JP2017049184A - Fluorescently labeled antibody with use of nucleotide binding site (nbs) of antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for preparing a fluorescently-labeled antibody, the fluorescence intensity of which varies depending on antigen-binding by a photochemical modification method with use of a nucleotide binding site in a variable region of the antibody, and the fluorescently-labeled antibody obtained.SOLUTION: A labeled antibody for fluoroimmunoassay in which a compound for labeling a nucleotide binding site (NBS) of an antibody is conjugated to a nucleotide binding site in the antibody, the compound having indolebutyric acid (IBA) and a fluorescent group being bound, the compound represented by the following formula (1), wherein F is a fluorescent group and R is an optionally substituted linker group selected from the group consisting of alkyl groups, alkenyl groups, alkynyl groups, C4 to C10 cycloalkyl groups, C4 to C10 heterocyclyl groups, C4 to C10 aryl groups, C4 to C10 heteroaryl groups, arylene groups, ether groups, ester groups, PEG groups, sulfide groups, amido groups, ketone groups, sulfamide groups and a combination thereof.SELECTED DRAWING: None

Description

  The present invention relates to a method of fluorescently labeling an antibody using the nucleotide binding site of the antibody, and a labeled antibody for fluorescent immunoassay that is fluorescently labeled by the method.

  A technique has been developed that can detect the binding of an antigen by a change in fluorescence by binding a fluorescent molecule to the N-terminal part of an antibody (see Patent Document 1 and Patent Document 2). An antibody that binds a fluorescent molecule used in this technique and whose fluorescence intensity changes due to antigen binding is called Quenchbody (Q-body (registered trademark)). In the disclosure, as an example, using a cell-free translation system, an amino acid to which a fluorescent molecule is bound is introduced into the N-terminal portion of the antibody to synthesize an antibody to which the fluorescent molecule is bound.

In addition to these examples, the conventional methods have problems that it takes time and effort to synthesize an antibody to which a fluorescent molecule is bound, and the amount of synthesis is low. This is because a step of introducing an amino acid having a fluorescent molecule bound thereto into the N-terminal portion of the antibody using a cell-free translation system is necessary for the synthesis.
In addition, there is a possibility that the N-terminus of the antibody may be modified after being translated into a protein, and there is a restriction that a label cannot be applied to the N-terminus after modification.

  On the other hand, another antibody-specific chemical modification method has been reported that binds NBS (Nucleotide binding site) of antibody and indolebutyric acid (IBA) derivative by photochemical modification by UV irradiation (UV cross-linking) ( (See Patent Literature 3 and Non-Patent Literature 1). In the conventional method of randomly labeling the amino group mainly present in the lysine residue in the antibody as a target, the function of the antibody may be impaired, but in the method using photochemical modification, the function of the antibody is not impaired. It has been reported that functional groups (biotin, fluorescent dyes, iRGD peptides, anticancer agents, etc.) can be bound in a site-specific manner.

Japanese Patent No. 5043237 International Publication No.2013 / 065314 International Publication No. 2014/152801

Alves, N, J et al., Biomaterials, 34, 5700-5710 (2013)

  In order to synthesize Q-body, an antibody whose fluorescence intensity changes due to antigen binding, it has been common to use recombinant antibody technology. The present invention provides a method for producing a Q-body that is an antibody whose fluorescence intensity changes due to antigen binding by photochemical modification using nucleotide binding sites present in the variable regions of many antibodies, and obtained The purpose is to provide a Q-body.

  The present inventors have developed the Q-body described in Japanese Patent No. 5043237 and International Publication No. 2013/065314 and used it for antigen detection. Q-body is a fluorescence that is located in the vicinity of a site with many tryptophan residues (hereinafter referred to as an antigen pocket) that exists from the antigen-binding site of the antibody to the inside of the variable region when no antigen is bound to the antibody. The antigen is detected by utilizing the fact that the dye becomes fluorescent when the antigen is bound or is more quenched and the fluorescence intensity changes.

  Q-body uses a genetic recombination technique to label the amino acid in the peptide added to the N-terminus of the light chain variable region and / or heavy chain variable region of the antibody with a fluorescent dye. It was over.

  The present inventors have examined a method for producing Q-body more simply, utilizing the fact that an indole group binds to a nucleotide binding site (NBS) present in the variable region of an antibody by photochemical modification, It was found that it can be labeled with a fluorescent dye. By using this method, antibody variable regions prepared in advance could be easily labeled with a fluorescent dye without using a genetic recombination technique.

In addition, since the nucleotide binding site of the antibody and the antigen pocket are close to each other, the Q-body produced using photochemical modification is more quenched when the antigen is not bound. As a result, the present inventors have found that the antigen can be detected with higher sensitivity than the conventional Q-body, and have completed the present invention.
That is, the present invention is as follows.

[1]
Following formula (1):

Wherein F is a fluorescent group, R is a substituted or unsubstituted alkyl group, alkenyl group, alkynyl group, C4 to C10 cycloalkyl group, C4 to C10 heterocyclyl group, C4 to C10 aryl group, C4 to An indole represented by a C10 heteroaryl group, an arylene group, an ether group, an ester group, a PEG group, a sulfide group, an amide group, a ketone group, a sulfamide group, and combinations thereof. A labeled antibody for fluorescent immunoassay in which a compound for labeling a nucleotide binding site (NBS) of an antibody in which butyric acid (IBA) and a fluorescent group are bound is conjugated to a nucleotide binding site in the antibody.

[2] The fluorescent group may be rhodamine, coumarin, oxazine, carbopyronine, cyanine, pyromesene, naphthalene, biphenyl, anthracene, phenanthrene, pyrene, carbazole, Cy, EVOblue and The labeled antibody for fluorescent immunoassay according to [1], which is at least one selected from the group consisting of these derivatives.

[3] The labeled antibody for fluorescent immunoassay according to [2], wherein the fluorescent group contains at least one selected from the group consisting of tetramethylrhodamine, rhodamine 110 and ATTO655 (registered trademark).
[4] The labeled antibody for fluorescence immunoassay according to any one of [1] to [3], wherein the antibody is selected from the group consisting of an scFv type antibody, a Fab type antibody and a complete type antibody.

[5] A method for producing a labeled antibody for fluorescence immunoassay,
Following formula (1):

Wherein F is a fluorescent group, R is a substituted or unsubstituted alkyl group, alkenyl group, alkynyl group, C4 to C10 cycloalkyl group, C4 to C10 heterocyclyl group, C4 to C10 aryl group, C4 to An indole represented by a C10 heteroaryl group, an arylene group, an ether group, an ester group, a PEG group, a sulfide group, an amide group, a ketone group, a sulfamide group, and combinations thereof. By irradiating ultraviolet light with a central wavelength of 254 nm to a solution containing an antibody nucleotide binding site (NBS) labeling compound in which butyric acid (IBA) and a fluorescent group are bound, an indole group of the compound and the antibody A method for producing a labeled antibody for fluorescent immunoassay, comprising a step of causing a photocrosslinking reaction between nucleotide binding sites and conjugating the compound with an antibody.

[6] The fluorescent group includes rhodamine, coumarin, oxazine, carbopyronine, cyanine, pyromesene, naphthalene, biphenyl, anthracene, phenanthrene, pyrene, carbazole, Cy, EVOblue, and The production method of [5], which is at least one selected from the group consisting of these derivatives.

