JP2011195744A - Substrate for entrapping acidic-sugar chain - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a substrate for entrapping a sugar chain that can immobilize an acidic sugar on the surface thereof and can stably entrap the acidic sugar only on the surface thereof by suppressing nonspecific adsorption of a substance other than the entrapped acidic sugar.SOLUTION: The substrate for immobilizing an acidic sugar is prepared by forming on one surface thereof a resin layer made of a copolymer of a monomer having a hydrophilic group, a monomer having a functional group capable of entrapping the acidic sugar, and a monomer having a hydrophobic cyclic alkyl group.

Description

  The present invention includes a method of capturing acidic saccharides on a substrate and the substrate.

In vivo, glycoproteins and glycopeptides exist on the surface of cell membranes and function as cell membrane receptors, are secreted into body fluids such as serum, and exist as components of extracellular matrix. In recent years, it has been revealed that these sugar chains play an important role in biological activities.
Once the relationship between such sugar chains and cell differentiation and proliferation, cell recognition, immune response, and cell carcinogenesis is clarified, this sugar chain is closely related to cell engineering or organ engineering, and a new sugar It is expected to develop chain engineering.
The most abundant saccharides in the body are glycosaminoglycans (GAGs), which are molecules that exist only in animals, not in plants, and may exist in any connective tissue in the body. Are known.

GAGs include heparin, heparan sulfate, hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratan sulfate, etc., mainly present in animal connective tissues, and heparin is famous as an anticoagulant. It is used for medicines.
GAGs are known to have specific interactions with various proteins in vivo, thereby regulating biological functions.
GAGs are formed from a repeating structure of disaccharides with sulfate groups added, and are many negatively charged molecules because they contain many sulfate groups, and are well known to be acidic sugars. .
Therefore, investigating the interaction between these acidic sugars and proteins and verifying functional adjustment is not only an academic meaning, but is an important theme for the development of medicine.
In order to investigate this, it is important to first fix each acidic sugar molecule on the surface of the substrate and investigate what kind of protein binds to it.

Patent Document 1 describes a method for immobilizing GAGs on the surface of a substrate using the characteristics. In Patent Document 1, in order to capture GAGs, a monomer having an amino group is plasma-polymerized on the substrate surface to form a substrate surface rich in positive electrification, and GAGs are electrostatically interacted with it. A method for immobilization is described. A substrate having a positive charge on the surface can be constructed by a single reaction, and according to this method, a positive charge can be easily introduced on the surface of the substrate. However, in this method, not only electrostatic interaction but also non-specific adsorption to the substrate body occurs at the same time, so that a blocking operation is required.

In Patent Document 2, an aldehyde prepared by reducing a sugar chain is reacted with a linker compound composed of H 2 N—X—SH (where X is an alkylene group having 1 to 3 carbon atoms or a phenylene group) or the like. To immobilize sugar chains on the solid surface. Furthermore, Patent Document 2 also mentions a method for measuring the interaction between a saccharide and a protein with a breaking force. However, in this method, since the sugar chain is reduced, a part of the sugar chain structure is changed, and the solid surface is made to be an apprentice in that state. is there. Moreover, measuring the interaction with the breaking force is one of the methods, and it is difficult to measure the function of the sugar chain alone.

Special table 2006-504822 JP2008-105978

Provided is a substrate capable of stably capturing only acidic sugar on the substrate surface by immobilizing acidic sugar on the substrate surface and suppressing non-specific adsorption.

  Such an object is achieved by the present invention described in the following (1) to (10).

