JP2014156428A - Antibody binding protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a fusion protein ligand that strongly binds to human antibody Fab fragment or other antibody IgG Fc fragments, and a fluorescent immune sensor including the fusion protein ligand.SOLUTION: Provided is a fusion protein in which Protein A or its antibody-binding domain is fused to Protein G or its antibody-binding domain via a peptide linker. Also provided is a fluorescent immune sensor being the fusion protein labeled with a fluorescent dye.

Description

  The present invention relates to an antibody-binding protein applicable to detection and purification of antibodies (immunoglobulins) having high industrial utility, and further to antigen detection based on the fluorescence quench principle.

  In recent years, there has been an increasing need for a carrier for purifying antibody proteins, including human antibodies, which are next-generation pharmaceuticals. Conventionally, natural antibody binding proteins Staphylococcus Protein A (PA) and Streptococcus Protein G (PG) have been used as ligands for IgG antibody affinity purification columns, but depending on the subclass of antibody IgG, the binding ability is low. It had problems such as high production costs.

On the other hand, the present inventors previously performed site-specific fluorescence labeling of the vicinity of the N-terminus of a single-chain antibody (scFv) using a non-natural amino acid introduction technique, so that the fluorescence intensity depends on antigen binding. Fluorescently labeled antibody (Quenchbody: Q-body), which is an antibody fragment with an increased number of nuclei, was developed (see Patent Document 1 and Non-Patent Document 1). This phenomenon is quenched by antigen binding when it interacts with a highly conserved tryptophan residue in the vicinity of the variable region V _H / _VL interface where the labeling dye constitutes scFv when antigen is independent. Happens to be.

  The fluorescently labeled antibody has high utility as a simple and rapid fluorescent immunoassay element (fluorescent immunosensor). However, the production of this fluorescently labeled antibody requires acquisition of antibody genes and site-specific fluorescence modification using a cell-free protein synthesis system, which has the problem of cost and time.

WO2011 / 061944 Publication

Abe et al., J. Am. Chem. Soc. 2011, 133, 17386-17394

  An object of the present invention is to provide a fusion protein ligand that strongly binds to a human antibody Fab fragment or many antibody IgG Fc fragments, and a fluorescent immunosensor containing the fusion protein ligand.

  The present inventor fused the IgG binding domains of Protein A and Protein G, which are antibody-binding proteins, at an optimal distance, thereby strongly binding to certain human Fab fragments and / or many IgG Fc fragments. It has been found that protein ligands can be made. As a result, it was expected that a ligand capable of binding specifically and strongly to IgG could be produced while being able to be produced at a low molecular weight and at a lower cost than a natural protein. Furthermore, we thought to construct a fluorescent immunosensor that was labeled with a fluorescent dye, bound to a Fab fragment, and whose fluorescence intensity varied depending on antigen binding.

  The Fc portion of antibody IgG is composed of a heavy chain dimer, and there are two protein A and protein G binding sites, one between each of the CH2-CH3 domains, that is, two per molecule. However, it was thought that the binding domains adjacent to each other in antibody binding proteins including those of natural origin can only bind to one of the binding sites in view of their molecular structure (short binding domain distance). .

  Therefore, the present inventors prepared a novel ligand PAxPG in which one IgG binding domain of Protein A and Protein G was bound via a long linker of about 15 to 20 amino acids, and the antibody binding ability was measured. As a result, it was found that PAxPG shows significantly higher binding activity to various IgG than not only one binding domain of Protein A and Protein G but also natural Protein A and Protein G having multiple binding domains. . That is, it was found that by optimizing the length and sequence of the linker between domains, it was possible to construct a novel ligand molecule capable of binding simultaneously to two binding sites of one IgG, and the present invention as a novel antibody binding protein was completed. I came to let you. Furthermore, it was found that PAxPG binds to a Fab fragment having a variable region belonging to human VH3 capable of binding Protein A with a dissociation constant of several nM or less by binding simultaneously with the CH1 domain. Utilizing these properties of PAxPG, a phenomenon in which fluorescence-labeled PAxPG is bound to an antigen-specific human Fab fragment, and the fluorescence that has been quenched by interacting with amino acids at the antigen-binding site is restored again by antigen binding. The present inventors have found that the presence / absence of an antigen can be detected by fluorescence using the above, and have completed the present invention.

That is, the present invention is as follows.
[1] A fusion protein in which Protein A or an antibody binding domain thereof is fused to Protein G or an antibody binding domain thereof via a peptide linker.
[2] The fusion protein of [1], wherein the antibody binding domain of Protein A is the D domain and the antibody binding domain of Protein G is the B1 domain.
[3] The fusion protein of [1] or [2], wherein the peptide linker consists of 10 to 20 amino acids.
[4] Peptide linkers include glycine (G), serine (S), alanine (A), threonine (T), aspartic acid (D), lysine (K), glutamic acid (E), leucine (L) and proline ( The fusion protein according to any one of [1] to [3], comprising an amino acid selected from the group consisting of P).
[5] The fusion protein according to any one of [1] to [3], wherein the peptide linker consists of an amino acid sequence represented by (G ₄ S) ₃ or (DDAKK) ₄ .
[6] Any of [1] to [5], wherein Protein A or an antibody binding domain thereof binds to a heavy chain VH region of an antibody, and Protein G or an antibody binding domain thereof binds to a heavy chain CH1 region of an antibody. Fusion protein.
[7] The fusion protein according to any one of [1] to [6], which binds to Fc.
[8] A method for purifying an antibody, comprising contacting the fusion protein of any one of [1] to [7] with the antibody.
[9] The fusion protein of [1] to [7], which is labeled with a fluorescent dye, a quenching dye, a fluorescent protein or an enzyme.
[10] A fluorescent immunosensor comprising a fusion protein of any one of [1] to [7] labeled with a fluorescent dye, wherein the fluorescence changes when the antibody binds to the antigen and binds to an antigen.
[11] The fluorescent immunosensor according to [10], wherein the N-terminal part of Protein A of the fusion protein or its antibody binding domain is labeled with a fluorescent dye.
[12] The fluorescent immunosensor according to [10] or [11], wherein ProX-tag is added to the N-terminal part of Protein A or an antibody binding domain thereof, and an amino acid in ProX-tag is labeled with a fluorescent dye.
[13] The fluorescent immunosensor according to [12], wherein ProX-tag labeled with a fluorescent dye, Protein A or an antibody binding domain thereof, a peptide linker, Protein G or an antibody binding domain thereof are fused in this order.
[14] The fluorescent immunosensor according to [10] to [13], wherein a peptide linker is interposed between ProX-tag and Protein A containing a fluorescent dye or an amino acid labeled with the fluorescent dye.
[15] A ProX-tag labeled with a fluorescent dye, a second peptide linker, Protein A or an antibody binding domain thereof, a first peptide linker, Protein G or an antibody binding domain thereof are fused in this order. 14] The fluorescence immunosensor of.
[16] Fluorescence in which fluorescence resonance energy transfer occurs between the fluorescent immunosensor of any one of [10] to [15] and an unlabeled antibody, an antibody labeled with a quencher, or a fluorescent dye in the fluorescent immunosensor A detection kit for an antigen to which the antibody specifically binds, comprising an antibody labeled with a dye.
[17] A method for detecting an antigen using an antibody specific for the antigen,
(a) contacting the fluorescent immunosensor of any one of [10] to [15] with an antibody to form a complex of the fluorescent immunosensor and the antibody,
(b) contacting the complex formed in (a) with an antigen in a test sample to bind the antibody and the antigen;
(c) measuring the change in fluorescence due to the binding between the antibody and the antigen, wherein the fluorescence quenching is canceled due to the binding between the antibody and the antigen;
And a method comprising.
[18] A method for detecting an antigen using an antibody specific for the antigen,
(a) contacting the fluorescent immunosensor of any one of [10] to [15] with an antibody labeled with a quencher in a liquid to form a complex of the fluorescent immunosensor and the labeled antibody;
(b) contacting the complex formed in (a) with an antigen in a test sample to bind the antibody and the antigen;
(c) measuring the change in fluorescence due to the binding between the antibody and the antigen, wherein the fluorescence quenching is canceled due to the binding between the antibody and the antigen;
And a method comprising.
[19] A method for detecting an antigen using an antibody specific for the antigen,
(a) contacting the fluorescent immunosensor of any one of [10] to [15] with an antibody labeled with a fluorescent dye in a liquid to form a complex of the fluorescent immunosensor and the labeled antibody, wherein The combination of the sensor fluorescent dye and the labeled antibody fluorescent dye is a combination of an energy donor dye and an energy acceptor dye capable of producing FRET;
(b) contacting the complex formed in (a) with an antigen in a test sample to bind the antibody and the antigen;
(c) measuring the change in fluorescence due to the binding between the antibody and the antigen, wherein the fluorescence from the energy acceptor dye decreases due to the binding between the antibody and the antigen;
And a method comprising.
[20] The method according to any one of [17] to [19], wherein the antibody is a Fab antibody fragment.

  According to the present invention, high-affinity ligands for various IgGs, which had been difficult until now by difficult mutagenesis and selection, can be obtained on a relatively simple principle. Furthermore, the present invention can be easily applied to antibody-binding ligands having properties superior to those of other natural substances, and can achieve higher affinity. Moreover, the possibility that a fluorescent immunoelement based on the principle of Quenchbody (WO2011 / 061944) can be produced simply by mixing with a purified antibody was also shown.

