WO2016104549A1

WO2016104549A1 - Fluorescence immunoassay method using antigen-binding protein including polypeptide including fluorescent-labeled antibody variable domain

Info

Publication number: WO2016104549A1
Application number: PCT/JP2015/085909
Authority: WO
Inventors: 亮二阿部; 富士男斎木; 典裕小林; 亨介山根
Original assignee: ウシオ電機株式会社
Priority date: 2014-12-24
Filing date: 2015-12-24
Publication date: 2016-06-30
Also published as: JP5949890B2; JP2016121919A

Abstract

Provided are an antigen measurement or detection kit whereby it is possible to measure an antigen concentration using as an indicator thereof a negative correlation between the fluorescence intensity of a fluorescent dye and an antigen concentration, and an antigen measurement or detection method which uses the kit. An antigen concentration measurement or detection kit including an antigen-binding protein comprising a polypeptide including an antibody light chain variable domain and a polypeptide including an antibody heavy chain variable domain, any one or both of the polypeptide including an antibody light chain variable domain and the polypeptide including an antibody heavy chain variable domain being labeled by a fluorescent dye, the antigen concentration measurement or detection kit characterized in that when the antigen-binding protein bonds with the antigen to be tested and forms a complex, the complex of the antigen and the antigen-binding protein becomes a quencher of the fluorescent dye, the antigen concentration in the liquid phase and the fluorescence intensity of the fluorescent dye are negatively correlated, and the fluorescent dye is more strongly quenched when the complex of the antigen and the antigen-binding protein is formed, whereby the fluorescence intensity is reduced and it is possible to measure the antigen concentration or visualize the antigen on the basis of measured or detected fluorescence, using as an indicator thereof the negative correlation between the antigen concentration in the liquid phase and the fluorescence intensity of the fluorescent dye.

Description

Fluorescence immunoassay method using an antigen-binding protein comprising a polypeptide containing a fluorescently labeled antibody variable region

The present invention relates to an antigen concentration measurement or detection kit capable of detecting a low-molecular compound or the like with high sensitivity without requiring an immobilization step and a washing step, and an antigen concentration measurement or detection method using the kit. .

The inventors of the present invention first increased the fluorescence intensity depending on antigen binding by site-specifically fluorescently labeling the vicinity of the N-terminus of a single-chain antibody (scFv) using an unnatural amino acid introduction technique. A fluorescent labeled antibody (Quenchbody: Q-body (registered trademark)), which is an antibody fragment, 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. That is, in the measurement using the fluorescently labeled antibody, the antigen concentration can be measured using as an index that the fluorescence intensity of the fluorescent dye and the antigen concentration are positively correlated.

In addition, the present inventors have not only quenched by tryptophan residues by making the single-chain antibody (scFv) in the above Q-body into a polypeptide complex containing a fluorescently labeled antibody variable region such as Fab. Further, it was found that a higher detection sensitivity can be obtained by the quenching effect (H-dimer) between the dyes, and this complex was named UQ-body (registered trademark) (Patent Document 2). Also in the measurement of antigen using UQ-body, it is possible to measure the antigen concentration using as an index that the fluorescence intensity of the fluorescent dye and the antigen concentration are positively correlated.

WO2011 / 061944 Publication WO2013 / 065314 Publication

The present invention relates to an antigen concentration measurement or detection kit that enables measurement of antigen concentration, using as an index that the fluorescence intensity of the fluorescent dye and antigen concentration are negatively correlated, and antigen concentration measurement or The purpose is to provide a detection method.

The inventors previously used a quenching (quenching) phenomenon using a complex of a polypeptide containing an antibody light chain variable region and a polypeptide containing an antibody heavy chain variable region labeled with a fluorescent dye, A method for detecting and measuring the antigen concentration using the positive correlation between the fluorescence intensity and the antigen concentration was developed (WO2011 / 061944 and WO2013 / 065314). In this method, when a complex of a polypeptide containing an antibody light chain variable region and a polypeptide containing an antibody heavy chain variable region is not bound to an antigen, a tryptophan residue present in the heavy chain variable region, Although it is quenched (quenched) by another fluorescent dye or quencher, when the complex binds to the antigen, the quenching of the fluorescent dye is weakened due to a change in conformation, and the fluorescence intensity increases.

