WO2018008596A1 - タンパク質解析用固相担体及びその製造方法 - Google Patents

タンパク質解析用固相担体及びその製造方法 Download PDF

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WO2018008596A1
WO2018008596A1 PCT/JP2017/024368 JP2017024368W WO2018008596A1 WO 2018008596 A1 WO2018008596 A1 WO 2018008596A1 JP 2017024368 W JP2017024368 W JP 2017024368W WO 2018008596 A1 WO2018008596 A1 WO 2018008596A1
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antibody
protein
immobilized
solid phase
amount
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French (fr)
Japanese (ja)
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剛史 大場
紗也加 野地
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Kao Corp
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Kao Corp
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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
    • G01N33/54306—Solid-phase reaction mechanisms
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/26—Synthetic macromolecular compounds
    • B01J20/262—Synthetic macromolecular compounds obtained otherwise than by reactions only involving carbon to carbon unsaturated bonds, e.g. obtained by polycondensation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/02—Peptides being immobilised on, or in, an organic carrier
    • C07K17/08—Peptides being immobilised on, or in, an organic carrier the carrier being a synthetic polymer
    • 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
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • 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
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to a solid phase carrier for protein analysis and a method for producing the same.
  • biomolecules and their derivatives are often detected and quantified using specific interactions between molecules such as antigen-antibody reactions and enzyme reactions.
  • an immunoassay such as an enzyme immunoassay (ELISA) as a method for analyzing a protein such as a physiologically active substance.
  • ELISA enzyme immunoassay
  • the presence or absence of protein interaction with an antibody immobilized on a measurement region is determined by enzyme labeling.
  • a fluorescently labeled secondary antibody and indirectly measured by enzymatic reaction or fluorescence detection.
  • proteins interacting with immobilized antibodies have the property that the refractive frequency changes using surface plasmon resonance (SPR) and the resonance frequency fluctuates (decreases) using quartz crystal microbalance (QCM).
  • SPR surface plasmon resonance
  • QCM quartz crystal microbalance
  • a technique for directly detecting an intermolecular interaction by using the measurement is also widely used. Thus, in the analysis using an antibody, it is important to fix the antibody in the measurement region.
  • Immobilization of proteins such as antibodies is generally divided into physical immobilization (adsorption) and chemical immobilization methods.
  • antibodies are immobilized.
  • the antibody molecules cannot be immobilized at specific sites in the molecule, and the orientation of the immobilized antibodies is random. Therefore, there is a problem that all the immobilized antibodies cannot function.
  • Non-patent Document 1 Non-patent Document 1
  • PS-tag polystyrene affinity peptide
  • Patent Document 1 a method of immobilizing biotinylated protein G via a streptavidin to a biotinylated solid phase carrier (Patent Document 1), suppression of non-specific adsorption using a material such as polyethylene glycol (PEG) and the amount of protein immobilized (Non-patent Document 4) and the like have been reported.
  • techniques such as PS-tags require synthesis of fusion proteins and preparation of affinity peptides for each substrate surface, which is not convenient, and is modified with polyethylene glycol or the like.
  • non-specific adsorption is suppressed on the substrate surface, it is difficult to introduce detection antibodies and the like, and the amount of detection antibody immobilized is reduced, so that it is difficult to increase detection sensitivity.
  • Patent Document 1 International Publication No. 2014/132629 (Non-Patent Document 1) Analytica Chimica Acta, 2012,728, 64-68 (Non-Patent Document 2) Anal. Chem., 2011, 83, 1969-1976 (Non-Patent Document 3) Anal. Bioanal. Chem., 2009, 395, 759-765 (Non-Patent Document 4) Anal. Chem., 2005, 77, 1075-1080
  • the present invention relates to the following 1) to 4).
  • a solid phase carrier for protein analysis in which a first antibody is immobilized on the surface of a substrate, and a second antibody having affinity for a substance to be analyzed is bound via protein A or G or a modified form thereof.
  • a method for producing a solid phase carrier for protein analysis in which a first antibody is bound to a substrate surface, then protein A or G or a variant thereof is allowed to act to bind to the first antibody, and then analyzed.
  • a method in which a second antibody having affinity for a target substance is allowed to act and bind to the protein A or G or a variant thereof.
  • a solid phase carrier for capturing an immunoglobulin wherein the first antibody is immobilized on the surface of the substrate, and protein A or G or a variant thereof is bound to the antibody.
  • the present invention provides a solid phase carrier capable of immobilizing an antibody for detection binding to a substance to be analyzed in a position-specific manner at an Fc site in the molecule, and immobilizing the antibody by controlling orientation in a measurement region. For the purpose.
  • the present inventors have an affinity for the first antibody immobilized on the substrate surface and the substance to be analyzed.
  • the second antibody (detection antibody) was position-specifically linked with protein A, G or a variant thereof, so that the second antibody was immobilized on the substrate surface in a controlled orientation state. It has been found that a solid support can be easily and efficiently produced.
  • the present invention it is possible to provide a solid phase carrier in which the detection antibody is position-specifically controlled at the Fc site in the molecule and fixed in a sufficient amount in the measurement region.
  • the solid phase carrier of the present invention is used in technical fields related to protein analysis and immunoglobulin capture, quantification or purification, for example, chromatography carriers, protein sensors, immunoaffinity carriers, antibody arrays, affinity analysis, affinity separation, etc. can do.
  • the first antibody is immobilized on the substrate surface, and the second antibody having affinity for the substance to be analyzed is the first antibody via protein A or G or a variant thereof. It is linked to the antibody.
  • FIG. 1 shows a schematic diagram thereof.