[7] The method according to [6], wherein the fluorescent group includes at least one selected from the group consisting of tetramethylrhodamine, rhodamine 110, and ATTO655 (registered trademark).
[8] The production method of any one of [5] to [7], wherein the antibody is selected from the group consisting of scFv type antibodies, Fab type antibodies, and complete antibodies.
[9] A method for detecting an antigen, comprising bringing the labeled antibody for fluorescent immunoassay according to any one of [1] to [4] into contact with an antigen and measuring a change in fluorescence intensity.

  The reagent for fluorescent immunoassay of the present invention uses a pre-synthesized antibody, and a nucleotide binding site that is a site in the vicinity of an antigen pocket that is not subjected to post-translational modification by ultraviolet irradiation in a short time in a short time. Since it can be specifically modified by a quencher, it can be applied to any antibody. Therefore, it is easy to obtain the antibody as a material, and genetic analysis is not necessary. As a result, it becomes possible to simplify the production of an antibody capable of detecting the binding of an antigen by a change in fluorescence and to improve the production efficiency.

  Furthermore, in the present invention, a nucleotide binding site labeling compound containing a fluorescent dye or a quencher binds to a nucleotide binding site adjacent to the antigen pocket. Therefore, compared to the labeled antibody described in Japanese Patent No. 5043237 and International Publication No. 2013/065314, the fluorescent dye or quencher is located closer to the antigen pocket and easily enters the antigen pocket. It becomes easier to be quenched when not. Therefore, since the change in fluorescence intensity when the antigen is bound is larger than when using the labeled antibody described in Japanese Patent No. 5043237 and International Publication No. 2013/065314, the antigen is measured with higher sensitivity. be able to.

It is a figure which shows the pattern of the structure of the fluorescence labeled antibody of this invention. It is a figure which shows the labeling pattern of the fluorescence labeled antibody of this invention. It is a figure which shows the principle that the fluorescence of the fluorescence labeled antibody of this invention changes. It is a figure which shows the result of having made the fluorescence labeled antibody and BGP peptide of this invention react, and detecting fluorescence. It is a figure which shows the result of having detected the fluorescence by making the fluorescence labeled antibody and the BGP peptide fluorescently labeled by the amber suppression method react.

Hereinafter, the present invention will be described in detail.
The present invention relates to a fluorescent immunoassay antibody labeled with a fluorescent dye, which is used in a method for measuring a test substance, which is an antigen contained in a test sample, using an antigen-antibody reaction using an antibody labeled with a fluorescent dye. And a fluorescent immunoassay antibody labeled with a fluorescent dye.

  In the method for producing the antibody of the present invention, a fluorescent dye is bound by a photochemical modification method or the like via an indole group having affinity for a nucleotide binding site (NBS) of the antibody.

1. Antibody used in the present invention In the present invention, a complex of a polypeptide containing an antibody light chain variable region (VL) and a polypeptide containing an antibody heavy chain variable region (VH) is used as an antibody.

  The antibody light chain variable region is not particularly limited as long as it contains an amino acid sequence specific to the antibody light chain variable region encoded by exons of the V region and J region of the antibody light chain gene. An arbitrary amino acid sequence may be further added to the N-terminal and / or C-terminal side of the amino acid sequence specific to the light chain variable region. The amino acid sequence specific to the antibody light chain variable region is preferably an amino acid sequence in which the 35th amino acid is tryptophan in the Kabat numbering system. A polypeptide containing an antibody light chain variable region only needs to contain an antibody light chain variable region, and can include an antibody light chain and a peptide consisting of any amino acid sequence in the antibody light chain. The chain variable region can be an antibody light chain constant region (Cκ) or a polypeptide further having a hinge portion. Among them, a polypeptide having an antibody light chain variable region with Cκ is preferred. Depending on the antigen to be detected, a polypeptide comprising an antibody light chain variable region capable of recognizing the antigen can be appropriately prepared.

  The antibody heavy chain variable region is not particularly limited as long as it contains an amino acid sequence specific to the antibody heavy chain variable region encoded by exons of the V region, D region, and J region of the antibody heavy chain gene. Alternatively, an arbitrary amino acid sequence may be added to the N-terminal and / or C-terminal side of the amino acid sequence specific to the antibody heavy chain variable region. The amino acid sequence specific to the antibody heavy chain variable region is an amino acid sequence in which the 36th, 47th, or 103rd amino acid is tryptophan in the Kabat numbering system. preferable. The polypeptide including the antibody heavy chain variable region only needs to contain the antibody heavy chain variable region, and can include an antibody heavy chain or a peptide consisting of any amino acid sequence in the antibody heavy chain. A polypeptide having an antibody heavy chain constant region (CH1) added to the chain variable region and a hinge region or Fc region can be used, and a polypeptide having CH1 added to the antibody heavy chain variable region is preferred. Depending on the antigen to be detected, a polypeptide containing an antibody heavy chain variable region capable of recognizing the antigen can be appropriately prepared.

  Preferably, the polypeptide containing the antibody light chain variable region and the polypeptide containing the antibody heavy chain variable region form a complex, and the amino acids that form a complex in the antibody light chain variable region and the antibody heavy chain variable region, respectively. There is no particular limitation as long as the peptide containing the sequence is bound thereto. As a peptide that forms a complex, in addition to the antibody constant region (CH1 and Cκ, etc.), one that forms a dimer can be imparted to the antibody light chain variable region and the other can be imparted to the antibody heavy chain variable region. It is also possible to select two types of proteins that interact to contribute to the formation of these complexes.

In the present invention, a complex consisting of a polypeptide containing an antibody light chain variable region and a polypeptide containing an antibody heavy chain variable region, which specifically binds to an antigen, is called an antibody. In addition, since the complex has the property of an antibody that binds to an antigen, it can also be called an antigen-binding protein. The antibodies of the present invention include VH + VL type antibodies, scFv type antibodies (single chain antibody: single chain variable fragment), Fab type antibodies, F (ab ′) type ₂ antibodies, complete type antibodies (complete antibodies), etc. Including.

  The antibody of the present invention is not limited as long as it comprises a polypeptide containing an antibody light chain variable region and a polypeptide containing an antibody heavy chain variable region as components and forms a complex. As long as the function of the antibody is not impaired, in addition to the polypeptide containing the antibody light chain variable region and the polypeptide containing the antibody heavy chain variable region, it may further contain a peptide, protein, lipid, metal or other compound as a constituent element. .

In addition, the antibody of the present invention may be a structure that can function as a combination of the polypeptides, and the presence or absence of a chemical bond between the polypeptides is not particularly problematic. Examples of the bond include a disulfide bond between the polypeptides, a bond formed using a cross-linking agent, and the like. These bonds may be used in combination in a single complex. Among these, a disulfide bond can be preferably exemplified. The antibody of the present invention preferably forms a complex in which the polypeptides are close to each other. A polypeptide containing an antibody light chain variable region containing a peptide having such a function and an antibody heavy chain variable region A complex comprising the polypeptide comprising is preferred. In the antibody molecule, the antibody light chain constant region and the antibody heavy chain constant region interact with each other to make the antibody light chain variable region and the antibody heavy chain variable region closer to each other, thereby forming a strong antigen-binding pocket. . Therefore, as the antigen-binding protein of the present invention, a polypeptide comprising an antibody light chain variable region and an antibody light chain constant region and a polypeptide comprising an antibody heavy chain variable region and an antibody heavy chain constant region are disulfide bonds. A Fab type antibody that is a single molecule antibody protein bound, an F (ab ′) ₂ type antibody in which two Fab type antibodies are linked by a disulfide bond via a hinge, and a complete type antibody are preferable. In this case, the polypeptide consisting of the antibody light chain variable region and the antibody light chain constant region is a polypeptide containing the antibody light chain variable region, and the polypeptide consisting of the antibody heavy chain variable region and the antibody heavy chain constant region is anti-antibody. A polypeptide comprising a weight chain variable region. The antibody of the present invention may be a VH + VL type antibody or scFv type antibody (single chain antibody: single chain variable region fragment) comprising an antibody light chain variable region and an antibody heavy chain variable region.

