein. May be.
The multifucose sugar chain is not particularly limited in its binding site, but can bind to asparagine in the protein portion of the glycoprotein.

(Glycoprotein)
There is no restriction | limiting in particular as a kind of glycoprotein of the multifucose glycoprotein made into the detection object in this invention, What is necessary is just to select suitable glycoprotein according to the objective of a detection. If the glycoprotein can be used as a marker for a disease by selecting a glycoprotein whose sugar chain structure changes due to the onset or progression of a specific disease and the sugar chain having multifucose can be significantly increased or decreased. preferable. Specific diseases include cancer, cerebral infarction, diabetes, rheumatism and the like.

In the present invention, examples of glycoproteins to be detected include blood secreted proteins having N-linked sugar chains.
In particular, when it is desired to detect hepatocellular carcinoma, the glycoproteins to be measured are ceruloplasmin, cellotransferrin, α1-acid glycoprotein, GP-73, hemopexin, HBsAg, α1-antichymotrypsin, α1-antitrypsin. , Α2-macroglobulin, α2-HS-glycoprotein, haptoglobin, fibrinogen γ chain precursor, immunoglobulin, APO-D, kininogen, histidine rich glycoprotein, complement factor 1 precursor, complement factor I heavy chain, complement Body factor I light chain, complement C1s, complement factor B precursor, complement factor BBa, complement factor BBb, complement C3 precursor, complement C3β chain, complement C3α chain, C3a anaphylatoxin, complement C3bα 'Chain, complement C3c, complement C3dg, complement C3g, complement C3d, complement C3f, complement C5, complement C5β chain Complement C5α chain, complement C4 binding protein, C5a anaphylatoxin, complement C5α ′ chain, complement C7, α1B-glycoprotein, B2-glycoprotein, vitamin D binding protein, inter α-trypsin inhibitor heavy chain H2, α1B -Glycoprotein, angiotensinogen precursor, angiotensin-1, angiotensin-2, angiotensin-3, GARP protein, β2-glycoprotein, crusterin (ApoJ), integrin α8 precursor glycoprotein, integrin α8 heavy chain, integrin α8 It is preferable to select light chain, hepatitis C virus particle, elf-5, kininogen, HSP33-homolog, lysyl endopeptidase or leucine rich repeat-containing protein, among which ceruloplasmin, cellotransferrin, 1-acid glycoprotein, hemopexin, alpha 1-antichymotrypsin, alpha2-macroglobulin, haptoglobin, it is preferable to select a complement 4 binding protein or leucine-rich repeat containing proteins.

At this time, for example, when the glycoprotein to be measured is α1-acid glycoprotein, the multi-marker sugar chains can be A4G4S4Fo2, A4G4S4Fo3, A4G4S3Fo2, A4G4S3Fo3, A3G3S3Fo2, and the like. In addition, when the glycoprotein to be measured is cellotransferrin, A3G3S2Fo2, A3G3S3Fo2, A3G3S3Fo3, A4G4S3Fo2, A4G4S4Fo2, A4G4S4Fo3, etc. can be detected as multimarker sugar chains.

[Detection method of the present invention]
Hereinafter, the detection method of the present invention will be described step by step.
If necessary, the specimen is pretreated before being subjected to steps (A) to (D) described later. Examples of the pretreatment include peptide fragmentation.

[Step (A)]
Step (A) is a step of bringing the body fluid into contact with a carrier on which an antibody that recognizes the protein portion of the glycoprotein is immobilized. In the present invention, the step (A) is not essential, but in the step (D), it is preferable to provide the step (A) from the viewpoint that the abundance can be measured for the multifucose glycoprotein having a specific protein portion.
In the step (A), first, an antibody that recognizes the protein part of the multifucose glycoprotein to be detected (hereinafter sometimes simply referred to as “protein part”) is immobilized on a carrier, or the antibody is previously prepared. A carrier on which is fixed is used.
The antibody may be either a monoclonal antibody or a polyclonal antibody, and a substance other than the antibody can be substituted as long as it has a function of recognizing the protein portion.

