WO2005075996A1 - Baseboard for constructing surface plasmon resonane antibody array sensor and method of constructing the same - Google Patents

Abstract

It is intended to provide a base material for constructing a surface plasmon resonance antibody array sensor on which antibody molecules can be immobilized under regulated orientation at a high density in a small region, and a surface plasmon resonance array sensor in which antibody molecules are immobilized thereon. A base material on which protein molecules or peptide molecules are regularly aligned and immobilized at a high density in a uniformly orientated state is obtained by binding the carboxyl end of an antibody-binding protein or a main peptide chain to a primary amino group on a base material having primary amino groups at a high density or a primary amino group in a polymer compound provided on a base material which has the polymer compound carrying primary amino groups in its repeating structure and having been introduced on the surface of a planar base material. A surface plasmon resonance antibody array sensor comprising an antibody immobilized on the base material, in which the antibody molecules are immobilized under regulated orientation at a high density, has an extremely high effective sensitivity compared with the existing antibody arrays.

Description

 Specification

 TECHNICAL FIELD The present invention relates to a substrate for producing a surface plasmon resonance antibody array sensor and a method for producing the same.

 The present invention relates to a substrate for producing an antibody array sensor based on surface plasmon resonance, an antibody array sensor based on surface plasmon resonance using the same, and a method for producing them.

 Background art

 Conventionally, many types of biomolecules have been immobilized on a specified number of minute regions of a planar solid base material (substrate), and the solution is contacted with a solution containing another biomolecule that is a candidate for a binding partner. Attempts have been made to rapidly and comprehensively test molecule-specific binding (interaction) phenomena, an important function of biomolecules, by observing the presence or absence of phenomena, their strength, and the like. In this method, a planar solid substrate (substrate) prepared by binding hundreds of thousands of different biomolecules to specified individual microregions on a surface several centimeters square is used as an array or The power of what is referred to as a chip The biomolecules that exist in a vast and diverse manner in a living unit such as a cell in recent years and constitute a system (genome when referring to genes, proteome when referring to proteins) The possibility of providing high-speed and comprehensive analysis required by a research method called genomics or proteomics, which aims to conduct a comprehensive and comprehensive analysis of the information, is becoming increasingly important.

[0003] As described above, the array technology integrates the detection of molecular bonding phenomena in a narrow area of a planar solid substrate (substrate) at a high level, thereby enabling high-speed and comprehensive analysis using an extremely small amount of biomolecule samples. It offers the possibility of performing a specific method, but on the other hand, the detection technology essential for this technology has been required to detect the binding phenomena of molecules extremely limited in quantity with high sensitivity . In fact, in genomic analysis, which is an advanced application of this array technology, a gene (DNA molecule) to be contacted is labeled with a fluorescent molecule capable of generating fluorescence. The presence or absence of binding to a specific gene immobilized on the DNA array is determined by irradiating the microregion where the gene is immobilized with a high-energy laser with the excitation wavelength of the fluorescent molecule to generate fluorescent light. Has been determined. [0004] With the progress of genome analysis of genes that can be compared to blueprints of life phenomena, comprehensive analysis of proteins that are the products of the blueprints and play a central role in dynamic life phenomena is performed. Proteome analysis has rushed to follow. Here, too, an array technology that has demonstrated its power in genomic analysis has been adopted in order to exhaustively and comprehensively analyze a vast and diverse variety of biomolecules. In a proteome in which the diversity of molecular species and their binding phenomena is far greater than that of the genome, as an analogy to DNA arrays, arbitrary proteins are integrated and immobilized on a planar solid substrate (substrate). Although protein arrays have been developed, it is practical to use antibody arrays in which antibody molecules that can specifically identify proteins with specific functions and provide useful molecular information are selected as immobilized proteins. Attention has been paid to these forms, and some of them have been improved.

 [0005] However, a protein array or an antibody array developed from the analogy of a DNA array has a new powerful problem that does not occur in a DNA array. That is, 1) the binding reaction of proteins is relatively weak compared to that of DNA, so the amount bound per unit area of the detection surface is reduced.2) Basically, linear molecules remain In the DNA to be bound, the interference with the binding phenomenon can be neglected if the fluorescent molecule labeling or immobilization on the surface is performed using the molecular ends avoiding the center of the chain contributing to the binding, For proteins whose linear macromolecules form a three-dimensional structure, the labeling site or immobilization site in the protein varies, and often overlaps with the site essential for protein binding function. Bound fluorescent molecules or molecular binding sites for immobilization can significantly inhibit the binding phenomena themselves. As a result, serious problems arise in the measurement of protein arrays or antibody arrays, in which effective sensitivity is not practically obtained, or measurement is performed at the lower end of the effective detectable range, resulting in lack of reproducibility. However, this has become a factor that has not spread as much as NA arrays.

[0006] In order to overcome such a problem, in recent years, the development of array sensors has been promoted. This uses the flat solid substrate (substrate) itself to constitute the array as the detection part of the sensor, and eliminates the use of fluorescent molecule labels, among the problems in 2) above. A sensor using a surface plasmon resonance sensor that can detect a molecule based on the mass of the molecule has been put to practical use. Surface plasmon resonance sensor is made of metal (mainly gold) The glass-side force on a thin film of a thin film deposited on the glass, the visible light is totally reflected, and at that time, the wave number (frequency) of the quantum wave called the evanescent wave that seeps into the interface between glass and metal and the medium in contact with the metal When the wave numbers of surface plasmon waves coincide, energy absorption called surface plasmon resonance occurs and the reflected light diminishes.This is based on the adsorption and dissociation phenomena of substances at the molecular level on the metal side surface. It is a sensor that can monitor the change in mass on the surface by measuring the resonance angle at which the change in the refractive index causes dimming. In the case of a surface plasmon resonance type sensor, it is possible to use this metal surface by dividing it into a large number of divided specific regions, and by immobilizing molecules in each specific region, an array sensor is obtained. Function can be exhibited.