  • protein analysis includes the concept of protein separation / purification, detection, and measurement.
  • the solid phase carrier of the present invention will be described.
  • the shape and material of the substrate (support) are not particularly limited as long as the first antibody can be bound to the surface by physical binding.
  • any Is also included.
  • plastics such as polyethylene, polypropylene, polystyrene, polymethacrylate, polyvinyl alcohol, hydrogel, agarose, dextran, cellulose, chitosan, latex, etc.
  • Natural materials, inorganic materials typified by silica, glass, ceramics, etc., and metal materials typified by gold, alumina, silver, etc. can be used widely.
  • a functional group such as an amino group, a carboxy group, or a vinyl group can be appropriately introduced into such a substrate using a known surface treatment technique.
  • the distance between the first antibody and the substrate is appropriate on the surface of the substrate for reasons such as ease of immobilization of the first antibody, or for the orientation control and reactivity of the second antibody.
  • Molecules (linkers) that can be secured to each other may be bound.
  • the molecule that can be a linker is usually selected according to the charge characteristics of the surface of the solid phase carrier, but preferably a thiol derivative such as alkanethiol that forms a self-assembled monolayer (SAM), or polyethylene glycol And a hydrophilic polymer containing a chain (PEG chain), MPC polymer (polymer of 2-methacryloyloxyethyl phosphorylcholine) and the like.
  • a substrate in which a self-assembled monolayer (SAM) is formed on a metal substrate such as gold, silver, aluminum, copper, platinum, or a substrate in which PEG is bonded is preferable as the substrate of the solid phase carrier of the present invention.
  • the self-assembled monolayer means that when a reactive organic molecule such as alkanethiol comes into contact with an appropriate substrate material and is left to stand, the organic molecule and the substrate material chemically react with each other on the substrate surface. It means an organic monomolecular film with uniform orientation of molecules formed on the substrate surface by adsorbed molecules being densely gathered by interaction between organic molecules as well as chemical adsorption, surface plasmon resonance (SPR), It is widely used in fields such as surface plasmon resonance excitation enhanced fluorescence spectroscopy (SPFS) and quartz crystal microbalance (QCM).
  • SPFS surface plasmon resonance excitation enhanced fluorescence spectroscopy
  • QCM quartz crystal microbalance
  • organic molecules that form a self-assembled monolayer that can be used on a substrate include, for example, the following formula: X—R (1) [wherein X is SH— (CH 2 ) n ⁇ . Or SH— (CH 2 ) n — (O—CH 2 —CH 2 ) m — (OCH 2 ) 1 —, wherein R is a hydrogen atom, a carboxy group, a hydroxyl group, a hydroxymethyl group, an amino group, an aminomethyl, Group, an aldehyde group, an amide group (—CONH 2 group), a phosphonic acid group, a sulfo group, a quaternary ammonium group such as a trimethylamino group, a vinyl group, an acetylene group, an azido group or a sulfobetaine group, and n is 2 to 18 represents an integer, m represents an integer of 1 to 18, and l represents 0 or 1. ]
  • X is SH— (
  • X is preferably SH— (CH 2 ) n — (O—CH 2 —CH 2 ) m — (OCH 2 ) 1 —, and n is preferably 2 to 16, 16 is more preferable. Further, m is preferably 1 to 12, and more preferably 3 to 6. R is preferably hydrogen, a carboxy group, an aminomethyl group, or a hydroxymethyl group.
  • organic molecules forming the self-assembled monolayer include, for example, 1-carboxyundecanethiol, 10-carboxy-1-decanethiol, 11-hydroxy-1-undecanethiol, carboxy-EG 6 -undecanethiol (Alternative name: 20- (11-mercaptoundecanyloxy) -3,6,9,12,15,18-hexaoxaeicosanoic acid).
  • the organic molecules forming the self-assembled monolayer can be used alone or in combination of two or more. In this case, it is preferable to combine carboxyalkanethiol and hydroxyalkanethiol, or carboxypolyethyleneoxyalkanethiol and hydroxypolyethyleneoxyalkanethiol.
  • the mixture ratio is arbitrary.
  • the number average molecular weight or the weight average molecular weight of the PEG chain [— (CH 2 —CH 2 —O) n — (n is an integer indicating the degree of polymerization)] is preferably 200 or more. More preferably, it is 500 or more, and preferably 30,000 or less, more preferably 10,000 or less, more preferably 5,000 or less. Further, it is preferably 200 to 30,000, more preferably 500 to 10,000, and more preferably 500 to 5,000.
  • the PEG chain in the hydrophilic polymer is preferably linear, but may form a branched chain. Also, multiple types of PEG polymers having different chain lengths can be introduced onto the substrate surface as linker molecules.
  • the hydrophilic polymer containing the PEG chain can have at least one functional group at one end of the PEG chain for chemically introducing the molecule into the substrate.
  • functional groups include amino groups, carboxyl groups, thiol groups, epoxy groups, aldehyde groups, maleimide groups, azido groups, cyano groups, active ester groups (succinimidyloxycarbonyl groups, 1H-benzotriazoles).
  • a molecule having affinity for the first antibody which will be described later, or a molecule thereof is bound to the PEG chain at a position different from the above one end (for example, the other end) in addition to an alkoxy group such as a methoxy group.
  • hydrophilic polymer containing a PEG chain can have a functional group (for example, a carboxyl group, an amino group, a maleimide group, an epoxy group, etc.)
  • a hydrophilic polymer containing a PEG chain when introducing a hydrophilic polymer containing a PEG chain onto a gold substrate, it has a thiol group at one end of the PEG chain and an alkoxy group such as a methoxy group, a carboxyl group, an amino group, etc. at the other end.