As the carrier, a plate, beads or the like can be used. The plate is preferably a polystyrene plate or the like, and the beads are preferably polystyrene or the like.
The purpose of immobilizing the antibody on the carrier is to bind the glycoprotein to be detected to the antibody, wash away unbound components, and isolate the glycoprotein to be detected. As long as it has a function, the antibody may be fixed to something other than a plate or a bead, and other methods can be used.

In the step (A), the method of binding the antibody to the carrier is not particularly limited, but a method of binding the antibody so that it is not detached from the carrier by subsequent treatment is preferable. For example, biotin / avidin binding is performed. And those that are directly bonded to the plates by hydrophobic bonding are preferred. When the antibody is directly hydrophobically bound to a carrier, the incubation temperature is usually 0 ° C. to 50 ° C., preferably 4 ° C. to 37 ° C., and the incubation time is usually 10 minutes to 24 hours. It is preferably 30 minutes to 16 hours.

The antibody bound to the carrier may be brought into contact with the body fluid as it is, but for the purpose of preventing the lectin described later from directly binding to the sugar chain of the antibody, the antibody is contacted before the body fluid is contacted. It is preferable to modify, decompose or cleave the sugar chain. Here, the periodate oxidation method is preferable as a method for decomposing the sugar chain of the antibody. Periodic acid oxidation of the antibody is carried out by contacting a phosphate buffer solution of sodium periodate 0.1 mM to 100 mM with the antibody, for example, by reacting at 0 ° C. to 50 ° C. for 10 minutes to 5 hours. In particular, it is preferable that a sodium phosphate 1 mM to 20 mM phosphate buffer is brought into contact with the antibody and reacted at 20 ° C. to 30 ° C. for 30 minutes to 2 hours.

Next, the body fluid is brought into contact with the antibody immobilized on the carrier. At this time, the number of moles of the antibody is not particularly limited, but the total number of moles of the multifucose glycoprotein and the number of moles of the non-multifucose glycoprotein is equal to or greater than the number of moles of the antibody. Is preferably 2 times or more, more preferably 10 times or more. Here, a glycoprotein having the same protein portion means a glycoprotein in which the primary sequence and tertiary structure of amino acids are identical.

The conditions for contacting the body fluid with the antibody are not particularly limited, but it is preferable to contact at 20 ° C. to 50 ° C. for 10 minutes to 3 hours.
After contacting the body fluid with the antibody fixed to the carrier, if necessary, the carrier is washed several times with a phosphate buffer, a Tris buffer, etc., to thereby remove a glycoprotein that does not bind to the antibody. After removing, it is preferable to proceed to the step (B ′) described later.

In some cases, the glycoprotein to be detected may be detached from the carrier before proceeding to the step (B ′). At this time, the method for desorbing the glycoprotein from the carrier is not particularly limited, but is preferably a condition that does not affect the later-described process method (B ′) by subsequent treatment, for example, 4 ° C. with 1 to 200 mM hydrochloric acid. Glycoprotein is desorbed from the carrier by reacting at -50 ° C. for 10 seconds to 30 minutes, and then the desorbed glycoprotein is neutralized to pH 6.0-9.0 with Tris buffer and recovered. However, after desorbing the glycoprotein from the carrier by reacting with 10-50 mM hydrochloric acid at 20 ° C.-40 ° C. for 1-20 min, the desorbed glycoprotein is adjusted to pH 7 with Tris buffer. More preferably, it is neutralized to 0.0 to 8.0 and recovered.

[Step (B) and Step (B ′)]
Step (B) is a step in which sialic acid bound to the sugar chains of all glycoproteins contained in body fluids is brought into contact with lectin, and is an essential step in the present invention.
When the step (A) is performed, the total glycoprotein in the step (B) is a glycoprotein bound to the antibody in the step (A) and binds to the antibody in the step (A). The glycoprotein may be allowed to proceed to step (B) as it is, or may be proceeded to step (B) after detaching the glycoprotein bound to the antibody from the antibody. In particular, when the step (C) described later is provided, it is necessary to desorb the glycoprotein bound to the antibody after the step (A).
Further, the (B) step after the (A) step is particularly referred to as a (B ′) step.