 [0007] As can be seen from the principle power, this array sensor can measure unlabeled molecules, so that labeling with fluorescent molecules is not necessary, but sensitivity is in principle lower than that of the detection method using fluorescent labeling. The adoption of the surface plasmon resonance method has not achieved a drastic improvement in effective sensitivity.

 Accordingly, the present invention relates to the improvement of the amount of binding per unit area of the above problem 1) in the conventional array sensor, particularly the practically useful antibody array sensor, and the improvement of the protein immobilization in the above problem 2). The aim is to provide a radical solution for overcoming binding function inhibition.

 Disclosure of the invention

 Problems to be solved by the invention

[0008] The present invention provides an antibody array sensor, in which an antibody-binding protein is used as an antibody molecule immobilizing means, the orientation of which is immobilized on a planar substrate surface at one carboxy terminus of a protein main chain. Means to prevent structural inhibition of the binding function of the immobilized antibody molecule by employing means for binding, and further develop this to increase the amount of immobilization per unit area. It is an object of the present invention to develop a means for immobilizing a protein with high density on a small area on an array substrate and thereby increasing the number of immobilized areas of the protein on the substrate. As a result, for example, it is possible to improve the practicality of antibody arrays and to expand the field of application through standardization of detection molecular systems in antibody array sensors, increase in detection sensitivity, etc. Than it is. Means for solving the problem

 In order to solve the above-mentioned problems, the present inventors firstly formed an amide bond formation reaction via a cyanocysteine residue on the surface of each minute region on a planar substrate also serving as a detection unit used in an array sensor. Using the antibody recognition protein, which has been ordered and immobilized via the carboxyl group at the carboxy terminus of the protein backbone, using as a scaffold for immobilization of the antibody molecule, and aligning the same antibody recognition molecule. To eliminate the deviation of the amount of immobilized antibody recognition molecules between each microregion, thereby reducing the amount of immobilized antibody molecules that are secondarily immobilized by binding to the antibody recognition molecules. We have found that it can be standardized between domains.

[0010] Furthermore, with the aim of increasing the amount of immobilization per unit area (ie, the immobilization density of the antibody) to 100 to 1000 times the level of single molecule adsorption (ie, about several / zg / mm ² ). As a result of intensive research, a polymer compound having a primary amino group in a repeating structure is introduced on the surface of the planar substrate, and the carboxyl terminus of the antibody-recognition protein main chain is bonded to the primary amino group of the introduced polymer compound. As a result, it has been found that proteins can be immobilized at extremely high density while orderly arranging proteins on a planar base material as a detection unit. It can be applied to the detection part of surface plasmon resonance sensor, which is an unlabeled sensor, which has been difficult, and the immobilization of polymers and antibody recognition molecules is achieved by strong covalent bonds. Utilizing the advantages that can be achieved, it is possible to reversibly desorb antibody molecules to the array sensor surface using an acid or salt solution, etc., thereby enabling repeated regeneration of the sensor surface. The present invention was completed by developing an application to a fully automatic measurement system based on a sensor that can automatically and repeatedly perform measurement using various antibody molecules ^ | lj, and completed the present invention.

 That is, the present invention is as described in the following (1)-(11).

 (1) An array substrate for producing a surface plasmon resonance antibody array sensor, wherein an antibody-binding protein or peptide is aligned and fixed in a specific region on the surface of a detection section.

(2) A surface plate characterized in that antibody proteins are aligned and immobilized on a substrate on which antibody-binding proteins or peptides are aligned and immobilized in a specific region on the detection surface. Rasmon resonance antibody array sensor.

 (3) An antibody-binding protein is obtained by immobilizing the carboxy terminus of a peptide via a linker sequence with a linker molecule on a sensor detection unit having a primary amino group with an amide bond, and Equation (1)

 NH— R to CO— NH— R— CO— NH— Y— Z (1)

 twenty two

An array substrate for producing a surface plasmon resonance antibody array sensor, characterized by being represented by:

[Wherein, R is the amino acid sequence of the antibody-binding protein or peptide, and R is any

 Amino acid sequence of linker sequence of 1 2, Y represents linker molecule, z represents surface material of sensor detector

]

 (4) a linker molecule having a primary amino group according to the above (3),

General formula (2)

 (NH-X) n (2)

 2

(Wherein, X represents a residue other than the primary amino group of the repeating unit of the polymer compound, and n represents an integer of 2 or more). An array substrate for producing a surface plasmon resonance antibody array sensor, comprising:

(5) An array substrate for producing a surface plasmon resonance antibody array sensor, wherein the polymer compound represented by the general formula (2) in the above (4) is polyallylamine.

 (6) An array substrate for producing a surface plasmon resonance antibody array sensor, wherein the polymer compound represented by the general formula (2) in the above (4) is polylysine.

 (7) In a specific region on the detection section surface having a primary amino group, a general formula (3)

NH-I R-C00H (3)

 twenty one

 [Wherein, R represents an amino acid sequence of a protein or peptide. ]

The method for producing an array substrate for producing a surface plasmon resonance antibody array sensor according to the above (1), wherein the antibody-binding protein or peptide is aligned and immobilized, the array being arranged on a substrate having a primary amino group. , Adsorbed, general formula (4)

 NH-R-CO-NH-CH (CH-SCN)-CO-NH-R-COOH

 2 2… · (4)

 twenty one

[Wherein, R represents the amino acid sequence of the antibody-binding protein or peptide; It represents an amino acid sequence that is strongly negatively charged in the vicinity and can make the isoelectric point of the protein or peptide represented by the general formula (3) acidic. ]

A peptide or a carboxy terminus of the protein main chain of the general formula (3) is bonded to a protein or peptide represented by the formula below and a primary amino group of a linker molecule on the sensor, and then the antibody is bound by the antibody-binding protein or peptide. A method for producing an array substrate for producing a surface plasmon resonance antibody array sensor, comprising binding molecules.

