Foreign code F180009505
File No. (S2017-0223-N0)
Posted date Nov 2, 2018
Country WIPO
International application number 2018JP001690
International publication number WO 2018135651
Date of international filing Jan 19, 2018
Date of international publication Jul 26, 2018
Priority data
  • P2017-008331 (Jan 20, 2017) JP
Abstract The purpose of the present invention is to provide an antibody-polysaccharide conjugate for immunoassay that is obtained by binding an antibody or antibody complex to at least one side chain of a water-soluble polysaccharide via a linker, and an immunoassay method using the same. Provided is an antibody-polysaccharide conjugate for high-sensitivity immunoassay such as a sandwich ELISA and immunochromatography, in which an antibody such as a VHH antibody or an antibody complex comprising an antibody and a cushion protein such as a stefin A mutant is bound, via a linker, to a functional group on a side chain of a water-soluble polysaccharide such as pullulan, whereby it is easier to control the orientation of the antibody when the water-soluble polysaccharide is adsorbed to a solid-phase surface for immunoassay, and it is possible for an antibody having a stable multivalent structure to bind to a target protein without the nonspecific adsorption of other proteins. Additionally provided is a high-sensitivity immunoassay method using the same.
Outline of related art and contending technology BACKGROUND ART
ELISA method, the sample solution contained in the target antigen (hereinafter, 'target antigen' is called.) To, these antigen-specific antibodies that specifically recognize (hereinafter, 'specific antibody' may also be referred to.) And captured, subsequent to the enzymatic reaction using a method that performs the detection or quantitation. The target antigen is, B cells produce antibodies to the antigen determinant production of a material having a (epitopes), for a predetermined length or more peptides, protein, sugar chain and the like. In the present specification, the 'antibody', including both artificial and natural antibodies. Here, 'native' is, any vertebrate produced by the same antibody having an amino acid sequence of the antibody. In addition, 'artificial' is, artificially constructed refers to an antibody, for example, the natural antibody amino acid sequence of the appropriate mutation was introduced into the other antibody, usually, a structure that does not exist in the natural world to include modifications made to the antibody.
And the antigen and the antibody as described above after the binding, which are used in the sample solution is added to the substrate, depending on the enzymatic activity generated by the dye, or fluorescent material or a chemiluminescent substance is generated. Then, when the dye absorbance is generated, in addition, a fluorescent material or a chemical is generated in the light-emitting substance, a fluorescent or chemiluminescent detection or the like are measured.
By a combination of an antigen-antibody, direct methods, indirect method, the sandwich method (hereinafter, 'sandwich ELISA method' may also be referred to.), Are classified into competition. Among these, a sandwich ELISA method, referred to as capture antibody and detection antibody antibody of the 2 type. The capture antibody, bound to a solid phase for use, also referred to as solid-phase antibody. This sandwich ELISA method, the solid-phase antibody and detection antibody antigen and antigen is inserted, is quantified.
Sandwich ELISA method, usually, an antibody in an immune globulin (hereinafter, 'IgG', or ' full-length antibodies' may also be referred to. See Fig. 1) is used. IgG antibody and a single molecule, immunoglobulin M and a pentamer (hereinafter, abbreviated as' IgM ' be.) As compared with the, biomolecules include a small molecular weight, molecular weight is approximately 17 million in the still. In addition, has a plurality of domains is IgG cannot be artificially synthesized. For this reason, a mammal and the antigen substance administered multiple times in a certain period of time (immune) and, at the time of the final dose of the fixed period has elapsed after the blood was collected, purified antibodies in the serum to the other, cannot be obtained.
In this way to immunize an animal to obtain the IgG, the period until the vote, which is essential for healthy animals raised for and, further, requires a large number of the antigen is administered. Further, the separation of the serum from blood collected, antibodies in the serum must be purified. Therefore, sufficient for the desired antigen antibody that can bind to in order to obtain, the time and expense is required.