VH + VL antibody, scFv antibodies and Fab type antibody consists polypeptide one comprising a polypeptide with one antibody heavy chain variable region comprising an antibody light chain variable region, F (ab ') ₂ type antibodies and full The somatic antibody consists of two polypeptides containing an antibody light chain variable region and two polypeptides containing an antibody heavy chain variable region.

  In the antibody comprising the polypeptide comprising the antibody light chain variable region of the present invention and the polypeptide comprising the antibody heavy chain variable region, the complex part comprising the antibody light chain variable region and the antibody heavy chain variable region is labeled with one fluorescent dye. This case is called a single label (for example, Fab type single label). In addition, the complex part composed of the antibody light chain variable region and the antibody heavy chain variable region may be labeled with two fluorescent dyes, may be labeled with the same type of fluorescent dye, or another type of fluorescent dye. It may be labeled with a dye. In the present invention, when a complex part consisting of an antibody light chain variable region and an antibody heavy chain variable region is labeled with two fluorescent dyes and both fluorescent dyes are of the same type, the same color double label (for example, Fab type same color double label) A different case is called a different color double label (for example, a Fab type different color double label). Furthermore, the complex part composed of the antibody light chain variable region and the antibody heavy chain variable region may be labeled with two dyes, one fluorescent dye and one quencher (quenching dye) for the fluorescent dye. Such an antibody is called fluorescence + quencher double label.

FIG. 1 shows the antibody structure as an antibody pattern. FIG. 1 shows the structures of VH + VL type antibody, scFv type antibody, Fab type antibody, F (ab ′) type ₂ antibody and complete type antibody. The complex part consisting of the antibody light chain variable region and the antibody heavy chain variable region of each type of antibody is a single label labeled with one dye.

In the present invention, a polypeptide containing an antibody light chain variable region, a polypeptide containing an antibody heavy chain variable region, an antibody that is a complex composed of these polypeptides, its constituents, etc. are known chemical synthesis. It can be prepared using a method, a gene recombination technique, a method for degrading antibody molecules with proteolytic enzymes, and the like. As a complete antibody, a monoclonal antibody produced by a hybridoma can be used as it is. Further, Fab-type antibodies and scFv-type antibodies can be obtained by degrading complete antibodies with proteolytic enzymes such as papain and pepsin. In addition, antibodies may be prepared by gene recombination techniques. When the polypeptide is prepared by genetic recombination technology, a recombinant vector is prepared by introducing DNA containing a base sequence encoding such a polypeptide into a suitable expression vector, so that bacteria, yeast, insects, animal and plant cells The target polypeptide can be expressed by an expression system using the above as a host or a cell-free translation 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. Of the polypeptide can be expressed. At this time, a polypeptide containing the antibody light chain variable region or a polypeptide containing the antibody heavy chain variable region may be added with a tag such as a ProX tag, a FLAG tag, or a His tag. It can be used for purification and the like. The polypeptide containing the antibody light chain variable region and the polypeptide containing the antibody heavy chain variable region thus obtained can form a complex in an appropriate solvent before and after labeling with a fluorescent dye. An example of forming a complex by bonding with a disulfide bond or a crosslinking agent can be given. For example, the gene encoding the polypeptide containing the antibody light chain variable region and the polypeptide containing the antibody heavy chain variable region is co-expressed in an E. coli cell-free synthesis system and then incubated at 4 ° C. for 16 hours to form disulfide bonds. To form a complex. Furthermore, disulfide bonds can be promoted by adding molecular chaperones such as protein disulfide isomerase and proline cis-trans isomerase to the E. coli cell-free synthesis reaction system. The crosslinking agent may be any compound that can crosslink and bond polypeptides together. Examples thereof include aldehydes (for example, glutaraldehyde), carbodiimides, imide esters, and the like. It can be obtained and used in a conventional manner. The complex of the present invention can also be prepared by cleaving an antibody with an enzyme or the like. For example, by treating the antibody with papain or pepsin, an Fab type antibody or F (ab ′) ₂ Type antibodies can also be produced.

  The antibody labeled with the fluorescent dye of the present invention is designed so that the presence or absence of fluorescence and the fluorescence intensity change when the antibody binds to the antigen, compared to when the antibody does not bind to the antigen. ing. That is, when the labeled antibody is not bound to the antigen, the fluorescent dye used for labeling is quenched (quenched) and does not emit fluorescence, or is in a state of generating fluorescence of a specific wavelength, and the antigen is bound to the antibody. In some cases, the fluorescence generation state of the fluorescent dye changes. For example, a fluorescent dye that has been in a quenched state when the antibody and the antigen are not bound to emit fluorescence when the antibody and the antigen are bound, or emits fluorescence when the antibody and the antigen are not bound. The wavelength of the fluorescence generated by the binding of the fluorescent dye and the antibody to the antigen shifts. Examples of such antibodies include antibodies that cause a change in fluorescence intensity due to a quencher (quenching dye), and antibodies that change the emission state of the fluorescence dye due to fluorescence resonance energy transfer (FRET). That is, in the present invention, an antibody that can change the fluorescence intensity when an antigen as a detection target substance and an antibody against the detection target substance form a complex is used.

  As an antibody whose fluorescence intensity can be changed, (1) quenching is resolved when an antibody comprising a polypeptide containing an antibody heavy chain variable region and a polypeptide containing an antibody light chain variable region binds to an antigen to form a complex. And (2) an antibody comprising a polypeptide containing the antibody heavy chain variable region and a polypeptide containing the antibody light chain variable region bound to an antigen to form a complex, When the complex of the antigen and the antibody becomes a quencher of the fluorescent dye, and the complex of the polypeptide including the antigen and the antibody heavy chain variable region and the polypeptide including the antibody light chain variable region is formed, the fluorescent dye An antibody whose fluorescence intensity decreases by being more strongly quenched can be mentioned. When the former antibody is used, the concentration of the antigen to be detected and the fluorescence intensity of the fluorescent dye have a positive correlation, and the concentration of the antigen to be detected and the fluorescence intensity of the fluorescent dye have a positive correlation. It is called an antibody. With the antibody, the antigen can be detected using as an indicator that the concentration of the antigen as the detection target substance in the test sample and the fluorescence intensity emitted when the antibody binds to the antigen are positively correlated. When the latter antibody is used, the antigen concentration to be detected and the fluorescence intensity of the fluorescent dye have a negative correlation, and the antigen concentration to be detected and the fluorescence intensity of the fluorescent dye have a negative correlation. It is called an antibody. With the antibody, the antigen can be detected by using as an indicator that the concentration of the antigen as the detection target substance in the test sample and the fluorescence intensity emitted when the antibody binds to the antigen have a negative correlation.

  In particular, fluorescently labeled antibodies that use tryptophan residues present in the VH region of the antibody as quenchers (quenching dyes) can be mentioned. There are tryptophan (W) residues at the 36th, 47th, and 103rd positions of the antibody VH region (according to the Kabat numbering system), and these tryptophan residues act as quenchers ( Patent No. 5043237). When an antibody labeled with a fluorescent dye binds to an antigen, the fluorescent dye is located in or near the antigen pocket, so that the fluorescent dye is located near the tryptophan residue and interacts with the tryptophan residue to fluoresce. Designed to quench the dye. That is, when the fluorescently labeled antibody is not bound to the antigen, it is quenched and does not emit fluorescence.