 (8) A surface plasmon resonance antibody array sensor, wherein the linker molecule having a primary amino group (7) is a molecule containing a polymer compound having a primary amino group represented by the general formula (2): Array substrate for fabrication.

 (9) A method for producing an array substrate for producing a surface plasmon resonance antibody array sensor, wherein the polymer compound represented by the general formula (2) is polyallylamine.

 (10) A method for producing an array substrate for producing a surface plasmon resonance antibody array sensor, characterized in that the polymer compound represented by the general formula (2) is polylysine.

 (11) The surface plasmon resonance antibody array sensor as described in (2) above, wherein the same quality can be repeatedly measured by performing a desorption / regeneration operation of the antibody molecule using an acid 'alkali' salt solution or the like. A method for measuring an antigen molecule using the method.

 (12) A feature of using the surface sensor described in (2) above for the detection unit and applying the repetitive reproduction operation described in (11) above, and automatically regenerating the surface of the detection unit every time the sample is measured. An automatic measurement system for a surface plasmon resonance antibody array sensor, which is equipped with an automatic liquid feeding device that performs the measurement and a data processing device that enables automatic collection and data processing of measurement data generated by detection.

The invention's effect

 According to the present invention, it is possible to provide an antibody array sensor in which antibody molecules have a high density and antibody proteins are immobilized in a controlled orientation on the surface of an array substrate such as a metal used for a surface plasmon resonance apparatus. If the antibody array sensor of the present invention is used in the detection section of the surface plasmon resonance sensor, the effective sensitivity is extremely remarkably increased as compared with the conventional antibody array, and the practicality of the antibody array sensor is improved. Enlargement can be achieved.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.

 According to the present invention, an antibody-binding protein is adsorbed to a primary amino group of a linker molecule on the surface of a detection metal of a surface plasmon resonance array sensor capable of detecting an unlabeled molecule, and the protein is further attached to the primary amino group. By binding the carboxy terminus of the main chain using an amide bond formation reaction via a cyanocystein residue, the proteins are aligned and immobilized on the substrate, and the antibody-binding protein is used as a scaffold for antibody capture. After preparing an array sensor substrate for antibody capture as a molecule, a plurality of arbitrary antibody molecules are captured by antibody-binding proteins arranged in specific small regions on the substrate, thereby constructing an antibody array sensor. Things.

 [0014] Further, a polymer compound having a primary amino group in the repeating structure is bonded to an arbitrary linker molecule on the metal surface of the detection unit to form a flat polymer-modified array sensor base material, By using the primary amino groups of the polymer compound in a plurality of specific micro-regions classified in the above, it is possible to further immobilize the antibody-binding protein having a higher density and orientation control by immobilization. Provided is an antibody array sensor in which the effective sensitivity is further improved by immobilizing high-density antibody molecules.

 In the present invention, in a very small area on the array sensor substrate, it can be fixed in a uniform orientation state while maintaining its function at a high density and can be captured by the antibody binding protein having the high density and a uniform orientation. The antibody molecule thus obtained can also be reliably used because the binding between the protein and the pile molecule can be reliably achieved not at the site responsible for the antigen binding function of the antibody molecule but at a site called the constant region, which is not related to the binding function. It can be fixed to a specific area on the array sensor with high orientation and high density.

 [0015] In addition, by increasing the density of primary amino groups on the surface of the base material by using the above-mentioned polymer compound, the density of the immobilized antibody-binding protein can be further amplified, thereby increasing the sensitivity. An antibody array sensor can be provided.

That is, according to the present invention which can achieve such highly uniform orientation and high density at the same time, the immobilized antibody molecule enables each molecule to exhibit the maximum antigen-binding function, and As the number of molecules increases, the sensitivity deficiency of the surface plasmon resonance array sensor can be improved, and the measurement can be performed with high reproducibility. In addition, all the bonds are formed by stable covalent bonds on the array sensor substrate for capturing the antibody, which is composed of an array sensor metal surface linker one molecule antibody binding protein or an array sensor metal surface linker one molecule polymer compound antibody binding protein A simple regenerating operation of the surface of the array sensor for desorbing the used antibody molecules by applying an appropriate acid or salt solution, etc., and binding and using new antibody molecules separately is performed. This makes it possible to provide the ability to fully contribute to the realization of an automatic measurement device using an antibody array sensor by automating a series of these operations.

 [0016] Hereinafter, the primary amino group of a polymer compound directly bonded to a linker molecule having a primary amino group on the metal surface of the detection portion of the array sensor or bonded to an arbitrary linker molecule on the metal surface of the detection portion will be described. Then, an antibody-binding protein array substrate is prepared by binding the antibody-binding protein by peptide bond, and a surface plasmon resonance antibody array sensor is prepared by further binding any target antibody molecule. The means and the measurement method using it are described in detail.

 [0017] 1. Array sensor substrate

 In order to achieve the above object of the present invention, first, a surface structure exposing a primary amino group sufficient to adsorb negatively charged antibody-binding protein by ionic interaction is required as an array sensor substrate. . The configuration of the substrate that satisfies this performance is to expose the primary amino groups on the metal (mainly gold) surface of the detection unit originally provided in the surface plasmon resonance array sensor on the Balta water side, and to form a covalent bond with the metal It is desirable to interpose a linker, which is a divalent low-molecular compound that can be bound by a single molecule. Such a linker molecule has been well studied and commercialized, but is represented by the following general formula.