It is a schematic diagram of the fluorescence immunosensor of the present invention. It is a figure which shows the principle of the detection method of the antigen using the fluorescence immunosensor of this invention. It is a figure which shows the arrangement | sequence structure of the constructed fluorescent probes A, B, and C. It is a figure which shows the result of SDS-PAGE (UV irradiation) of the probe A (8.6 kDa), the probe B (16.0 kDa), and the probe C (17.3 kDa) which were synthesize | combined. It is a figure which shows the result of the fixed_quantity | quantitative_assay of the probe by the calibration curve of the density | concentration of the concentration of TAMRA produced using GENios Pro of the probe A, the probe B, and the probe C which were synthesized. It is a figure which shows the result of binding ELISA (n = 3) of the synthesized probes A, B and C and mouse IgG1. It is a figure which shows the arrangement | sequence structure of constructed | assembled fluorescent probe DI. It is a figure which shows the result of SDS-PAGE (green light irradiation) of the probe C, F, and I which were synthesize | combined. It is a figure which shows the result of the fixed_quantity | quantitative_assay of the probe by the calibration curve of the density | concentration of TAMRA produced using GENios Pro of the synthesized probes D to I, and fluorescence intensity measurement. It is a figure which shows the result of SDS-PAGE of the prepared anti- BGP Fab antibody fragment. FT shows the result of the flow through of the Talon column purification, and E shows the result of the elution fraction of the Talon column. It is a figure which shows the result of the antigen binding ability confirmation ELISA (n = 3) of the prepared anti- BGP Fab antibody fragment. It is a figure which shows the result of the binding evaluation (n = 3) of the synthesized probes C, F and I, mouse IgG1 (FIG. 12A), and anti-BGP Fab fragment (FIG. 12B). It is a figure which shows the result of the binding evaluation (n = 3) with the probe and the anti-BGP Fab fragment solid-phased through the immobilized BGP peptide. It is a figure which shows the sensorgram which uses the unlabeled probe C (PAxPG) as a ligand, and a phage library-derived human anti-vimentin Fab fragment as an analyte. Results of comparing the binding constant Ka calculated using the Langmuir model with PA, PG 'when using unlabeled probe C (PAxPG) as the ligand and phage library-derived human anti-vimentin Fab fragment as the analyte. FIG. It is a figure which shows the sensorgram which used PAxPG as a ligand and mouse | mouth IgG1 as an analyte. The results of comparing Ka calculated using the Langmuir model with PA, PG 'when PAxPG is used as the ligand and mouse IgG1 is used as the analyte are shown. It is a figure which shows the presumed binding model of PAxPG and a mouse antibody. It is a figure which shows the result (n = 3) which detected polyclonal mouse | mouth IgG (mIgG) fix | immobilized on the microplate, IgG1, and IgG2a using the HRP labeled probe. It is a figure which shows the fluorescence intensity change at the time of adding a human anti-vimentin Fab fragment or a buffer solution to the probe F. FIG. 6 shows changes in fluorescence intensity when a human anti-vimentin Fab fragment or a buffer is added to probe I. It is the figure which represented the result of FIG. 21 as an average and the standard deviation (n = 3) of the relative fluorescence amount from Fab fragment addition time. It is a figure which shows the fluorescence intensity change at the time of adding antigen vimentin or a buffer solution to the sample which added the Fab fragment to the probe I. It is the figure which showed the result of FIG. 23 with the fluorescence intensity ratio with and without antigen addition.

Hereinafter, the present invention will be described in detail.
The present invention is a fusion protein of two types of antibody binding proteins, Protein A and Protein G, and a fluorescence immunosensor that can be used as a fluorescence immunoassay element containing the fusion protein.

1. Antibody Binding Protein Fusion Protein The antibody binding protein fusion protein of the present invention is a protein A and protein G fusion protein, and contains both protein A and protein G antibody binding sites.

  Protein A is a 46.7 kDa protein present in the cell wall of Staphylococcus aureus and specifically binds to the Fc region (heavy chain constant region) of immunoglobulin. Previously, Protein A has been reported to bind to the Fc region of three subclasses except IgG3 among the four subclasses of human IgG (IgG1, IgG2, IgG3, IgG4) and some heavy chain variable regions (VH3). It had been. Protein A has been reported to be relatively weak to mouse IgG and to bind rabbit IgG fairly strongly in the Fc region. The binding site of Protein A to IgG has five domains whose amino acid sequences of E domain, D domain, A domain, B domain, and C domain are homologous, all of which specifically bind to the Fc region of an antibody.

  Protein G is a protein present in the cell wall of group G streptococci and specifically binds to the Fc region and CH1 region of many species of IgG. The binding site of Protein G to IgG is 2 sites of B1 domain and B2 domain (GX7809 strain, SR Fahnestock, et al. J. Bacteriol., 167, 870, 1986), or 3 sites of C1, C2, and C3 domains ( G148 strain, A Olsson, et al., Eur. J. Biochem. 168, 319-324, 1987).

  Protein A constituting the fusion protein of the present invention may be full-length protein A or an antibody binding domain. Moreover, you may be comprised from the some antibody binding domain. Preferably the D domain is used. The amino acid sequence of full-length protein A (M. Uhlen et al., J. Biol. Chem. 259, 1695, 1984)) is shown in SEQ ID NO: 1 (Genbank J01786.1), and the amino acid sequence of the D domain is shown in SEQ ID NO: 2. .

  Protein G constituting the fusion protein of the present invention may be full-length protein G or an antibody binding domain. Moreover, you may be comprised from the some antibody binding domain. Preferably, a B1 domain derived from GX7809 strain is used. The amino acid sequence of full-length protein G is shown in SEQ ID NO: 3 (Genbank M13825.1), and the amino acid sequence of the B1 domain is shown in SEQ ID NO: 4.

  The fusion protein of the present invention contains two antibody binding proteins, Protein A and Protein G, and both bind to the antibody, so that it has a strong binding ability to the antibody as a fusion protein. The fusion protein of the present invention binds not only to a complete antibody consisting of two heavy chains and two light chains, but also to Fab fragments and Fab ′ fragments having a heavy chain CH1 region and a heavy chain variable region. Bind with great affinity. That is, since the Protein A part constituting the fusion protein binds to the heavy chain VH region of the antibody and the Protein G part binds to the heavy chain CH1 part, it can bind simultaneously to the Fab fragment at two positions. Moreover, since the Protein A part and Protein G part which comprise a fusion protein couple | bond with two heavy chains of an antibody Fc area | region simultaneously at two places, in a full length antibody, it can couple | bond with a total of three places. In particular, since Protein A binds strongly to the heavy chain VH region of certain human antibodies, the fusion protein of the present invention can bind strongly to human IgG and human Fab fragments.

  In the fusion protein, the two antibody binding proteins are fused in tandem via a linker. The linker is a peptide linker consisting of an amino acid sequence having a specific length, and the number of amino acids is not limited, but is 5 to 30, preferably 10 to 25, and more preferably 15 to 20. Depending on the length of the linker, the positional relationship, that is, the distance between the two types of antibody binding proteins constituting the fusion protein changes. The distance between two binding sites of the antibody binding protein constituting the fusion protein can be adjusted by the length of the linker. As a result, it is possible to perform fusion so that the protein A binding domain and the protein G binding domain constituting the fusion protein have an optimum distance for binding to the antibody. The optimal linker length is about 3 to 10 nm, preferably about 4 to 8 nm, and more preferably about 6 nm. For example, when Protein A binds to one of the heavy chains of the Fc region and Protein G binds to the other heavy chain, the number of amino acids in the linker is set to 10-20, so that Protein A of the fusion protein It binds to the Fc region of the heavy chain in an appropriate positional relationship, and the protein G of the fusion protein simultaneously and cooperatively binds to another heavy chain that constitutes the Fc region in an appropriate positional relationship. Synergistically increases the binding ability.

  The linker may be a 3-10 repeat sequence of 2-10 amino acid sequences. The type of amino acid is not limited, but it is preferable to use an amino acid that has few side chains and is not highly reactive, or an amino acid that has a linear α-helix structure when linked. Examples of amino acids with small side chains and small sizes include glycine (G), serine (S), alanine (A), and threonine (T). Examples of amino acids that contribute to the formation of the α-helix structure include alanine (A), aspartic acid (D), lysine (K), glutamic acid (E), leucine (L), and methionine (M). Moreover, proline (P) which can form a polyproline helix structure is mentioned.

Examples of the linker mainly composed of amino acids having few side chains include a linker consisting of an amino acid sequence consisting of G and S. For example, a sequence in which the amino acid sequence represented by GGGGS (SEQ ID NO: 5) is repeated 2 to 6 times (G ₄ S) _{2-6, and} the sequence (G ₄ S) ₃ repeated three times is preferable. Furthermore, the arrangement | sequence which consists of polyglycine which 5-30 pieces, Preferably 10-20 glycine (G) connected is also mentioned. Moreover, as an amino acid sequence mainly composed of amino acids forming an α helix, there is a sequence (DDAKK) _{2-6 in} which the amino acid sequence represented by DDAKK (SEQ ID NO: 6) is repeated 2 to 6 times, and is repeated 4 times. The sequence (DDAKK) ₄ is preferred. Moreover, the sequence (EAAAK) _2-6 which repeated 2-6 times of the amino acid sequence represented by EAAAK (sequence number 7) is mentioned, The sequence (EAAAK) ₄ repeated 4 times is preferable. Furthermore, the sequence which consists of a polyproline which 5-30 pieces, Preferably 10-20 pieces of proline (P) connected is also mentioned.

  The fusion protein containing the protein A binding site and protein G binding site of the present invention is also referred to as PAxPG. In addition, the fusion protein of the present invention is sometimes referred to as a fusion protein ligand.

  An antibody-binding protein or a fusion protein of the antibody-binding site can be prepared by a method for producing a known fusion protein, for example, a chemical synthesis method or a method by gene recombination technology. preferable. When producing the fusion protein of the present invention by gene recombination technology, two antibody binding proteins and a DNA encoding a linker are linked in-frame, and the DNA encoding the fusion protein containing the linker is introduced into an expression vector. Thus, a recombinant vector may be prepared and expressed by an expression system using bacteria, yeast, insect cells, plant cells, animal cells and the like as hosts.

Any vector can be used as long as it can be replicated in a host cell such as a plasmid, phage, or virus. The vector contains a replication origin, a selectable marker, and a promoter, and may contain an enhancer, a transcription termination sequence (terminator), a ribosome binding site, a polyadenylation signal, and the like as necessary.
The

  At this time, the fusion protein may be added with tags such as ProX tag, FLAG tag, His tag, GST (glutathione S transferase) and the like. These tags can be used for fluorescent dye addition, fusion protein purification, and the like.

  The produced fusion protein can be separated and purified by various separation and purification methods. For example, ammonium sulfate precipitation, gel filtration, ion exchange chromatography, affinity chromatography and the like can be used alone or in appropriate combination. In this case, when the expression product is expressed as a fusion protein with GST or the like, it can be purified using the properties of the protein or peptide fused with the target protein. For example, when expressed as a fusion protein with GST, since GST has an affinity for glutathione, it can be efficiently purified by affinity chromatography using a column in which glutathione is bound to a carrier. In addition, when expressed as a fusion protein with a histidine tag, a protein having a histidine tag binds to the chelate column and can be purified using the chelate column.

  The fusion protein of the present invention can also be expressed in a cell-free translation system (cell-free system). When expressing a target polypeptide in a cell-free translation system, for example, in a reaction solution in which nucleotide triphosphates and various amino acids are added to a cell-free extract such as E. coli, wheat germ, rabbit reticulocyte, etc. Can be expressed.

  The fusion protein of the present invention can be used for antibody purification. Since the fusion protein has two binding sites, it binds to the VH region and CH1 region of the antibody simultaneously, or binds to the FC region of the antibody at two sites simultaneously, so it binds to the antibody with high affinity and makes the antibody efficient. The antibody can be purified by capture and eluting the capture antibody. The fusion protein of the present invention can be purified by contacting with a sample containing the antibody, allowing the antibody to bind to the fusion protein, and then elution.

  For example, the fusion protein of the present invention can be immobilized on a chromatography carrier and used as a solid phase carrier for affinity chromatography for antibody purification. Chromatographic carriers include, but are not limited to, cellulose, polysaccharides such as agarose and dextran, silica, vinyl polymers, acrylamide polymers and the like that can be used as carriers in various chromatography. Commercially available chromatography carriers may be used, such as Sephadex, Sephacel, Sepharose (above Pharmacia), Bio-Gel, Macro-Prep, Affi-Gel (above Bio Rad) and the like.