  When an antibody with a positive correlation between the antigen concentration and the fluorescence intensity of the fluorescent dye is used, when the antigen binds to the antibody, the three-dimensional structure of the antibody changes, and the fluorescent dye located near tryptophan moves away from tryptophan. , No longer interacts with tryptophan, the quench is released and fluorescence is emitted. Furthermore, if the antibody is labeled with two fluorescent dyes (perhaps both fluorescent dyes enter the antibody's antigen-binding pocket), an interaction occurs between the two fluorescent dyes, and the quenching effect between the fluorescent dyes. (H-dimer formation) is obtained, but when the antigen binds to the antibody, the three-dimensional structure of the antibody changes, the two fluorescent dyes are separated, and the quenching effect disappears. That is, when an antigen is present, the antibody and the antigen are combined, the three-dimensional structure of the antibody is changed, the quenching of the fluorescent dye is released, and fluorescence is emitted. Further, when the antibody is labeled with one fluorescent dye and one quencher, the fluorescent dye and the quencher interact when there is no antigen, and the fluorescent dye is quenched by the quencher. When the antigen is bound to the antibody, the three-dimensional structure of the antibody changes, the quencher and the fluorescent dye of the fluorescently labeled antibody are separated and do not interact, the quench 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.

  Thus, when the antigen concentration is measured using an antibody having a positive correlation between the antigen concentration and the fluorescence intensity of the fluorescent dye, or when detecting the antigen, the more the antigen bound to the antigen-binding protein, the more the fluorescence intensity becomes. To increase.

  On the other hand, when an antibody in which the antigen concentration and the fluorescence intensity of the fluorescent dye have a negative correlation is used, the complex of the antigen and antibody acts as a quencher on the fluorescent dye, and the fluorescent dye is further quenched to generate a fluorescent dye. The fluorescence intensity of the fluorescence to be weakened. At this time, the fluorescent dye used to label the antibody is located in the antigen-binding pocket of the antibody and is located closer to the tryptophan of the heavy chain variable region, resulting in stronger interaction with tryptophan and quenching. Is done. When an antibody is labeled with two fluorescent dyes, both fluorescent dyes enter the antigen-binding pocket of the antibody, causing an interaction between the two fluorescent dyes, and the quenching effect between the fluorescent dyes (H-dimer formation) ) Is obtained. At this time, the antibody is labeled using two fluorescent dyes, the two fluorescent dyes are different fluorescent dyes, and a donor dye and an energy acceptor (acceptor) that serve as an energy donor (donor) for fluorescence resonance energy transfer. When the antibody binds to the antigen, the orientation of both fluorescent dyes, ie the energy donor and the energy acceptor, changes from the energy emitted by the energy donor to the energy acceptor. Fluorescence resonance energy transfer (FRET) no longer occurs, and the fluorescence intensity of the generated fluorescence is weakened. An antibody comprising an antibody light chain variable region polypeptide and an antibody heavy chain variable region polypeptide, wherein the antibody light chain variable region polypeptide and the antibody heavy chain variable region polypeptide are labeled with one or two fluorescent dyes In the case of measuring and detecting an antigen by using a quenching effect due to fluorescence resonance energy transfer (FRET) effect in addition to quenching by tryptophan residues and quenching between fluorescent dyes, quenching becomes larger. The fluorescent dye interacts with the antigen-antibody complex by hydrophobic interaction, electrostatic interaction, or the like, and the degree of quenching is increased.

  As described above, when the antigen concentration is measured using an antibody having a negative correlation between the antigen concentration and the fluorescence intensity of the fluorescent dye, or when the antigen is detected, the more antigen bound to the antigen binding protein, the more The generated fluorescence is quenched and the fluorescence intensity decreases. That is, when the antibody binds to the antigen to be detected to form a complex, the antigen-antibody complex becomes a quencher of the fluorescent dye, and the antigen concentration in the liquid phase and the fluorescence intensity of the fluorescent dye Are negatively correlated, and when the complex of antigen and antibody is formed, the fluorescent dye is more strongly quenched, thereby reducing the fluorescence intensity.

  An example of an antibody having a negative correlation between the antigen concentration and the fluorescence intensity of the fluorescent dye is a monoclonal antibody produced by hybridoma A-04. NITE BP-01970 (“Indication of identification”) at NITE Patent Microorganisms Depository (Room 2-5-8 Kazusa-Kamashita, Kisarazu-shi, Chiba, Japan) Is deposited internationally in "A-04").

  As the polypeptide comprising the antibody light chain variable region and the polypeptide comprising the antibody heavy chain variable region constituting the antibody of the present invention, those derived from monoclonal antibodies can be used. That is, a polypeptide containing an antibody light chain variable region of a monoclonal antibody produced by the hybridoma using a antigen to be tested as an immunogen using an antigen as an immunogen, and an antibody heavy chain variable region produced by the hybridoma Polypeptides containing can be utilized. In addition, a monoclonal antibody produced by the hybridoma can be used as a complete antibody. Furthermore, DNA encoding the antibody light chain variable region and DNA encoding the antibody heavy chain variable region are obtained from the hybridoma, and the polypeptide and antibody heavy chain containing the antibody light chain variable region are used as recombinant proteins using the DNA. Antibodies consisting of a polypeptide containing a chain variable region can also be produced.

  In the present invention, an antibody comprising a polypeptide containing an antibody light chain variable region (VL) and a polypeptide containing an antibody heavy chain variable region (VH) is labeled with one or two fluorescent dyes. Trademark).

2. Fluorescent labeling of antibody by photochemical modification method etc. In the present invention, a nucleotide binding site (NBS) formed by an aromatic amino acid, which exists inside the variable region of an antibody, is used, photochemical modification method etc. The antibody is labeled with a fluorescent dye by a binding method to produce an antibody for fluorescence immunoassay (Q-body).

  The nucleotide binding site in the variable region of an antibody is formed by four aromatic amino acids, a light chain variable region and a heavy chain variable region. The four aromatic amino acids are tyrosine or phenylalanine at position 42 of the light chain variable region, tyrosine or phenylalanine at position 103 of the light chain variable region, tyrosine at position 103 of the heavy chain variable region, and position 118 of the heavy chain variable region. Tryptophan (location is numbered by IMGT® (the international ImmunoGeneTics database)) (Alves, N, J et al., Biomaterials, 34, 5700-5710 (2013)). The nucleotide binding site of an antibody is located near the antigen pocket to which the antigen binds.

For labeling an antibody with a fluorescent dye, the fluorescent dye may be bound to the antibody via an indole group having affinity for the nucleotide binding site. Specifically, a fluorescent dye is bound to indolebutyric acid (IBA) to produce a compound for labeling the nucleotide binding site of an antibody, the compound for labeling the nucleotide binding site and the antibody are mixed, and the antibody is prepared by a photochemical modification method. An indole group may be bonded to the nucleotide binding site. Further, even if not using a photochemical modification method, both can be bound by the interaction between the nucleotide binding site and the indole group.
A fluorescent dye to be bound to a compound for labeling a nucleotide binding site is also referred to as a fluorescent group.

