JP2009098108A - Refinement method of material having sugar chain and utilization of the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for less-invasively identifying or estimating a sugar chain modification due to a disease and a clinical condition related to a variation at an early stage. <P>SOLUTION: An albumin occupying a majority of blood is excluded by less-invasively sampling the blood by using a material for recognizing a particular sugar chain such as lectin, and fractionating and concentrating the blood. The minute quantity of a disease marker is detected by comprehensively fractionating and concentrating only a protein having the particular sugar chain. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention is a method for fractionating a saccharide such as sialic acid (Neu5Ac α2-3Galβ1-4GlcNAc) or fucose or a glycoprotein having such a sugar chain, and the sample state or disease state in a specific sugar chain It relates to a method for identifying. More specifically, the present invention relates to a method for estimating that there is a possibility of a pathological condition related to a specific sugar chain when a specific sugar chain is significantly present as compared with a healthy (non-pathological condition) sample.

There are methods to identify the pathologic state and sample state by analyzing the invasively excised and removed tissues using DNA array or tiling array. However, genome analysis alone translates phosphorylation state and sugar chain modification. It is very difficult to analyze the post-modification.
In general, when a pathological condition such as a malignant tumor is analyzed by biopsy or the like, it is necessary to surgically remove and remove the affected area affected by the malignant tumor tissue. This method is invasive and is very likely to be discovered at a time when the stage of the tumor is advancing.
Considering the fact that if tumors can be discovered before metastasis, they are likely to be completely cured by local surgical operations or chemotherapy such as anticancer drugs, it is particularly important to use samples such as blood collected minimally invasively. It is thought that it can be reflected in the early detection of the pathological condition of the disease and its complete cure.

It is known that sugar chain modification mutations occur with canceration. In fact, prominent cancer antigens such as CA19-9 are sugar chain antigens, and the presence or absence of these is actually used clinically in distinguishing the pathology of healthy individuals and cancer patients. In particular, it is known that sialic acid addition has occurred, but there are few reports of blood fractionation methods focusing on sialic acid accompanying canceration.
An antibody is a substance that selectively binds to a protein having a specific sugar chain, and is actually used in many clinical diagnoses. However, it is not only extremely difficult to produce an antibody against a specific sugar chain, but it often exhibits false positives because there are few antibodies against effective disease markers.

Other substances that selectively bind to proteins with specific sugar chains include lectins, which can be extracted from natural products or synthesized as artificial proteins. Although the binding ability is generally inferior to that of an antibody, it can selectively bind to a specific sugar chain, and thus is suitable for exhaustive and selective analysis.
In addition, most of the proteins collected in a minimally invasive manner are glycoproteins and do not bind to albumin, which is a simple protein occupying 50% or more of them, so that only proteins having specific sugar chains can be efficiently fractionated.
In particular, since many cytokines, hormones, disease markers, etc. are in very small amounts of several ng / ml or less, effective fractionation methods and concentration methods have been sought, but proteins with specific sugar chains are fractionated. This can solve these problems.

  Comparative analysis of proteins is generally performed by fractionation with gels, capillaries, columns, etc., and identified with a mass spectrometer such as MALDI-TOF-MS (Matrix assisted laser desorption time of flight Mass Spectrometer). It is possible to identify a (at) mol level protein in both accuracy and sensitivity.

  A technology that can comprehensively fractionate and comparatively analyze proteins can be expected in screening, clinical diagnosis, and application to drugs, and is expected to discover more important disease markers that could not be screened by genomics.

Although the accuracy and sensitivity of mass spectrometers (MS) have been dramatically improved and protein identification technology has been generalized, the dynamic range of proteins in blood and so on is very wide, and more complex in analysis when post-translational modifications are considered Increase.
In particular, albumin, which is a simple protein in blood, preferentially exists, making comparative analysis difficult, so it is possible to exclude albumin that does not have a sugar chain and fractionate and concentrate only a protein that has a specific sugar chain. If possible, the possibility of identifying a trace amount of a disease marker increases dramatically.