  The fusion protein of the present invention may be labeled with a labeling substance. Examples of the labeling substance include fluorescent dyes, quenching dyes, fluorescent proteins, enzymes such as alkaline phosphatase (ALP) and horseradish peroxidase (HRP). Labeling with a labeling substance can be performed by a known protein labeling method. The fusion protein labeled with these labeling substances can be used as a probe in an immunoassay utilizing an antigen-antibody reaction. When a fluorescent dye is used, it can be used as a fluorescent immunosensor described later. The labeling method using a fluorescent dye will be described later. The labeled fusion protein of the present invention is referred to as a labeled probe, and in particular, the fusion protein labeled with a fluorescent dye is sometimes referred to as a fluorescent probe.

2. Fluorescent immunosensor comprising a fusion protein of an antibody binding protein By labeling the above fusion protein with a fluorescent dye, it can be used as a fluorescent immunosensor capable of detecting the binding between an antigen and its specific antibody.

  A fluorescent immunosensor is a substance obtained by labeling the above antibody-binding protein with a fluorescent dye, and is used by binding to an antibody. A schematic diagram of the fluorescent immunosensor of the present invention is shown in FIG. By using the fluorescent immunosensor and the antibody of the present invention, an antigen to which the antibody specifically binds can be detected as a substance to be detected. The fluorescent immunosensor of the present invention is also called a fluorescent probe.

The antibody to which the fluorescent immunosensor of the present invention is bound is a specific antibody against the target antigen to be detected, and may be a complete antibody, Fab antibody, F (ab ′) ₂ antibody, one Fab and It may be a Fab / c antibody having complete Fc. Further, it may be an antibody fragment or an artificial antibody that has a region to which Protein A binds and a region to which Protein G binds and has a structure capable of binding to an antigen. Examples thereof include antibody fragments and artificial antibodies having a VH region to which Protein A binds and a CH1 region to which Protein G binds. In the present invention, the term “antibody” includes functional fragments of such antibodies and artificial antibodies. The antibody may be a monoclonal antibody produced by an antibody-producing hybridoma or a fragment thereof, or an antibody prepared by genetic recombination technology based on the DNA information of the monoclonal antibody produced by the hybridoma. Among these antibodies, Fab fragments having one heavy chain variable region and one heavy chain CH1 region, and one light chain variable region and one light chain CL region are preferred. The animal from which the antibody is derived is not limited, and humans, mice, rats, rabbits, goats, horses, musk rats, sheep and the like can be used. Since Protein A of the fusion protein of the fluorescent immunosensor of the present invention can bind strongly to the VH region of a human antibody, the immunofluorescent sensor can bind strongly to a human antibody or a human antibody Fab fragment.

  The fluorescent immunosensor of the present invention is the presence or absence of the occurrence of fluorescence and the fluorescence intensity between when the fluorescent immunosensor is bound to an antibody and when the antibody bound to the fluorescent immunosensor is bound to an antigen. Is designed to change. That is, when the fluorescent immunosensor is bound to the antibody, the fluorescent substance used for the label is quenched (quenched) and does not emit fluorescence or is in a state of generating fluorescence of a specific wavelength. When an antigen is bound to the antibody, the fluorescence generation state of the fluorescent dye can change. For example, a fluorescent dye that has been quenched when the antibody and the antigen are not bound to fluoresce when the antibody and the antigen are bound, or fluoresce when the antibody and the antigen are not bound. The fluorescent dye that has been used causes the wavelength of the fluorescence generated by the binding of the antibody and the antigen to shift.

  Thus, as a system in which the fluorescence state is changed by binding an antigen to the antibody to which the antibody-binding fusion protein of the present invention is bound, a system in which a fluorescence intensity is changed by a quenching dye (quencher), a fluorescence resonance energy transfer ( FRET) is a system in which the emission state of the fluorescent dye changes. Specifically, the following systems (1) to (3) are mentioned.

(1) A system that uses tryptophan residues present in the VH region of an antibody as a quencher dye The 36th, 47th, and 103rd of the VH region of an antibody (according to the Kabat numbering system) There are tryptophan (W) residues, and these tryptophan residues act as quenchers (WO2011 / 061944). When a fluorescent immunosensor, a fusion protein containing an antibody-binding protein labeled with a fluorescent dye, binds to the antibody, the fluorescent dye is located near the tryptophan residue and interacts with the tryptophan residue to quench the fluorescent dye. To design. That is, in a state where the fluorescent immunosensor is bound to the antibody, it is quenched and does not emit fluorescence. When the antigen is bound to the antibody, the three-dimensional structure of the antibody is changed, and the fluorescent dye located in the vicinity of tryptophan is separated from tryptophan, does not interact with tryptophan, is quenched, and emits fluorescence. A schematic diagram of this principle is shown in FIG.

  When an antigen is measured by an antigen-antibody reaction in this system, the fluorescent immunosensor of the present invention is bound to an antibody against the antigen to be measured to form a complex of the antibody and the fluorescent immunosensor. At this time, the fluorescent dye is quenched by tryptophan present in the VH region. When a sample that may contain the antigen is mixed with a complex of an antibody and a fluorescent immunosensor, if the antigen is present, the antibody and antigen are bound, the three-dimensional structure of the antibody is changed, and the fluorescent dye is quenched. Is released, and fluorescence is emitted. By measuring this fluorescence, the presence of the antigen can be detected, and the antigen can also be quantified by the fluorescence intensity.

(2) System using quenching dye (quencher) and fluorescent dye In this system, the antibody to which the fluorescent immunosensor of the present invention is bound is labeled with a quencher. The labeling of the antibody with the quencher is performed such that when the antibody and the fluorescent immunosensor are bound, the fluorescent dye and the quencher are located in the vicinity and interact with each other so that the fluorescent dye is quenched by the quencher. That is, the fluorescence immunosensor is quenched by the antibody quencher in a state of binding to the antibody. When the antigen is bound to the antibody, the three-dimensional structure of the antibody changes, the quencher of the antibody and the fluorescent dye of the fluorescent immunosensor are separated from each other, no longer interact with each other, the quench is released, and fluorescence is emitted.

When an antigen is measured by an antigen-antibody reaction in this system, the fluorescent immunosensor of the present invention is bound to an antibody against the antigen to be measured and labeled with a quencher. At this time, the fluorescent dye of the fluorescent immunosensor is quenched by the quencher of the antibody. When a sample that may contain the antigen and an antibody and a complex of a fluorescent immunosensor are mixed, if the antigen is present, the antibody and the antigen are bound, the quench of the fluorescent dye is released, and fluorescence is emitted. It becomes like this. By measuring this fluorescence, the presence of the antigen can be detected, and the antigen can also be quantified by the fluorescence intensity.
In this system, the fluorescent immunosensor of the present invention may be labeled with a quencher and the antibody labeled with a fluorescent dye.

(3) System Using Fluorescence Resonance Energy Transfer (FRET) In this system, an antibody to which the fluorescent immunosensor of the present invention is bound is labeled with a fluorescent dye different from the fluorescent dye of the fluorescent immunosensor. In this case, the combination of the fluorescent dye of the fluorescent immunosensor and the fluorescent dye of the antibody is a combination of a donor dye that becomes an energy donor (donor) for fluorescence resonance energy transfer and an acceptor dye that becomes an energy acceptor (acceptor). To choose. Either the immunofluorescent sensor or the antibody is labeled with an energy donor dye and the other is labeled with an energy acceptor dye. The labeling of an antibody with a fluorescent dye is such that when the antibody and the fluorescent immunosensor are combined, the fluorescent dye of the fluorescent immunosensor and the fluorescent dye of the antibody, that is, the energy donor and the energy acceptor are located nearby and interact with each other. This is done at a position where the energy emitted by the donor moves to the energy acceptor. For example, when a fluorescent immunosensor labeled with an energy donor dye is present alone, fluorescence at a specific wavelength is generated from the energy donor dye due to the excitation wavelength of the energy donor dye. When a fluorescent immunosensor labeled with an energy donor dye is combined with an antibody labeled with an energy acceptor dye to form a complex, the energy donor dye and the energy acceptor dye are located in the vicinity. Energy transfer can occur to the energy acceptor dye. Therefore, excitation energy is transferred from the energy donor dye to the energy acceptor dye depending on the excitation wavelength of the energy donor dye, and fluorescence having a specific wavelength is generated from the energy donor dye. When the antigen is bound to the antibody, the three-dimensional structure of the antibody is changed, and the energy donor dye of the fluorescent immunosensor and the energy acceptor dye of the antibody are separated from each other and do not interact, and fluorescence resonance energy transfer does not occur. As a result, fluorescence emitted from the energy acceptor is not observed. A similar phenomenon occurs when the fluorescent immunosensor is labeled with an energy acceptor dye and the antibody is labeled with an energy donor dye.

  When antigen is measured by antigen-antibody reaction in this system, the antibody against the antigen to be measured and labeled with the energy donor dye or energy acceptor dye is labeled with the energy acceptor dye or energy donor dye. The fluorescent immunosensor of the present invention is bound. At this time, fluorescence resonance energy transfer occurs between the fluorescent dye of the fluorescent immunosensor and the fluorescent dye of the antibody, that is, the energy donor dye and the energy acceptor dye, and fluorescence is emitted from the energy acceptor dye. When the sample that may contain the antigen and the complex of the antibody and the fluorescent immunosensor are mixed, if the antigen is present, the antibody and the antigen are bound, and the energy donor dye and the energy acceptor dye are separated. , They no longer interact and the fluorescence emitted by the energy donor is no longer observed. By measuring this change in fluorescence, the presence of the antigen can be detected, and the antigen can also be quantified by the fluorescence intensity.

  In the present invention, the method for labeling a fusion protein containing an antibody-binding protein with a fluorescent dye for producing a fluorescent immunosensor is not limited, and various known methods can be used. For example, a method of labeling by chemical modification directly or indirectly through a cross-linking agent using functional groups at both ends or side chains of the fusion protein, a cell-free translation system, and synthesizing a fusion protein or a recombinant antibody. However, a site-specific labeling method or the like may be used. As a method of labeling using a cell-free translation system, an amber suppression method (Ellman J et al. (1991) Methods Enzymol. 202: 301-36), a 4-base codon method (Hohsaka T., et al., J Am. Chem. Soc., 118, 9778-9779, 1996), artificial base codon method (Hirao, I., et al. Narute Biotech., 20, 177-182, 2002), C-terminal labeling method (JP 2000-139468), N-terminal labeling method (US Pat. No. 5,643,722, Olejnik et al. (2005) Methods 36: 252-260) and the like. The amber suppression method creates DNA or mRNA by replacing the codon encoding the amino acid of the target site of the label with an amber codon that is one of the stop codons, and synthesizes the protein from the DNA or mRNA using a cell-free translation system At the time of synthesis, a protein with a labeled amino acid introduced at the site substituted for the amber codon is synthesized by adding a suppressor tRNA to which a labeled unnatural amino acid is bound to the protein synthesis reaction solution. . In the 4-base codon method, a codon is expanded to a 4-base codon such as CGGG, a codon encoding an amino acid is replaced with CGGG, and a protein is obtained from the DNA or mRNA using a cell-free translation system. Synthesize. At that time, by adding tRNA CGGG to which the labeled unnatural amino acid is bound to the protein synthesis reaction solution, a protein in which the labeled amino acid is introduced at the site substituted with the 4-base codon can be synthesized. In the C-terminal labeling method, a protein having a label introduced specifically is synthesized by translating DNA or mRNA into protein in a cell-free translation system to which labeled puromycin is added at an optimum concentration. can do.