  • a hydrophilic polymer (PEG thiol reagent) having, and the polymer can be introduced by bringing it into contact with a gold substrate. It is also possible to introduce a plurality of hydrophilic polymers containing PEG chains having such thiol groups at the ends.
  • the substrate surface of the present invention (including the above-described surface into which the functional group or linker has been introduced) has an affinity for the first antibody in order to facilitate immobilization by physical binding of the first antibody. It is preferable that the molecule to be introduced is introduced.
  • the molecule having affinity for the first antibody include antibody-binding molecules.
  • the antibody-binding molecule is a molecule containing an antibody antigen, and specifically includes the following molecules.
  • biotin derivatives include biotin ethylenediamine monoamide and N- (5-aminopentyl) biotinamide.
  • any functional group for example, carboxy group
  • the substrate or a linker on the substrate surface or appropriately introduced.
  • spacer molecules for example, the carboxy group on the substrate or linker is appropriately activated using water-soluble carbodiimide (WSC), sodium N-hydroxysulfosuccinimide (NHS), etc., and then the molecule having affinity for the first antibody is dissolved.
  • WSC water-soluble carbodiimide
  • NHS sodium N-hydroxysulfosuccinimide
  • the concentration of the molecule having affinity for the first antibody is preferably from 0.0001 to 10 mM, more preferably from 0.0001 to 10 mM, from the viewpoint of introduction efficiency of the molecule having affinity for the first antibody. It is about 001 to 1 mM.
  • Examples of the spacer molecule include a PEG derivative, and the chain length of — (CH 2 —CH 2 —O—) n — is preferably such that the number of n is 1 to 24 and is 2 to 24. Is more preferable, and 4 to 24 is more preferable.
  • Examples of PEG derivatives include H 2 N— (CH 2 —CH 2 —O) n —CH 2 —CH 2 —COOH and H 2 N— (CH 2 —CH 2 —O) n —CH 2 —CH 2.
  • a molecule having affinity for the first antibody can be introduced in advance into a molecule that can serve as a linker, and the molecule can be bound to the substrate.
  • a method of immobilizing a thiol derivative or PEG thiol reagent having a thiol group at one end and biotin bonded at the other end by an ester bond or an amide bond on the gold substrate on the thiol group side, or A monobiotinylated PEG derivative in which biotin is introduced into a spacer molecule for example, Biotin-NH 2 —CH 2 —CH 2 —NH—OC— (CH 2 —CH 2 —O—) n —CH 2 CH 2 —NH 2
  • proteins such as albumin, casein, globulin, gelatin, skim milk, etc., which are known as proteins forming a non-specific adsorption preventing layer (so-called blocking agents), as molecules having affinity for the first antibody
  • blocking agents proteins forming a non-specific adsorption preventing layer
  • the first antibody is immobilized on the surface of the substrate.
  • the antibody used is not particularly limited as long as it has a constant region (Fc domain) that binds to a molecule on the substrate surface and binds to protein A or G or a variant thereof.
  • Antiserum prepared from the serum of an animal immunized with an antigen recognized by the antibody, an immunoglobulin fraction purified from the antiserum, and a monoclonal antibody obtained by cell fusion using spleen cells of an animal immunized with the antigen Alternatively, a polyclonal antibody can be used.
  • disconnection of the disulfide bond between the heavy chains (H chain) may be sufficient.
  • Immobilization of the first antibody on the surface of the substrate is a physical bond other than a covalent bond such as affinity bond, van der Waals force, Coulomb force, or hydrogen bond between the first antibody and a molecule on the substrate surface.
  • a physical bond other than a covalent bond such as affinity bond, van der Waals force, Coulomb force, or hydrogen bond between the first antibody and a molecule on the substrate surface.
  • binding by physical interaction with a self-assembled monolayer on the substrate surface affinity binding with PEG on the substrate surface, or linker on the substrate surface or substrate surface (self-assembled monolayer, PEG, etc. And binding to the affinity molecule introduced above.
  • an anti-PEG antibody is used as the first antibody and has an affinity for a low molecular compound such as biotin or digoxigenin or a protein such as albumin or streptavidin.
  • a low molecular compound such as biotin or digoxigenin or a protein such as albumin or streptavidin.
  • an antibody against the molecule for example, an anti-biotin antibody, an anti-digoxigenin antibody, an anti-albumin antibody, an anti-streptavidin antibody or the like is used as the first antibody.
  • an anti-PEG antibody either an antibody that forms an affinity bond with a substituent such as a methoxy group at the end of the PEG chain or an antibody that forms an affinity bond with a PEG chain can be used. .
  • a commercially available antibody may be purchased and used. If a monoclonal antibody is used, a hybridoma is prepared by cell fusion after immunization using a corresponding immunogen, and an antibody secreted from the cell is used. It may be used after purification.
  • a biotinylated antigen can be prepared by allowing biotin or a derivative thereof to act on a known carrier protein.
  • an anti-biotin antibody can be prepared using biotinylated denatured Ovalbumin as an antigen. It can be prepared by using an antigen in which a PEG derivative is conjugated to Keyhole Limpet Haemocyanin (KLH) or the like.
  • KLH Keyhole Limpet Haemocyanin
  • the concentration of the antibody when the first antibody is allowed to act on molecules on the substrate surface by standing or by feeding is not particularly limited, but is preferably 1 to 1000 ⁇ g / ml.
  • a solution containing the antibody is added to the self-assembled monolayer, and the solution is added at 0 to 50 ° C. for several minutes to several This can be achieved by standing for a time or feeding.
  • Protein A or G or a modification thereof is bound to both the first antibody and the second antibody.