Although there is a method for discriminating the presence or absence of prostate cancer-specific antigen PSA (Japanese Patent Application Laid-Open No. 2002-55108), for example, there has been no report on exhaustive fractionation and concentration of sialic acid using canine endurectin.
Since the method for searching for a disease marker by using a substance that binds to a specific sugar chain (lectin, etc.), for example, fractionation of a protein having a comprehensive specific sugar chain using blood, is immature, the specific sugar chain related to the disease An exhaustive search method is desired.

In addition, even when there is no antibody against a specific sugar chain, a disease state can be identified or estimated by comparing a protein having the specific sugar chain with a healthy sample.
That is, an object of the present invention is to provide a method for identifying or estimating a disease state related to sugar chain modification accompanying a disease minimally invasively and at an early stage.

  As a result of intensive studies on the problems of the prior art, the present inventor has developed a method capable of fractionating and comparing proteins having specific sugar chains in a minimally invasive and comprehensive manner by using proteins that bind to specific sugar chains. I found it.

More specifically, only a protein having a specific sugar chain is fractionated by using lectin, and albumin that occupies most of the blood, which is a simple protein, is separated as an unbound fraction. It is possible to concentrate only a small amount of protein.
Lectin is a general term for proteins that recognize sugar chains, and is widely found in animals and plants and microorganisms. The present invention is characterized in that, for example, a protein having a high concentration of sialic acid is obtained by using a lectin having affinity for sialic acid such as canine endurectin as a support for the affinity column.

  When a sample containing sialic acid is passed through such a lectin affinity column using canine endurectin as a support, only the protein having sialic acid is selectively bound to the lectin (binding step). Next, when the washing solution is passed, contaminants flow out of the lectin, and only the protein having sialic acid is retained as a lectin-binding fraction (washing step). Furthermore, by passing the eluate through a lectin column, the protein having sialic acid in the lectin-binding fraction is eluted, and it becomes possible to obtain a protein having a selective and high concentration of sialic acid (elution step).

By comparing proteins that have been fractionated and concentrated using a lectin affinity column, such as healthy samples and pathological samples, by two-dimensional electrophoresis, etc., it is possible to identify or estimate the pathological condition as a marker related to the pathological condition. become.
This method means that a disease state can be identified or estimated even in the absence of an antibody against the marker.

That is, the present invention
(1) A method for purifying a substance having a sugar chain in a sample by bringing the sample into contact with a lectin and separating a fraction that binds to the lectin, wherein sialic acid is used when the lectin is canine endurectin A method comprising purifying a substance having a sugar chain containing a sugar chain containing fucose when the lectin is a lentil lectin or a yellow bamboo lectin;
(2) The method according to (1), wherein the lectin is immobilized on a support;
(3) The method according to (2), wherein the support is at least one selected from the group consisting of sepharose, agarose, and dextran;
(4) The method according to any one of (1) to (3), wherein the step of bringing the sample into contact with the lectin and separating the fraction bound to the lectin is performed by affinity chromatography;
(5) The method according to any one of (1) to (4), wherein the substance having a sugar chain is a glycoprotein;
(6) The sample according to (1) to (5), wherein the sample is at least one selected from the group consisting of blood, serum, plasma, cell extract, urine, lymph, tissue fluid, ascites, spinal fluid, and body fluid The method according to any one of the above;
(7) The method according to any one of (1) to (6), wherein the sample is derived from a healthy person and / or a diseased patient;
(8) A step of separating a substance having a sugar chain purified by the method according to any one of (1) to (7) by two-dimensional electrophoresis, and a result obtained in the step, Comparing between samples;
A method for detecting a substance having a sugar chain containing sialic acid or fucose, the expression level of which differs between samples,
(9) a step of identifying a substance detected by the method according to (8) with a mass spectrometer,
A method for identifying a substance having a sugar chain containing sialic acid or fucose, the expression level of which differs between samples,
(10) The mass spectrometer is at least one selected from the group consisting of MALDI-TOF-MS, ESI-MS, SELDI-MS, LC-MS, Q-TOF-MS, and membrane MS. ) Method described in;
(11) A method for searching for a disease marker,
(9) A method for searching using a result obtained by the method according to any one of (10);
(12) A method for estimating a disease state,
(9) A method for inferring using the result obtained by the method according to any one of (10);
About.