  Alternatively, fluorescent dyes can be introduced site-specifically by genetic recombination techniques using E. coli or animal cells as hosts. Using an aminoacyl-tRNA synthetase that recognizes azidotyrosine and Escherichia coli introduced with suppressor azidotyrosyl-tRNA as a host, azidotyrosine can be introduced site-specifically and a fluorescent dye can be bound to the introduced azido group. it can. Furthermore, using an animal cell introduced with archaebacterial pyrrolidyl-tRNA synthetase and suppressor pyrrolidyl-tRNA as a host, site-specifically introduce azide Z-lysine into the polypeptide, and bind the fluorescent dye to the introduced azide group Can do.

  Among these methods, amino acids modified with fluorescent dyes can be assigned to codons that are not assigned amino acids in nature, and proteins can be labeled with fluorescent dyes during protein synthesis. The codon method is preferred.

For these methods, ProX (trademark) -tag may be used. ProX-tag is a partial modification of the gene sequence in the N-terminal region of a protein that is highly expressed in a cell-free translation system of E. coli. The position where the unnatural amino acid is incorporated and the sequence before and after it are optimized. Yes. ProX-tag can be added to the N-terminus of the fusion protein or antibody to be labeled. That is, when synthesizing a fusion protein or antibody, DNA encoding ProX-tag may be introduced into the N-terminal side and synthesized. The amino acid sequence of ProX-tag is represented by MSKQIEVNXSNET (X is a fluorescently labeled amino acid, SEQ ID NO: 8). The method using ProX-tag can be performed using, for example, CloverDirect (trademark) reagent (Protein Express).
Further, a chemical modification method capable of producing a large amount of a label at a time is also preferable.

  A fusion protein containing an antibody binding protein is located near the tryptophan residue in the HV region of the antibody to which the fluorescent dye binds when the fusion protein binds to the antibody, or the antibody is labeled with a fluorescent dye or quencher If so, it is labeled so that it is located in the vicinity of this fluorescent dye or quencher. Preferably, Protein A or its antibody binding domain of a fusion protein consisting of Protein A or its antibody binding domain and Protein G or its antibody binding domain is labeled, and more preferably Protein A or its antibody binding domain is N-terminal or N-terminal Mark the neighborhood. Alternatively, a peptide tag such as ProX-tag may be added to the N-terminal part of Protein A or its antibody binding domain, and the tag may be labeled.

  In addition, an antibody used in combination with the fluorescent immunosensor of the present invention can be labeled with a fluorescent dye or a quencher in the same manner. In addition, as described below, the antibody is not directly labeled with a fluorescent dye or quencher, but the antibody is indirectly labeled by binding an antibody binding protein such as Protein L labeled with a fluorescent dye or quencher to the antibody. Is also possible.

  When producing a fluorescent immunosensor by labeling a fusion containing an antibody-binding protein, the fluorescent dye used for the label takes an appropriate spatial arrangement when the fluorescent immunosensor and the antibody are bound, and the VH region of the antibody It may be designed so that there is a certain distance between the binding domain of the fluorescent dye and the antibody binding protein so that it takes an appropriate spatial arrangement with the tryptophan residue or the fluorescent dye or quencher used to label the antibody. Good. For this purpose, for example, an appropriate linker may be provided between the amino acid labeled with the fluorescent dye in the fluorescent immunosensor and the binding domain of the fusion protein. As the linker, a peptide linker comprising an appropriate number of amino acid sequences can be used.

  For example, when a peptide tag such as ProX-tag is added to a fusion protein containing an antibody binding protein and an amino acid in the tag is labeled, a linker may be provided between the tag and the fusion protein. For example, FIG. 7 shows the case where ProX-tag is added to a fusion protein containing an antibody binding protein and the amino acids in ProX-tag are labeled with a fluorescent dye, and a peptide linker comprising an amino acid sequence is placed between the tag and the fusion protein. Contains. As the linker used at this time, the same linker as the linker existing between Protein A and Protein G in the above fusion protein can be used. In particular, a linker consisting of an amino acid sequence consisting of G and S can be mentioned. An amino acid sequence composed of proline that forms a characteristic helix structure can also be used as a linker. For example, polyproline composed of 5 to 20 prolines, preferably polyproline composed of 5 to 10 prolines.

  In the present invention, the peptide linker that intervenes between Protein A or its antibody binding domain and Protein G or its antibody binding domain in the fusion protein is referred to as the first peptide linker, and the fusion protein containing the antibody binding protein and the fluorescent dye The peptide linker that intervenes between them may be referred to as a second peptide linker.

  In the present invention, the fluorescent dye or quencher used for labeling the fluorescent immunosensor or antibody is a fluorescent dye that undergoes quenching and dequenching according to the principle of (1) above, or is quenched and quenched by the principle of (2) above. A combination of a fluorescent dye and a quencher in which the release of the fluorescence occurs, and a combination of a fluorescent dye in which fluorescence resonance energy transfer occurs according to the principle of (3) above.

  Examples of fluorescent dyes used for fluorescent labeling include fluorescent dyes having rhodamine, coumarin, Cy, EvoBlue, oxazine, Carbopyronin, naphthalene, biphenyl, anthracene, phenenthrene, pyrene, carbazole and the like as basic skeletons and derivatives of the fluorescent dyes. Specifically, CR110: carboxyrhodamine 110: Rhodamine Green (trademark), TAMRA: carbocytetremethlrhodamine: TMR, Carboxyrhodamine 6G: CR6G, ATTO655 (trademark), BODIP YFL (trademark): 4,4-difluoro-5,7-dimethyl- 4-bora-3a, 4a-diaza-s-indancene-3-propionicacid, BODIPY 493/503 (trademark): 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a, 4a- diaza-s-indancene-8-propionicacid, BODIPY R6G (trademark): 4,4-difluoro-5- (4-phenyl-1,3-butadienyl) -4-bora-3a, 4a-diaza-s-indancene- 3-propionic acid, BODIPY 558/568 (trademark): 4,4-difluoro-5- (2-thienyl) -4-bora-3a, 4a-diaza-s-indancene-3-propionicacid, BODIPY 564/570 ( Trademark): 4,4-difluoro-5-styryl-4-bora-3a, 4a-diaza-s-indancene-3-propionic acid, BODIPY 576/589 (trademark): 4,4-difluoro-5- (2 -pyrrolyl) -4-bora-3a, 4a-diaza-s-indancene-3-propionicacid, BODIPY 581/591 (trademark): 4,4-difluoro-5- (4-phenyl-1,3-butadienyl)- 4-bora-3a, 4a-diaza-s-indancene-3-propionic acid, Cy3 ™, Cy3B ™, Cy3.5 ™, Cy5 ( Mark), Cy5.5 (trademark), EvoBlue10 (trademark), EvoBlue30 (trademark), MR121, ATTO 390 (trademark), ATTO 425 (trademark), ATTO 465 (trademark), ATTO 488 (trademark), ATTO 495 (trademark) ), ATTO 520 (TM), ATTO 532 (TM), ATTO Rho6G (TM), ATTO 550 (TM), ATTO 565 (TM), ATTORho3B (TM), ATTO Rho11 (TM), ATTO Rho12 (TM), ATTO Thio12 (TM), ATTO 610 (TM), ATTO 611X (TM), ATTO 620 (TM), ATTO Rho14 (TM), ATTO 633 (TM), ATTO 647 (TM), ATTO647N (TM), ATTO 655 (TM) ), ATTO Oxa12 (TM), ATTO 700 (TM), ATTO 725 (TM), ATTO 740 (TM), Alexa Fluor 350 (TM), Alexa Fluor 405 (TM), Alexa Fluor 430 (TM), Alexa Fluor 488 (Trademark), Alexa Fluor 532 (trademark), Alexa Fluor 546 (trademark), Alexa Fluor 555 (trademark), Alexa Fluor 568 (trademark), Alexa Fluor 594 (trademark), Alexa Fluor 633 (trademark), A lexa Fluor 647 (TM), Alexa Fluor 680 (TM), Alexa Fluor 700 (TM), Alexa Fluor 750 (TM), AlexaFluor 790 (TM), Rhodamine Red-X (TM), Texas Red-X (TM), 5 (6) -TAMRA-X (trademark), 5TAMRA (trademark), SFX (trademark), etc. can be used. Among these, rhodamine fluorescent dyes TAMRA and CR110, and oxazine fluorescent dyes ATTO 655 are preferable.

  Examples of the quenching dye (quencher) include quenching dyes having a basic skeleton such as NBD: 7-nitrobenzofurazan, DABCYL, BHQ, ATTO, QXL, QSY, Cy, Lowa Black, IRDYE, and derivatives of the quenching dye. Specifically, NBD, DABCYL, BHQ-1 (trademark), BHQ-2 (trademark), BHQ-3 (trademark), ATTO540Q (trademark), ATTO580Q (trademark), ATTO612Q (trademark), QXL490 (trademark), QXL520 (TM), QXL570 (TM), QXL610 (TM), QXL670 (TM), QXL680 (TM), QSY-35 (TM), QSY-7 (TM), QSY-9 (TM), QSY-21 ( (Trademark), Cy5Q (trademark), Cy7Q (trademark), Lowa Black FQ (trademark), LowaBlack RQ (trademark), IRDYE QC-1 (trademark), etc. can be used. Among these, NBD is preferable.

  Examples of the combination of the antibody immunosensor of the present invention, the fluorescent dye for labeling the antibody, and the quencher include a combination of TAMRA and NBD. The antibody immunosensor of the present invention is labeled with an antibody, and the combination of an energy donor and an energy acceptor that causes fluorescence resonance energy transfer, for example, BODIPY (registered trademark) FL as an energy donor and BODIPY as an energy acceptor (Registered trademark) 558/568 may be selected. Alternatively, combinations of energy donors and energy acceptors include BODIPY FL and BODIPY 576/586, BODIPY FL and TAMRA, BODIPY FL and Cy3, Fluorescein and BODIPY 558/568, Alexa488 and BODIPY 558/568, and BODIPY 558/568. Combinations such as Cy5 can also be used.

  As described above, the antibody can also be indirectly labeled using a labeled antibody binding protein such as Protein L. In the system based on the above principles (2) and (3), it is necessary to label the antibody to be used with a fluorescent dye or a quencher. By using a labeled antibody binding protein, the antibody to be used is labeled with the fluorescent dye. Without using the fluorescent immunosensor of the present invention, the antigen can be detected.

  In this case, an antibody binding protein different from the antibody binding protein may be used. Such an antibody binding protein includes Protein L. Protein L is an antibody-binding protein produced by Peptostreptococcus magnus and binds to the kappa light chain of the antibody. Protein L may be a full length or an antibody binding site.