  • Protein A is a protein of the cell wall component produced by Staphylococcus aureus, recognizes the constant region (Fc domain) of immunoglobulin G, immunoglobulin A, and immunoglobulin M, and is not shared It has an activity to bind to these by binding (antibody binding activity).
  • Protein A is a multidomain membrane protein composed of a plurality of domains, and some of the extracellular domains exhibit binding activity (antibody binding activity) to a protein having an Fc region of immunoglobulin G.
  • the Z domain is a modified protein in which mutations for substituting Ala at position 1 and Gly at position 29 with Val and Ala, respectively, relative to the B domain (Tashiro M, Montelione GT. (1995) Structures of bacterial immunoglobulin). Curr Opin Struct Biol. 5, 471-481.), alkaline resistance and binding ability are improved compared to B domain.
  • Protein G is also a protein produced by streptococci having specific binding activity to the Fc region of immunoglobulin G.
  • Protein G is also a multi-domain membrane protein composed of a plurality of domains, and it is a part of the extracellular domain that shows binding activity (antibody binding activity) to the Fc region of immunoglobulin.
  • binding activity antibody binding activity
  • protein G derived from the G148 strain three domains B1, B2, and B3 exhibit antibody binding activity (sometimes referred to as C1, C2, and C3 domains).
  • C1, C2, and C3 domains there are three antibody binding domains in protein G of GX7805 strain and two antibody binding domains in protein G of GX7809 strain.
  • recombinant protein A or recombinant protein G (referred to as “modified”) modified with protein engineering has been created in order to enhance antibody binding activity of protein A or G.
  • modification is made. It is preferable to use the body.
  • a variant in addition to the above-mentioned Z domain of protein A, a variant having a total domain number of 2 or more having an immunoglobulin binding active domain of protein A or protein G, and an immunoglobulin binding active domain of protein A and protein G are fused. And variants having a total number of domains of 2 or more, preferably 2 to 20, more preferably 2 to 12.
  • such a variant is a protein G having a total domain number of 2 or 3 from which a part other than the immunoglobulin binding active domain has been removed (manufactured by Thermo, BioVision), and the number of domains. 5 protein A (Thermo, BioVision), or Protein A / G (total, 6 or 8) fused with Protein A and G immunoglobulin binding domains (Thermo, BioVision) ) And the like.
  • the concentration at which protein A or G or a variant thereof is allowed to act on the first antibody on the substrate surface is not particularly limited, but is preferably 1-1000 ⁇ g / mL.
  • the second antibody is an antibody that binds to the compound to be analyzed, and is immobilized by binding to the first antibody via protein A or G or a variant thereof.
  • the second antibody is not particularly limited as long as it reacts specifically with the compound to be measured.
  • antiserum prepared from the serum of an animal immunized with a compound recognized by the antibody purified from antiserum A monoclonal antibody or a polyclonal antibody obtained by cell fusion using spleen cells of an animal immunized with the immunized immunoglobulin fraction and the compound can be used. Further, it may be modified as in the case of a chimeric antibody or the like.
  • disconnection of the disulfide bond between the heavy chains (H chain) may be sufficient.
  • the second antibody can be used regardless of the animal species.
  • antibodies derived from monkeys, mice, rats, rabbits, chickens, goats, sheep, guinea pigs, etc. specifically mouse IgG, rat IgG Rabbit IgG, goat IgG, sheep IgG and the like can be used, and any of polyclonal antibody and monoclonal antibody may be used.
  • Any antibody subclass can be used as long as it can bind to protein A or G or a variant thereof.
  • the concentration at which the second antibody is allowed to act on protein A or G on the substrate surface or a variant thereof is not particularly limited, but is preferably 1 to 1000 ⁇ g / mL.
  • a linker molecule to which a sexual compound is bound is immobilized. Such immobilization is not necessary if the substrate surface itself has an affinity for the first antibody.
  • the binding order is Either may be immobilized on the substrate surface in the order of the first antibody, protein A or G or a variant thereof and the second antibody, or the protein may be immobilized on the first antibody immobilized on the substrate surface.
  • a complex of A or G or a variant thereof and the second antibody may be bound, or a complex of the first antibody, protein A or G or variant thereof, and the second antibody is prepared in advance. Alternatively, it may be immobilized on the substrate surface.
  • the first antibody is bound to the substrate surface, then 2) protein A or G or a variant thereof is allowed to act to bind to the first antibody, and then 3) the second antibody is It is a method of binding to the protein A or G or a variant thereof by acting.
  • the binding between the substrate surface and the first antibody, the binding between the first antibody and protein A or G or a variant thereof, and between the protein A or G or variant thereof and the second antibody All bonds are physical bonds based on intermolecular interactions other than covalent bonds. Therefore, immobilization of each molecule of the first antibody, protein A or G or a variant thereof, and the second antibody is performed by, for example, dissolving each molecule in a running buffer (for example, DPBS (Dulbecco ⁇ s Phosphate Buffered Saline)).
  • a running buffer for example, DPBS (Dulbecco ⁇ s Phosphate Buffered Saline)
  • the flow rate is 1-30 ⁇ L / min per flow cell (length 2.9 mm, width 0.5 mm, height 0.04 mm) It can be carried out by feeding the solution on the substrate for 5 to 60 minutes, or by immersing the measurement substrate in the above solution of each molecule.
  • the second antibody can be obtained by utilizing a physical interaction based on an intermolecular interaction other than a covalent bond by utilizing protein A or G or a variant thereof. In the Fc region in the molecular structure, non-covalent bond is immobilized on the substrate surface in a position-specific manner, so that the orientation of the antibody molecule is controlled. This is useful compared to the method adopted for the part.