First, sialic acid that binds to the sugar chain of the glycoprotein is removed from the sugar chain. The method for desorbing sialic acid is not particularly limited, but desorption with sialidase is preferable, and among sialidases, desorption with neuraminidase is preferable. Sialic acid includes N-acetylneuraminic acid and N-glycolylneuraminic acid. Of these, neuraminidase is the enzyme that eliminates N-acetylneuraminic acid, and only N-acetylneuraminic acid is present in the human body, so neuraminidase is preferred as described above.

The sialidase digestion conditions are not particularly limited as long as sialic acid is eliminated, but it is desirable that the pH is 4.0 to 7.0 and the temperature is 20 to 40 ° C. When sialic acid is eliminated in this manner, in principle, the lectin described later does not bind to a single fucose sugar chain but only binds to a multifucose sugar chain.
Even if the sialic acid in the sugar chain of the glycoprotein is removed after the glycoprotein to be detected is bound to the antibody in the step (A), the bound glycoprotein is removed from the antibody. It may be performed after separating and collecting (that is, step (B ′)). In particular, in the lectin sandwich ELISA method described later, the step (B ′) is performed on the glycoprotein bound to the antibody in the step (A) without detaching the glycoprotein from the antibody. On the other hand, in the two-stage extraction method described later, the glycoprotein bound to the antibody in the step (A) is desorbed from the antibody, and the step (B ′) is performed on the desorbed glycoprotein. Do.

Further, sialic acid can be eliminated from the sugar chain of the glycoprotein without performing step (A) in advance (ie, step (B)).
Next, the lectin is brought into contact with the glycoprotein from which sialic acid has been eliminated. The lectin is not particularly limited as long as it is a lectin that recognizes fucose, but AAL lectin, AOL lectin, lentil lectin and the like are preferable, and among them, AAL lectin is particularly preferable. AAL lectin refers to Aleuria aurantia Lectin. Any artificially produced AAL lectin may be used as long as it exhibits the same effect as a natural AAL lectin, and a recombination protein may be used as long as the homology is 80% or more.
Thus, when a lectin is brought into contact with a glycoprotein from which sialic acid has been eliminated, a multifucose glycoprotein of the glycoprotein can be specifically bound to the lectin.

[Step (C)]
Step (C) is a step of eluting the multifucose glycoprotein bound to the lectin from the lectin. In the present invention, the step (C) is not essential. For example, in the step (B), the multifucose glycoprotein to be detected is bound to the lectin. If the abundance of the multifucose glycoprotein can be measured as it is (the step (D) described later can be performed), the step (C) can be omitted.
The multifucose glycoprotein bound to the lectin is not particularly limited as long as it can be eluted from the lectin. For example, it can be eluted with a fucose solution (concentration is preferably 10 mM to 200 mM).

[Step (D)]
Step (D) is a step of measuring the abundance of multifucose glycoprotein bound to the lectin, and is an essential step in the present invention.
The method for measuring the abundance of multifucose glycoprotein is not particularly limited as long as it can be measured correctly.

In the lectin sandwich ELISA method described later, in order to quantify the three-molecule conjugate consisting of the antibody, multifucose glycoprotein, and lectin, a luminescent substance is directly bound to the lectin in advance, or peroxidase or An abundance of multifucose glycoprotein contained in a body fluid can be measured by binding an enzyme that induces color development, such as alkaline phosphatase, and measuring luminescence and color development.
In the two-stage extraction method described later, the abundance of the multifucose glycoprotein eluted and recovered can be measured using a commercially available ELISA kit or a self-made ELISA.

[Specific Example of Detection Method of the Present Invention]
A specific example of the detection method of the present invention described above will be described below. The following description is merely a specific example, and embodiments of the present invention are not limited to the following.

(Sandwich ELISA method)
(A) Process:
An antibody (for example, 1 μg) capable of recognizing a glycoprotein to be detected is immobilized on a carrier (ELISA plate, immunoassay beads, etc.), and after the antibody immobilization reaction, the excess antibody solution is removed. . Here, it is preferable to add a blocking agent to the carrier on which the antibody is immobilized under the above conditions, and coat the surface on which the antibody is not immobilized with the blocking agent. Further, for example, the sugar chain bound to the antibody is decomposed by the periodate oxidation method described above, and then the sugar chain is brought into contact with a phosphate buffer containing, for example, 0.25 M dimethylamine-borane. It is preferred to reduce and protect the detached moiety.