（式中Ｒ１、Ｒ２、Ｒ３は水素原子またはメチル基を、Ｒ４は疎水性基を示す。Ｘは炭素数１〜１０のアルキレンオキシ基を示し、ｐは１〜２０の整数を示す。ｐが２以上２０以下の整数である場合、繰り返されるＸは、同一であっても、または異なっていてもよい。Ｙはアルキレングリコール残基を含むスペーサーであり、Ｚは酸素原子である。ｌ、ｍ、ｎは自然数である。）
（５）前記親水性を保持するためのモノマーがホスホリルコリン基を含む（４）に記載の酸性糖捕捉基材。
（６）前記疎水性基、Ｒ４が環状アルキル基である（４）に記載の酸性糖捕捉基材。
（７）前記環状アルキル基がシクロヘキシル基である（６）に記載の酸性糖捕捉基材。
（８）前記酸性糖が、グリコサアミノグリカン、ヘパリン、ヘパラン硫酸、デルマタン硫酸、ヒアルロン酸から選択される少なくとも一つの酸性糖である（１）−（７）いずれか記載の酸性糖捕捉基材。
（９）（１）−（８）いずれか記載の捕捉基材であって、
（ａ）固相表面に官能基を含む高分子物質をコートする工程
（ｂ）酸性糖を有する物質を正電荷と反応させて、表面に固相化する工程
からなる、酸性糖捕捉方法。
（１０）（９）に記載の捕捉方法であって、酸性糖を捕捉後、乾燥させる工程を含む酸性糖捕捉方法
(1) An acidic sugar capturing base material for capturing acidic sugar,
A monomer having a hydrophilic group;
A monomer having a functional group capable of capturing the acidic sugar;
A resin layer composed of a copolymer of monomers having a hydrophobic group,
An acidic sugar-capturing base material that is formed.
(2) The acidic sugar capturing substrate according to (1), wherein the functional group of the monomer having a functional group capable of capturing the acidic sugar is a primary amino group.
(3) The acidic sugar capturing substrate according to (2), wherein the primary amino group is an aminooxyl group.
(4) The acidic sugar capturing substrate according to (1), wherein the copolymer is a copolymer represented by the following general formula [1].

(Wherein R1, R2 and R3 represent a hydrogen atom or a methyl group, R4 represents a hydrophobic group, X represents an alkyleneoxy group having 1 to 10 carbon atoms, and p represents an integer of 1 to 20. p is an integer. When X is an integer of 2 or more and 20 or less, the repeated Xs may be the same or different, Y is a spacer containing an alkylene glycol residue, and Z is an oxygen atom. , N is a natural number.)
(5) The acidic sugar-trapping substrate according to (4), wherein the monomer for maintaining hydrophilicity contains a phosphorylcholine group.
(6) The acidic sugar-trapping substrate according to (4), wherein the hydrophobic group and R4 are cyclic alkyl groups.
(7) The acidic sugar-trapping substrate according to (6), wherein the cyclic alkyl group is a cyclohexyl group.
(8) The acidic sugar capturing substrate according to any one of (1) to (7), wherein the acidic sugar is at least one acidic sugar selected from glycosaminoglycan, heparin, heparan sulfate, dermatan sulfate, and hyaluronic acid. .
(9) The capture substrate according to any one of (1) to (8),
(A) A step of coating a solid phase surface with a polymer substance containing a functional group (b) A method of capturing an acidic sugar, comprising a step of reacting a substance having an acidic sugar with a positive charge to solidify the surface.
(10) The capturing method according to (9), wherein the capturing method includes the step of drying after capturing the acidic sugar.

  According to the present invention, acidic sugar can be efficiently and easily captured on a solid phase carrier, and nonspecific adsorption of foreign substances and other substances that react with the acidic sugar can be effectively suppressed. Therefore, it is possible to manufacture and evaluate a simple and high-throughput test container or biochip.

The present invention provides a method for capturing acidic sugars as follows:
(A) a functional group capable of capturing an acidic sugar and a polymeric substance having hydrophilicity at the same time are introduced into the base material; (b) the acidic sugar is captured via the functional group;
(C) It is characterized by suppressing non-specific adsorption due to hydrophilicity.

Specifically, in order to acquire hydrophilicity, an ethylenically unsaturated polymerizable monomer having a phosphorylcholine residue, an ethylenically unsaturated polymerizable monomer having a functional group, and a monomer having a hydrophobic group are copolymerized. Use the resulting polymeric material.
The hydrophilic unit contained in the polymer compound of the present invention is represented by a phosphorylcholine group, and the structure is not particularly limited. However, the unit of the following general formula [1] is a component of the left part of the structural unit. As shown by the unit, it is most preferable that the (meth) acrylic residue and the phosphorylcholine group are bonded via a chain of an alkyleneoxy group X having 1 to 10 carbon atoms. Of these, X is most preferably an ethyleneoxy group. The repeating number of the alkyleneoxy group in the formula is an integer of 1 to 20, and when the repeating number is 2 or more and 20 or less, the carbon number of the repeating alkyleneoxy group may be the same or different. Although l is naturally a natural number, it may be expressed as a composition ratio of each component.