  Instead of binding the antibody with a fluorescent dye or quencher, Protein L may be labeled with a fluorescent dye or quencher to label the antibody. When the labeled Protein L and the antibody are mixed with the above fluorescent immunosensor, the labeled Protein L and the fluorescent immunosensor bind to the same antibody at different sites, and the fluorescent substance of the antibody immunosensor and the fluorescent substance or quencher of Protein L Interact with each other and the fluorescent material is quenched or fluorescence energy transfer occurs between the fluorescent materials, and the antigen can be detected by the principle of (2) or (3) above. In the present invention, an antibody labeled using an antibody binding protein such as Protein L is also referred to as a labeled antibody.

  The present invention relates between a fluorescent immunosensor that is a fusion protein containing an antibody-binding protein and labeled with the above-described fluorescent dye, and an unlabeled antibody, an antibody that is labeled with a quencher, or a fluorescent dye in a fluorescent immunosensor. Also included are kits that combine antibodies labeled with fluorescent dyes that produce fluorescence resonance energy transfer, or kits that combine immunosensors, labeled antibodies, and protein L labeled.

  These kits can be used as detection kits that use an antigen to which an antibody specifically binds as a substance to be detected.

3. Method for detecting antigen using immunofluorescent sensor of the present invention The principle of antigen detection using the fluorescent immunosensor of the present invention is as follows.
(i) The fluorescent immunosensor of the present invention and a labeled antibody are contacted and bound to form a complex of the fluorescent immunosensor and a labeled or unlabeled antibody. In this case, instead of using a labeled antibody as an antibody, a complex of a fluorescent immunosensor, an antibody, and a labeled protein L may be formed by using unlabeled antibody and labeled protein L.

(ii) Next, the complex formed in (i) and the test sample are mixed and contacted. At this time, a complex of the fluorescent immunosensor and the antibody may be formed in advance, and then contacted with the test sample, or the test sample, the fluorescent immunosensor, and the labeled antibody may be mixed and contacted at the same time.
When an antigen that is a substance to be detected exists in the test sample, the complex containing the fluorescent immunosensor and the antibody binds to the antigen in the test sample. When the antigen-antibody reaction occurs, the three-dimensional structure of the antibody changes, and the fluorescent substance of the immunofluorescent sensor and the tryptophan residue of the antibody, the fluorescent dye and the quencher of the labeled antibody, The fluorescent dye of the labeled antibody does not interact.

(iii) The presence of the antigen can be detected by measuring the fluorescence emitted from the fluorescent dye and detecting the change in fluorescence such as the disappearance of the fluorescence or the fluorescence generated from the energy acceptor dye.
At this time, it is preferable to prepare a calibration curve by previously bringing a test sample containing a complex of a fluorescent immunosensor and an antibody and a known amount of an antigen to be detected into contact with the mixture and measuring a change in fluorescence at that time. . Alternatively, when performing detection, a control test sample containing a plurality of known amounts of the antigen to be detected may be prepared, and a calibration curve may be created by simultaneously measuring the control test sample. The amount of the antigen to be detected in the test sample can be calculated from the measured fluorescence and the calibration curve. There is a correlation between the measured fluorescence intensity and the amount of the detected antigen in the test sample, and the amount of the detected antigen can be measured using the fluorescence intensity as an index.

Detection of an antigen to be detected using the fluorescent immunosensor of the present invention can be performed by the following steps.
(a) contacting the fluorescent immunosensor with the antibody in a liquid to form a complex of the fluorescent immunosensor and the antibody,
(b) contacting the complex formed in (a) with an antigen in a test sample to bind the antibody and the antigen;
(c) A step of measuring a change in fluorescence due to the binding between the antibody and the antigen, wherein the fluorescence quenching is canceled due to the binding between the antibody and the antigen.

  According to this method, the presence of the antigen can be detected by eliminating the disappearance of fluorescence or increasing the fluorescence intensity, and the antigen can be quantified by measuring the fluorescence intensity.

In addition, detection of an antigen to be detected using the fluorescent immunosensor of the present invention can be performed by the following steps.
(a) contacting a fluorescent immunosensor with an antibody labeled with a quencher in a liquid to form a complex of the fluorescent immunosensor and the labeled antibody;
(b) contacting the complex formed in (a) with an antigen in a test sample to bind the antibody and the antigen;
(c) A step of measuring a change in fluorescence due to the binding between the antibody and the antigen, wherein the fluorescence quenching is canceled due to the binding between the antibody and the antigen.

  According to this method, the presence of the antigen can be detected by eliminating the disappearance of fluorescence or increasing the fluorescence intensity, and the antigen can be quantified by measuring the fluorescence intensity.

Furthermore, detection of an antigen to be detected using the fluorescent immunosensor of the present invention can be performed by the following steps.
(a) A step in which a fluorescent immunosensor is brought into contact with an antibody labeled with a fluorescent dye in a liquid to form a complex of the fluorescent immunosensor and the labeled antibody, wherein the fluorescent dye of the fluorescent immunosensor and the fluorescent dye of the labeled antibody are The combination is a combination of an energy donor dye and an energy acceptor dye capable of producing FRET;
(b) contacting the complex formed in (a) with an antigen in a test sample to bind the antibody and the antigen;
(c) A step of measuring a change in fluorescence due to the binding between the antibody and the antigen, wherein the fluorescence from the energy acceptor dye decreases due to the binding between the antibody and the antigen.

  According to this method, the presence of the antigen can be detected by the disappearance of the fluorescence from the energy acceptor dye or the decrease in the fluorescence intensity, and the antigen can be quantified by measuring the fluorescence intensity.

  For the measurement of fluorescence in the antigen detection method using the fluorescence immunosensor of the present invention, a light source or a measurement device used for fluorescence detection can be usually used. Any light source may be used as long as it can irradiate an excitation light wavelength. Specific examples include a mercury lamp, a xenon lamp, an LED (light emitting diode), and a laser beam. At this time, excitation light having a specific wavelength can be obtained using an appropriate fluorescent filter. As the fluorescence measuring apparatus, for example, a fluorescence microscope equipped with a light source of excitation light and its irradiation system, a fluorescence image acquisition system, and the like can be used. For example, MF20 / FluoroPoint-Light (manufactured by Olympus) or FMBIO- III (manufactured by Hitachi Software Engineering). Moreover, you may use the small and portable fluorescence detection apparatus provided with the light source, the irradiation system, and the measurement system. By using such a small device, it is possible to detect the antigen at the collection site without collecting the test sample and carrying it to the laboratory for measurement. The fluorescence detection may be a fluorescence spectrum detection or a fluorescence intensity detection at a specific wavelength.

  Further, in the method for detecting an antigen using the fluorescent immunosensor of the present invention, the excitation light to be irradiated and the wavelength of the fluorescence to be measured and / or detected may be appropriately selected according to the type of fluorescent dye to be used. For example, when CR110 is used as the fluorescent dye, an excitation light wavelength of 480 nm and a fluorescence wavelength of 530 nm are used, when TAMRA is used, an excitation light wavelength of 530 nm and a fluorescence wavelength of 580 nm are used, and when ATTO655 is used, an excitation light wavelength of 630 nm and fluorescence are used. A wavelength of 680 nm may be used. Also, when two different fluorescent dyes are used, a combination of excitation light wavelength and fluorescence wavelength that can measure the antigen concentration and / or detect the antigen may be appropriately selected and used.

  In the method of the present invention, the substance to be detected is an antigen or antibody that can be measured by an immunoassay, that is, an assay using an antigen-antibody reaction. The antigen may be any antigen that can produce an antibody, and examples thereof include proteins, polysaccharides, lipids, glycolipids and the like. Protozoa, fungi, bacteria, mycoplasma, rickettsia, chlamydia, viruses, animal tissues and the like containing these substances can also be detected. In addition, chemical substances including low-molecular compounds such as narcotics, explosives, agricultural chemicals, fragrances, and pollutants can also be measured. Examples of such substances include stimulants such as amphetamine, methamphetamine, morphine, heroin, and codeine, and narcotics, aflatoxins, sterigmatocystins, neosolaniol, nivalenol, fumonisins, ochratoxins, and endotoxin-producing toxins. Poisons, sex hormones such as testosterone and estradiol, additives illegally used in feeds such as clenbuterol and ractopamine, harmful substances such as PCB, gossypol, histamine, benzpyrene, melamine, acrylamide and dioxin, acetamiprid, imidacloprid, chlorfenapyr, malathion, Residual pesticides such as carbaryl, clothianidin, triflumizole, chlorothalonil, spinosad, lannate, methamidophos, chlorpyrifos, bisphenol Such as the environment Mormon, such as Nord A can be mentioned.

Samples are not limited, and biological fluid samples such as blood, serum, plasma, urine, saliva, spinal fluid, culture supernatant, cell extract, fungal extract, waste water, animal tissue-derived substances such as allergen, narcotics Examples include a sample obtained by wiping a substance that may have adhered to the surface with paper or the like.
Since the detection of the present invention is performed in a liquid system, the sample may be detected by suspending, dissolving, or immersing the sample in a physiological saline or a buffer as appropriate.

  The present invention will be specifically described by the following examples, but the present invention is not limited to these examples.

Example 1 Construction and synthesis of antibody-binding fluorescent probe using cell-free system, and verification of binding ability with antibody Conventional fluorescent-labeled antibody Quenchbody (WO2011 / 061944) whose fluorescence intensity increases in an antigen-dependent manner There is a problem that it takes time to construct, and in this example, an attempt was made to solve this problem by using a fluorescent probe based on an antibody-binding protein. For this purpose, an attempt was made to produce an antibody-binding fluorescent probe using a cell-free protein synthesis system. Specifically, a fluorescent dye (TAMRA) is introduced near its N-terminal, and Protein G (B1 domain, PG-B1), and Protein A (D domain, PA-D) and PG-B1 are two types of linkers. A total of three types of fluorescent probes were prepared by binding via the. In the probe having PA-D and PG-B1, two kinds of linker sequences (G ₄ S) ₃ and (DDAKK) ₄ were inserted between them. (G ₄ S) ₃ Linker is a flexible linker with few side chains consisting of 3 repeats of 1 unit composed of 4 glycine and 1 serine amino acid sequences, and (DDAKK) ₄ Linker is more This linker is expected to have a linear α-helix structure. PA-D can be found in _VH sites of certain human antibodies (VH3) (JL Hillson, NS Karr, IR Oppliger, M Mannik, and EH Sasso, J. Exp. Med. 178 (1), 331-336, 1993. ;....... M Graille et al, Proc Natl Acad Sci 97 (10) 5399-5404, 2000), PG-B1 binds to C _H 1 region of most IgG antibody (JP Derrick, and DB Wigley, J. Mol. Biol. 243 (5), 906-918, 1994.) Therefore, it is expected that the binding ability is synergistically increased by arranging them in tandem at an appropriate distance. Therefore, a cell-free expression system for a total of three types of fluorescent probes was constructed, probes in which TAMRA dyes were introduced by the aforementioned unnatural amino acid introduction method were synthesized, and the binding ability between these and antibodies was verified.