Next, the specimen is brought into contact with the carrier on which the antibody is immobilized, and the antibody and the glycoprotein to be detected contained in the specimen are bound. Thereafter, the carrier on which the antibody in which the glycoprotein to be detected is bound is immobilized is washed with a Tris buffer (25 mM Tris, 100 mM NaCl, 0.05% Tween), and the carrier other than the glycoprotein is washed. It is preferred to remove the components.

(B ') Process:
A sialidase solution is added to the carrier prepared in the step (A), and sialic acid is eliminated by, for example, reacting at 37 ° C. for 30 minutes.
Next, an AAL lectin solution to which biotin is bound is added to the carrier after the sialidase reaction and brought into contact therewith. Subsequently, the multifucose glycoprotein contained in the glycoprotein to be detected is bound to AAL lectin (the temperature condition at this time is preferably 4 ° C. to 25 ° C.). Thereafter, the carrier is preferably washed with a Tris buffer to remove components other than the multifucose glycoprotein.

(D) Process:
(B ′) The amount of antibody, multifucose glycoprotein, and AAL lectin conjugate present on the carrier after completion of the step is measured. Although there is no restriction | limiting in particular in a measuring method, For example, the following methods are mentioned.
Avidin solution conjugated with horseradish peroxidase (HRP) is added to the carrier, and contacted at 20 to 25 ° C. to bind AAL and avidin, and binding of antibody, multifucose glycoprotein, AAL lectin, and avidin Form the body. A coloring reagent is added thereto, and after reaction at 20 to 25 ° C., coloring is stopped with a 1N sulfuric acid solution or the like. Thereafter, the amount of the multifucose glycoprotein can be measured by measuring the color development amount with a measuring instrument such as a plate reader capable of measuring the absorbance.

(Two-stage extraction method)
(A) Process:
An antibody (for example, 50 μg) that recognizes a glycoprotein to be detected and is bound to biotin is subjected to a temperature condition of 20 ° C. to 25 ° C. with respect to a carrier (for example, agarose beads) to which avidin is bound. And the antibody is immobilized on a carrier. Next, the specimen is brought into contact with the carrier on which the antibody is immobilized, and the antibody and the glycoprotein to be detected contained in the specimen are bound. The carrier on which the antibody to which the glycoprotein to be detected is bound is immobilized is washed with a Tris buffer solution to remove components other than the glycoprotein to be detected, and then contacted with 20 mM HCl for 3 minutes. For example, the glycoprotein to be detected is desorbed from the antibody, and the desorbed glycoprotein to be detected is neutralized with, for example, Tris buffer to pH 7.5 and collected.

(B ') Process:
Sialidase is added to the solution containing the glycoprotein to be detected (hereinafter referred to as “glycoprotein solution”) collected in the step (A) and reacted at 37 ° C. for 30 minutes, for example. Release sialic acid.
Next, the glycoprotein solution after the sialidase reaction is brought into contact with the agarose beads on which the AAL lectin is immobilized, and the multifucose glycoprotein and the AAL lectin contained in the glycoprotein to be detected are bound (the temperature at this time is 20 ° C. to 25 ° C. is preferable.

(C) Process:
(B ′) After the step, the carrier in which the multifucose glycoprotein is bound is washed with, for example, Tris buffer (10 mM, pH 7.5) to remove components other than the multifucose glycoprotein, and then fucose The multifucose glycoprotein is eluted and collected by, for example, contacting with a solution (10 mM Tris-HCl pH 7.5 100 mM Fucose) for 5 minutes.

(D) Process:
The abundance of the recovered multifucose glycoprotein is measured by, for example, a commercially available ELISA kit that can measure the glycoprotein to be detected.
[analysis method]
In the present invention, the amount of multifucose glycoprotein present in the body fluid is measured, but the ratio of the amount of multifucose glycoprotein present to the amount of total glycoprotein present in the body fluid can also be determined. . This method is particularly effective in cases where the amount of multifucose sugar chains increases as a result of increased protein expression even if the abundance ratio of multifucose sugar chains does not change due to inflammation or the like.