  • the solid phase site where the antibody has not been immobilized may be blocked using a blocking agent such as BSA (bovine serum albumin), block ace, skim milk, casein and the like.
  • BSA bovine serum albumin
  • stabilization such as the synthesis of polyethylene glycol and polysaccharides, natural polymers, surfactants, and commercially available Immunoassay® Stabilizer (ABI) stabilizes the immobilization.
  • An agent can be added.
  • a surfactant is added to each solution used during the above-described immobilization operation for the purpose of reducing the loss of each protein due to adhesion of each solution used to the flow path or the like. Can be added.
  • the solid phase carrier produced by the above-described immobilization operation in which the first antibody is immobilized on the substrate surface, and protein A or G or a variant thereof is bound to the first antibody is protein A or It can be used as a solid phase carrier for purification or supplementation or quantification of an antibody molecule having an Fc site capable of binding to G or a variant thereof, specifically an immunoglobulin, preferably IgG.
  • kits containing at least a substrate, a first antibody, protein A or G or a modified form thereof, and a second antibody may be used. If this kit is used, the detection antibody is position-specifically controlled at the Fc site in the molecule and can be immobilized in a sufficient amount in the measurement region.
  • a solid phase carrier for protein analysis in which a first antibody is immobilized on a substrate surface, and a second antibody having affinity for a substance to be analyzed is bound via protein A or G or a modified form thereof A solid phase carrier linked to a first antibody.
  • a first antibody is immobilized on a substrate surface, and a second antibody having affinity for a substance to be analyzed is bound via protein A or G or a modified form thereof A solid phase carrier linked to a first antibody.
  • the molecule having affinity for the first antibody is biotin, digoxigenin or a derivative thereof.
  • ⁇ 4> The solid phase carrier according to ⁇ 2>, wherein the molecule having affinity for the first antibody is a protein selected from albumin, casein, globulin, gelatin, skim milk, fibronectin and lysozyme.
  • ⁇ 5> The solid phase carrier according to ⁇ 2>, wherein the molecule having affinity for the first antibody is avidin, streptavidin, neutravidin, or a complex of these with biotin or a derivative thereof.
  • ⁇ 6> The solid phase carrier according to any one of ⁇ 1> to ⁇ 5>, wherein the substrate surface is a surface into which a hydrophilic polymer containing a polyethylene glycol chain is introduced.
  • ⁇ 7> The solid phase carrier according to ⁇ 6>, wherein the hydrophilic polymer containing a polyethylene glycol chain is a hydrophilic polymer containing a PEG chain having an average molecular weight of 500 to 5000.
  • the substrate surface is a surface on which a self-assembled monolayer is formed.
  • the self-assembled monolayer is an alkanethiol derivative.
  • ⁇ 10> A variant in which protein A or G variant has a protein A or protein G immunoglobulin-binding active domain, or a total of two or more domains in which protein A or protein G immunoglobulin-binding active domain is fused
  • ⁇ 12> A method for producing a solid phase carrier for protein analysis, comprising binding a first antibody to a substrate surface, then allowing protein A or G or a variant thereof to act and binding the first antibody; A method in which a second antibody having affinity for an analysis target substance is allowed to act and bind to the protein A or G or a variant thereof.
  • ⁇ 14> A solid phase carrier for capturing or quantifying immunoglobulin, wherein the first antibody is immobilized on the surface of the substrate, and protein A or G or a variant thereof is bound to the antibody.
  • ⁇ 15> The solid phase carrier according to ⁇ 14>, wherein the immunoglobulin is IgG.
  • ⁇ 16> A method for capturing or quantifying immunoglobulin using the solid phase carrier according to ⁇ 14> or ⁇ 15>.
  • Example 1 (1) Preparation of SAM Carboxy-EG 6 -undecanthiol (20- (11-mercaptoundecanyloxy) -3,6,9,12,15,18-hexaoxaeicosanoic acid, Dojindo Laboratories, C445) is immersed in a 100 ⁇ M ethanol solution (10mL) for the Biacore T-200 sensor chip (GE Healthcare, SIA Kit Au, BR-1004-05) overnight at 25 ° C, and then on the gold surface of the chip. A self-assembled monolayer (SAM) was prepared. The sensor chip after the immersion in shaking was washed with ethanol and Milli-Q water and then dried with nitrogen.
  • SAM self-assembled monolayer
  • the above-described droplet deposition operation was repeated to convert the carboxyl group on the SAM surface into an active ester group.
  • the chip was immersed in a 1 ⁇ M DMF solution (10 mL) in which N- (5-aminopentyl) biotinamide (trifluoroacetate) was neutralized with an equimolar amount of triethylamine, and shaken at 25 ° C. for 2 hours. Then, after washing the chip with Milli-Q water, 300 ⁇ L of 1M-ethanolamine solution is dropped on the chip surface for 10 minutes to hydrolyze the unreacted active ester. This dropping operation is repeated twice. went.
  • the chip was washed with Dulbecco's phosphate-buffered saline (Life Technology, DPBS, 14190-144, pH 7.0-7.3), dried with nitrogen, and subjected to SPR measurement. SPR measurement was performed at 25 ° C. using Biacore T200.
  • Immobilization of anti-biotin antibody to the biotinylated sensor chip surface is performed by 100 ⁇ g / mL of anti-biotin antibody (Abcam, ab53494, Rabbit polyclonal) dissolved in DPBS as a running buffer. The solution was fed on the sensor chip at a flow rate of 10 ⁇ L / min for 15 minutes, and then the running buffer was again fed for 15 minutes. The amount of anti-biotin antibody immobilized was 3376.4RU.