I. Construction and synthesis of antibody-bound fluorescent probes Construction of constructs of fluorescent probes A, B and C
The following three types of fluorescent probe constructs were constructed through appropriate linker sequences in the D domain of Protein A and the B1 domain of Protein G. FIG. 3 shows the arrangement structure of the fluorescent probes A, B and C. In the following sequences, PA-D is referred to as PA and PG-B1 is referred to as PG.
A ProX-PG-His ₆
B ProX-PA- (G ₄ S) ₃ -PG-His ₆
C ProX-PA- (DDAKK) ₄ -PG-His ₆

The following primers were used for constructing the construct.
ProX-PGB1back: 5'-TAGTCTAATGAGACCGCCCGCGACTTACAAATTAATC-3 '(SEQ ID NO: 9)
His-PGB1for: 5'-TGATGATGAGAACCCCCCCCTTCCAGTAACTGTAAAGGTC-3 '(SEQ ID NO: 10)
ProX-PA: 5'-TAGTCTAATGAGACCGCGCAACAAAATAACTTC-3 '(SEQ ID NO: 11)
PA-GS3: 5'-GCTGCCACCTCCGCCTGAACCGCCTCCACCTGCTTGAGATTCGTTTAAT-3 '(SEQ ID NO: 12)
PA-DDAKK4: 5'-CGCGTCGTCCTTCTTGGCATCATCTTTTTTTTAGCATCATCTGCTTGAGATTCGTTTAATTT-3 '(SEQ ID NO: 13)
GS3-PG: 5'-GGCGGAGGTGGCAGCGGCGGTGGCGGGTCGGCCGCGACTTACAAATTAATCCT-3 '(SEQ ID NO: 14)
DDAKK4-PG: 5'-GCCAAGAAGGACGACGCGAAAAAAGATGATGCTAAAAAAGCCGCGACTTACAAATTAATCCT-3 '(SEQ ID NO: 15)
PGNcoBack: 5'-TCGCGCCATGGCGACTTACAAATTAATCCTTAAT-3 '(SEQ ID NO: 16)
ProxNcofor: 5'-GACGTCCATGGTCTCATTAGACTAGTTTAC-3 '(SEQ ID NO: 17)

The E. coli used in the experiment is as follows.
XL10-Gold (Kan ^r ): Tet ^r , Δ (mcrA) 183, TYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTE (mcrCB-hsdSMR-mrr) 173, endA1, supE44, thi-1, recA1, gyrA96, relA1, lac, te lacI ^q ZΔM15, Tn10 (Tet ^r ), Tn5 (Kan ^r ), Amy]
JM109: recA1, endA1, gyrA96, thi-1, hsdR17 (rK - mK +), e14 - (mcrA -), supE44, relA1, Δ (lac-proAB) / F ' ^{[traD36, proAB +, lac I q} , lacZΔM15)

For culture of E. coli, LB medium or SOC medium containing an appropriate antibiotic was used. Transformation was performed by the calcium chloride method or the electroporation method. Plasmid purification was performed using the PureYield ^™ Plasmid Miniprep System (Promega).

1-1. Construction of constructs of fluorescent probes B and C PG-V _L (C Suzuki, H Ueda, W Mahoney and T Nagamune, Anal. Biochem. 286 (2), which is a template DNA containing part of Protein G and Protein A, respectively. 238-246, 2000.) and PA LcLuc (Y Maeda et al., Anal. Biochem. 249, 147-152, 1997.) using the above primers as appropriate, and PCR reaction with KOD FX polymerase (Takara-Bio). went. This product was electrophoresed on a 1.2% agarose gel, and the target band was excised and purified by Wizard SV gel extraction kit (Promega). Thereby, the following DNA fragments were synthesized.
1 ProX-PA- (G ₄ S) ₃
2 ProX-PA- (DDAKK) ₄
3 (G ₄ S) ₃ -PG-His ₆
4 (DDAKK) ₄ -PG-His ₆
5 ProX-PG- His ₆

  Next, by performing overlap extension PCR using two types of outer primers and two types of templates, a DNA fragment of fluorescent probe B is synthesized from 1 and 3 of the above synthesized DNA fragments, and the above DNA A DNA fragment of fluorescent probe C was synthesized from fragments 2 and 4. These were mixed with pROX-FL92.1amber (Protein Express) cleaved with restriction enzymes NcoI and SmaI, and subjected to In-fusion reaction. Then, JM109 or XL-10 Gold was transformed, and 37 ° C in an LBA plate medium. Incubated overnight. The next day, several clones were selected from the grown colonies, cultured in 4 ml LBA medium, a small amount of plasmid was extracted, and the nucleotide sequence was examined. Concentration measurement was also performed at the same time, which was used as an indicator of the amount of DNA during the fluorescent probe synthesis. When measuring the concentration of the synthesized fluorescent probe, a TAMRA dye with a known concentration was used as a calibration curve, excitation was performed at 535 nm using a fluorescence plate reader (TEEn GEnNios Pro), and measurement was performed at 590 nm.

The In-Fusion reaction is a characteristic reaction different from general homologous recombination in which a homologous sequence (15 bases) set at the end of the vector and the insert is fused. By making the end of the vector and the end of the insert 15 bases into homologous sequences by designing the primer, the insert can be incorporated into the vector without any restriction enzyme treatment. In-Fusion Advantage PCR Cloning Kit and In-Fusion HD Cloning Kit (Clontech) were used with the following protocols, respectively.
In-Fusion Advantage PCR Cloning Kit
PCR product (insert) ・ ・ ・ 100 ng
Linear Vector ・ ・ ・ 100 ng
5 × In-Fusion Reaction Buffer ・ ・ ・ 2μl
In-Fusion Enzyme ・ ・ ・ 1μl
Nuclease Free Water: up to 10 μl
The mixture was allowed to stand at 37 ° C. for 15 minutes, and immediately the temperature was changed to 50 ° C. and left for 15 minutes.
In-Fusion HD PCR Cloning Kit
PCR product (insert) ・ ・ ・ 10-100 ng
Linear vector ・ ・ ・ 50-100 ng
5 × In-Fusion HD Enzyme Premix ・ ・ ・ 2μl
Nuclease-free Water: up to 10 μl
The mixture was left at 50 ° C. for 15 minutes. The reaction was performed on a Thermal Cycler.

  The target fluorescence is obtained by electrophoresis of PCR to synthesize 1 to 5 DNA fragments, overlap PCR using them, in-fusion reaction with pROX-FL92amber, and confirmation of the nucleotide sequence of the plasmid after transformation into E. coli. It was confirmed that plasmid DNAs for probes B and C were prepared.

1-2. Construction of fluorescent probe A A plasmid of fluorescent probe A was prepared by removing Protein A and the linker sequence from the plasmid in which fluorescent probes B and C were incorporated into the pROX vector. First, the plasmids of the fluorescent probes B and C were subjected to PCR with KOD FX polymerase using PGNcoBack and ProxNcofor primers, unnecessary sequences were removed, and NcoI cleavage sites were introduced at both ends. This product was purified by electrophoresis through a 1.2% agarose gel. The amplified band was treated with the restriction enzyme NcoI at 37 ° C. for 2 hours. After purification, self ligation was performed using Ligation Mix (Nippon Gene). After transformation into XL-10 Gold cells, the cells were cultured overnight at 37 ° C. in LBA plate medium. On the next day, several clones were selected from the grown colonies, cultured in 4 ml LBA medium, a small amount of plasmid was extracted, and the nucleotide sequence was examined. Concentration measurement was also performed at the same time, which was used as an indicator of the amount of DNA during the fluorescent probe synthesis.

  Confirmation that the plasmid of the target fluorescent probe A was prepared by PCR using the plasmid synthesized in 1-1, restriction enzyme treatment, ligation, and confirmation of the base sequence of the plasmid after transformation into E. coli. It was done.

1-3. Synthesis of fluorescent probe proteins of A, B, and C Using the plasmids of fluorescent probes A, B, and C, using a cell-free synthetic system RYTS kit (Protein Express), the following solutions are mixed and synthesized as fluorescent dyes A probe incorporating TAMRA was obtained.
(1) E. coli Lysate: 16.5μl
(2) 2 × Reaction Mix: 25μl
(3) Methionine: 0.5μl
(4) Enzyme Mix: 2.5μl
(5) TAMRA-tRNA: 5 μl
(6) Template plasmid: 160 ng
(7) Nuclease-free Water: up to 50μl

  TAMRA-tRNA (5) is obtained by dissolving TAMRA-tRNA in 30 μl of buffer per tube. This mixture was mixed by pipetting and then reacted at 25 ° C. to 30 ° C. for 2 hours.

Subsequently, purification was performed by immobilized metal affinity purification using a His SpinTrap ^TM column, and PBS containing 0.05% Tween20 with Amicon Ultra-0.5 device (10 mM sodium phosphate, 147 mM NaCl, 2.7 mM KCl, pH 7.2, below) After exchanging the buffer with PBST), protein expression was confirmed by SDS-PAGE, a calibration curve of TAMRA concentration was created using GENios Pro, and the concentration of the synthesized fluorescent probe was calculated.

  The results of SDS-PAGE UV irradiation observation are shown in FIG. This confirmed that each probe protein was synthesized. Moreover, the result of having drawn the calibration curve using TAMRA is shown in FIG.

II. Verification of binding ability of antibody-binding fluorescent probe Binding of fluorescent probes A, B, and C to IgG antibody was measured by ELISA.
The binding ability of each probe synthesized as follows and IgG was examined.

  Immobilize mouse anti-domoic acid monoclonal antibody (subclass IgG1) diluted in ELISA plate, block with PBS containing 20% Immunoblock (20% IPBS), add each fluorescent probe diluted 20-fold with PBST overnight 4 Allow to stand at ℃, and add Anti-His HRP diluted 2000 times with PBS containing 5% Immunoblock on the next day (hereinafter referred to as 5% IPBS), let stand for 3 hours, add enzyme reaction solution and react for 5 minutes. After that, the reaction was stopped and the absorbance was measured.

  A graph of the results of the ELISA experiment is shown in FIG. From this, it was confirmed that each synthesized fluorescent probe was capable of binding to mouse IgG1 antibody.

Example 2 Construction and synthesis of improved fluorescent probe and verification of binding ability with antibody In Example 1, the binding ability of the synthesized fluorescent probe and IgG antibody was shown. In this example, the constructs of the fluorescent probes B and C prepared in Example 1 are applied, and the dye and the three-dimensional structure are linked via a linker between the sequence of ProX tag and Protein A into which the fluorescent dye is introduced. Probes were created that could interact better. In addition, a plurality of linker sequences were examined, and a GS linker using different lengths and a proline linker in which a proline forming a left-handed helix in the protein was continuous were used.

  In Example 1, the binding ability was examined only with the IgG antibody. In this example, an anti-BGP Fab antibody fragment having high responsiveness as Quenchbody (WO2011 / 061944) was synthesized and newly synthesized. The binding ability with the prepared and synthesized probe was examined.