X-A-Y (4)

[X in the above formula (4) is thiol (HS-), disulfide (Y-A-SS-, Y, -A, -SS-), sulfide (YAS-, Y, -A, -S-), Selenol (HSe-), diselenide (Y-A-SeSe-, Y, -A, -SeSe-), selenide (Υ-Se-Se-, Y, -A, -Se-) and the like. In the example, a spontaneous covalent bond is formed between the sulfur or selenium atom and gold. A is a saturated hydrocarbon chain ((CH2) n) or ethylene glycol polymer ((CH2-CH2-0-) n) (n is an integer of 2 or more), but partially unsaturated bonds (double bonds, etc.) are inserted Sometimes. Y is here a primary amino group. In addition, Y ′ may be a residue which may be a residue compatible with Y, or may be another hydrophilic group. In the latter case, it does not participate in the binding reaction. ]

[0018] The linker molecule represented by the above formula (4) exhibits amphipathic property although there is a difference in strength. Therefore, in an aqueous solution in which an antibody array sensor is used, Y is directed toward the Balta water side for a plurality of times. A spontaneously formed cohesive monolayer structure on the gold surface, where the A of the child is bound. This is a structure called a self-assembly monolayer (SAM), in which the covalent bond between the sulfur atom or selenium atom and gold is further strengthened by agglomeration, and the primary amino acid used for the bond reaction. Since the group is highly oriented and exposed on the Balta water side, it is efficient as a linker molecule.

 Further, in the present invention, when it is desired to further increase the amount of immobilization by using a polymer compound, the primary amino group in the polymer is bonded to the force using one linker molecule of the above formula (4). Various hydrophilic functional groups can be used for Y in the above formula (4). For example, when Y is a carboxyl group, if the carboxy group is previously converted to an activated form using an active form reagent such as hydroxysuccinimide, the reaction proceeds only by contact with the primary amino group in the polymer. In the case where Y is a -amino group, it is possible to react by performing activation with dartaldehyde, which is the same structure, and other hydroxyl groups and the like can be reacted through activation with epichlorohydrin and cyanogen bromide. Available.

 [0020] The polymer compound according to the present invention has a primary amino group in a repeating structure, and has a partial force other than the primary amino group, a side chain of the protein to be fixed, an α-amino group at the amino terminal, or a carboxyl group at the carboxy terminal. Any substance can be used as long as it does not react with the substance.

 Examples of the polymer compound having a primary amino group in the repeating structure include those having a polyalkylene chain, a polyamide chain, a polyester chain, a polystyrene chain, and the like, and have a repeating structure represented by the following general formula.

(Χ-ΝΗ) η (5) [In the above formula (5), X represents, for example, a monomer residue constituting a polyalkylene chain, a polyamide chain, a polyester chain, a polystyrene chain, or the like. Further, the NH2 group may be a group contained in the monomer residue, or may be a group contained in the side chain of the main chain of the polymer compound. n indicates an integer of 2 or more. ]

In the present invention, among these polymer compounds, those having a polyalkylene chain include, for example, polyallylamine. This polymer compound has a high primary amine content per unit mass according to the present invention. It can be used as a thing. The present invention is not limited to this, and there is, for example, a copolymer of a vinyl compound having a primary amino group in a side chain and another vinyl compound! For / ヽ, various polymer compounds such as polylysine can be used.

[0022] 2. Protein capable of binding to an antibody molecule

 In the present invention, any antibody-binding protein or peptide used for immobilization can be used as long as it has a binding ability to an antibody molecule.

 As a protein capable of binding to an antibody molecule,

Staphylococcus aureus-derived Aguchi Aino A (described in A. Forsgren and J. Sjoquist, J. Immunol. (1966) 97, 822-827.), A protein G derived from Streptococus sp.Group CIG (EP013 1 142A2 (1983)) ), Protein L from pg / ο <: ο «:" 5, "agm <.s (described in US5 65390 (1992)), protein H from group AS e /? To" 5 ti (Described in US5 180810 (1993)), Haemophilus influenzae Kamakura's Pine (described in US6025484 (1990)), Septococcwi. AP4-derived protein Arp (Protein Arp 4) (described in US5210183 (1987)) Streptococcal FcRc from group C Streptococcus (described in US4900660 (1985)), protein from group A streptococcus, Type II strain (described in US5556944 (1991)), and protein from Human Colonic Mucosal Epithelial Cell (US6271362 (1994)) Description), St tapping hvhcoccus aureus, protein from strain 8325-4 (described in US6548639 (1997)), derived from Pseudomonas maltophilia Protein (described in US5245016 (1991)) and the like are known.

[0023] In addition, it has been revealed that these proteins often have a repeating sequence, and fragmented proteins also have an ability to bind to antibody molecules. Examples of the protein or peptide capable of binding to the antibody molecule targeted by the present invention include these naturally occurring antibody binding proteins, partial proteins, their sequence-modified proteins, and partial proteins. Peptides, their mimetic peptides, artificial peptides capable of binding to antibody molecules, and the like can be mentioned. For a protein capable of binding to such an antibody molecule, the general formula (6) NH-R-COOH (6)

 twenty one

 [In the above formula, R is an amino acid of a protein or peptide capable of binding to an antibody molecule.

 1

 Represents an array]

 Can be represented by

 [0024] In the present invention, a protein or a protein having the binding ability represented by the general formula (6) NH-R-COOH may be used.

 twenty one

 Is to be able to immobilize the peptide,

 General formula (7)

 NH-R-CO-NH-R-CO-NH-CH (CH-SH) -CO-NH-R-COOH (7)

 It is necessary to prepare a protein for immobilization shown in 2 122. In these general formulas, R

 3 is

, Strongly negatively charged near neutral, and NH-R-CO-NH-R-CO-NH-CH (CH

 2 1 2 2

 -SH) -CO-NH-R -A chain of arbitrary amino acid residues that can make the isoelectric point of -COOH acidic

 Three

 . R

 1 is an amino acid sequence of a protein or peptide capable of binding to the above-mentioned antibody molecule. R

 2 is a linker peptide extending between the protein to be immobilized represented by the general formula (1) and the amphipathic linker molecule on the metal surface. R

 The amino acid sequence of 2 is arbitrary and its type and number are not limited. For example, Gly-Gly-Gly-Gly or the like can be used. Such a fusion protein is combined with a gene encoding the protein represented by the general formula (6).