In addition, the change in the expression level of multifucose can be expressed more accurately by determining the ratio of the abundance of the multifucose glycoprotein to the abundance of the non-multifucose glycoprotein bound to the antibody in the step (A). Can do. This method is also particularly effective in cases where the amount of multifucose sugar chains increases as a result of increased protein expression even if the abundance ratio of multifucose sugar chains does not change due to inflammation or the like. Here, the glycoprotein having the same protein portion is as described above.

In addition, when the multifucose glycoprotein to be detected is significantly increased or decreased due to the onset or progression of a specific disease, the present invention can detect the onset or progress of the specific disease. For example, when the specific disease is cancer, if necessary, the abundance of the multifucose glycoprotein contained in a body fluid collected from a cancer patient and the body fluid collected from a non-cancer patient By providing a step of comparing the abundance of multifucose glycoprotein and the like, the detection method of the present invention detects the onset and progression of cancer using the abundance of the multifucose glycoprotein contained in body fluid as an index. be able to.

EXAMPLES Next, although an Example demonstrates this invention still in detail, this invention is not limited to a following example, unless the summary is exceeded.
[Reference Example 1]
First, it was confirmed that a single outer fucose sugar chain having no sialic acid does not bind to AAL lectin (manufactured by J-Oil Mills) as follows.

An N-linked sugar chain labeled with pyridylamination (manufactured by Takara Bio Inc., specifically, A4G4Fo1, A2G2, A2G0Fc1, A2G2Fc1, A3G3Fo1, A3G3, A3G3S3Fo1, A3G3S3) was converted into an AAL lectin column (manufactured by J-Oil Mills, (4 mm ID, 150 mm), 2 pmol each, and separated by chromatography, the relationship between the sugar chain structure and the AAL lectin binding was examined.

The conditions for chromatography using an AAL lectin column are shown below. Eluent A solution: 10 mM ammonium acetate aqueous solution, Eluent B solution: 10 mM ammonium acetate, 30 mM fucose aqueous solution (pH = 7) were used. After injecting the sugar chain to be measured, 100% solution A was passed through for 40 minutes, and then the ratio was linearly changed to solution B 100% over 40 minutes (40 to 80 minutes after injection). . The flow rate was 0.4 mL / min. The eluate was collected every 10 minutes, and after concentrating the solution, the presence or absence of binding of each sugar chain to the AAL column was examined by measuring reverse phase liquid chromatography under the following conditions.

(Measurement conditions for reversed-phase liquid chromatography)
Column: Develosil (Nomura Kagaku) 4.6 mm ID × 150 mm
Oven: 30 ° C
Eluent A: 5 mM ammonium acetate (pH = 4)
Eluent B: 10% acetonitrile, 5 mM ammonium acetate (pH = 4)
Gradient: B20% (0 minutes)-B42% (60 minutes)
Flow rate: 0.5 mL / min
Excitation wavelength: 320 nm
Detection wavelength: 400 nm

As a result, in the sugar chain in which one Lewis X-type fucose is attached to three branched chains, the asialo type (A3G3Fo) to which sialic acid is not bonded does not bind to the AAL column, but A3G3S3F0 to which sialic acid is bonded is It was found to bind strongly to the AAL column (see FIG. 1). This indicates that the AAL lectin that recognizes fucose also recognizes sialic acid, which is a new fact.

[Example 1]
A commercially available α1-acid glycoprotein (manufactured by Sigma) was dissolved in urea and Tris buffer (pH 8.5), and the disulfide bond was reductively aminated. Thereafter, peptide fragmentation was performed using trypsin and lysyl endopeptidase, and the following experiment was performed using the resulting glycopeptide.

Experiment I: The obtained glycopeptide derived from α1-acid glycoprotein was bound to a column (manufactured by Varian, 3 mL) on which AAL lectin (manufactured by Vector) was fixed. Next, 10 mM TrisHCL (pH 7.4) solution was passed through to sufficiently elute unbound components from the column, and then 100 mM fucose solution was passed through to recover the AAL lectin-binding glycopeptide. .

Experiment II: The obtained α1-acid glycoprotein-derived glycopeptide was bound to a column on which AAL lectin was immobilized after sialic acid was desorbed using sialidase (manufactured by Nacalai Tex). Next, 10 mM TrisHCL (pH 7.4) solution was passed through to sufficiently elute unbound components from the column, and then 100 mM fucose solution was passed through to recover the AAL lectin-binding glycopeptide. .