I. Construction and Synthesis of Improved Fluorescent Probe The following construct in which a linker sequence is inserted between ProXtag and PA-D or PG-B1 using the plasmid DNA of the fluorescent probes B and C prepared in Example 1 as a template Constructed (Figure 7).
_{D ProX- (G 4 S) 2} -PA- (G 4 S) 3 -PG-His 6
E ProX-P ₈ -PA- (G ₄ S) ₃ -PG-His ₆
F ProX- (G ₄ S) ₂ -PA- (DDAKK) ₄ -PG-His ₆
G ProX-P ₈ -PA- (DDAKK) ₄ -PG-His _{6
H ProX- (G 4 S) 5} -PA- (G 4 S) 3 -PG-His 6
I ProX- (G ₄ S) ₅ -PA- (DDAKK) ₄ -PG-His ₆

The following primers were used for constructing the construct.
GSx2PAback: 5'-GGGCGGCGGCAGCGGTGGAGGTTCAACCGCGCAACAAAATAACTTC-3 '(SEQ ID NO: 18)
GS3x1PXfor: 5'-CGCTGCCGCCGCCCTCATTAGACTAGTTTACTTC-3 '(SEQ ID NO: 19)
Px8PAback: 5'-GCCGCCGCCGCCGCCTCCACCTCCAACCGCGCAACAAAATAACTTC-3 '(SEQ ID NO: 20)
Px4ProXfor: 5'-GCGGCGGCGGCGGCTCATTAGACTAGTTTACTTC-3 '(SEQ ID NO: 21)
G3Sx3PAback: 5'-GGGCGGCGGCAGCGGTGGAGGTTCAGGAGGTGGATCAACCGCGCAACAAAATAACTTC-3 '(SEQ ID NO: 22)
G3Sx3PXfor: 5'-CGCTGCCGCCGCCCGAACCGCCTCCGCTACCACCACCCTCATTAGACTAGTTTACTTC-3 '(SEQ ID NO: 23)

  First, PCR reaction was performed with KOD FX polymerase using fluorescent probes B and C plasmid DNA as templates and using the above primers as appropriate. This product was electrophoresed on a 1.2% agarose gel, and the target band was excised and purified. By treating this with the restriction enzyme DpnI at 37 ° C. for 1 hour, the methylated template DNA remaining in the solution was cleaved. XL-10Gold was transformed with restriction enzyme-treated DNA and cultured overnight at 37 ° C. in LBA plate medium. The primer set used in PCR is designed so that the terminal sequences are complementary to each other in advance, so that the linear DNA is transformed with the ends annealed into a circular shape and is completely double-stranded in the cell. Repaired to plasmid. On the next day, several clones were selected from the grown colonies, cultured in 4 ml LBA medium, a small amount of plasmid was extracted, and the nucleotide sequence was examined. Concentration measurement was also performed at the same time, which was used as an indicator of the amount of DNA during the fluorescent probe synthesis.

Probe proteins C, F and I were synthesized in the same manner as in Example 1.
As a result of confirmation of the base sequence of the plasmid inserted into the pROX vector by the PCR reaction using the plasmid DNAs of the fluorescent probes B and C as a template and transformed into Escherichia coli, the construction of the target plasmid DNAs D to I was confirmed.

  The results of SDS-PAGE of the synthesized fluorescent probes D to I are shown in FIG. This confirmed that each probe protein was synthesized. Moreover, the result of having drawn the calibration curve using TAMRA is shown in FIG.

II. Preparation of anti-osteocalcin (BGP) Fab fragment and confirmation of antigen binding ability
(1) Preparation of anti-BGP Fab fragment Preparation of Fab antibody fragment of KTM219, which is an anti-osteocalcin (Bone Gla protein, BGP) antibody, was carried out by JH Dong, M Ihara, and H Ueda, Anal. Biochem. 386, 36-44, 2009. Went according to.

  FIG. 10 shows the results of SDS-PAGE after the protein was expressed using pDong1 (KTM219) and purified with a Talon column. From this, it was confirmed that a protein having a molecular weight (30 kd) corresponding to the target Fab fragment was expressed in the periplasm in E. coli and obtained.

(2) Confirmation of antigen binding ability of anti-BGP Fab antibody By the ELISA measurement method of Example 1, the binding ability of the synthesized Fab antibody and the BGP antigen thereto was examined.

  Streptavidin (10 μg / ml) was immobilized on an ELISA plate, blocked, biotinylated BGP antigen diluted 4000 times with 5% IPBS was added, and allowed to stand for 1 hour. Thereafter, the Fab antibody (2 μg / ml) synthesized in (1) diluted with 5% IPBS was added, and after standing for 1 hour, α-His HRP diluted 8000 times with 5% IPBS and 3000 with 5% IPBS Double-diluted α-FLAG HRP was added and allowed to stand for 1 hour. Thereafter, the enzyme reaction solution was added and allowed to react for 5 minutes. Then, the reaction was stopped, and the absorbance was measured.

  The results are shown in FIG. Since it is detected with a secondary antibody against the FLAG tag (FIG. 11A) and His tag (FIG. 11B) present in the synthesized Fab fragment, the Fab synthesized therefrom has the ability to recognize and bind to the antigen BGP peptide. I understood.

III. Confirmation of binding ability between fluorescent probes C, F and I and IgG and Fab fragments According to the ELISA measurement method of Example 1, probes C, F and I synthesized as follows, human IgG and synthesized anti-BGP Fab antibody The binding ability to was investigated.

(1) ELISA for binding fluorescent probes to human IgG and Fab (antibody immobilization)
Human IgG (2 μg / ml) and anti-BGP Fab (3.3 μg / ml) diluted with PBS were immobilized on an ELISA plate overnight at 4 ° C., blocked with 20% IPBS, and 1.5 μl of fluorescent probe per well , Diluted with PBST and added overnight at 4 ° C. Next, a rabbit anti-TAMRA antibody (2.5 μg / ml) diluted with 5% IPBS was added, allowed to stand for 1 hour, HRP-labeled anti-rabbit IgG diluted 6000 times with 5% IPBS was added, and allowed to stand for 2 hours. After the enzyme reaction solution was added and reacted, the reaction was stopped and the absorbance was measured.

  A graph of the ELISA results is shown in FIG. Both probes were found to have the ability to bind to human IgG and anti-BGP Fab (mouse human chimeric antibody). The enzyme reaction time is 7 minutes.

(2) Fluorescent probe and Fab binding ELISA (antigen immobilization)
Streptavidin (10μg / ml) was immobilized on an ELISA plate at 4 ° C overnight in PBS. After blocking with 20% IPBS, biotinylated BGP-C12 peptide antigen diluted 4000 times with 5% IPBS was added and allowed to stand for 1 hour. did. Thereafter, anti-BGP Fab (3.3 μg / ml) in PBST was added and allowed to stand for 1 hour. The fluorescent probe was diluted with PBST so that 1.5 μl of each well was added and allowed to stand overnight at 4 ° C. Next, add rabbit anti-TAMRA antibody (2.5 μg / ml) diluted with 5% IPBS, let stand for 1 hour, add HRP-labeled anti-rabbit IgG diluted 6000 times with 5% IPBS, let stand for 2 hours, After adding the reaction solution, the reaction was stopped and the absorbance was measured.

  The resulting graph is shown in FIG. This suggests that any of the probes can bind to the anti-BGP Fab in an antigen-bound state, and that fluorescent probe I has the highest binding ability.

Example 3 Synthesis of fluorescent probe using E. coli, binding to various antibodies, and fluorescence measurement In Examples 1 and 2, the fluorescent probe was synthesized in a cell-free system using RYTS kit. This method has a merit that a fluorescent label can be formed at the time of translation by a simple operation, but has a problem that the amount of protein obtained is small, and the possible analysis and the number of times are limited. Therefore, an expression vector was changed to one suitable for a large-scale expression system in Escherichia coli (pET), and an unlabeled probe was expressed in Escherichia coli, and a method of labeling with a fluorescent dye by chemical modification was attempted. At this time, the amber codon that was an unnatural amino acid introduction site in pROX was changed in advance to a codon TGC encoding cysteine (Cys). Thereafter, kinetic analysis of the interaction between the expressed and purified unlabeled probe and various IgG and fragments thereof was performed using an SPR biosensor.

  Further, by labeling cysteine at the fluorescent dye introduction site with horseradish peroxidase (HRP), the binding ability to various antibodies was evaluated by ELISA.

  Further, the fluorescent dye was chemically modified at the Cys site, and the change in fluorescence intensity due to the addition of Fab antibody to the fluorescent probe solution and subsequent addition of the antigen was measured.

In the present example, the following E. coli was used.
BL21 (DE3) pLysS: E. coli B F- dcm ompT hsdS (r _B -m _B- ) gal λ (DE3) [pLysS Cam ^r ] ^a
The following primers were used.
ProCXback: 5'-cgaaGTAAACTGCTCTAATGAG-3 '(SEQ ID NO: 24)
pROX2FabQ-Bamfor: 5'- CGTCGTCCTTGTAGTCGGATCCACTAGTAACGGC -3 '(SEQ ID NO: 25)

1. Replacement of fluorescent probes A to I from pROX to pET vector Replace the Open Reading Frame of the fluorescent probe A to I plasmid from pROX to pET vector and replace the amber codon in the ProX tag with Cys codon by the following method. went.

  Each plasmid vector was subjected to PCR reaction using ProCXback and pROX2FabQ-Bamfor as primers. This product was electrophoresed on a 1.2% agarose gel, and the target band was excised and purified. Further, the Fab fragment expression vector pET FabQ Hd ++ with pET32-derived ProX (Cys) tag was treated with restriction enzymes with AgeI and BamHI to excise the Fab-encoding fragment, and the longer band was collected and purified. Each PCR fragment and the vector were subjected to In-fusion reaction, then transformed with BL21 (DE3) pLysS, and cultured overnight at 37 ° C. on an LBAC (LBA agar containing 34 μg / ml chloramphenicol) plate. Several clones were selected from the obtained colonies, cultured in 4 ml LBAC medium, and a small amount of plasmid was extracted to confirm that the nucleotide sequence was the target. Note that the ORF of the protein obtained by this operation is the same as before except for the codon in the ProX tag.

2. Preparation of unlabeled probe protein One clone was selected from colonies of fluorescent probes C, F and I that had been successfully recombined, and cultured overnight in 4 ml LBAC. Then, 1 ml of this medium was taken and 0.1% for the main culture. Place in 100 ml LBAC containing glucose and shake culture at 200 rpm and 37 ° C with an incubator until OD ₆₀₀ reaches about 0.5, add isopropylthiogalactopyranoside IPTG to a final concentration of 1 mM, 37 Cultured with shaking at 4 ° C for 4 hours. Thereafter, the culture solution was transferred to a centrifuge tube, centrifuged at 6000 rpm, 10 min, 4 ° C., the precipitate was suspended in 20 ml of Talon extraction buffer, and soluble protein was prepared using an ultrasonic crusher. Thereafter, this was centrifuged at 11000 rpm, 20 min, 4 ° C. to obtain an extract. The protein was purified using a Talon column by the same method as in Example 1, and the amount and purity of the target protein were confirmed by SDS-PAGE.