  One molecule of the nucleotide binding site labeling compound may have one fluorescent dye or a plurality of fluorescent dyes. Moreover, you may have one fluorescent dye and one quencher. A labeling pattern when a compound for labeling a nucleotide binding site having one fluorescent dye is bound to a complex part consisting of an antibody light chain variable region and an antibody heavy chain variable region of an antibody is called a single label, and two fluorescent dyes are The labeling pattern in the case where the compound for labeling a nucleotide binding site is bound to a complex part consisting of an antibody light chain variable region and an antibody heavy chain variable region is called a double label. When two fluorescent dyes are the same type, it is called the same color double label, and when two fluorescent dyes are different types, it is called a different color double label. Furthermore, the labeling pattern when a compound for labeling a nucleotide binding site having one fluorescent dye and one quencher is bound to a complex part consisting of an antibody light chain variable region and an antibody heavy chain variable region of the antibody is fluorescence + quencher. This is called a char double label.

  FIG. 2 shows each labeling pattern when a Fab type antibody is used as the antibody. In the figure, NBS represents a nucleotide binding site, and IBA represents indolebutyric acid. In the figure, each label pattern of a single label, the same color double label, a different color double label, and a fluorescence + quencher double label is shown.

  By providing a linker between indolebutyric acid and the fluorescent dye, the distance between the fluorescent dye and indolebutyric acid can be adjusted. When there is one linker, there is one fluorescent dye that binds to the nucleotide binding site labeling compound. However, two fluorescent dyes or one fluorescent dye and 1 fluorescent dye can be added to the nucleotide binding site labeling compound by branching the linker. Two quenchers can be combined. Binding of indolebutyric acid and the fluorescent dye can be performed by a known chemical synthesis method.

  When the compound for labeling the nucleotide binding site is bound to the antibody, the fluorescent dye or quencher of the compound for labeling the nucleotide binding site is hydrophobic. It is located in a certain antigen pocket or in the vicinity of the antigen pocket and is structurally stabilized. As a result, the tryptophan constituting the antigen pocket and the fluorescent dye come close to each other, and the fluorescent dye is quenched. In addition, when there are a plurality of fluorescent dyes, they gather in the antigen pocket or in the vicinity thereof, and therefore are quenched by the interaction of fluorescent dyes such as H-dimer formation. Further, in the case of a combination of a fluorescent dye and a quencher, the fluorescent dye and the quencher are collected in the antigen pocket or in the vicinity thereof, so that the fluorescent dye is quenched by the quencher.

  The principle that the intensity of the fluorescence from the fluorescent dye changes due to the binding of the antigen is shown in FIG. In the figure, the antibody is an scFv type antibody. When a compound for labeling a nucleotide binding site in which indolebutyric acid and a fluorescent dye are combined with an antibody, the indole group binds to the nucleotide binding site (NBS), and the fluorescent dye of the compound for labeling the nucleotide binding site is located within the nucleotide binding site of the antibody. And quenched. When the antigen binds to the antibody, the fluorescent dye is released from the nucleotide binding site, the quench is released, and fluorescence is emitted.

  In the present invention, a nucleotide binding site labeling compound containing a fluorescent dye or a quencher binds to a nucleotide binding site adjacent to the antigen pocket. Therefore, compared to the labeled antibody described in Japanese Patent No. 5043237 and International Publication No. 2013/065314, since the fluorescent dye or quencher is located closer to the antigen pocket, the antigen is more likely to enter the antigen pocket. The quenching effect when not bonded is increased. Therefore, since the change in fluorescence intensity when the antigen is bound is larger than when using the labeled antibody described in Japanese Patent No. 5043237 and International Publication No. 2013/065314, the antigen is measured with higher sensitivity. be able to.

  Depending on the type of antibody, there is a difference in the distance from the nucleotide binding site to the antigen pocket. Therefore, depending on the distance from the nucleotide binding site to the antigen pocket, the length from the nucleotide binding site of the nucleotide binding site labeling compound to the fluorescent dye or quencher so that the fluorescent dye is located in or near the antigen pocket. Need to be adjusted. For this purpose, the length from the indole group moiety bound to the nucleotide binding site in the compound for labeling the nucleotide binding site to the fluorescent dye or quencher may be adjusted by the length of the linker. As the linker, a substituted or unsubstituted alkyl group, alkenyl group, alkynyl group, C4 to C10 cycloalkyl group, C4 to C10 heterocyclyl group, C4 to C10 aryl group, C4 to C10 heteroaryl group, arylene group, ether group A linker group selected from the group consisting of ester groups, PEG groups, sulfide groups, amide groups, ketone groups, sulfamide groups and combinations thereof can be used.

  Examples of the compound for labeling a nucleotide binding site in which a fluorescent dye and indolebutyric acid are combined, and in which one fluorescent dye is combined, include the following compounds.

Following formula (1):

Wherein F is a fluorescent dye, R is a substituted or unsubstituted alkyl group, alkenyl group, alkynyl group, C4 to C10 cycloalkyl group, C4 to C10 heterocyclyl group, C4 to C10 aryl group, C4 to An indole represented by a C10 heteroaryl group, an arylene group, an ether group, an ester group, a PEG group, a sulfide group, an amide group, a ketone group, a sulfamide group, and combinations thereof. A compound for labeling the nucleotide binding site (NBS) of an antibody, wherein butyric acid (IBA) and a fluorescent dye are bound.

  Examples of the nucleotide binding site labeling compound in which a fluorescent dye and indolebutyric acid are combined and the two fluorescent dyes are combined include the following compounds.

Following formula (2):

(Wherein F1 and F2 are fluorescent dyes, F1 and F2 may be the same type of fluorescent dyes or different types of fluorescent dyes, and R, R ′ and R ″ are independently Substituted or unsubstituted alkyl group, alkenyl group, alkynyl group, C4 to C10 cycloalkyl group, C4 to C10 heterocyclyl group, C4 to C10 aryl group, C4 to C10 heteroaryl group, arylene group, ether group, ester group, A nucleotide of an antibody in which indolebutyric acid (IBA) and a fluorescent dye are bound to each other, which is represented by a PEG group, a sulfide group, an amide group, a ketone group, a sulfamide group, and a combination thereof. Compound for binding site (NBS) labeling.

  Examples of the compound for labeling a nucleotide binding site in which a fluorescent dye and indolebutyric acid are bound and in which one fluorescent dye and one quencher are bound include the following compounds.

Following formula (3):

Wherein F is a fluorescent dye, Q is a quencher, R, R ′ and R ″ are independently substituted or unsubstituted alkyl, alkenyl, alkynyl, C4 to C10 cycloalkyl. Group, C4 to C10 heterocyclyl group, C4 to C10 aryl group, C4 to C10 heteroaryl group, arylene group, ether group, ester group, PEG group, sulfide group, amide group, ketone group, sulfamide group and combinations thereof A compound for labeling a nucleotide binding site (NBS) of an antibody, wherein indolebutyric acid (IBA) and a fluorescent dye are bound to each other.