 General formula (8)

 NH-R-C0-NH-CH (CH-SH) —CO—NH—R-COOH (8)

 2 2 2 3

 [Wherein, R and R have the above meanings. ]

 twenty three

 By binding to a gene encoding a peptide sequence represented by the general formula (7) NH

 2

— R -CO-NH-R -C0-NH-CH (CH— SH) — C〇— NH— R— C 融合 H

1 2 2 3

 The gene can be obtained by preparing a gene encoding a protein, expressing the gene in a host organism such as Escherichia coli, and then separating and purifying the expressed protein.

[0025] Such a fusion protein can be obtained by a known technique (for example, M. Iwakura et al., J. Biochem. Ill. 37-45 (1992)). Alternatively, the fusion protein can be produced by a combination of a genetic engineering technique and a conventional protein synthesis technique, or by only a protein synthesis technique.

 As for R in the above general formulas (7) and (8), aspartic acid and glutamic acid are often used.

 Three

 Preferred sequences include. Preferably, a sequence containing a large amount of aspartic acid or glutamic acid should be designed such that the isoelectric points of the substances of the above general formulas (7) and (8) are between 4 and 5. A suitable class of such sequences is Ararael-polyaspartic acid. By changing the amino acid residue next to the cyanocysteine residue to alanine, an amide bond-forming reaction is likely to occur via the cyanocysteine residue, and the carboxyl group of aspartic acid is most frequently present in the amino acid side chains. This is because it is acidic.

 [0026] In order to more specifically show the above, a description will be given below using Protein A derived from Staphylococcus aureus as an example.

 Protein A from Staphylococcus aureus is composed of five domains named A, B, C, D, and E, which have remarkably similar amino acid sequences, and a sequence associated therewith. Each of these domains is composed of 57 amino acids, but each has a stable structure alone and can be expressed in large amounts in, for example, Escherichia coli. In addition, each domain can exert its own binding ability to an antibody molecule. Its binding strength is almost the same as that of the naturally-occurring whole protein A when two force domains that are weaker than the naturally-occurring whole protein A are joined. Therefore, focusing on the domain of protein A, we designed a sequence for immobilization of two types, a single domain (this is called a monomer) and two linked domains (this is called a dimer). did.

 [0027] SEQ ID NO: 1 is an amino acid sequence of an immobilization protein prepared for subjecting the A domain monomer of protein A to immobilization reaction. SEQ ID NO: 2 is an immobilization of the A domain monomer of protein A. 2 shows the amino acid sequence of a protein for immobilization prepared for use in the dani reaction. SEQ ID NO: 1

Ala Asp Asn Asn Phe Asn Lys Glu Gin Gin Asn Ala Phe Tyr Glu lie Leu Asn Met Pro Asn Leu Asn Glu Glu Gin Arg Asn Gly Phe lie Gin Ser Leu Lys Asp Asp Pro Ser Gin Ser Ala Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn Glu Ser Gin Ala Pro Lys Gly Gly Gly Gly Cys Ala Asp Asp Asp Asp Asp Asp

Sequence Listing 2

 Ala Asp Asn Asn Phe Asn Lys Glu Gin Gin Asn Ala Phe Tyr Glu lie Leu Asn Met Pro Asn Leu Asn Glu Glu Gin Arg Asn Gly Phe lie uln Ser Leu Lys Asp Asp Pro Ser Gin Ser Ala Asn Leu Leu Ser ulu Ala Lys Lys Leu Asn Glu Ser Gin Ala Pro Lys

Ala Asp Asn Asn Phe Asn Lys Glu Gin Gin Asn Ala Phe Tyr Glu He Leu Asn Met Pro Asn Leu Asn Glu Glu Gin Arg Asn Gly Phe lie uln Ser Leu Lys Asp Asp Pro Ser Gin Ser Ala Asn Leu Leu Ser ulu Ala Lys Lys Leu Asn Glu Ser Gin Ala Pro Lys

Gly Gly Gly Gly Cys Ala Asp Asp Asp Asp Asp Asp Asp

 The respective sequences are represented by the polyglycine cysteine residue and alanine residue shown in SEQ ID NO: 5 at the carboxy-terminal side of the A domain monomer sequence of protein A and the A domain dimer sequence of protein A shown in SEQ ID NOs: 3 and 4. This is a sequence obtained by adding the sequence of base-polyaspartic acid. SEQ ID NO: 3

 Ala Asp Asn Asn Phe Asn Lys Glu Gin Gin Asn Ala Phe Tyr Glu lie Leu Asn Met Pro Asn Leu Asn Glu Glu Gin Arg Asn Gly Phe lie uln Ser Leu Lys Asp Asp Pro Ser Gin Ser Ala Asn Leu Leu Ala ulu Ala Lys Lys Leu Asn Glu Ser Gin Ala Pro Lys SEQ ID NO: 4

Ala Asp Asn Asn Phe Asn Lys Glu Gin Gin Asn Ala Phe Tyr Glu lie Leu Asn Met Pro Asn Leu Asn Glu Glu Gin Arg Asn Gly Phe lie uln Ser Leu Lys Asp Asp Pro Ser Gin Ser Ala Asn Leu Leu Ser ulu Ala Lys Lys Leu Asn Glu Ser Gin Ala Pro Lys Ala Asp Asn Asn Phe Asn Lys Glu Gin Gin Asn Ala Phe Tyr Glu He Leu Asn Met Pro Asn Leu Asn Glu Glu Glu Gin Arg Asn Gly Phe lie uln Ser Leu Lys Asp Asp Pro Ser Gin Ser Ala Asn Leu Leu Ser ulu Ala Lys Lys Leu Asn Glu Ser Gin Ala Pro Lys Gly Gly Gly Gly Cys Ala Asp Asp Asp Asp Asp Asp Asp

 SEQ ID NO: 5

 Gly Gly Gly Gly Cys Ala Asp Asp Asp Asp Asp Asp Asp

 [0029] In SEQ ID NO: 5, four glycine residues are shown as the sequence of a part of the linker, but the sequence of the linker is arbitrary and is not limited in length or type. The cysteine residue is an essential residue for converting the SH group in the side chain into a cyanocysteine and utilizing it for the immobilization reaction. The subsequent sequence of alanine-polyasnogic acid was a sequence introduced to promote the immobilization reaction and increase the reaction efficiency, and the isoelectric point of the protein shown in SEQ ID NOS: 1 and 2 was changed from 4 to 5. Any array can be used as long as it can be a value between them.