The inventors of the present invention further studied the antigen measurement and detection method using quenching, and as a result, found that the fluorescent dye was quenched by the binding of the antigen and the fluorescence intensity emitted by the fluorescent dye decreased. It was. That is, it was found that the fluorescence intensity of the fluorescent dye and the antigen concentration have a negative correlation. This phenomenon occurs when the antigen to be tested is capable of interacting with the fluorescent dye mainly hydrophobicly and electrostatically, and as a result of the higher affinity between the antigen pocket and the fluorescent dye, the quenching is increased. Arise.

Based on the above principle, the present inventors comprise a polypeptide comprising an antibody light chain variable region and a polypeptide comprising an antibody heavy chain variable region, wherein the polypeptide comprising the antibody light chain variable region and the antibody heavy chain variable region And an antigen-binding protein in which either or both of the polypeptides containing the protein are labeled with a fluorescent dye, and the antigen-binding protein capable of negative correlation between the fluorescence intensity of the fluorescent dye and the antigen concentration, A method for measuring or detecting the concentration of an antigen using an antigen-binding protein has been developed and the present invention has been completed.

That is, the present invention is as follows.
[1] A polypeptide comprising an antibody light chain variable region and a polypeptide comprising an antibody heavy chain variable region, either one of the polypeptide comprising the antibody light chain variable region and the polypeptide comprising the antibody heavy chain variable region, or An antigen concentration measurement or detection kit comprising an antigen binding protein, both of which are labeled with a fluorescent dye,
When the antigen binding protein binds to the antigen to be tested to form a complex, the complex of the antigen and the antigen binding protein becomes the quencher of the fluorescent dye,
There is a negative correlation between the concentration of the antigen in the liquid phase and the fluorescence intensity of the fluorescent dye. When the complex of antigen and antigen-binding protein is formed, the fluorescence intensity is decreased by quenching the fluorescent dye more strongly. And
It is possible to measure the antigen concentration or visualize the antigen based on the fluorescence measured or detected by using as an index that the antigen concentration in the liquid phase and the fluorescence intensity of the fluorescent dye have a negative correlation. An antigen concentration measurement or detection kit.
[2] The antigen concentration measurement or detection kit according to [1], wherein the antigen-binding protein comprising a polypeptide containing an antibody light chain variable region and a polypeptide containing an antibody heavy chain variable region is an scFv antibody.
[3] An antigen-binding protein comprising a polypeptide containing an antibody light chain variable region and a polypeptide containing an antibody heavy chain variable region is a Fab antibody, and two Fab antibodies are joined by a disulfide bond via a hinge F (ab ′) The antigen concentration measurement or detection kit according to [1], which is selected from the group consisting of ₂ antibodies and complete antibodies.
[4] The antigen concentration measurement or detection kit according to [3], wherein the polypeptide containing the antibody light chain variable region and the polypeptide containing the antibody heavy chain variable region are labeled with the same fluorescent dye.
[5] The antigen concentration measurement or detection kit according to any one of [1] to [4], wherein the fluorescent dye is a rhodamine fluorescent dye.
[6] The antigen concentration measurement or detection kit according to [5], wherein the fluorescent dye is carboxytetramethylrhodamine.
[7] An antigen concentration measurement or detection method comprising sequentially performing the following steps (a) to (c):
(A) a step of bringing an antigen-binding protein into contact with an antigen to be examined in a liquid phase,
The antigen-binding protein comprises a polypeptide comprising an antibody light chain variable region and a polypeptide comprising an antibody heavy chain variable region, and any one of the polypeptide comprising the antibody light chain variable region and the polypeptide comprising the antibody heavy chain variable region One or both are labeled with a fluorescent dye that is quenched in a state labeled with a polypeptide containing an antibody heavy chain variable region or a polypeptide containing an antibody light chain variable region, and the antigen binding protein and the antigen to be tested are combined with the antigen. A process in which a complex of an antigen to be tested and an antigen-binding protein acts as a quencher of a fluorescent dye when forming a complex of an antigen-binding protein;
(B) measuring or detecting fluorescence of the fluorescent dye; and (c) measuring antigen concentration or detecting antigen,
When the antigen binding protein consisting of the polypeptide containing the antibody heavy chain variable region and the polypeptide containing the antibody light chain variable region binds to the antigen to form a complex of the antigen and the antigen binding protein, quenching of the fluorescent dye is eliminated. Based on the measured or detected fluorescence, using as an indicator that the antigen concentration in the liquid phase and the fluorescence intensity of the fluorescent dye have a negative correlation due to a decrease in fluorescence intensity due to stronger quenching. A step of calculating the amount of the antigen to be examined or detecting the antigen to be examined.
[8] The method for measuring or detecting an antigen concentration according to [7], wherein the antigen-binding protein comprising a polypeptide containing an antibody light chain variable region and a polypeptide containing an antibody heavy chain variable region is an scFv antibody.
[9] An antigen-binding protein comprising a polypeptide containing an antibody light chain variable region and a polypeptide containing an antibody heavy chain variable region is a Fab antibody, and two Fab antibodies are joined by a disulfide bond via a hinge F (ab ′) [7] The antigen concentration measurement or detection method according to [7], wherein the antigen concentration is selected from the group consisting of ₂ antibodies and complete antibodies.
[10] The method for measuring or detecting an antigen concentration according to [9], wherein the polypeptide containing the antibody light chain variable region and the polypeptide containing the antibody heavy chain variable region are each labeled with the same fluorescent dye.
[11] The method for measuring or detecting an antigen concentration according to any one of [7] to [10], wherein the antigen is a low molecular compound.
[12] The method for measuring or detecting an antigen concentration according to any one of [7] to [11], wherein the antigen is a cannabis component.
[13] The method for measuring or detecting an antigen concentration according to [12], wherein the cannabis component is selected from the group consisting of tetrahydrocannabinol (THC), tetrahydrocannabinolic acid (THC-A), and cannabinol (CBN). .
[14] A hybridoma that produces an antibody that binds to tetrahydrocannabinol (THC) or a derivative thereof deposited internationally under the deposit number NITE BP-01970.
[15] A monoclonal antibody that binds to tetrahydrocannabinol (THC) or a derivative thereof produced by the hybridoma of [14].
[16] An antigen-binding protein comprising a polypeptide comprising an antibody light chain variable region and a polypeptide comprising an antibody heavy chain variable region is a polypeptide comprising the antibody light chain variable region of the monoclonal antibody of [15] and the monoclonal antibody The kit for measuring or detecting an antigen concentration according to any one of [1] to [6], which is an antigen-binding protein comprising a polypeptide containing an antibody heavy chain variable region.
[17] An antigen-binding protein comprising a polypeptide containing an antibody light chain variable region and a polypeptide containing an antibody heavy chain variable region is a polypeptide comprising the antibody light chain variable region of the monoclonal antibody of [15] and the monoclonal antibody The method for measuring or detecting an antigen concentration according to any one of [7] to [13], which is an antigen-binding protein comprising a polypeptide containing an antibody heavy chain variable region.