  As fluorescent dyes used for fluorescent labeling of antibodies, rhodamine, coumarin, oxazine, carbopyronine, cyanine, pyromesene, naphthalene, biphenyl, anthracene, phenanthrene, pyrene, carbazole, Cy, Examples thereof include EVOblue-based fluorescent dyes and derivatives of the fluorescent dyes. Here, for example, a rhodamine-based fluorescent dye refers to a fluorescent dye having a rhodamine skeleton. Specifically, TAMRA (carboxytetramethylrhodamine: CR6G (carboxyrhodamine 6G), ATTO655 (trade name), CR110 (carboxyrhodamine 110) (rhodamine 110), Rhodamine Green (trade name) ), BODIPY FL (trade name), 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indancene-3-propionic acid, BODIPY 493/503 (trade name), 4 , 4-difluoro-1,3,5,7-tetramethyl-4-bora-3a, 4a-diaza-s-indancene-8-propionicacid, BODIPY R6G (trade name), 4,4-difluoro-5- (4 -phenyl-1,3-butadienyl) -4-bora-3a, 4a-diaza-s-indancene-3-propionic acid, BODIPY 558/568 (trade name), 4,4-difluoro-5- (2-thienyl ) -4-bora-3a, 4a-diaza-s-indancene-3-propionic acid, BODIPY 564/570 (trade name), 4,4-difluoro-5-styryl-4-bora-3a, 4a-diaza- s-indancene-3-propionic acid, BODIPY 576/589 (trade name), 4,4-difluoro-5- (2-pyrrolyl) -4-bora-3a, 4a-diaza-s-indancene-3-propionic acid , BODIPY 581/591 (trade name), 4,4-difluoro-5- (4-phe nyl-1, 3-butadienyl) -4-bora-3a, 4a-diaza-s-indancene-3-propionic acid, Cy3 (trade name), Cy3B (trade name), Cy3.5 (trade name), Cy5 ( Trade name), Cy5.5 (trade name), EvoBlue 10 (trade name), EvoBlue 30 (trade name), MR121, ATTO 390 (trade name), ATTO 425 (trade name), ATTO 465 (trade name), ATTO 488 (trade name) Name), ATTO 495 (trade name), ATTO 520 (trade name), ATTO 532 (trade name), ATTO Rho6G (trade name), ATTO 550 (trade name), ATTO 565 (trade name), ATTO Rho3B (trade name) ), ATTO Rho11 (trade name), ATTO Rho12 (trade name), ATTO Thio12 (trade name), ATTO 610 (trade name), ATTO 611X (trade name), ATTO 620 (trade name), ATTO Rho 14 (trade name) , ATTO 633 (trade name), ATTO 647 (trade name), ATTO 647N (trade name), ATTO 655 (trade name), ATTO Oxa12 (trade name), ATTO 700 (trade name), ATTO 725 (trade name), ATTO 740 (trade name), Alexa Fluor 350 (trade name), Alexa Fluor 405 (trade name), Alexa Fluor 430 (trade name) Trade name), Alexa Fluor 488 (trade name), Alexa Fluor 532 (trade name), Alexa Fluor 546 (trade name), Alexa Fluor 555 (trade name), Alexa Fluor 568 (trade name), Alexa Fluor 594 (trade name) ), Alexa Fluor 633 (trade name), Alexa Fluor 647 (trade name), Alexa Fluor 680 (trade name), Alexa Fluor 700 (trade name), Alexa Fluor 750 (trade name), Alexa Fluor 790 (trade name), Examples include Rhodamine Red-X (trade name), Texas Red-X (trade name), 5 (6) -TAMRA-X (trade name), 5TAMRA (trade name), and SFX (trade name). Cy3, EvoBlue10, rhodamine-based fluorescent dyes TAMRA and CR110, and oxazine-based fluorescent dye ATTO655 can be particularly preferably exemplified.

  Among the fluorescent dyes, the combination of TAMRA and TAMRA is particularly preferable for the same color double label, and the combination of TAMRA and CR110 and the combination of TAMRA and ATTO 655 are particularly preferable for the different color double label.

  Some fluorescent dyes have polarity sensitivity that changes fluorescence intensity according to polarity (M. Renard et al., J. Mol. Biol. (2002) 318, 429-442). For example, IANBD, CNBD, Acrylodan, 5-IAF and the like can be mentioned. When performing measurements based on fluorescence quenching using antibodies labeled with these fluorescent dyes, the binding of the antigen shields the fluorescent dye from the solvent and further reduces the chance of the fluorescent dye contacting the quencher. The quench progresses. In the present invention, the fluorescent dye having polarity sensitivity as described above is excluded, and the antigen is measured or detected by the quench principle not based on polarity sensitivity.

  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.

  Among the labeled antibodies of the present invention, examples of combinations of fluorescent dyes and quenchers of antibodies whose labeling pattern is fluorescence + quencher double label include a combination of TAMRA and NBD.

  The coupling | bonding via the indole group of the compound for labeling a nucleotide binding site and the nucleotide binding site of an antibody is performed using a photochemical modification method. Specifically, a compound for labeling a nucleotide binding site and an antibody may be mixed and irradiated with ultraviolet light having a central wavelength of 254 nm to cause a photocrosslinking reaction between the compound and the antibody.

  For the photochemical modification method, an ultraviolet light source having a peak wavelength of 254 nm may be used. For example, a UV irradiation apparatus such as a UV crosslinker CL-1000 series (UVP LLC) can be used.

The amount of light to be irradiated depends on the amount of the compound to be treated, but is, for example, about 0.5 to 10 J / cm ^{2 on the} irradiation surface. Further, the required irradiation time is not limited, but irradiation may be performed for several tens of seconds to several minutes.

  The present invention relates to a nucleotide binding site labeling compound having one fluorescent dye, a nucleotide binding site labeling compound having two fluorescent dyes, and a nucleotide binding site labeling compound having one fluorescent dye and one quencher. Is included.

Furthermore, the present invention includes antibodies (V + L type antibody, scFv type antibody, Fab type antibody, F (ab ') type ₂ antibody and complete type antibody) labeled with these nucleotide binding site labeling compounds. An antibody labeled with a nucleotide binding site labeling compound is also referred to as a labeled antibody for fluorescent immunoassay in which a nucleotide binding site (NBS) labeling compound is conjugated to a nucleotide binding site in the antibody.

  It is also possible to conjugate the binding of a compound for labeling a nucleotide binding site having a fluorescent dye or a quencher to an antibody. An antibody labeled with a compound for labeling a nucleotide binding site may be combined with a compound for labeling a nucleotide binding site and an antibody. Also called a conjugate. Conjugation includes binding by a covalent bond and non-covalent binding by an interaction such as electrostatic interaction.

3. Measurement of antigen using labeled antibody for fluorescent immunoassay Contacting the labeled antibody for fluorescent immunoassay of the present invention with an antigen, and measuring a change in fluorescence intensity emitted by a fluorescent dye before and after contacting the antigen Thus, the antigen can be detected. When an antibody consisting of a polypeptide containing an antibody heavy chain variable region and an antibody consisting of a polypeptide containing an antibody light chain variable region binds to an antigen to form a complex and the quenching is canceled and the fluorescence intensity increases As the antigen binds to the antibody, the fluorescence intensity increases. On the other hand, when a polypeptide comprising an antibody heavy chain variable region and a polypeptide comprising an antibody light chain variable region bind to an antigen to form a complex, the complex of the antigen and antibody serves as a quencher for the fluorescent dye. An antibody whose fluorescence intensity decreases when a complex of a polypeptide comprising the antigen and the antibody heavy chain variable region and a polypeptide comprising the antibody light chain variable region is formed and the fluorescent dye is more strongly quenched. When used, the fluorescence intensity decreases as the antigen binds to the antibody.

  At this time, it is preferable to prepare a calibration curve in advance by bringing a test sample containing a fluorescent immunoassay labeled antibody and a known amount of antigen into contact with each other and measuring the change in fluorescence at that time. Alternatively, when performing detection, a control sample containing a plurality of known amounts of antigen may be prepared, and a calibration curve may be created by simultaneously measuring the control sample. The amount of antigen in the test sample can be calculated from the measured fluorescence and calibration curve.