In order to solve such a problem as one of means 1, as described above can be used in place of the new antigen IgG binding molecules have been researched. Such binding molecules include, for example, composed of a single-chain polypeptide, and recognizes the target material itself, which can be a single chain antibody (single chain Fv: scFv) can be cited, these may be referred to as a single chain antibody. Single-chain antibodies, naturally occurring, e.g., camelid produced by a single-chain antibody (hereinafter, single domain antibody, 'VHH antibody' or 'VHH' may also be referred to. See Fig. 1. Zhang L., et al., 1993, Nature, 362: 446-448) made by genetic recombination and non-natural images which are not classified. Non-natural single chain antibody, the antigen binding site to one variable region fragment 2 (heavy chain variable region (VH) and a light chain variable region (VL) ) are connected by a flexible linker made recombinant antibody fragment, for example, single-chain Fv (scFv: can be single chain Fragment of variable region) (Pierce Catalog and Handbook, 1994-1995, Pierce Chemical Co., Rockford, IL) and the like. These single-chain antibodies, microorganism relatively easily can be expressed.
In fact, the molecular weight is less than 1/10 of the IgG VHH, and phage display library that do not utilize other immune responses (hereinafter, 'non-immune library' may be referred to.) Is used can be artificially produced.
However, typical of the IgG (dissociation constant) and Kd 10-6-10-9 M is, among these, the 10-9 M has a high affinity for the antigen IgG can be determined. On the other hand, non-immune library of antibody obtained from the Kd 10-5-10-8 IgNAR VHH antibody or the order of the (non-patent document 1 and non-patent reference 2), as compared with the low affinity IgG antigen has been known.
Quantitative ELISA method in order to perform a stable, constant or more antigen affinity is required. Then, as a method to enhance the affinity for the antigen, polyvalent due to avidity (specific antigen binding force) is the introduction of known. Such enhanced avidity is, the expression system of an animal cell, VHH antibody tag such as a single domain antibody molecule expressed as a fusion protein is fuzed, then, via a tag molecule antibody multimer by self-association to construct some example have been reported (or less, 'prior art 1' is called. See Non-Patent Document 3).
ELISA method, the ability to bind to the antigen is immobilized is important to increase the measurement sensitivity. Heretofore, such as Escherichia coli and VHH antibody produced recombinant, such as polystyrene and the physical adsorption to the substrate directly, the VHH antibody antigen binding force is not originally lose known to have a very high (see Non-Patent Document 4-6). In addition, solid phase direct physical adsorption of the VHH antibody, IgG antibodies compared to the solid phase, by ELISA method included in the washing operation can be easily peeled off from the substrate (drop) has been known.
VHH antibody direct physical adsorption as described above improve the deterioration of ability to bind antigen by the method, a method of using biotin and streptavidin. Specifically, the surface of the substrate adsorbed streptavidin, biotin antibody VHH VHH and biotinylated coupled to, and adsorbed on the surface of the substrate through a streptavidin biotinylated indirectly VHH solid-phase method (hereinafter, 'prior art 2' is called. See Non-Patent Document 7) has been known. In addition, the end of the VHH antibody specific for the peptides that bind to the substrate (hereinafter, 'substrate binding peptide specific' is called.) Fused, this substrate by passing through a specific binding peptide, while controlling the orientation of the VHH method of the solid phase (hereinafter, 'prior art 3' is called. See Non-Patent Document 8) and the like are known.
In addition, the protein or peptide such as an enzyme, a metal or metal oxide or the like adsorbed onto the solid surface activity is lowered (that is, a decrease in binding ability) are known, such a reduction in activity of the protein or peptide has been proposed a method of improving. 'Peptide of a specific array, and at least one selected from the group consisting of protein cushion one of 1, the adsorbent can adsorb on the surface of the cushioning property of the solid' by using, when the enzyme immobilized on the remaining activity when not in use can be made larger than have been reported (or less, 'prior art 4' is called. See Patent Document 1). In addition, the target substance for coupling using a polysaccharide as a scaffold, a solid phase surface-(chemical bonding) -polysaccharide-(chemical bonding) -purpose material (protein, nucleic acid and the like) in the molecule and the configuration has been proposed a system (hereinafter, 'prior art 5' is called. See Patent Document 2.)
Scope of claims (In Japanese)請求の範囲
  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • Inventor
  • MATSUSHITA, Takahiko
  • MATSUOKA, Kouji
  • HATANO, Ken
  • NEMOTO, Naoto
  • ARAI, Hidenao
IPC(International Patent Classification)
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