3. Analysis of binding between probe and various antibodies using SPR biosensor
The kinetic analysis using Biacore 2000 (GE Healthcare) was performed as follows. PA (Merck Millipore 539202), PG '(Fluka 08062) (CR Goward, JP Murphy, T Atkinson, and DA Barstow, Biochem. J. 267, 171-177, 1990) and 10-50 μg of unlabeled probe C as ligands The solution was diluted in a 10 mM NaOAc solution (pH 4.5, but PG was pH 3.0) at a concentration of / ml. These were immobilized on the sensor chip CM5 by the amine coupling method (however, the fluorescent probe C was a thiol coupling method using Cys in the ProX tag) at 1000 to 5000 RU. As the analyte, various IgGs and Fabs were diluted in HBS-EP solution to a concentration of 12.5 nM to 200 nM, and this was sequentially passed through the sensor chip at a rate of 20 μl / min to measure the interaction with each ligand. After that, kinetic analysis was performed by BIAevaluation software 4.1 by Global fitting method based on Langmuir model or Langmuir model considering mass transfer rate limiting.

FIG. 14 shows a sensorgram using an unlabeled probe C (PAxPG) as a ligand and a phage library-derived human anti-vimentin Fab (Morphosys AG, Martinsried, Germany) as an analyte. FIG. 15 shows the result of comparing the coupling constant Ka calculated using the Langmuir model with PA and PG ′. This indicates that PAxPG binds to this Fab fragment with a significantly higher binding constant than PA and PG ', which have two or more binding domains. That is, it was suggested that each domain of PAxPG was bound more strongly by binding to _VH and CH1 of the Fab fragment (Avidity effect). The error bars in the figure indicate the SEs derived from the standard errors SE of the binding rate and dissociation rate.

  Next, FIG. 16 shows a sensorgram using PAxPG as a ligand and mouse IgG1 (9E10, OEM Concepts, Toms River, NJ) as an analyte. FIG. 17 shows the results of comparing Ka calculated from each sensorgram including this using the Langmuir model considering the mass transfer rate control with PA and PG ′. From this, it was shown that PAxPG binds significantly stronger than PA and PG 'to mouse IgG1 and IgG2a, which are often used in various detection experiments. It was also shown that IgG3 has a strong binding ability almost equal to IgG1 and IgG2a.

  It is known that PAxPG has a stronger binding ability to various mouse IgG than PA and PG ', since PA is not bound to mouse Fab. This is probably because the two binding domains of PAxPG are linked cooperatively and an avidity effect is obtained.

4). ELISA using HRP labeled probe
Probe C was labeled with HRP according to the instructions of Peroxidase Labeling Kit (Dojindo). Using the obtained HRP-labeled probe, binding to various antibodies was measured by ELISA as follows.

  Various kinds of mouse IgG (1 μg / ml) diluted with PBS were adsorbed and immobilized at 4 ° C. overnight on an ELISA plate. After blocking, an HRP-labeled probe diluted 1000-fold with 5% IPBS was added and allowed to stand for 1 hour. Thereafter, the enzyme reaction solution was added and allowed to react for 5 to 30 minutes. Then, the reaction was stopped and the absorbance was measured.

  As a result, it was found that the HRP-labeled probe prepared as shown in FIG. 19 can detect polyclonal mouse IgG (mIgG), IgG1, and IgG2a. In particular, IgG1 could be detected significantly more strongly than commercially available HRP-labeled protein A (PA-HRP). The error bar in the figure indicates the standard deviation SD of three measurements.

5. Fluorescence measurement using TAMRA fluorescently labeled probe 50 μl of each of unlabeled probes F and I and TAMRA-C5-maleimide (Biotium) were mixed at a molar ratio of approximately 3: 1 and kept in the dark for 2 hours at room temperature. Left to stand. Thereafter, ultrafiltration was performed several times with a spin column (cut-off molecular weight 3 K) to remove unreacted TAMRA-maleimide. After SDS-PAGE of the obtained solution, it was confirmed that the probe was modified with TAMRA by irradiating with green LED light.

  The labeled probe F thus obtained (final concentration 100 nM, determined by comparing the density of each concentration with bovine serum albumin (BSA) as standard protein by SDS-PAGE) contains 250 μl of 1 mg / mL BSA. FIG. 20 shows the results of dilution with PBS + 0.1% Tween20 (PBST0.1), excitation with 530 nm using a Hitachi F-2500 fluorescence spectrophotometer, and measurement of fluorescence intensity at 580 nm. 30 minutes after the start of the measurement, an anti-vimentin Fab fragment (12.5 μl) having a final concentration of 100 nM was added, and thereafter a decrease in fluorescence intensity was observed over about 1 hour. On the other hand, when PBST0.1 was added instead of Fab, no obvious decrease was observed. In addition, all the fluorescence intensities are corrected and shown for the decrease accompanying the change in the amount of the solution.

  FIG. 21 shows the result of the same experiment performed for a labeled probe I having a longer dye-PA linker. Based on this, the fluorescence intensity change after the Fab fragment was introduced was normalized and expressed with the fluorescence intensity at the time of the Fab fragment being taken as 1, which was significantly greater in the sample in which the Fab fragment was introduced than in the sample that was not added. A decrease in fluorescence intensity (up to about 9%) was observed (FIG. 22). The error bar in the figure indicates the standard deviation SD of three measurements.

  Next, FIG. 23 shows the results of measuring changes in fluorescence intensity with stepwise addition of 13.6 μl each of antigen vimentin (indicating final concentration) or PBST0.1 to the two types of samples used in FIGS. 21-22. . Furthermore, this was normalized by the fluorescence intensity at the time of antigen injection and expressed as a ratio with or without antigen input (FIG. 24). As a result, the fluorescence intensity increased by adding the antigen to the mixture (complex) of labeled probe I and Fab. You can see that That is, it has been clarified that this labeled probe I can bind to a human Fab having a particularly high binding ability and exhibit the properties as Q-body.

  PAxPG, which is a fusion protein of the antibody-binding protein of the present invention, has a lower molecular weight than natural PA and PG, but can be applied to higher antigen-binding ability and a wide range of subclass IgGs. Application to is expected. Further, by labeling PAxPG with a fluorescent dye or enzyme, it can be applied as a simple detection reagent as a fluorescent immunosensor. Furthermore, various human-type Fabs can be used for detection of antigens just by mixing with fluorescently labeled PAxPG as in Quenchbody WO2011 / 061944.

SEQ ID NOs: 5-8 Synthetic SEQ ID NOs: 9-25 Primers

Claims (20)

  A fusion protein in which Protein A or an antibody binding domain thereof is fused to Protein G or an antibody binding domain thereof via a peptide linker.   The fusion protein according to claim 1, wherein the antibody binding domain of Protein A is a D domain and the antibody binding domain of Protein G is a B1 domain.   The fusion protein according to claim 1 or 2, wherein the peptide linker consists of 10 to 20 amino acids.   Peptide linkers from glycine (G), serine (S), alanine (A), threonine (T), aspartic acid (D), lysine (K), glutamic acid (E), leucine (L) and proline (P) The fusion protein according to any one of claims 1 to 3, comprising an amino acid selected from the group consisting of: The fusion protein according to any one of claims 1 to 3, wherein the peptide linker consists of an amino acid sequence represented by (G ₄ S) ₃ or (DDAKK) ₄ .   The fusion according to any one of claims 1 to 5, wherein Protein A or an antibody binding domain thereof binds to the heavy chain VH region of the antibody, and Protein G or an antibody binding domain thereof binds to the heavy chain CH1 region of the antibody. protein.   The fusion protein according to any one of claims 1 to 6, which binds to Fc.   A method for purifying an antibody, comprising contacting the fusion protein according to any one of claims 1 to 7 with the antibody.   The fusion protein according to claim 1, which is labeled with a fluorescent dye, a quenching dye, a fluorescent protein or an enzyme.   A fluorescent immunosensor comprising the fusion protein according to any one of claims 1 to 7, which is labeled with a fluorescent dye, which binds to an antibody and changes fluorescence when the antibody binds to an antigen.   The fluorescent immunosensor according to claim 10, wherein the N-terminal part of Protein A of the fusion protein or its antibody binding domain is labeled with a fluorescent dye.   The fluorescent immunosensor according to claim 10 or 11, wherein ProX-tag is added to the N-terminal part of Protein A or an antibody binding domain thereof, and an amino acid in ProX-tag is labeled with a fluorescent dye.   The fluorescent immunosensor according to claim 12, wherein ProX-tag labeled with a fluorescent dye, Protein A or an antibody binding domain thereof, a peptide linker, Protein G or an antibody binding domain thereof are fused in this order.   The fluorescent immunosensor according to claim 10, wherein a peptide linker is interposed between ProX-tag and Protein A containing a fluorescent dye or an amino acid labeled with the fluorescent dye.   15. A fluorescent dye-labeled ProX-tag, a second peptide linker, Protein A or an antibody binding domain thereof, a first peptide linker, Protein G or an antibody binding domain thereof are fused in this order. Fluorescent immunosensor.   Fluorescent dye in which fluorescence resonance energy transfer occurs between the fluorescent immunosensor according to any one of claims 10 to 15 and an unlabeled antibody, an antibody labeled with a quencher, or a fluorescent dye in the fluorescent immunosensor And a detection kit for an antigen that specifically binds to the antibody. A method of detecting an antigen using an antibody specific for the antigen,
(a) contacting the fluorescent immunosensor according to any one of claims 10 to 15 with an antibody to form a complex of the fluorescent immunosensor and the antibody,
(b) contacting the complex formed in (a) with an antigen in a test sample to bind the antibody and the antigen;
(c) measuring the change in fluorescence due to the binding between the antibody and the antigen, wherein the fluorescence quenching is canceled due to the binding between the antibody and the antigen;
And a method comprising. A method of detecting an antigen using an antibody specific for the antigen,
(a) contacting the fluorescent immunosensor according to any one of claims 10 to 15 with an antibody labeled with a quencher in a liquid to form a complex of the fluorescent immunosensor and the labeled antibody;
(b) contacting the complex formed in (a) with an antigen in a test sample to bind the antibody and the antigen;
(c) measuring the change in fluorescence due to the binding between the antibody and the antigen, wherein the fluorescence quenching is canceled due to the binding between the antibody and the antigen;
And a method comprising. A method of detecting an antigen using an antibody specific for the antigen,
(a) contacting the fluorescent immunosensor according to any one of claims 10 to 15 with an antibody labeled with a fluorescent dye in a liquid to form a complex of the fluorescent immunosensor and the labeled antibody, The combination of the fluorescent dye of the fluorescent immunosensor and the fluorescent dye of the labeled antibody is a combination of an energy donor dye and an energy acceptor dye capable of producing FRET;
(b) contacting the complex formed in (a) with an antigen in a test sample to bind the antibody and the antigen;
(c) measuring the change in fluorescence due to the binding between the antibody and the antigen, wherein the fluorescence from the energy acceptor dye decreases due to the binding between the antibody and the antigen;
And a method comprising.   The method according to any one of claims 17 to 19, wherein the antibody is a Fab antibody fragment.

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