  • BSA blocking Blocking with Bovine Serum Albumin (SIGMA, BSA, A3059-10G) on the surface of the sensor chip on which the anti-adiponectin antibody is immobilized is a PBS solution (1 mg / mL) in which BSA is dissolved in DPBS as a running buffer. The solution was fed on the sensor chip at a flow rate of 10 ⁇ L / min for 20 minutes, and then the running buffer was again fed for 40 minutes. The amount of BSA adsorption was 674.7RU.
  • Antigen (adiponectin) binding amount SPR measurement of the antigen binding amount on the surface of the sensor chip prepared by the operations of (1) to (6) was also performed at 25 ° C. using Biacore T200.
  • a running buffer a DPBS solution containing 0.05% (V / W) Tween 20 having a BSA concentration of 1 mg / mL was used.
  • Adiponectin (Enzo Life Sciences ALX-522-063-C050, Human, Recombinant, HEK293 cells) is also used by dissolving it in the running buffer, and 1.0 ⁇ g / mL solution is sent onto the sensor chip at a flow rate of 10 ⁇ L / min for 20 minutes.
  • the running buffer was again fed for 20 minutes to measure the amount of antigen binding.
  • the amount of antigen binding was determined by correcting with reference data.
  • reference data in the step of immobilizing the anti-adiponectin antibody, a detection interface was prepared by immobilizing the anti-biotin antibody instead of the anti-adiponectin antibody, and the amount of antigen binding at that interface was subtracted from the measured value and corrected. .
  • the corrected antigen binding amount was 336.2 RU. The results are shown in Table 1.
  • Example 2 Preparation of SAM A SAM was prepared according to the method of Example (1).
  • BSA blocking BSA blocking was performed according to the method of (1) of Example 1.
  • the amount of BSA adsorption was 360.8RU.
  • Antigen (adiponectin) binding amount According to the method of (7) of Example 1, the antigen binding amount was measured. The corrected antigen binding amount was 285.8 RU. The results are shown in Table 1.
  • Example 3 (1) Preparation of mixed SAM 10-carboxy-1-decanethiol (Dojindo Laboratories, C385) in 500 ⁇ M ethanol solution and 11-hydroxy-1-undecanthiol (Dojindo Laboratories, H337) in 500 ⁇ M ethanol solution
  • the sensor chip (GE Healthcare, SIA Kit Au, BR-1004-05) used in the Biacore T-200 was immersed in a mixed solution (10 mL) mixed at a ratio of 1: 9 at 25 ° C. for 10 minutes.
  • a self-assembled monolayer (SAM) was created on the gold surface of the sensor chip.
  • the surface of the sensor chip after the immersion in shaking was washed with ethanol and Milli-Q water and then dried with nitrogen.
  • BSA blocking BSA blocking was performed according to the method of (1) of Example 1.
  • the amount of BSA adsorption was 946.4RU.
  • Antigen (adiponectin) binding amount According to the method of (7) of Example 1, the antigen binding amount was measured. The corrected antigen binding amount was 275.7 RU. The results are shown in Table 1.
  • Example 4 Preparation of mixed SAM A mixed SAM was prepared according to the method of Example 3 (1). However, a mixed solution in which a solution of thiol reagent 10-carboxy-1-decanethiol and 11-hydroxy-1-undecanethiol was mixed at a ratio of 1:99 was used.
  • BSA blocking BSA blocking was performed according to the method of (1) of Example 1.
  • the amount of BSA adsorption was 907.6RU.
  • Example 5 Preparation of mixed SAM A mixed SAM was prepared according to the method of Example 3 (1). However, a mixed solution in which a solution of 10-carboxy-1-decanethiol and 11-hydroxy-1-undecanethiol was mixed at a ratio of 1: 1999 was used.
  • BSA blocking According to (6) of Example 1, BSA blocking was performed. The amount of BSA adsorption was 1184.1RU.
  • BSA blocking Blocking with BSA on the surface of the sensor chip on which the anti-adiponectin antibody is immobilized is performed by using a PBS solution (1 mg / mL) in which BSA is dissolved in DPBS as a running buffer at a flow rate of 10 ⁇ L / min. After feeding for 15 minutes, the running buffer was fed again for 5 minutes. The amount of BSA adsorption was 112.9RU.
  • Amount of antigen (adiponectin) binding SPR measurement of the amount of antigen binding on the surface of the sensor chip prepared by the operations of (1) to (3) was performed at 25 ° C. using Biacore T200.
  • a running buffer a DPBS solution containing 0.05% (V / W) Tween 20 having a BSA concentration of 1 mg / mL was used.
  • Adiponectin was also used by dissolving in the solution, and the 1.0 ⁇ g / mL solution was fed onto the sensor chip at a flow rate of 10 ⁇ L / min for 15 minutes and then the running buffer was again fed for 10 minutes.
  • the amount of antigen binding was determined by correcting with reference data.
  • anti-interleukin 6 (IL6) antibody R & D SYSTEM, Human IL-6 Antibody, Monoclonal Mouse IgG 2B , MAB2061
  • IL6 antibody R & D SYSTEM, Human IL-6 Antibody, Monoclonal Mouse IgG 2B , MAB2061
  • a detection interface was prepared, and correction was performed by subtracting the amount of antigen binding at the interface from the measured value.
  • the corrected antigen binding amount was 100.7 RU.
  • Table 1 The results are shown in Table 1.
  • Example 6 Creation of SAM A SAM was created according to the method of Example 1 (1).