The glycopeptides collected in Experiment I and Experiment II were subjected to LC-MS analysis under the following conditions, and the structure and abundance of glycopeptides derived from α1-acid glycoprotein bound to AAL lectin were compared.
LC-MS analysis was performed under the following conditions using Agilent HP1200 (manufactured by Agilent technologies) for liquid chromatography and Q-TOF 6520 (manufactured by Agilent technologies) as a mass spectrometer. As a column for liquid chromatography, inert sill ODS4 (inner diameter 1.5 mm, length 100 mm, particle size 2 μm) was used. As eluent, liquid A: 0.1% formic acid aqueous solution, liquid B: 0.1% formic acid, 90% acetonitrile aqueous solution was used, and the ratio of liquid B changed linearly from 10% to 56% over 40 minutes. After that, the B liquid ratio was maintained at 56% for another 10 minutes. The column oven temperature was 40 ° C. and the flow rate was 0.1 ml / min. Mass spectrometry was performed in a negative mode, and capillary voltage: 4000 V, nebulizer gas amount: 45 psi, dry gas 10 L / min (350 ° C.). The collision energy of MSMS measurement for peptide identification was optimized between 20 eV and 70 eV depending on each peptide.

As a result, as shown in Table 1, Experiment II (which was contacted with AAL lectin after sialic acid was desorbed) was detected in comparison with Experiment I (which did not desorb sialic acid). The percentage of glycopeptides that bind two or more fucose (multi-fucose) contained in the whole glycopeptide greatly increases, while glycopeptides that bind one fucose contained in the whole detected glycopeptide The ratio of (single fucose) was greatly reduced. Thus, when sialic acid is eliminated from the sugar chain of glycoprotein, glycoprotein having single fucose tends to be less likely to bind to AAL lectin, whereas multifucose glycoprotein tends to be more likely to bind to AAL lectin. You can see that

[Example 2]
20 sera collected from hepatocellular carcinoma patients who have obtained informed consent and 10 sera collected from healthy individuals may be referred to as α1-acid glycoproteins having multifucose sugar chains (hereinafter referred to as “AGP”). ) Was present. As will be described later, two types of extraction, a two-stage extraction method and a mass spectrometry method, were used, and the measured values were compared.

(Two-stage extraction method)
A spin-column was filled with 100 μL of avidin-labeled agarose beads, and 50 μg of biotinylated anti-AGP antibody (Abcam) was added. After incubating for 15 minutes to remove unbound antibody, 50 μL of 10-fold diluted serum was added. After incubation at room temperature for 30 minutes, the unbound fraction was washed, and 80 μL of 0.02N hydrochloric acid aqueous solution was added to elute the antibody binding component (AGP glycoprotein). Next, 3 μL of sialidase 1 mU / mL was added to the eluted AGP glycoprotein and reacted at 37 ° C. for 30 minutes to remove sialic acid. The obtained AGP glycoprotein was passed through an AAL column equilibrated with Tris-HCl buffer at pH 7.5, and incubated at room temperature for 30 minutes. Thereafter, 100 μL of 100 mM fucose solution was passed through to elute the AAL binding protein. The eluted AAL-binding protein (considered to be an AGP glycoprotein having a multifucose sugar chain) was quantified with a commercially available AGP measurement kit (manufactured by Abcam).

(Mass spectrometry)
After adding 400 μL of acetone to 100 μL of serum, the mixture was centrifuged at 12,000 rpm for 20 minutes at 4 ° C. to precipitate the protein. After removing the supernatant, a denaturing agent containing urea was added to the precipitate, the protein was denatured, and reductive alkylation was performed. After removing the denaturing agent and reducing agent, trypsin was added to fragment the protein into peptides, and the fucose-containing glycopeptide was concentrated using an AAL lectin column. The prepared fucose-containing glycopeptide was measured under the following conditions using liquid chromatography (Agilent HP1200, manufactured by Agilent Technologies) and a mass spectrometer (Q-TOF 6520, manufactured by Agilent technologies).