  For the measurement of fluorescence in the method for detecting an antigen using the labeled antibody for fluorescence immunoassay 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.

  Moreover, in the method for detecting an antigen using the labeled antibody for fluorescent immunoassay of the present invention, the excitation light to be irradiated and the wavelength of the fluorescence to be measured and / or detected can be appropriately selected according to the type of fluorescent dye used. Good. 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.

The detection target substance in the detection of an antigen using the labeled antibody for fluorescent immunoassay of the present invention is an antigen that can be detected by an antigen-antibody reaction, and the antigen includes a polypeptide containing an antibody heavy chain variable region and the above antibody light chain variable. The antigen is not particularly limited as long as it is an antigen specifically recognized by a polypeptide containing a region, and examples thereof include proteins, peptides, carbohydrates, lipids, glycolipids, low molecular compounds, and the like. That is, in the method of the present invention, the antigen to be detected is an antigen or antibody that can be measured by an immunoassay, that is, an assay utilizing 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 cannabinoids called cannabinoids such as tetrahydrocannabinol (THC), tetrahydrocannabinolic acid (THC-A), cannabinol (CBN), and cannabidiol (CBD), amphetamine, methamphetamine, morphine, Stimulants and narcotics such as heroin and codeine; mold toxins such as aflatoxin, sterigmatocystin, neosolaniol, nivalenol, fumonisin, ochratoxin, and endophyte-producing toxin; sex hormones such as testosterone and estradiol; clenbuterol and ractopamine Additives illegally used in animal feeds; harmful substances such as PCB, gossypol, histamine, benzpyrene, melamine, acrylamide, dioxin; acetamiprid, imidacloprid, chlorfenapyr, ma It can be like that include environmental hormones such as bisphenol A; thiones, carbaryl, clothianidin, triflumizole, chlorothalonil, spinosad, Ran'neto, methamidophos, pesticide residues, such as chlorpyrifos. Tetrahydrocannabinol (THC) has double bond regioisomers, Δ ⁸ -THC and Δ ⁹ -THC. Reference to THC includes Δ ⁸ -THC and Δ ⁹ -THC. The above substances also include derivatives of each substance.

The test sample is also not limited, 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, Examples include a sample obtained by wiping with a paper or the like a substance to which a drug or the like may adhere.
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 buffer as appropriate.
The present invention includes a method for detecting an antigen using the labeled antibody for fluorescent immunoassay.
Furthermore, the present invention also includes a kit for detecting an antigen containing the above labeled antibody for fluorescent immunoassay.

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

Example 1 Preparation of Qbody by photocrosslinking method (1) Preparation of 5 (6) -TAMRA-C8-IBA (TAMRA-IBA)

(i) Boc-C8-NH ₂ synthesis

Diaminooctane (Sigma-Aldrich) (500 mg, 3.5 mmol) was dissolved in 5 mL of Methylene chloride, and a Boc anhydride (151 mg, 0.69 mmol, 1 mL in CH ₂ Cl ₂ ) solution was added dropwise at 4 ° C. After standing at 4 ° C. for 30 minutes, the reaction was continued overnight at room temperature. After the reaction, the solvent was dried by an evaporator, washed with a saturated aqueous Na ₂ CO ₃ solution, and dehydrated with NaSO ₄ to obtain Boc-C8-NH ₂ (formula I).

(ii) Synthesis of Boc-C8-IBA

Indole-3-butanoic acid, IBA (43 mg, 0.24 mmol) and 1,1'-Carbonyldiimidazole, CDI (59 mg, 0.36 mmol) were dissolved in anhydrous THF (150 mL) and allowed to stand at room temperature for 1.5 h. -C8-NH ₂ (119 mg, 049 mmol) was mixed and allowed to react overnight at room temperature. After the reaction, the solvent was dried with an evaporator, dissolved in ethyl acetate, washed with 4% NaHCO ₃ and 5% KHSO ₄ , dehydrated with NaSO _{4, and} dried with an evaporator to give Boc-C8-IBA (Formula II) was obtained.

(iii) Synthesis of NH ₂ -C8-IBA

Boc-C8-IBA (30 mg) was mixed with TFA (200 μL) on ice, allowed to stand for 15 minutes, and dried by an evaporator to obtain NH ₂ —C8-IBA (formula III).

(iv) Synthesis of 5 (6) -TAMRA-C8-IBA

Add 20 μL NH ₂ -C8-IBA / DMSO (100 mM), 20 μL 5 (6) -TAMRA-SE (Setareh biotech) / DMSO, 40 μL of 100 mM NaHCO ₃ aqueous solution to a 1.5 mL tube, and mix by mixing on ice. It was. The synthesized product was separated from this solution by high performance liquid chromatography (HPLC) and dried to obtain 5 (6) -TARMAR-C8-IBA (formula IV). This dried product was dissolved in DMSO to a predetermined concentration.

(2) Preparation of anti-BGP (human Bone Gla Protein) antibody (scFv)
(i) Material
KOD-Plus, T4 DNA polymerase and Ligation-high ligation kit were obtained from Toyobo. Restriction enzymes and E. coli SHuffle T7 Express lysY were obtained from New England Biolabs. Oligonucleotides were obtained from Operon-Eurofins. The PureYield plasmid miniprep kit was obtained from Promega. Talon metal affinity resin and Talon disposable gravity column were obtained from Takara Bio Inc. The ultrafiltration device (centrifugal filter tube Ultra-4, MWCO 3 k) was obtained from Millipore. TCEP-immobilized TCEP disulfide reducing gel and Zeba spin desalting column (MWCO 7 k) were obtained from Thermo Pierce. His Mag Sepharose Ni was obtained from GE Healthcare. Anti-DYKDDDDK tag antibody magnetic beads, DYKDDDDK (SEQ ID NO: 1) peptide, Supersep PAGE gel and silver staining kit were obtained from Wako Pure Chemical Industries. The Nanosep centrifugal-3K ultrafiltration device was obtained from Pall Corporation.

(ii) Construction of scFv type Q-body gene Fab type Q-body expression vector pUQ1H (KTM219) containing DNA encoding heavy chain variable region and light chain variable region of anti-BGP antibody (Abe R. et al., SCientific Reports, 4: 4640) was digested with EcoRV and BamHI, blunted with T4 DNA polymerase, and then ligated to remove the L chain fragment. In order to delete the heavy chain fragment, it was digested with AgeI and EagI and ligated with the scFv fragment of KTM219 digested with AgeI and EagI using the Ligation-high ligation kit. Next, with the primers KTMAgeBack (5′-GGAATTCACCGGTCAAGTAAAGCTGCAGCAGTC-3 ′) (SEQ ID NO: 2) and T7term (5′-TGCTAGTTATTGCTCAGCGG-3 ′) (SEQ ID NO: 3), PROX-FL92.1amber (KTM219) as a template and KOD plus Amplified using DNA polymerase. To insert the Flag tag, the ligated gene was amplified with primers KTMAgeBack and pROXHis6Bamfor (5'-GTCGGATCCGCCATGATGATGATGATGATGATAAC-3 ') (SEQ ID NO: 4), then digested with AgeI and BamHI, then digested with AgeI and EagI Ligation with pUQ1H (KTM219) gave pSQ (KTM219). The resulting plasmid was prepared with the PureYield plasmid miniprep system and the entire sequence of the coding region was identified.