  • the anti-biotin antibody was immobilized on the biotinylated sensor chip surface by dissolving the anti-biotin monoclonal antibody (Abcam, ab36406, Mouse monoclonal) in DPBS as a running buffer at 25 ° C. The 5.0 ⁇ g / mL solution was fed onto the sensor chip at a flow rate of 10 ⁇ L / min for 30 minutes, and then the running buffer was added again for 20 minutes. The amount of anti-biotin antibody immobilized was 4493.2RU.
  • BSA blocking The blocking of the surface of the sensor chip on which the anti-CRP antibody is immobilized with BSA is a solution of BSA dissolved in DPBS as a running buffer (1 mg / mL) on the sensor chip at a flow rate of 10 ⁇ L / min. After feeding for 20 minutes, the running buffer was fed again for 10 minutes. The amount of BSA adsorption was -194.3RU.
  • Antigen (CRP) binding amount SPR measurement of the antigen binding amount on the surface of the sensor chip prepared by the operations of (1) to (6) was performed at 25 ° C. using Biacore T200.
  • a running buffer a DPBS solution containing 0.05% (V / W) Tween 20 having a BSA concentration of 1 mg / mL was used.
  • Example 7 Creation of SAM A SAM was created according to the method of Example 1 (1).
  • biotin derivative N- (5-aminopentyl) biotinamide (trifluoroacetate salt) was immobilized according to the method of Example 6 (2).
  • Immobilization of Protein A / G Immobilization of Protein A / G (Pierce 21186, Recombinant Protein A / G from E.coli, 6 domain) on the surface of the sensor chip on which the anti-biotin antibody is immobilized is 25 A 25 ⁇ g / mL solution of Protein A / G dissolved in DPBS as a running buffer was fed at 30 ° C. for 30 minutes at a flow rate of 10 ⁇ L / min, and then the running buffer was again fed for 30 minutes. The amount of Protein A / G immobilized was 622.4 RU.
  • Example 8 Creation of SAM A SAM was created according to the method of Example 1 (1).
  • biotin derivative N- (5-aminopentyl) biotinamide (trifluoroacetate salt) was immobilized according to the method of Example 6 (2).
  • Comparative Example 2 (1) Preparation of SAM It was carried out according to the method of Comparative Example 1 (1).
  • BSA blocking BSA on the surface of the sensor chip on which the anti-CRP antibody is immobilized is blocked with a PBS solution (1 mg / mL) in which BSA is dissolved in PBS as a running buffer at a flow rate of 10 ⁇ L / min. After feeding for 15 minutes, the running buffer was fed again for 5 minutes. BSA adsorption amount was 73.6RU.
  • Amount of antigen binding SPR measurement of the amount of antigen binding on the surface of the sensor chip prepared by the operations of (1) to (3) was performed at 25 ° C. using Biacore T200.
  • a running buffer a DPBS solution containing 0.05% (V / W) Tween 20 having a BSA concentration of 1 mg / mL was used.
  • C-Reactive protein (CRP, R & D SYSTEM 1707-CR-200, Mouse myeloma cell line) is also used by dissolving in that solution, and 1.0 ⁇ g / mL solution is sent over the sensor chip at a flow rate of 10 ⁇ L / min for 15 minutes. After the solution, the running buffer was again fed for 10 minutes.
  • the antigen-antibody reaction amount was obtained by correcting with reference data.
  • reference data in the process of immobilizing the anti-CRP antibody, a detection interface with anti-interleukin 6 (IL6) antibody immobilized instead of the anti-CRP antibody was created, and the amount of antigen-antibody reaction at that interface was determined from the measured value Correction was made by subtracting.
  • the corrected CRP binding amount was 284.2 RU. The results are shown in Table 2.
  • Example 9 (1) Preparation of mPEGylated interface
  • the mPEG (methoxypolyethylene glycol) interface was prepared at 37 ° C. using Biacore T-200.
  • As a running buffer a solution in which 14.61 g of sodium chloride was added to Dulbecco's phosphate-buffered saline (Life Technologies, DPBS, 14190-144, pH 7.0-7.3, 250 g) was used.
  • a running buffer solution of mPEG thiol 5k (Creative PEG works, mPEG-SH, MW 5000, PLS-604, 1 mg / mL) dissolved in the running buffer is fed onto the sensor chip at a flow rate of 15 ⁇ L / min for 20 minutes.
  • the running buffer was fed for 10 minutes, the 0.05N-NaOH solution for 1 minute, the running buffer for 3 minutes, the 0.05N-NaOH solution for 1 minute, and then the running buffer at a flow rate of 15 ⁇ L / min for 11 minutes.
  • This series of operations was repeated twice to immobilize mPEG thiol 5k on the sensor chip surface.
  • the total amount of mPEG thiol 5k immobilized was 2940.1 RU.
  • mPEG thiol 2k manufactured by Creative PEG works, mPEG-SH, MW 2000, PLS-605 was further immobilized on the same chip under the same conditions.
  • the total immobilized amount of mPEG thiol 2k was 65.6RU.
  • the anti-mPEG antibody was immobilized on the surface of the mPEG-modified sensor chip at 25 ° C. by anti-mPEG monoclonal antibody (abcam ab51257, Mouse monoclonal, Anti-Polyethylene glycol antibody [PEG-B -47]) was carried out by feeding a 10.0 ⁇ g / mL solution dissolved in DPBS as a running buffer onto the sensor chip at a flow rate of 10 ⁇ L / min for 30 minutes, and then again running the running buffer for 20 minutes. The amount of anti-mPEG antibody immobilized was 3906.9RU.
  • BSA blocking It carried out according to the method of Example 6 (6).
  • the amount of BSA adsorption was -38.5RU.