本発明の高分子化合物に含まれるホスホリルコリン基を有するユニットの組成割合は（ｌ，ｍ，ｎの和に対するｌの比率）は、高分子の全ユニットに対して、５〜９８ｍｏｌ％が好ましくより好ましくは１０〜８０ｍｏｌ％、最も好ましくは、１０〜８０％である。組成比が下限値を下回ると親水性が弱くなり非特異吸着が多くなる。一方、上限値を上回ると水溶性が高まり、アッセイ中に高分子化合物が溶出してしまう可能性がある。

The composition ratio of the unit having a phosphorylcholine group contained in the polymer compound of the present invention (ratio of l to the sum of l, m, n) is preferably 5 to 98 mol% with respect to all units of the polymer. Is 10 to 80 mol%, most preferably 10 to 80%. When the composition ratio is below the lower limit, the hydrophilicity becomes weak and non-specific adsorption increases. On the other hand, when the upper limit is exceeded, water solubility increases, and the polymer compound may be eluted during the assay.

The composition ratio of the unit having a hydrophobic group contained in the polymer compound of the present invention (ratio of m to the sum of l, m, and n) is preferably 10 to 90 mol% with respect to all units of the polymer. Preferably it is 10-80 mol%, Most preferably, it is 20-80%. If the upper limit is exceeded, non-specific adsorption may increase.

The unit having a primary amino group contained in the polymer compound of the present invention is not particularly limited in structure, but as represented by the structural unit on the right side of the structural unit of the general formula [1], A structure in which a spacer Y containing a (meth) acrylic residue and an oxylamino residue is resolved is preferable. In the case of an oxylamino group, Z represents an oxygen atom, and in the case of a hydrazide group, Z represents NH. n is originally a natural number, but may be expressed as a composition ratio of each component. The structure of the spacer Y containing an alkylene glycol residue is not particularly limited, but is preferably the following general formula [2] or [3], more preferably [2]. q or r is a natural number, preferably 1-20.

各成分の組成割合は（ｌ，ｍ，ｎの和に対するｎの比率）は、高分子化合物の全ユニットに対して、１〜９４ｍｏｌ％が好ましくより好ましくは２〜９０ｍｏｌ％、最も好ましくは、２０〜４０ｍｏｌ％である。組成値が下限地を下回ると糖、糖鎖、および／またはこれらを有する生理活性物質を十分量固定化できなくなる。また、上限値を上回ると非特異吸着が増加する。

The composition ratio of each component (ratio of n to the sum of l, m, n) is preferably 1 to 94 mol%, more preferably 2 to 90 mol%, most preferably 20 to the total unit of the polymer compound. ˜40 mol%. When the composition value falls below the lower limit, it is impossible to immobilize a sufficient amount of sugar, sugar chain, and / or physiologically active substance having these. Moreover, when it exceeds an upper limit, nonspecific adsorption | suction increases.

The method for synthesizing the polymer compound of the present invention is not particularly limited, but at least a monomer having a phosphorylcholine group, a monomer having a hydrophobic group, and a primary amino group are previously protected with a protecting group for ease of synthesis. A production method comprising a step of radical copolymerization of the monomer obtained and a step of removing a protecting group from the polymer compound obtained by the step is preferred. Alternatively, a step of radical copolymerizing a monomer having at least a phosphorylcholine group, a monomer having a hydrophobic group, and a monomer having a functional group capable of introducing a primary amino group, and the polymer compound obtained by the step having a primary amino group The manufacturing method including the step of introducing is preferable.

  The monomer having a phosphorylcholine group is not particularly limited in structure. For example, 2- (meth) acryloyloxyethyl phosphorylcholine, 2- (meth) acryloyloxyethoxyethyl phosphorylcholine, 6- (meth) acryloyloxyhexyl phosphorylcholine, 10 -(Meth) acryloyloxyethoxynonyl phosphorylcholine, 2- (meth) acryloyloxypropyl phosphorylcholine and the like can be mentioned, and 2-methacryloyloxyethyl phosphorylcholine is preferable from the viewpoint of availability.

    Specific examples of the monomer having a hydrophobic group include n-butyl (meth) acrylate, iso-butyl (meth) acrylate, sec-butyl (meth) acrylate, t-butyl (meth) acrylate, n-neopentyl ( (Meth) acrylate, iso-neopentyl (meth) acrylate, sec-neopentyl (meth) acrylate, neopentyl (meth) acrylate, cyclohexyl (meth) acrylate, n-hexyl (meth) acrylate, iso-hexyl (meth) acrylate, heptyl ( (Meth) acrylate, n-octyl (meth) acrylate, iso-octyl (meth) acrylate, 2-ethylhexyl (meth) acrylate, n-nonyl (meth) acrylate, iso-nonyl (meth) acrylate, n-decyl (meth) Acrylate is o-decyl (meth) acrylate, n-dodecyl (meth) acrylate, iso-dodecyl (meth) acrylate, n-tridecyl (meth) acrylate, iso-tridecyl (meth) acrylate, n-tetradecyl (meth) acrylate, iso -Tetradecyl (meth) acrylate, n-pentadecyl (meth) acrylate, iso-pentadecyl (meth) acrylate, n-hexadecyl (meth) acrylate, iso-hexadecyl (meth) acrylate, n-octadecyl (meth) acrylate, iso-octadecyl (Meth) acrylate, isobonyl (meth) acrylate, etc. are mentioned.

