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WO2005075996A1 - Plateau pour construire un détecteur à barrettes pour anticorps utilisant la résonance des plasmons de surface et procédé de construction de ce plateau - Google Patents

Plateau pour construire un détecteur à barrettes pour anticorps utilisant la résonance des plasmons de surface et procédé de construction de ce plateau

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Publication number
WO2005075996A1
WO2005075996A1 PCT/JP2005/001579 JP2005001579W WO2005075996A1 WO 2005075996 A1 WO2005075996 A1 WO 2005075996A1 JP 2005001579 W JP2005001579 W JP 2005001579W WO 2005075996 A1 WO2005075996 A1 WO 2005075996A1
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WIPO (PCT)
Prior art keywords
antibody
protein
surface plasmon
plasmon resonance
array sensor
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Ceased
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PCT/JP2005/001579
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English (en)
Japanese (ja)
Inventor
Masahiro Iwakura
Kiyonori Hirota
Hiroyuki Sota
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National Institute of Advanced Industrial Science and Technology AIST
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National Institute of Advanced Industrial Science and Technology AIST
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Publication of WO2005075996A1 publication Critical patent/WO2005075996A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/55—Specular reflectivity
    • G01N21/552—Attenuated total reflection
    • G01N21/553—Attenuated total reflection and using surface plasmons

Definitions

  • 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.
  • a planar solid 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)
  • genomics or proteomics which aims to conduct a comprehensive and comprehensive analysis of the information, is becoming increasingly important.
  • 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.
  • a high-energy laser with the excitation wavelength of the fluorescent molecule to generate fluorescent light.
  • Proteome analysis has rushed to follow.
  • 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.
  • 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.
  • 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.
  • this has become a factor that has not spread as much as NA arrays.
  • 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
  • the wave numbers of surface plasmon waves coincide, energy absorption called surface plasmon resonance occurs and the reflected light diminishes.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a planar substrate also serving as a detection unit used in an array sensor.
  • 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.
  • it can be standardized between domains.
  • 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 ⁇
  • 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.
  • 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.
  • 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)
  • An array substrate for producing a surface plasmon resonance antibody array sensor characterized by being represented by:
  • R is the amino acid sequence of the antibody-binding protein or peptide, and R is any
  • An array substrate for producing a surface plasmon resonance antibody array sensor comprising:
  • R represents an amino acid sequence of a protein or peptide.
  • 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 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.
  • 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.
  • 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.
  • the proteins are aligned and immobilized on the substrate, and the antibody-binding protein is used as a scaffold for antibody capture.
  • 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,
  • 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.
  • an antibody array sensor in which the effective sensitivity is further improved by immobilizing high-density antibody molecules.
  • 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.
  • the density of the immobilized antibody-binding protein can be further amplified, thereby increasing the sensitivity.
  • An antibody array sensor can be provided.
  • 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.
  • 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.
  • 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
  • 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 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 ( ⁇ -Se-Se-, Y, -A, -Se-) and the like.
  • 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.
  • 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.
  • 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.
  • SAM self-assembly monolayer
  • 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).
  • Y is a carboxyl group
  • an active form reagent such as hydroxysuccinimide
  • 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 ⁇ -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.
  • X represents, for example, a monomer residue constituting a polyalkylene chain, a polyamide chain, a polyester chain, a polystyrene chain, or the like.
  • 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.
  • 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 / ⁇ , various polymer compounds such as polylysine can be used.
  • any antibody-binding protein or peptide used for immobilization can be used as long as it has a binding ability 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 / ⁇ ⁇ : ⁇ «:" 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.
  • proteins often have a repeating sequence
  • fragmented proteins also have an ability to bind to antibody molecules.
  • 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.
  • the general formula (6) NH-R-COOH (6)
  • R is an amino acid of a protein or peptide capable of binding to an antibody molecule.
  • a protein or a protein having the binding ability represented by the general formula (6) NH-R-COOH may be used.
  • 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.
  • amino acid sequence of 2 is arbitrary and its type and number are not limited.
  • 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).
  • 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.
  • Such a fusion protein can be obtained by a known technique (for example, M. Iwakura et al., J. Biochem. Ill. 37-45 (1992)).
  • 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.
  • Preferred sequences include.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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 is a sequence obtained by adding the sequence of base-polyaspartic acid.
  • 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.
  • 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.
  • 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.
  • SEQ ID NO: 1 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 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.
  • 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.
  • 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.
  • pUC-type and PBR-type high copy number vectors are suitable as commercially available vectors.
  • 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.
  • a chromatography operation usually used for protein purification.
  • anion exchange chromatography, gel filtration chromatography, etc. are effective.
  • Affinity chromatography using a carrier on which immunoglobulin is immobilized is most effective.
  • 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.
  • 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.
  • 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 ⁇ l. Will be done.
  • 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.
  • 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.
  • 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.
  • 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).
  • R and R R are the same as R and R R in the general formula (7), respectively.
  • R is a linker peptide, R is a strongly negative charge near neutrality and R
  • This cyanation reaction can be performed using a commercially available cyanation reagent.
  • cyanation reagent 2-nitro-5-thiocyanobenzoic acid (NTCB) (see Y.Degani, A. Ptchornik, Biochemistry, 13, 1-11 (1974)) )
  • NTCB 2-nitro-5-thiocyanobenzoic acid
  • Shiano-dani using NTCB can be performed efficiently in a 10 mM phosphate buffer at pH 7.0.
  • 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.
  • 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.
  • 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.
  • 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.
  • poly-L-lysine commercially available from Sigma-Aldrich was used.
  • 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).
  • Ala-Asp-Asp-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.
  • 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.
  • 100 ⁇ g / mL anti-hidge IgG antibody dissolved in HEPES buffer 0.05 M HEPES (pH 7.4) + 0.2 M NaCl
  • HEPES buffer 0.05 M HEPES (pH 7.4) + 0.2 M NaCl
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.