This specification includes the disclosure of Japanese Patent Application No. 2014-261183, which is the basis of the priority of the present application.

The polypeptide comprising an antibody light chain variable region and a polypeptide comprising an antibody heavy chain variable region according to the present invention, and any one of the polypeptide comprising the antibody light chain variable region and the polypeptide comprising the antibody heavy chain variable region Alternatively, when an antigen-binding protein, both of which are labeled with a fluorescent dye, is brought into contact with the antigen, the fluorescent dye is quenched and the fluorescence intensity decreases as the antigen concentration increases. That is, there is a negative correlation between the fluorescence intensity of the fluorescent dye and the antigen concentration. When this principle is used, the antigen concentration can be measured or the antigen can be detected simply by bringing the antigen-binding protein into contact with and binding to the antigen. In addition, when a negative correlation is established, if there is a target antigen in the test object, it is detected by a decrease in the amount of fluorescence, and there is no target antigen in the test object, and there is a fluorescent component as some impurity Sometimes the amount of fluorescence increases, making it easy to distinguish.

According to the method of the present invention, an antigen concentration is measured or an antigen is detected based on a decrease in fluorescence intensity of a labeled fluorescent dye by forming a complex by bringing an antigen-binding protein into contact with an antigen. be able to. Therefore, the method of the present invention does not require an immobilization step or a washing step, and can detect an antigen with high sensitivity.