Among these, a cyclic polyolefin group represented by cyclohexyl (meth) acrylate is particularly preferable, and cyclohexyl (meth) acrylate is most preferable.

The structure of the monomer in which the primary amino group is previously protected with a protecting group is not particularly limited, but the following general formula [4] (wherein R 3 is a hydrogen atom or a methyl group, Y is a spacer containing an alkylene glycol residue) , Z represents an oxygen atom or NH, and W represents a protecting group.) As represented by (meth) acrylic group and oxylamino group or hydrazide group via a spacer Y containing an alkylene glycol residue. A structure is preferred.

保護基Ｗ としてはアミノ基を保護基できるものであれば何ら制限を受けるものではなく、任意に用いることができる。なかでもｔ―ブトキシカルボニル基（Ｂｏｃ基）やベンジロキシカルボニル基（Ｚ基、Ｃｂｚ基） 、９−フルオレニルメトキシカルボニル基（Ｆｍｏｃ基） などが好適に用いられる。

The protecting group W 1 is not limited as long as it can protect an amino group, and can be arbitrarily used. Of these, t-butoxycarbonyl group (Boc group), benzyloxycarbonyl group (Z group, Cbz group), 9-fluorenylmethoxycarbonyl group (Fmoc group) and the like are preferably used.

  Specific examples of the monomer are those represented by the following formula.

  Deprotection can be performed under general conditions using trifluoroacetic acid, hydrochloric acid, or anhydrous hydrogen fluoride.

  On the other hand, the method for introducing a primary amino group after polymerizing a polymer compound is not limited at all, but at least a monomer having a phosphorylcholine group, a monomer having a hydrophobic group, and a monomer having an alkoxy group are radicalized. A method of producing a hydrazide group by reacting an alkoxy group introduced into the polymer compound with hydrazine after copolymerization is preferred. As the alkoxy group, a methoxy group, an ethoxy group, a propoxy group, a t-butoxy group and the like are preferable.

  Specific examples of the monomer having an alkoxy group are those represented by the following formula.

As the synthetic solvent for the polymer compound of the present invention, any solvent may be used as long as each monomer can be dissolved. For example, alcohols such as methanol, ethanol, isopropanol, n-butanol, t-butyl alcohol, and n-pentanol are used. , Benzene, toluene, tetrahydrofuran, dioxane, dichloromethane, chloroform, cyclohexanone, N, N-dimethylformamide, dimethyl sulfoxide, methyl acetate, ethyl acetate, butyl acetate, methyl ethyl ketone, methyl butyl ketone, ethylene glycol monoethyl ether, ethylene glycol monomethyl ether And ethylene glycol monobutyl ether. These solvents are used alone or in combination of two or more.

    The polymerization initiator may be any ordinary radical initiator, such as 2,2′-azobisisobutylnitrile (hereinafter referred to as “AIBN”), 1,1′-azobis (cyclohexane-1-carbonitrile), etc. Examples thereof include organic peroxides such as azo compounds, benzoyl peroxide, and lauryl peroxide.

  The molecular weight of the polymer compound of the present invention is preferably 5,000 or more and more preferably 10,000 or more because the polymer compound and unreacted monomer can be easily separated and purified.

Deprotection can be performed under general conditions by using the above-described trifluoroacetic acid, hydrochloric acid, and anhydrous hydrogen fluoride, but the timing of deprotection is as follows.
Usually, the polymerization is completed and the polymer compound is generally prepared. In order to obtain the polymer compound of the present invention, the polymer substance is removed by deprotection after the polymerization is completed. Obtainable.

On the other hand, in the present invention, it is possible to easily impart the property of suppressing the nonspecific adsorption of the physiologically active substance and the property of immobilizing the physiologically active substance by coating the polymer surface on the substrate surface. It is shown that.