 [0030] The proteins shown in SEQ ID NO: 1 and SEQ ID NO: 2 can also be produced by using a chemical synthesis technique, but are obtained by expressing the gene encoding the sequence in a host such as Escherichia coli and separating and purifying from the expressing cells. Can be

 Examples of the sequences encoding the proteins shown in SEQ ID NO: 1 and SEQ ID NO: 2 include the nucleotide sequences shown in SEQ ID NO: 6 and SEQ ID NO: 7, respectively. SEQ ID NO: 6

 GATGACTAA

SEQ ID NO: 7 TGCGCTGATGACGATGACGATGACTAA

[0031] These sequence listings show the sequences with ATG as the start codon and TAA as the stop codon at the 5 'end and the 3' end, respectively! /

 Since the nucleotide sequence encoding an amino acid is degenerate and corresponds to one amino acid residue having multiple codons, the sequences encoding the proteins shown in Sequence Listing 1 and Sequence Listing 2 are shown in Sequence Listing 6 and Sequence Listing. Not limited to 7, but as many as possible codon combinations

[0032] In order to express the gene sequence encoding the protein shown in SEQ ID NO: 1 or SEQ ID NO: 2 in a host cell such as Escherichia coli, a sequence necessary for transcription and translation of the gene is replaced with the sequence encoding the protein. It is necessary to add it upstream. Examples of gene sequences which can be added to such a sequence and introduced into a vector include the sequences shown in SEQ ID NO: 8 and SEQ ID NO: 9. SEQ ID NO: 8

SEQ ID NO: 9

SEQ ID NO: 10

Attach the sequence shown in GCAGCAAAAGGAGGAACGACT upstream of the initiation codon, and connect the recognition cleavage sequence of the restriction enzyme BamHI at the 5 'end and the recognition cleavage sequence of the restriction enzyme EcoRI at the 3' end so that it can be introduced into the vector DNA. Here is the array.

 The sequences shown in SEQ ID NO: 8 and SEQ ID NO: 9 can be artificially synthesized by chemically synthesizing V and some fragments, and then using a PCR method or an enzyme such as DNA ligase.

 [0034] The synthetic gene thus obtained is inserted into an appropriate vector using a restriction enzyme site, and is expressed in a host cell. Any vector can be used as long as an appropriate restriction enzyme site can be used. For example, pUC-type and PBR-type high copy number vectors are suitable as commercially available vectors. By expressing the recombinant into which Sequence Listing 6 and Sequence Listing 7 have been introduced, for example, in E. coli, the proteins shown in Sequence Listing 1 and Sequence Listing 2 can be soluble in about 5 to 30% of the bacterial protein. In the state of 'expression can be accumulated.

[0035] In this way, the protein which has been expressed and accumulated can be purified to homogeneity from a cell-free extract of the expressed cells by a chromatography operation usually used for protein purification. As the chromatography to be used, anion exchange chromatography, gel filtration chromatography, etc. are effective. Affinity chromatography using a carrier on which immunoglobulin is immobilized is most effective.

 [0036] 3. Spot method for protein array sensor

 Next, in the present invention, the antibody binding protein for immobilization prepared as described above is arranged and adsorbed on a protein array substrate, but the method is not particularly limited. Any method can be used as long as the protein solution can be spotted on the region. For example, there is a method using a needle-like object such as a pin, an ink jet, a capillary, or the like. Any of these methods may be used. It is also possible to use a picking robot. Hereinafter, as an example, a method of spotting using a capillary will be described in detail.

 [0037] A solution of the protein for immobilization represented by the general formula (7) is filled in the capillaries, and an appropriate amount of the protein solution can be spotted at an intended place by applying a suitable pressure to the upward force. is there. When the substrate for immobilization has a water-absorbing property, the solution can be quickly absorbed into the substrate without applying pressure if the protein solution has a volume of about 10 μl. Will be done. At this time, the solvent of the protein solution diffuses in all directions around the spot, but remains at the spot because the protein is adsorbed to primary amine by electrostatic interaction. Therefore, it is possible to adsorb proteins to small regions at high density. Further, by controlling the spotting position, proteins can be aligned and fixed in an arbitrary pattern shape. This can be performed by computer control, for example, by printing a pattern drawn on a computer with an ink jet printer. Therefore, it is obvious that any method used for alignment can be applied, and this does not limit the present invention.

 [0038] 4. Protein immobilization

As described as another method, the protein array may be directly formed by adsorbing and immobilizing the spotted proteins, but at this stage, the proteins are bound to the substrate by non-covalent bonds such as electrostatic interactions. Since the bond strength is low, in order to firmly immobilize the protein, an amide bond is further formed between the carboxy group at the carboxy terminus of the protein and the primary amino group of the polymer on the substrate. . In order for this reaction to take place, the cysteine residue introduced at the carboxy terminus of the protein for immobilization was used. The sulfhydryl group of the group needs to be cyanated and converted to cyanocysteine.

 In order to achieve this bond, the sulfhydryl group of the cysteine residue in the protein of the general formula (7) needs to be converted to cyanocysteine by converting the sulfhydryl group to a cyanocysteine. The cyanated protein obtained by the conversion is a protein represented by the following general formula (9).

 NH—R—CO-NH—R—CO—NH—CH (CH—SCN) —CO—NH—R—COOH · '· (9)

 2 1 2 2 3

 [In the above formula, R and R R are the same as R and R R in the general formula (7), respectively.