  In the present invention, for example, according to a method for fractionating a specific sugar chain (such as sialic acid) in blood, regardless of the presence or absence of an antibody against the specific sugar chain, the pathological condition associated with the modification of the specific sugar chain can be minimally invasive and early. Can be identified or estimated.

  More specifically, a large amount of albumin that makes comparative analysis in blood difficult can be excluded, and only a protein with a specific sugar chain can be fractionated and concentrated. It is expected to have a tremendous effect on the search and comparative analysis.

  Therefore, the present invention not only greatly contributes to clinical diagnosis as a fractionation method and a disease state identification method that have largely solved the problems of the prior art, but also an antibody drug and a sugar chain drug targeting specific sugar chains involved in diseases. There is great expectation to contribute to the development of the pharmaceutical field.

  Hereinafter, preferred embodiments of the present invention will be specifically described.

  The substance capable of binding to the specific sugar chain used in the present invention is not limited as long as it can identify the presence or absence of sialic acid (Neu5Ac α 2-3Gal β 1-4GlcNAc), fucose, etc. Particularly suitable are lectins derived from canaencia (Mackia amurensis), lentil (Lens culinaris), and Aleuria aurantia.

  Samples containing sialic acid (Neu5Acα2-3Galβ1-4GlcNAc) and fucose used in the present invention include blood, serum, plasma, urine, body fluid, cell extract, lymph fluid, tissue fluid, ascites, cerebrospinal fluid, etc. Can be mentioned.

Sample fractionation is performed by affinity chromatography. Basically, a support obtained by crosslinking a substance that binds to a specific sugar chain is used as a stationary phase. The order in which these substances are used or the mixing ratio is not particularly specified.
The packed carrier as the support is preferably sepharose, agarose, dextran, etc. The shape of the carrier may include all shapes such as beads, sheets, and meshes.
As the crosslinking conditions, an appropriate amount of lectin and NHS carrier (N-hydroxysuccinylated activated Sepharose: GE Healthcare) are mixed in a HEPES solution and rotated. After crosslinking, unreacted groups are blocked with a Tris solution, equilibrated again with a HEPES solution, and used as a lectin column.
An appropriate amount of a blood sample or cell extract is added to the prepared lectin column and allowed to bind overnight, the unbound fraction is washed out with the binding solution, and the bound fraction is eluted with glycine.

  After further concentrating the eluted bound fraction, the amount of protein is quantified, and a few hundred μg of that is compared and analyzed by two-dimensional electrophoresis to detect proteins that differ from healthy samples.

  It is desirable to identify a protein having a difference with a mass spectrometer (MS), and it can be determined that the identified protein is very likely to be involved in a disease state.

  In the present invention, the substance having a sugar chain may be any substance having a sugar chain, such as a glycoprotein, a glycolipid, and a free sugar chain.

  In the present invention, as the sample, both a healthy subject and a diseased patient can be used, but only one of them can be used. The sample derived from a patient with a disease is not limited to a person apparently having a disease, but also includes a sample derived from a person with a possibility of a disease. Further, samples collected from a plurality of persons may be mixed.

  Examples of the disease in the present invention include cancer, lifestyle-related diseases such as diabetes, and metabolic syndrome.

In the present invention, a disease marker refers to a marker that can be used to estimate the progression or pathology of a specific disease based on the quantitative or qualitative difference.
In the present invention, a method for searching for a disease marker is performed by purifying a protein by lectin column chromatography using a sample derived from a healthy subject and a sample derived from a patient having a specific disease, and separating the protein by two-dimensional electrophoresis. Compare the results. Therefore, a spot detected only in a patient sample or a spot darker than a spot in a healthy sample is selected, and such a protein having a difference in expression level is identified by a mass spectrometer. From the identified results, a novel disease marker candidate related to a specific disease is extracted. As an object to be compared, it can be performed between a healthy person and a patient having a specific disease, or can be performed between patients having a specific disease with different degrees of progression.
The disease marker obtained by using the present invention can be used not only for diagnosis and drug discovery but also as an index for estimating a specific disease state.