In this case, it is possible to coat a polymer compound having a primary amino group which has been deprotected, but in some cases, coating a polymer material having a highly reactive primary amino group in a solution may be It is conceivable that the primary amino group reacts and inactivates during the work.
Therefore, it is desirable to purify the polymer compound immediately before deprotection, coat the surface of the substrate, and then perform a deprotection reaction to form a state in which a primary amino group is present on the surface of the substrate.

For coating the base material surface with a polymer compound, for example, a polymer solution in which a polymer compound is dissolved in an organic solvent so as to have a concentration of 0.05 to 50% by weight is prepared, and a known method such as immersion or spraying is used. After coating on the surface of the substrate, drying is performed at room temperature or under heating.

    Examples of organic solvents include ethanol, methanol, isopropanol, n-butanol, t-butyl alcohol, n-pentanol, cyclohexanol, and other alcohols, benzene, toluene, tetrahydrofuran, dioxane, dichloromethane, chloroform, acetone, methyl acetate, ethyl acetate, Examples thereof include butyl acetate, methyl ethyl ketone, methyl butyl ketone, ethylene glycol monoethyl ether, ethylene glycol monomethyl ether, ethylene glycol monobutyl ether, and cyclohexanone. These solvents are used alone or in combination of two or more. Of these, alcohols such as ethanol, methanol, isopropanol, n-butanol, t-butyl alcohol, and n-pentanolcyclohexanol are preferable because they do not denature the plastic substrate and can be easily dried.

(Substrate material and shape)
In the present invention, glass, plastic, metal or the like can be used as the material for the base material to which the copolymer is applied. However, the base material used in the present invention is from the viewpoint of ease of surface treatment and mass productivity. Plastic is preferred. For example, it is more preferable to use linear polyolefins such as polyethylene, polypropylene, polypentene, polycarbonate, polystyrene, polyamide, and saturated cyclic polyolefin. Examples of the shape of the substrate used in the present invention include a plate-like substrate typified by a slide glass, a multi-well plate shape typified by 96 holes and 384 holes, a bead shape, or a composite thereof. Specific examples of the plastic substrate include substrates made of polystyrene, cycloolefin polymer, polypropylene, polyethylene, polysulfone, polyimide, polycarbonate, and polymethyl methacrylate.

(Capturing acidic sugar)
In the present invention, as a method for capturing acidic sugar, a solution containing acidic sugar is added to the base material prepared as described above, and the primary amine and acidic sugar built on the base material are coordinated by ionic bonds. This can be achieved.

  Examples of the acidic sugar chain include glycosaminoglycan, heparin, heparan sulfate, dermatan sulfate, and hyaluronic acid. You may capture | acquire the acidic sugar chain which consists of those 1 type or a combination of multiple types simultaneously.

The solution used in this case is not particularly limited. For example, physiological saline, phosphate buffer, carbonate buffer, HEPES buffer, MOPS buffer, Tris hydrochloric acid buffer, and the like can be used.
The type of the solution is not limited, but the interaction between the primary amino group and the acidic sugar of the base material shows a dependency on the pH value of the solution, and the lower the solution pH, the more the primary amino group and the acidic sugar This interaction is advantageous. Specifically, pH 6 or less is preferable, and pH 4 or less is more preferable.

  In addition, the capture of acidic sugar by the capture base of this patent increases the bond between the base and the acidic sugar by drying in a state where the acidic sugar is captured by the base after capture of the acidic sugar. This effect can be an effective means for producing, for example, a sugar chain array.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example and a comparative example, this invention is not limited to this.

<Example 1> Immobilization of acidic polysaccharide (96 well plate)
(Production of sugar chain-trapping substrate)
A 96-well plate was used as a substrate on which the acidic sugar chain-trapping polymer material was formed on the surface. The 96-well plate used was molded from a cyclic polyolefin resin at Sumitomo Bakelite.

2-methacryloyloxyethyl phosphorylcholine-butylmethacrylate-N- [2- [2- [2- (t-butoxycarbonylaminooxy-acetylamino) ethoxy] ethoxy] ethyl] -methacrylamide as acidic sugar chain-trapping polymer Using a polymer, 150 uL of a 1.0 wt% ethanol solution of the polymer compound is dispensed into each well of a 96-well plate and allowed to stand for 30 minutes, then the solution is removed and dried, and the solvent is evaporated to the substrate surface. Applied.

  After drying, 2M HCl was dispensed and treated at 37 ° C. for 2 hours to prepare a 96-well plate having a Boc group deprotected and an acidic sugar chain-trapping polymer compound on the surface.