It is a figure which shows the structure of the antigen binding protein of this invention, and the principle of the measuring method using an antigen binding protein. It is a figure which shows the reactivity with THCA, THC, and CBN of the monoclonal antibody which hybridoma A-04 produces. It is a figure which shows the BGP measurement result using the scFv type | mold antigen binding protein couple | bonded with BGP labeled with Cy3. It is a figure which shows the BGP measurement result using scFv type | mold antigen binding protein couple | bonded with BGP labeled with EvoBlue10. It is a figure which shows the tetrahydrocannabinol (THC) measurement result using different color double label Fab type | mold antigen binding protein. It is a figure which shows the cannabinol (CBN) measurement result using different color double label Fab type | mold antigen binding protein. It is a figure which shows the measurement result of tetrahydrocannabinol (THC), tetrahydrocannabinolic acid (THC-A), and cannabinol (CBN) using the same color double label Fab type | mold antigen binding protein.

Hereinafter, the present invention will be described in detail.

The present invention comprises a polypeptide comprising an antibody light chain variable region (VL) and a polypeptide comprising an antibody heavy chain variable region (VH), comprising the polypeptide comprising the antibody light chain variable region and the antibody heavy chain variable region A kit for measuring or detecting an antigen concentration, comprising an antigen-binding protein in which either one or both of polypeptides are labeled with a fluorescent dye.

The present invention is also a method for detecting an antigen using the above-described antigen concentration measurement or detection kit.
1. Kit for antigen concentration measurement or detection The antibody light chain variable region is particularly limited as long as it contains an amino acid sequence specific to the antibody light chain variable region encoded by the V region and J region exon of the antibody light chain gene. In addition, 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 antibody 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 examined, 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 examined, a polypeptide comprising 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 composed of a polypeptide containing an antibody light chain variable region and a polypeptide containing an antibody heavy chain variable region is called an antigen-binding protein. Further, since the complex has the characteristic of an antibody that binds to an antigen, it can also be called an antibody molecule. Antigen-binding proteins also include antibodies, and include, for example, scFv antibodies (single chain antibodies), Fab antibodies, F (ab ′) ₂ antibodies and the like described later. In the present invention, scFv antibody and Fab antibody are sometimes referred to as scFv type antigen binding protein and Fab type antigen binding protein.

The antigen-binding protein of the present invention may be any one as long as it contains 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. In addition to the polypeptide containing the antibody light chain variable region and the polypeptide containing the antibody heavy chain variable region, the peptide, protein, lipid, metal, other compounds, etc. May be included.

In addition, the antigen-binding protein of the present invention may be a structure that can function as a single body by combining 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 antigen-binding protein 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 A complex consisting of a polypeptide comprising a region 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, the antigen-binding protein of the present invention includes a polypeptide comprising an antibody light chain variable region and an antibody light chain constant region, and a polypeptide chain comprising an antibody heavy chain variable region and an antibody heavy chain constant region having disulfide bonds. Antibody, which is a single molecule antibody protein bound in 1), F (ab ′) ₂ antibody in which _two Fab antibodies are bound by a disulfide bond via a hinge, and a complete antibody are preferable, and Fab antibody is most preferable.

The antibody-binding protein of the present invention may be an scFv antibody (single chain antibody: single chain variable fragment) consisting of an antibody light chain variable region and an antibody heavy chain variable region.

The scFv antibody and the Fab antibody are composed of one polypeptide containing the antibody light chain variable region and one polypeptide containing the antibody heavy chain variable region. The F (ab ′) ₂ antibody and the complete antibody are antibody light chain variable It consists of two polypeptides containing regions and two polypeptides containing antibody heavy chain variable regions. In scFv antibody and Fab antibody, only the polypeptide containing the antibody light chain variable region may be fluorescently labeled, or only the polypeptide containing the antibody heavy chain variable region may be fluorescently labeled. Both the polypeptide containing the antibody and the polypeptide containing the antibody heavy chain variable region may be fluorescently labeled. The F (ab ′) ₂ antibody and the complete antibody are composed of four polypeptides, ie, two polypeptides including the antibody light chain variable region and two polypeptides including the antibody heavy chain variable region. As a pattern of labeling, one or two polypeptides containing an antibody light chain variable region are labeled, one polypeptide containing an antibody heavy chain variable region, or two are labeled, an antibody light chain One polypeptide comprising a variable region and one polypeptide comprising an antibody heavy chain variable region are labeled, two polypeptides comprising an antibody light chain variable region and polypeptide 1 comprising an antibody heavy chain variable region Three polypeptides labeled, one polypeptide containing antibody light chain variable region and two polypeptides containing antibody heavy chain variable region labeled Are things include those polypeptides two four polypeptide comprising a polypeptide two and antibody heavy chain variable region comprising an antibody light chain variable regions are labeled.