  In the present invention, the method for estimating the pathological condition uses a sample derived from a patient and compares it with a sample derived from a healthy person or a patient having a different pathological condition when it is desired to examine in detail what pathological condition a patient has. Thus, proteins having different expression levels are detected and identified. If the protein thus identified is clearly related to a specific disease state including the aforementioned marker, the specific disease state can be estimated from the information.

The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited thereto.
1. Comparison between unfractionated and canine-end-treated fractions Normal human serum was brought into contact with a lectin column prepared by cross-linking canine endurectin with NHS carrier, and only the bound fraction was eluted with glycine and concentrated.
Thereafter, two-dimensional electrophoresis was performed using the same amount of serum from a healthy person who had not been treated and a serum sample containing a protein having sialic acid (Neu5Acα2-3Galβ1-4GlcNAc) on a canine endurectin column.
As a result, not only did albumin, which is the biggest obstacle that makes comparative analysis difficult, was successfully excluded, but protein groups that could not be detected with untreated serum were clearly detected as spots (FIG. 1).
These results indicate that fractionation and concentration with canine endurectin is very useful for blood treatment.

2. Comparison of unfractionated and lentil-treated fractions Normal human serum was brought into contact with a lectin column prepared by crosslinking lentil lectin to NHS carrier, and only the bound fraction was eluted with glycine and concentrated.
Thereafter, two-dimensional electrophoresis was performed with the same amount of serum from a healthy person who had not been treated and a serum sample containing a protein having fucose in a lentil lectin column.
As a result, not only was the albumin, which was the biggest obstacle making comparative analysis difficult, successfully excluded, but a protein group that could not be detected by untreated serum was clearly detected as spots (FIG. 2).
These results indicate that fractionation and concentration with lentil lectin is very useful for blood treatment.

  According to the present invention, it is possible to exhaustively purify a protein having a specific sugar chain. If the purification method of the present invention is used, it is useful for clinical diagnosis of cancers and diseases caused by sugar chain mutations or modifications, and development of antibody drugs targeting proteins with specific sugar chains.

  It is a photograph which shows the result of the two-dimensional electrophoresis of the protein in the untreated healthy serum sample (A) and the healthy serum sample (B) fractionated with canine endlectin.   It is a photograph which shows the result of the two-dimensional electrophoresis of the protein in an untreated healthy serum sample (A) and the healthy serum sample (B) fractionated with lentil lectin.

Claims (12)

A method for purifying a substance having a sugar chain in a sample by contacting the sample with a lectin and separating a fraction that binds to the lectin, and when the lectin is canine endurectin, a sugar containing sialic acid A method comprising purifying a substance having a chain, and a substance having a sugar chain containing fucose when the lectin is a lentil lectin or a yellow bamboo lectin. The method according to claim 1, wherein the lectin is immobilized on a support. The method according to claim 2, wherein the support is at least one selected from the group consisting of sepharose, agarose, and dextran. The method according to any one of claims 1 to 3, wherein the step of contacting the sample with the lectin and separating the fraction bound to the lectin is performed by affinity chromatography. The method according to any one of claims 1 to 4, wherein the substance having a sugar chain is a glycoprotein. The sample according to any one of claims 1 to 5, wherein the sample is at least one selected from the group consisting of blood, serum, plasma, cell extract, urine, lymph, tissue fluid, ascites, spinal fluid, and body fluid. The method described. The method according to any one of claims 1 to 6, wherein the sample is derived from a healthy person and / or a diseased patient. A step of separating a substance having a sugar chain purified by the method according to any one of claims 1 to 7 by two-dimensional electrophoresis, and comparing a result obtained in the step among a plurality of samples The process of
A method for detecting a substance having a sugar chain containing sialic acid or fucose, the expression level of which differs between samples. Identifying the substance detected by the method according to claim 8 with a mass spectrometer,
A method for identifying a substance having a sugar chain containing sialic acid or fucose and having a difference in expression level between samples. The mass spectrometer is at least one selected from the group consisting of MALDI-TOF-MS, ESI-MS, SELDI-MS, LC-MS, Q-TOF-MS, and membrane MS. the method of. A method for searching for a disease marker,
The search method using the result obtained by the method of any one of Claims 9-10. A method of guessing the pathology,
The method of estimating using the result obtained by the method of any one of Claims 9-10.

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