 1 2, 3 1 2, 3 1

 R is a linker peptide, R is a strongly negative charge near neutrality and R

, twenty three

 7 represents an amino acid sequence capable of making the isoelectric point of the compound of 7) acidic. ]

[0040] This cyanation reaction can be performed using a commercially available cyanation reagent. As the cyanating reagent, 2-nitro-5-thiocyanobenzoic acid (NTCB) (see Y.Degani, A. Ptchornik, Biochemistry, 13, 1-11 (1974)) ) Or 1-cyano-4-dimethylaminopyridi-dimethyltetrafluoroboric acid

 (1— cyano— 4-dimethylaminopyridinium tetrafluoroborate (CDAP)) etc.

 Shiano-dani using NTCB can be performed efficiently in a 10 mM phosphate buffer at pH 7.0. After the cyanation reaction, the fixing reaction proceeds by making the solvent weak alkaline. That is, an amide bond is formed between the carboxyl group of the amino acid residue immediately before the cyanocysteine residue and the primary amino group of the carrier. This can be achieved, for example, by replacing the buffer with a 10 mM boric acid buffer at pH 9.5.

[0041] The conversion of the cyanocysteine of the sulfhydryl group of the cysteine residue necessary for the immobilization reaction is carried out before the protein is adsorbed to the substrate on which the immobilization is carried out, as has already been clarified by the present inventors. However, it may be performed later or simultaneously with the adsorption (see Japanese Patent Application No. 2002-148950). Since the protein after cyanation represented by the general formula (9) also has a strongly negatively charged amino acid sequence near neutrality, the protein after cyanation is aligned and adsorbed on a substrate. In addition, the carboxy terminal side of the protein main chain is adsorbed to the primary amino group side of the polymer compound on the carrier, and the amide formation reaction binds to the primary amino group only at the carboxy terminal of the protein main chain, thereby homogenizing the protein. With high alignment and high density A protein array that has been aligned and fixed can be obtained.

 [0042] In the reaction involving cyanocysteine used in the present invention, the potential for a hydrolysis reaction to occur as a side reaction. Since all the reactants generated from such a side reaction are soluble in a solvent, the By washing the protein array after the protein immobilization reaction with an appropriate solvent, side reaction products can be removed.

As described above, the antibody-binding protein was immobilized on the protein array substrate of the present invention and the immobilizing protein by performing the immobilization by the above-described operation. An antibody array sensor immobilized at a high density of about several g / mm ² can be produced. Example

 Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to these examples.

 In the present embodiment, an NH2-chip or COOH chip attached to Toyobo equipment, Multi SPRinter is used as a surface plasmon resonance array sensor exposing the primary amine on the surface, and this is attached to the equipment. investigated. Each of these chips divides the metal surface on the chip into 98 microscopic areas, each of which has an amino or carboxyl group at the end and a thiol group at the opposite end. Polyethylene glycol (PEG) molecules provide a single layer of stable SAM surface. As a polymer compound having a primary amino group in the repeating structure, poly-L-lysine commercially available from Sigma-Aldrich was used.

[0045] In this example, the antibody-binding protein prepared for use in immobilization was prepared by adding the amino acid sequence of the linker-peptide portion to protein A (SEQ ID NO: 1).

 Gly-Gly-Gly-Gly-Gly-Gly), cysteine (Cys) and an amino acid sequence to strongly acidify the isoelectric point of the resulting strongly charged protein around neutrality (

Ala-Asp-Asp-Asp-Asp-Asp-Asp) are sequentially added proteins (SEQ ID NO: 2), all of which have been prepared by the present inventor (Japanese Patent Application No. 2003-352937). Described in). Further, the antibody immobilized on the binding protein is an anti-hige IgG antibody. This antibody was derived from a heron (Funakoshi), and the reaction was observed by applying an anti-Pein 'albumin hidge antibody (Funakoshi) to the surface to which this antibody was bound.

 Example 1

 [L] Fabrication of a conventional array sensor constructed by direct non-alignment immobilization of antibody molecules for comparative experiments using COOH chips

 COOH—0.2 M EDC on chip

 (N-ethyl-N '-[3- (dimethylamino) propyl] carbodiimide) /0.05M NHS

 A mixed aqueous solution of (N-Hydroxysuccinimide) was dropped so as to cover the whole, and allowed to stand for 1 hour to activate the surface carboxylic acid. After rinsing the surface with pure water, 100 μg / mL anti-hidge IgG antibody dissolved in HEPES buffer (0.05 M HEPES (pH 7.4) + 0.2 M NaCl) was dropped for 2 hours. I let it. After rinsing the surface with pure water, a 1.0 M aqueous solution of ethanolamine hydrochloride (pH 8.5) was allowed to act for 30 minutes to inactivate the unreacted carboxylic acid activated compound.

 [2] Preparation of the array sensor of the present invention by direct antibody molecule orientation fixation using a COOH chip and a polymer

 COOH—0.2 M EDC on chip

 (N-ethyl-N '-[3- (dimethylamino) propyl] carbodiimide) /0.05M NHS

A mixed aqueous solution of (N-Hydroxysuccinimide) was dropped so as to cover the whole and allowed to stand for 1 hour to activate the surface carboxylic acid. After rinsing the surface with pure water, an aqueous solution containing 1% poly-L-lysine (molecular weight: 150,000 to 300,000) was applied to a small area of the surface plasmon resonance array sensor using a pin-type automatic spotting device manufactured by Toyobo. And kept for 2 hours by keeping the humidity at 80% in a sealed container. After washing the surface with 1.0 M KC1 five times, a 1.0 M ethanolamine hydrochloride aqueous solution (pH 8.5) was allowed to act for 30 minutes to inactivate the unreacted carboxylic acid-activated amide. A polymer-modified SPR sensor chip constructed in this way was mounted on a Multi SPRinter flow cell, and a recombinant protein A molecule containing 10 g / mL of negatively charged amino acids and cysteine residues was added to the flow cell. The liquid was circulated for 1 hour to promote the binding between the protein terminal and the primary amino group in the polymer by ionic interaction. Thereafter, a 5 mM NTCB solution (pH 7.0) was circulated for 1 hour to After cyanating the cysteine residue in A, the solution is circulated through a 10 mM borate buffer (pH 9.5) for 1 hour to immobilize the C-terminal backbone of the protein A protein via the cyanated cysteine residue. The reaction was accelerated. Finally, the surface was washed 5 times with 1.0M KC1 to wash unreacted proteins that could not form a covalent bond! While the substrate is kept in the flow cell, the surface plasmon resonance antibody array according to the present invention is obtained by circulating 10 / zg / mL anti-hidden IgG antibody and a heron-derived antibody to bind to the protein A molecule on the surface. The sensor was completed.