In the present invention, a polypeptide containing an antibody light chain variable region, a polypeptide containing an antibody heavy chain variable region, an antigen-binding protein that is a complex composed of these polypeptides, its components, and the like are known chemicals. It can be prepared using a synthesis method, a gene recombination technique, a method for degrading an antibody molecule with a proteolytic enzyme, etc., among others, by a gene recombination technique that can be prepared in a large amount by a relatively easy operation. It is preferable to prepare. 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 addition and purification of polypeptides. The polypeptide containing the antibody light chain variable region and the polypeptide containing the antibody heavy chain variable region thus obtained form a complex in an appropriate solvent during or before 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, the Fab antibody or the F (ab ′) ₂ antibody, respectively. Can also be produced.

In the antigen-binding protein comprising a polypeptide comprising an antibody light chain variable region and a polypeptide comprising an antibody heavy chain variable region of the present invention, any of a polypeptide comprising an antibody light chain variable region and a polypeptide comprising an antibody heavy chain variable region Either or both are labeled with a fluorescent dye. A case where either one is labeled is called a single-label antigen-binding protein (for example, a single-label Fab antibody). Moreover, when both are label | marked, the same kind of fluorescent dye may be sufficient and another kind of fluorescent dye may be sufficient. In the present invention, when both a polypeptide containing an antibody light chain variable region and a polypeptide containing an antibody heavy chain variable region are labeled with a fluorescent dye and both fluorescent dyes are of the same type, the same color double-label antigen binding protein ( For example, it is called the same color double-label Fab antibody), and the different case is called a different color double-label antigen binding protein (for example, a different color double-label Fab antibody).

The polypeptide comprising an antibody light chain variable region and a polypeptide comprising an antibody heavy chain variable region according to the present invention, and any one of the polypeptide comprising the antibody light chain variable region and the polypeptide comprising the antibody heavy chain variable region Alternatively, an antigen-binding protein that is both labeled with a fluorescent dye has the labeled fluorescent dye not quenched or weakly quenched with no antigen bound. There are tryptophan (W) residues at the 36th, 47th, and 103rd positions of the antibody heavy chain variable region (according to the Kabat numbering system), and these tryptophan residues act as quenchers. (WO2011 / 061944 publication). When an antigen-binding protein labeled with a fluorescent dye is not bound to an antigen, if the fluorescent dye is located in the vicinity of a tryptophan residue, the fluorescent dye is quenched (quenched) by interacting with the tryptophan residue. Only weak fluorescence is generated. On the other hand, when the fluorescent dye is not located in the vicinity of the tryptophan residue and is separated from the tryptophan residue, it does not interact with the tryptophan residue, so that the fluorescent dye is not quenched and can generate fluorescence.

When an antigen-binding protein consisting of a polypeptide containing the antibody light chain variable region and a polypeptide containing the antibody heavy chain variable region binds to the antigen, the complex of the antigen and antigen-binding protein acts as a quencher on the fluorescent dye, and the fluorescence The dye is further quenched, and the fluorescence intensity of the fluorescence generated by the fluorescent dye is weakened. At this time, the fluorescent dye used for labeling the polypeptide containing the antibody light chain variable region of the antigen binding protein and / or the polypeptide containing the antibody heavy chain variable region is located in the antigen binding pocket of the antigen binding protein, Located closer to the tryptophan of the heavy chain variable region, the interaction with tryptophan becomes stronger and quenched. When both the polypeptide containing the antibody light chain variable region and the polypeptide containing the antibody heavy chain variable region are labeled with a fluorescent dye, both fluorescent dyes enter the antigen-binding pocket of the antigen-binding protein, and the two fluorescent dyes Interaction occurs, and a quenching effect (H-dimer) between fluorescent dyes is obtained. In this case, the fluorescent dye used for labeling the polypeptide containing the antibody light chain variable region and the fluorescent dye used for labeling the polypeptide containing the antibody heavy chain variable region are different fluorescent dyes, providing energy for fluorescence resonance energy transfer. In the case of a combination of a donor dye serving as a body (donor) and an acceptor dye serving as an energy acceptor (acceptor), when an antigen-binding protein binds to an antigen, both of the fluorescent dye, ie, the energy donor and the energy acceptor The orientation changes, and fluorescence resonance energy transfer (FRET) from the energy emitted by the energy donor to the energy acceptor does not occur, and the fluorescence intensity of the generated fluorescence becomes weak.