  • Example 10 (1) Creation of mPEGylated interface
  • the mPEGylated interface was created at 37 ° C. using Biacore T-200.
  • As a running buffer a solution in which 14.61 g of sodium chloride was added to Dulbecco's phosphate-buffered saline (Life Technologies, DPBS, 14190-144, pH 7.0-7.3, 250 g) was used.
  • a running buffer solution of mPEG thiol 2k dissolved in the running buffer (Creative PEG works, mPEG-SH, MW 2000, PLS-605, 1 mg / mL) is fed onto the sensor chip at a flow rate of 15 ⁇ L / min for 20 minutes.
  • the running buffer was fed for 20 minutes, the 0.05N-NaOH solution for 1 minute, the running buffer for 3 minutes, the 0.05N-NaOH solution for 1 minute, and then the running buffer at a flow rate of 15 ⁇ L / min for 11 minutes.
  • This series of operations was repeated 5 times to immobilize mPEG thiol 2k on the sensor chip surface.
  • the total immobilized amount of mPEG thiol 2k was 1959.7RU.
  • poly (ethylene glycol) methyl ether thiol 800 (mPEG thiol 800, Sigma, 729108, average Mn 800, 1 mg / mL) was further immobilized on the same chip by repeating the same conditions 5 times.
  • the total amount of mPEG thiol 800 immobilized was 90.6 RU.
  • the anti-mPEG antibody was immobilized on the mPEG-modified sensor chip surface at 25 ° C. by anti-mPEG antibody (Abcam ab51257, Mouse monoclonal, Anti-Polyethylene glycol antibody [PEG-B- 47]) was carried out by feeding a 5.0 ⁇ g / mL solution dissolved in DPBS as a running buffer onto the sensor chip at a flow rate of 10 ⁇ L / min for 30 minutes, and then feeding the running buffer again for 30 minutes.
  • the immobilized amount of anti-mPEG antibody was 3126.6RU.
  • Example 11 Creation of mPEGylated interface This was performed according to the method of Example 9 (1).
  • the total immobilized amount of mPEG thiol 5k was 3055.1 RU, and the immobilized amount of mPEG thiol 2k was 63.4 RU in total.
  • BSA blocking It carried out according to the method of Example 6 (6).
  • the amount of BSA adsorption was -119.8RU.
  • Example 12 (1) Preparation of biotin PEGylated interface Biotin PEGylated interface was prepared at 37 ° C using Biacore T-200. A PBS solution (pH 7.4, 0.05M) having a sodium chloride concentration of 1M was used as a running buffer.
  • the total amount of biotinylated PEG thiol 5k immobilized was 1781.2 RU.
  • mPEG thiol 2k manufactured by Creative PEG works, mPEG-SH, MW 2000, PLS-605, 1 mg / mL was further immobilized on the same chip under the same conditions.
  • the total amount of mPEG thiol 2k immobilized was 545.5 RU.
  • Example 6 (2) Immobilization of anti-biotin antibody This was carried out according to the method of Example 6 (3).
  • the anti-biotin antibody solution was a solution having a concentration of 10.0 ⁇ g / mL.
  • the amount of anti-biotin antibody immobilized was 3604.5 RU.
  • BSA blocking It carried out according to the method of Example 6 (6).
  • the amount of BSA adsorption was -62.5RU.
  • Example 13 (1) Preparation of biotin PEGylated interface Biotin PEGylated interface was prepared at 37 ° C using Biacore T-200. A PBS solution (pH 7.4, 0.05M) having a sodium chloride concentration of 1M was used as a running buffer.
  • the total amount of biotinylated PEG thiol 2k immobilized was 2121.6 RU.
  • poly (ethylene glycol) methyl ether thiol 800 (mPEG thiol 800, manufactured by Sigma, 729108, average Mn 800, 1 mg / mL) was further immobilized on the same chip under the same conditions.
  • the total immobilized amount of mPEG thiol 800 was 533.1RU.
  • BSA blocking It carried out according to the method of Example 6 (6).
  • the adsorption amount of BSA was -48.9RU.
  • Example 14 Preparation of SAM The SAM was prepared according to the method of Example 1 (1).
  • Antigen (CRP) binding amount SPR measurement of the antigen binding amount on the surface of the sensor chip prepared by the operations of (1) to (6) was performed at 25 ° C. using Biacore T200.
  • Example 15 Adsorption of BSA (Bovine Serum Albumin)
  • BSA Bovine Serum Albumin
  • the BSA adsorption amount on the measurement sensor chip is measured using Biacore T-200, and the measurement sensor chip (GE Healthcare, SIA Kit Au, BR-1004) -05)
  • DPBS Dulbecco's phosphate-buffered saline, Life Technologies, 14190-144, pH 7.0-7.3
  • Example 16 (1) Preparation of SAM Sensor chip (GE Healthcare, SIA Kit Au, BR-) used in Biacore T-200 in 100 ⁇ M ethanol solution (10 mL) of 10-carboxy-1-decanethiol (Dojindo Laboratories, C385) 1004-05) was immersed overnight at 25 ° C., and a self-assembled monolayer (SAM) was formed on the gold surface of the substrate.
  • SAM self-assembled monolayer
  • BSA Bovine Serum Albumin
  • Example 17 Preparation of SAM The SAM was prepared according to the method of Example 16 (1).
  • BSA Bovine Serum Albumin
  • Antigen binding amount was measured according to the method of Example 14 (7).
  • the amount of CRP bound was 480.6RU. The results are shown in Table 6.

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JP2002520618A (ja) * 1998-07-14 2002-07-09 ザヨミックス， インコーポレイテッド タンパク質のアレイおよびその使用方法
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