(iii) Synthesis and protein purification
SHuffle T7 Express lysY cells were transformed with PSQ (KTM219) and cultured in LBA medium (LB medium containing 100 μg / mL ampicillin) and 1.5% agar at 30 ° C. for 16 hours. A single colony was picked and grown at 30 ° C. in 4 mL LBA medium until OD ₆₀₀ reached 0.9, and 1.6 ml was inoculated into 100 ml LBA medium. Cells were cultured at 30 ° C. until OD ₆₀₀ reached 0.6, and 0.4 mM galactopyranoside isopropyl was added. The solution was further incubated at 16 ° C. for 16 hours and then centrifuged (8,000 × g, 20 minutes, 4 ° C.). The pellet was suspended in 10 ml Talon wash buffer (50 mM phosphoric acid, 0.3 M sodium chloride (NaCl), 5 mM imidazole, pH 7.4) and then sonicated. After centrifugation (8,000 × g, 20 minutes, 4 ° C.), the supernatant was incubated with 0.2 mL of Talon metal resin on a rotating wheel for 30 minutes at 25 ° C. The beads were washed 3 times with 8 mL Talon wash buffer. After adding 4 mL of Talon elution buffer (50 mM phosphate, 0.3 M NaCl, 0.5 M imidazole, pH 7.4) and incubating for 30 minutes at 25 ° C., the eluate was collected using a Talon disposable gravity column. The eluate was applied to an ultrafiltration device, equilibrated with PBST (10 mM phosphate, 137 mM sodium chloride, 2.7 mM potassium chloride, 0.05% Tween 20, pH 7.4) and concentrated to 250 μL. Protein expression and purification was confirmed using SDS-PAGE analysis, and protein concentrations were determined using ImageJ software (National Institutes of Health, Bethesda, MD) using various concentrations of bovine serum albumin (BSA) as a standard. Determined.

  The amino acid sequence of the scFv with a tag in pSQ (KTM219) is shown in SEQ ID NO: 5. In SEQ ID NO: 5, the sequence represented by GGSHHHHHHGGSDYKDDDDK (SEQ ID NO: 6) is the tag portion sequence.

(3) Preparation of labeled antibody for fluorescence immunoassay by photocrosslinking method and assay of the antibody 3 nmol TAMRA-IBA prepared in (1) and 0.2 nmol (5 μg) anti-BGP antibody (scFv) prepared in (2) Was placed in 10 μL of PBS and incubated at 25 ° C. for 1 hour. Next, the mixed solution of anti-BGP antibody and TAMRA-IBA was irradiated with UV light having a wavelength of 254 nm for 2 minutes and 40 seconds so as to obtain a light amount of 1 J / cm ^{2 on} the irradiated surface. At this time, the irradiation apparatus used was UVP CL-1000 (manufactured by UVP LLC).

  Next, the protein was purified using His-tag, the buffer was exchanged by Ultra filtration (membrane filter), and the protein was further purified using Flag-tag.

Antigen peptide BGP-C7 (BGP C-terminal amino acid sequence NH ₂ -RRFYGPV-COOH) (SEQ ID NO: 7) concentration of 0M to 1 × 10 ^-5 M in 8 steps was mixed with purified antibody Then, irradiation was performed at an excitation wavelength of 546 nm, and changes in fluorescence intensity were examined.

  The results are shown in FIG. FIG. 5 shows the use of an anti-BGP antibody (scFv) labeled with TAMRA by the amber suppression method (Ellman J et al., (1991) Methods Enzymol. 202: 301-36; International Publication No. 2011/061944). Is the result of In the experiment whose results are shown in FIG. 5, a TAMRA-labeled anti-BGP antibody (scFv) with or without a spacer was reacted with BGP-C7. The spacer sequences of G3S (1), G3S (2) and G3S (3) were GGGS (SEQ ID NO: 8), GGGSGGGS (SEQ ID NO: 9) and GGGSGGGSGGGS (SEQ ID NO: 10), respectively. As shown in FIG. 4, the fluorescence intensity increased as the concentration of BGP-C7 increased. Compared with the results in FIG. 5, the antibody (scFv) labeled by the photocrosslinking method showed a significant increase in fluorescence as compared with the antibody (scFv) labeled by the amber suppression method. This result indicates that the antibody (scFv) labeled by the photocrosslinking method is a labeled antibody for fluorescence immunoassay capable of analyzing the presence of BGP-C7 with high sensitivity.

  By using the labeled antibody for fluorescent immunoassay of the present invention, various antibodies can be detected with high sensitivity.

Claims (9)

Following formula (1):

Wherein F is a fluorescent group, R is a substituted or unsubstituted alkyl group, alkenyl group, alkynyl group, C4 to C10 cycloalkyl group, C4 to C10 heterocyclyl group, C4 to C10 aryl group, C4 to An indole represented by a C10 heteroaryl group, an arylene group, an ether group, an ester group, a PEG group, a sulfide group, an amide group, a ketone group, a sulfamide group, and combinations thereof. A labeled antibody for fluorescent immunoassay in which a compound for labeling a nucleotide binding site (NBS) of an antibody in which butyric acid (IBA) and a fluorescent group are bound is conjugated to a nucleotide binding site in the antibody.   The fluorescent group is rhodamine, coumarin, oxazine, carbopyronine, cyanine, pyromesene, naphthalene, biphenyl, anthracene, phenanthrene, pyrene, carbazole, Cy, EVOblue, and derivatives thereof. The labeled antibody for fluorescent immunoassay according to claim 1, which is at least one selected from the group consisting of:   The labeled antibody for fluorescent immunoassay according to claim 2, wherein the fluorescent group contains at least one selected from the group consisting of tetramethylrhodamine, rhodamine 110 and ATTO655 (registered trademark).   The labeled antibody for fluorescence immunoassay according to any one of claims 1 to 3, wherein the antibody is selected from the group consisting of an scFv type antibody, a Fab type antibody and a complete antibody. A method for producing a labeled antibody for fluorescent immunoassay, comprising:
Following formula (1):

Wherein F is a fluorescent group, R is a substituted or unsubstituted alkyl group, alkenyl group, alkynyl group, C4 to C10 cycloalkyl group, C4 to C10 heterocyclyl group, C4 to C10 aryl group, C4 to An indole represented by a C10 heteroaryl group, an arylene group, an ether group, an ester group, a PEG group, a sulfide group, an amide group, a ketone group, a sulfamide group, and combinations thereof. By irradiating ultraviolet light with a central wavelength of 254 nm to a solution containing an antibody nucleotide binding site (NBS) labeling compound in which butyric acid (IBA) and a fluorescent group are bound, an indole group of the compound and the antibody A method for producing a labeled antibody for fluorescent immunoassay, comprising a step of causing a photocrosslinking reaction between nucleotide binding sites and conjugating the compound with an antibody.   The fluorescent group is rhodamine, coumarin, oxazine, carbopyronine, cyanine, pyromesene, naphthalene, biphenyl, anthracene, phenanthrene, pyrene, carbazole, Cy, EVOblue, and derivatives thereof. The manufacturing method of Claim 5 which is at least 1 sort (s) selected from the group which consists of.   The production method according to claim 6, wherein the fluorescent group contains at least one selected from the group consisting of tetramethylrhodamine, rhodamine 110 and ATTO655 (registered trademark).   The production method according to any one of claims 5 to 7, wherein the antibody is selected from the group consisting of an scFv type antibody, a Fab type antibody and a complete antibody.   A method for detecting an antigen, comprising contacting the labeled antibody for fluorescent immunoassay according to any one of claims 1 to 4 with an antigen and measuring a change in fluorescence intensity.

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