[3] Performance comparison test of conventional and antibody array sensors according to the present invention

 In order to compare the surface plasmon resonance antibody array sensor according to the present invention and the conventional method prepared in the above [1] and [2], two array sensors were separately placed in a flow cell and 10 IX g / mL anti-albumin hitch was used. The reaction was observed by applying diantibodies (Funakoshi) through liquid circulation (Fig. 1). As is clear from the figure, on the surface (curve A in the figure) on which the antibody molecules were immobilized by the conventional immobilization method (non-oriented), the detection limit was reached! However, in the array sensor according to the present invention (curve B in the figure), a much larger signal was observed reflecting the improvement in the effective sensitivity due to the orientation control fixing method, and the surface plasmon resonance array according to the present invention was observed. This shows the validity of the sensor construction technology.

 Brief Description of Drawings

 [FIG. 1] FIG. 1 shows that a surface plasmon resonance antibody array sensor was immobilized with an anti-hidden IgG antibody and a heron-derived antibody according to the conventional method and the present invention, respectively, and a 10 g / mL anti-human albumin antibody was detected. FIG. 3 is a diagram in which a reaction was observed with (Funakoshi) acting. The vertical axis is the signal value of surface plasmon resonance, and the horizontal axis is time. Thick horizontal lines indicate the time during which 10 g / mL anti-serum / albumin antibody (Funakoshi) was circulated through the flow cell, and during the other times, the HEPES buffer was circulated.

Claims

The scope of the claims  [1] An array substrate for producing a surface plasmon resonance antibody array sensor, wherein an antibody-binding protein or peptide is aligned and fixed in a specific region on the surface of a detection unit.  [2] The surface plasmon resonance, wherein the antibody binding protein on the array substrate for producing a surface plasmon resonance antibody array sensor according to claim 1 is obtained by aligning and fixing the antibody protein via a peptide. Antibody array sensor. [3] An antibody-binding protein is obtained by immobilizing the carboxy terminus of a peptide via a linker sequence to a linker molecule on a sensor detection unit having a primary amino group with an amide bond, and Equation (1)  NH—R—CO—NH—R—CO—NH—Y—Z (1)  2 1 2  The array substrate for producing a surface plasmon resonance antibody array sensor according to claim 1, wherein the array substrate is represented by:  [Wherein, R is the amino acid sequence of the antibody-binding protein or peptide, and R is any  Amino acid sequence of linker sequence of 1 2, Y represents linker molecule, z represents surface material of sensor detector ]  [4] The linker molecule having a primary amino group according to claim 3,  General formula (2)  (NH-X) n (2)  2  (Wherein, X represents a residue other than the primary amino group of the repeating unit of the polymer conjugate, and n represents an integer of 2 or more). 4. The array substrate for producing a surface plasmon resonance antibody array sensor according to claim 3, wherein:  [5] The array substrate for producing a surface plasmon resonance antibody array sensor according to claim 4, wherein the polymer compound represented by the general formula (2) in claim 4 is polyallylamine.  [6] The array substrate for producing a surface plasmon resonance antibody array sensor according to claim 4, wherein the polymer compound represented by the general formula (2) in claim 4 is polylysine.  [7] The specific formula (3) NH-I-COOH (3) [In the formula, represents an amino acid sequence of a protein or peptide. ]  A method for preparing an array substrate for preparing a surface plasmon resonance antibody array sensor in which antibody-binding proteins or peptides are aligned and immobilized, which is arranged and adsorbed on a substrate having a primary amino group, General formula (4)  NH-R-CO-NH-CH (CH-SCN)-CO-NH-R-COOH  2 1 2 2… · (4)  [Wherein, R represents the amino acid sequence of the antibody-binding protein or peptide;  It represents an amino acid sequence that is strongly negatively charged around 12 and can make the isoelectric point of the protein or peptide represented by the general formula (3) acidic. ]  And a carboxy terminus of the protein main chain of the general formula (3) is bonded to the primary amino group of a linker molecule on the sensor by a peptide bond, and then to the antibody-binding protein or peptide bond. A method for producing an array substrate for producing a surface plasmon resonance antibody array sensor, comprising: [8] The linker molecule having a primary amino group according to claim 7,  The method for producing an array substrate for producing a surface plasmon resonance antibody array sensor according to claim 7, wherein the molecule is a molecule containing a polymer compound having a primary amino group represented by the general formula (2).  [9] The method for producing an array substrate for producing a surface plasmon resonance antibody array sensor according to claim 7, wherein the polymer compound represented by the general formula (2) in claim 7 is polyallylamine.  [10] The method for producing an array substrate for producing a surface plasmon resonance antibody array sensor according to claim 7, wherein the polymer compound represented by the general formula (2) in claim 7 is polylysine.  [11] The surface plasmon resonance antibody array sensor according to claim 2, wherein the desorption / regeneration operation of the antibody molecule using an acid 'alkali' salt solution or the like enables repeated measurement of the same quality. A method for measuring an antigen molecule using the method. [12] The surface sensor according to claim 2 is used for the detection unit, and the repetitive reproduction operation according to claim 11 is applied, and an automatic regeneration process of the detection unit surface is performed every time the sample is measured. Automatic collection and measurement of measurement data generated by the pump Automatic measurement system for surface plasmon resonance antibody array sensor, equipped with an integrated data processing device that enables data processing.