As the column for liquid chromatography, inert sill ODS4 (inner diameter 1.5 mm, length 100 mm, particle size 2 μm) was used. As eluent, liquid A: 0.1% formic acid aqueous solution, liquid B: 0.1% formic acid, 90% acetonitrile aqueous solution was used, and the ratio of liquid B changed linearly from 10% to 56% over 40 minutes. After that, the B liquid ratio was maintained at 45% for another 10 minutes. The column oven temperature was 40 ° C. and the flow rate was 0.1 ml / min. Mass spectrometry was performed in a negative mode, and capillary voltage: 4000 V, nebulizer gas amount: 45 psi, dry gas 10 L / min (350 ° C.).

The abundance of multifucose glycoprotein measured by the two-stage extraction method showed a high correlation (R = 0.89) with the abundance of multifucose glycoprotein measured by a mass spectrometer (see FIG. 2). On the other hand, the abundance of the single fucose glycoprotein measured by the two-stage extraction method did not correlate with the abundance of the single fucose glycoprotein measured by the mass spectrometer (R = 0.45) (FIG. 3).

[Comparative Example 1]
An attempt was made to measure AGP glycoprotein having multifucose under the same conditions as in Example 2 (two-step extraction method) except that sialic acid removal by sialidase was not performed.
However, the AGP protein bound strongly to the AAL column and could not be eluted. This is probably because the affinity between sialic acid and the AAL column is very high.
It should be noted that the entire content of the specification, claims, drawings and abstract of Japanese Patent Application No. 2013-043075 filed on March 5, 2013 is cited herein as the disclosure of the specification of the present invention. Incorporated.

Claims (12)

A method for detecting multifucose glycoprotein contained in a body fluid, comprising:
(B) contacting the lectin with a product obtained by eliminating sialic acid that binds to the sugar chains of all glycoproteins contained in the body fluid; and
(D) measuring the abundance of multifucose glycoprotein bound to the lectin,
A detection method characterized by comprising:
However, the multifucose glycoprotein is a glycoprotein having at least one N-linked sugar chain having at least two fucose per sugar chain per molecule of glycoprotein. The detection method according to claim 1, wherein the lectin is AAL lectin (Hirochawantake lectin). The detection method according to claim 1 or 2, wherein in the step (B), sialic acid is eliminated by bringing the total glycoprotein into contact with a sialidase enzyme. Prior to the step (B),
(A) contacting the body fluid with a carrier on which an antibody that recognizes a protein portion of a glycoprotein is immobilized,
And said (B) process,
(B ′) a step of contacting a lectin with a product from which sialic acid bound to the sugar chain of the glycoprotein bound to the carrier in step (A) has been eliminated,
The detection method according to any one of claims 1 to 3, wherein: Between the step (B) or the step (B ′) and the step (D),
(C) eluting multifucose glycoprotein bound to the lectin from the lectin;
The detection method according to any one of claims 1 to 4, characterized by comprising: The step (B ') is performed in the step (A) without desorbing the glycoprotein from the antibody on the glycoprotein bound to the antibody. 6. The detection method according to 5. 5. In the step (A), the glycoprotein bound to the antibody is desorbed from the antibody, and the step (B ′) is performed on the desorbed glycoprotein. Or the detection method of Claim 5. In the step (A),
The number of moles of the multifucose glycoprotein with respect to the number of moles of the antibody, the glycoprotein having the same protein portion as the multifucose glycoprotein, and the sugar chain portion is not a multifucose sugar chain (hereinafter referred to as “non-multiple So that the total number of moles of fucose glycoproteins "
The detection method according to any one of claims 4 to 7, wherein the concentration of the antibody and / or the glycoprotein is adjusted. The step (D) further comprises determining the ratio of the abundance of the multifucose glycoprotein to the abundance of the total glycoprotein contained in the body fluid. The detection method according to item. In the step (D), the ratio of the abundance of the multifucose glycoprotein to the abundance of the non-multifucose glycoprotein bound to the antibody in the step (A) is further obtained. 10. The detection method according to any one of items 9 to 9. The detection method according to any one of claims 1 to 10, wherein the multifucose glycoprotein is significantly increased or decreased by the onset or progression of a specific disease. The specific disease is cancer;
The detection method according to claim 11, wherein cancer is detected using an abundance of the multifucose glycoprotein contained in a body fluid as an index.

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