(Acid sugar chain immobilization)
Heparin-derived oligosaccharide (Iduron HO16) was adjusted to 10 μg / mL with 300 mM sodium acetate buffer (pH 5.0) to prepare an acidic polysaccharide solution. 100 μL of the prepared solution was dispensed into the 96-well plate for capturing sugar chains and reacted at room temperature for 2 hours. After the reaction, it was washed with pure water to remove unreacted acidic polysaccharide.

(Detection of fixed sugar chains)
FGF2 (Peprotech, 100-18B), which has been reported to bind to heparin, was prepared to 25 ng / mL with the following solvent.
Solvent: 50 mM Tris.HCl (pH 7.5), 100 mM NaCl, 1 mM CaCl2, MnCl2, MgCl2, 0.05% Tween20.

100 μL each was dispensed to each of the wells to which the acidic sugar chain had been immobilized and the wells to which the acidic sugar chain had not been immobilized, and reacted at room temperature for 2 hours.
After the reaction, the well was washed three times with the following washing solution to remove unreacted FGF2.

Washing solution: 50 mM Tris.HCl (pH 7.5), 100 mM NaCl,
1 mM CaCl2, MnCl2, MgCl2,
0.05% Triton X-100

Anti-FGF2 antibody (Peprotech, 500-M38) was prepared to 2 μg / mL with the following solvent.
Solvent: 50 mM Tris.HCl (pH 7.5), 100 mM NaCl, 1 mM CaCl2, MnCl2, MgCl2, 0.05% Tween20.

  100 μL of the antibody solution was dispensed into each well and reacted at room temperature for 1 hour. After completion of the reaction, each well was washed three times with a washing solution.

An HRP-labeled anti-mouse IgG antibody (DakoCytomation, P0260) was prepared at 1.3 μg / mL with the following solvent.
Solvent: 50 mM Tris.HCl (pH 7.5), 100 mM NaCl, 1 mM CaCl2, MnCl2, MgCl2, 0.05% Tween20.

  100 μL of the antibody solution was dispensed into each well and reacted at room temperature for 1 hour. After completion of the reaction, each well was washed three times with a washing solution.

In order to measure the amount of peroxidase immobilized, the color was developed with TMB (Bio-Rad, 172-1067) for 15 minutes, and the absorbance at a measurement wavelength of 450 nm was measured with a microplate reader (Infinit 200 manufactured by TECAN).
The results are shown in the table below.

<Example 2> Immobilization method of acidic polysaccharide (production of sugar chain-trapping substrate)
A 96-well plate was used as a substrate on which the acidic sugar chain-trapping polymer material was formed on the surface. The 96-well plate used was molded from a cyclic polyolefin resin at Sumitomo Bakelite.

2-methacryloyloxyethyl phosphorylcholine-butylmethacrylate-N- [2- [2- [2- (t-butoxycarbonylaminooxy-acetylamino) ethoxy] ethoxy] ethyl] -methacrylamide as acidic sugar chain-trapping polymer Using a polymer, 150 uL of a 1.0 wt% ethanol solution of the polymer compound is dispensed into each well of a 96-well plate and allowed to stand for 30 minutes, then the solution is removed and dried, and the solvent is evaporated to the substrate surface. Applied.
After drying, 2M HCl was dispensed and treated at 37 ° C. for 2 hours to prepare a 96-well plate having a Boc group deprotected and an acidic sugar chain-trapping polymer compound on the surface.

(Acid sugar chain immobilization)
Chondroitin sulfate (Seikagaku Biobusiness, 400650) was immobilized by two methods. First, chondroitin sulfate was adjusted to 50 μg / mL with 300 mM sodium acetate buffer (pH 5.0), and 100 μL was dispensed into a 96-well plate and reacted at room temperature for 2 hours. Next, chondroitin sulfate was adjusted to 50 μg / mL with a solution of 2% AcOH / CH 3 CN: water = 75: 25, dispensed 100 μL into another well, and reacted at 80 ° C. for 1 hour.
After the reaction, it was washed with pure water to remove unreacted acidic polysaccharide.

(Detection of fixed sugar chains)
Anti-chondroitin sulfate antibody (Seikagaku Biobusiness Co., Ltd., 370710) was prepared to 125 ng / mL with the following solvent.
Solvent: 50 mM Tris.HCl (pH 7.5), 100 mM NaCl, 1 mM CaCl2, MnCl2, MgCl2, 0.05% Tween20.

  100 μL of the antibody solution was dispensed into each well and reacted at room temperature for 1 hour. After completion of the reaction, each well was washed three times with a washing solution.