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  • Peptides Or Proteins (AREA)

Abstract

Il est prévu de fournir un matériau de base pour construire un détecteur à barrettes pour anticorps utilisant la résonance des plasmons de surface sur lequel les molécules d’anticorps peuvent être immobilisées selon une orientation ajustée à une densité élevée dans une petite région, et un détecteur à barrettes utilisant la résonance des plasmons de surface dans lequel les molécules d’anticorps sont immobilisées. Un matériau de base sur lequel les molécules de protéines ou les molécules de peptides sont régulièrement alignées et immobilisées à une densité élevée selon un état uniformément orienté est obtenu en liant l’extrémité carboxyle d’une protéine se liant aux anticorps ou une chaîne principale d’un peptide à un groupe amine primaire sur un matériau de base comportant des groupes amines primaires à une densité élevée ou un groupe amine primaire dans un composé polymère fourni sur un matériau de base qui comporte le composé polymère portant les groupes amines primaires dans sa structure récurrente et ayant été introduit sur la surface d’un matériau de base plan. Un détecteur à barrettes pour anticorps utilisant la résonances des plasmons de surface comprenant un anticorps immobilisé sur le matériau de base, dans lequel les molécules d’anticorps sont immobilisées selon une orientation ajustée à une densité élevée, a une sensibilité d’une efficacité très élevée comparé aux barrettes pour anticorps existantes.
PCT/JP2005/001579 2004-02-06 2005-02-03 Plateau pour construire un détecteur à barrettes pour anticorps utilisant la résonance des plasmons de surface et procédé de construction de ce plateau Ceased WO2005075996A1 (fr)

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JP2004030700A JP3937020B2 (ja) 2004-02-06 2004-02-06 表面プラズモン共鳴抗体アレイセンサ作製用基板及びその作製方法
JP2004-030700 2004-02-06

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EP3462174A3 (fr) * 2013-03-15 2019-05-22 Arizona Board of Regents on behalf of Arizona State University Compositions et procédés de microréseaux de biocapteurs
CN110221059A (zh) * 2019-07-18 2019-09-10 大连理工大学 一种调控硅纳米材料表面hcg抗体取向的方法

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EP1826565B8 (fr) 2006-02-23 2015-11-25 FUJIFILM Corporation Biocapteur et procédé pour immobiliser une substance active physiologiquement
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JP5004165B2 (ja) * 2006-10-10 2012-08-22 独立行政法人産業技術総合研究所 タンパク質の配向制御固定化に適したタンパク質
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8697349B2 (en) 2007-11-01 2014-04-15 Kabushiki Kaisha Toyota Chuo Kenkyusho Method of producing solid-phase body having immobilized microobject and the use thereof
EP3462174A3 (fr) * 2013-03-15 2019-05-22 Arizona Board of Regents on behalf of Arizona State University Compositions et procédés de microréseaux de biocapteurs
US10983118B2 (en) 2013-03-15 2021-04-20 Arizona Board Of Regents On Behalf Of Arizona State University Biosensor microarray compositions and methods
US11828753B2 (en) 2013-03-15 2023-11-28 Arizona Board Of Regents On Behalf Of Arizona State University Biosensor microarray compositions and methods
CN110221059A (zh) * 2019-07-18 2019-09-10 大连理工大学 一种调控硅纳米材料表面hcg抗体取向的方法

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