Washing solution: 50 mM Tris.HCl (pH 7.5), 100 mM NaCl,
1 mM CaCl2, MnCl2, MgCl2,
0.05% Triton X-100

An HRP-labeled anti-mouse IgM antibody (SANTA CRUZ, SC-2873) was prepared to 200 ng / mL with the following solvent.
Solvent: 50 mM Tris.HCl (pH 7.5), 100 mM NaCl, 1 mM CaCl2, MnCl2, MgCl2, 0.05% Tween20.

  100 μL of the antibody solution was dispensed into each well and reacted at room temperature for 1 hour. After completion of the reaction, each well was washed three times with a washing solution.

  In order to measure the amount of peroxidase immobilized, the color was developed with TMB (Bio-Rad, 172-1067) for 15 minutes, and the absorbance at a measurement wavelength of 450 nm was measured with a microplate reader (Infinit 200 manufactured by TECAN).

The results are shown in the table below.

<Example 3> Immobilization of acidic polysaccharide (microarray)
(Production of sugar chain-trapping substrate)
For the production of the microarray, a glass substrate made of glass slide made of Sumitomo Bakelite and cyclic polyolefin resin was used.

2-methacryloyloxyethyl phosphorylcholine-cyclohexyl methacrylate-N- [2- [2- [2- [t-butoxycarbonylaminooxyacetylamino) ethoxy] ethoxy] ethyl] -methacrylamide copolymer 1.0 wt% ethanol The slide was immersed in the solution to apply the polymer compound.
After coating, the Boc group was deprotected by treatment with 2M HCl at 37 ° C. for 2 hours.

(Immobilization of sugar chains)
Three types of heparin with different molecular weights (Iduron, HO16, HO30, HEP001) were prepared in the following solutions to 100 ug / mL, spotted on a substrate, reacted at 80 ° C. for 1 hour, and acid sugar chains were immobilized on the substrate. .
Solution: 100 mM NaOAc buffer (pH 5.0) + surfactant (0.01% Triton X-100, 0.01% PVA (degree of polymerization = 1500))
After immobilization, it was washed with pure water to remove unreacted acidic polysaccharide.

(Detection of fixed sugar chains)
FGF2 (Peprotech, 100-18B), which has been reported to bind to heparin, was prepared to 100 ng / mL with the following solvent.
Solvent: 50 mM Tris.HCl (pH 7.5), 100 mM NaCl, 1 mM CaCl2, MnCl2, MgCl2, 0.05% Tween20.

The solution was reacted on the substrate and reacted at room temperature for 2 hours.
After the reaction, the substrate was washed with a washing solution to remove unreacted FGF2.
Washing solution: 50 mM Tris.HCl (pH 7.5), 100 mM NaCl,
1 mM CaCl2, MnCl2, MgCl2,
0.05% Triton X-100

Anti-FGF2 antibody (Peprotech, 500-M38) was prepared to 2 μg / mL with the following solvent.
Solvent: 50 mM Tris.HCl (pH 7.5), 100 mM NaCl, 1 mM CaCl2, MnCl2, MgCl2, 0.05% Tween20.

The solution was reacted on the substrate and reacted at room temperature for 1 hour.
After the reaction, the substrate was washed with a washing solution to remove unreacted antibodies.

Cy3-labeled anti-mouse IgG antibody (GE Healthcare, PA43002) was prepared to 2 ug / mL with the following solution.
Solution: 50 mM Tris.HCl (pH 7.5), 100 mM NaCl,
1 mM CaCl2, MnCl2, MgCl2, 0.05% Tween20

The solution was reacted on the substrate and reacted at room temperature for 1 hour.
After the reaction, the substrate was washed with a washing solution to remove unreacted antibodies.

The fluorescence intensity of Cy3 was measured using an X-100 microarray reader, ScanArrayLite (manufactured by PerkinElmer). (Laser = 90, PMT = 60)

The results are shown in the table below.

<Comparative Example 1>
(Acid sugar chain immobilization)
A commercially available substrate for acidic polysaccharide immobilization (Iduron, H / G Plates) was prepared. Heparin-derived oligosaccharide (Iduron HO16) was prepared to 10 μg / mL with the following solution. 100 μL of the prepared solution was dispensed into a 96-well plate for immobilizing acidic polysaccharide and allowed to react overnight at room temperature. After the reaction, it was washed with the following solution to remove unreacted acidic polysaccharide.
After washing, 150 μL was dispensed into wells in which PBS containing 1% BSA was written, and a blocking operation was performed at room temperature for 1 hour.
After 1 hour, it was washed with the following solution.

Solution: 100 mM NaCl, 50 mM Sodium acetate, 0.2% v / v Tween 20, pH 7.2

(Detection of fixed sugar chains)
FGF2 (Peprotech, 100-18B), which has been reported to bind to heparin, was prepared to 25 ng / mL with the following solvent.
Solvent: 50 mM Tris.HCl (pH 7.5), 100 mM NaCl, 1 mM CaCl2, MnCl2, MgCl2, 0.05% Tween20.

100 μL each was dispensed to each of the wells to which the acidic sugar chain had been immobilized and the wells to which the acidic sugar chain had not been immobilized, and reacted at room temperature for 2 hours.
After the reaction, the well was washed three times with the following washing solution to remove unreacted FGF2.

  Washing solution: Solution: 100 mM NaCl, 50 mM Sodium acetate, 0.2% v / v Tween 20, pH 7.2

Anti-FGF2 antibody (Peprotech, 500-M38) was prepared to 2 μg / mL with the following solvent.
Solvent: 50 mM Tris.HCl (pH 7.5), 100 mM NaCl, 1 mM CaCl2, MnCl2, MgCl2, 0.05% Tween20.

  100 μL of the antibody solution was dispensed into each well and reacted at room temperature for 1 hour. After completion of the reaction, each well was washed three times with a washing solution.

An HRP-labeled anti-mouse IgG antibody (DakoCytomation, P0260) was prepared at 1.3 μg / mL with the following solvent.
Solvent: 50 mM Tris.HCl (pH 7.5), 100 mM NaCl, 1 mM CaCl2, MnCl2, MgCl2, 0.05% Tween20.

  100 μL of the antibody solution was dispensed into each well and reacted at room temperature for 1 hour. After completion of the reaction, each well was washed three times with a washing solution.

  In order to measure the amount of peroxidase immobilized, the color was developed with TMB (Bio-Rad, 172-1067) for 15 minutes, and the absorbance at a measurement wavelength of 450 nm was measured with a microplate reader (Infinit 200 manufactured by TECAN).

The results are shown in the table below.

According to the present invention, acidic sugar can be efficiently and easily bound to a solid phase carrier, and non-specific adsorption of another substance that reacts with the acidic sugar is effectively suppressed. It can be used for analysis of binding proteins and other acidic sugar-binding molecules.

Claims (10)

An acidic sugar capturing substrate for capturing acidic sugar,
A monomer having a hydrophilic group;
A monomer having a functional group capable of capturing the acidic sugar;
A resin layer composed of a copolymer of monomers having a hydrophobic group,
An acidic sugar-capturing base material that is formed.   The acidic sugar-trapping substrate according to claim 1, wherein the functional group of the monomer having a functional group capable of capturing the acidic sugar is a primary amino group.   The acidic sugar-trapping substrate according to claim 2, wherein the primary amino group is an aminooxyl group. The acidic sugar-trapping substrate according to claim 1, wherein the copolymer is a copolymer represented by the following general formula [1].

(Wherein R1, R2 and R3 represent a hydrogen atom or a methyl group, R4 represents a hydrophobic group, X represents an alkyleneoxy group having 1 to 10 carbon atoms, and p represents an integer of 1 to 20. p is an integer. When X is an integer of 2 or more and 20 or less, the repeated Xs may be the same or different, Y is a spacer containing an alkylene glycol residue, and Z is an oxygen atom. , N is a natural number.)   The acidic sugar-trapping substrate according to claim 4, wherein the monomer for maintaining hydrophilicity contains a phosphorylcholine group. The acidic sugar-trapping substrate according to claim 4, wherein the hydrophobic group, R4, is a cyclic alkyl group.   The acidic sugar-trapping substrate according to claim 6, wherein the cyclic alkyl group is a cyclohexyl group.   The acidic sugar capturing substrate according to any one of claims 1 to 7, wherein the acidic sugar is at least one acidic sugar selected from glycosaminoglycan, heparin, heparan sulfate, dermatan sulfate, and hyaluronic acid. A capture substrate according to any one of claims 1-8,
(A) A step of coating a solid phase surface with a polymer substance containing a functional group (b) A method of capturing an acidic sugar, comprising a step of reacting a substance having an acidic sugar with a positive charge to solidify the surface.   The capturing method according to claim 9, which includes a step of drying after capturing the acidic sugar.