That is, it comprises a polypeptide comprising an antibody light chain variable region and a polypeptide comprising an antibody heavy chain variable region, and either or both of the polypeptide comprising the antibody light chain variable region and the polypeptide comprising the antibody heavy chain variable region When antigen is measured and detected using an antigen-binding protein labeled with a fluorescent dye, in addition to quenching by tryptophan residues and quenching between fluorescent dyes, quenching by fluorescence resonance energy transfer (FRET) effect The effect is obtained and the quench is increased. In the present invention, a fluorescent dye interacts with a complex of an antigen and an antigen-binding protein 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 the antigen-binding protein comprising the polypeptide containing the antibody light chain variable region of the present invention and the polypeptide containing the antibody heavy chain variable region, or when detecting the antigen, the antigen-binding protein As more antigens bind to, the fluorescence generated from the fluorescent dye is quenched, and the fluorescence intensity decreases. That is, the generated fluorescence intensity has a negative correlation with the antigen concentration. In the present invention, the antigen-binding protein can also be referred to as Q-body or UQ-body in which the generated fluorescence intensity has a negative correlation with the antigen concentration.

Even when one or both of the polypeptide containing the antibody light chain variable region and the polypeptide containing the antibody heavy chain variable region constituting the antigen-binding protein of the present invention are labeled with a fluorescent dye, the antigen concentration The type of fluorescent dye can be appropriately selected based on the principle that the fluorescence intensity of the fluorescent dye is negatively correlated.

Illustrative examples of fluorescent dyes used for fluorescent labeling include rhodamine, coumarin, Cy, EvoBlue, oxazine, Carbopyronin, naphthalene, biphenyl, anthracene, phenenthrene, pyrene, carbazole, etc. Specifically, CR110: carboxyrhodamine 110: Rhodamine Green (trade name), TAMRA: carbocytetremethlrhodamine: TMR, Carboxyrhodamine 6G: CR6G, ATTO655 (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-prop ionic 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-phenyl-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), ATTO 495 (trade name), ATTO 520 (trade name), ATTO 532 (trade name), ATTO Rho6G (trade name), ATTO 550 (trademark) Name), ATTO 565 (trade name), ATTOATTRho3B (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 Rho14 (trade name), ATTO 633 (trade name), ATTO 名 647 (trade name), ATTO 647N (trade name), ATTON655 (trade name), AT TO 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) ), 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 568 (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), Rhodamine Red -X (trade name), Texas® Red-X (trade name), 5 (6) -TAMRA-X (trade name), 5TAMRA (trade name), SFX (trade name), Cy3 EvoBlue 10, CR110 and TAMRA that are rhodamine-based fluorescent dyes, and ATTO655 that is an oxazine-based fluorescent dye can be particularly preferably exemplified.

Among the above 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 the fluorescence intensity according to the 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 material from the solvent, further reducing 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.

In the present invention, the method for labeling a polypeptide containing an antibody light chain variable region or a polypeptide containing an antibody heavy chain variable region with a fluorescent dye is not particularly limited, and functional groups at both ends or side chains of the polypeptide are used. For example, a method of labeling directly or indirectly through a crosslinking agent, a method of labeling site-specifically while synthesizing a polypeptide using a cell-free translation system, and the like can be used. As a labeling method using a cell-free translation system, an amber suppression method (Ellman J et al. (1991) Methods Enzymol.202: 301-36), a four-base codon method (Hohsaka T., et al., J Am. Chem. Soc., 118, 9778-9779, 1996), C-terminal labeling method (Japanese Patent Laid-Open No. 2000-139468), N-terminal labeling method (US Pat. No. 5,643,722, Olejnik et al. (2005) In the amber suppression method, DNA or mRNA is prepared 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. Proteins are synthesized from the DNA or mRNA using a cell translation system. At that time, by adding a suppressor tRNA to which a 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 for the amber codon can be synthesized. In the 4-base codon method, a codon is mainly expanded to CGGG, a DNA or mRNA in which a codon encoding an amino acid is replaced with CGGG is prepared, and a protein is synthesized from the DNA or mRNA using a cell-free translation system. At that time, by adding tRNA CGGG to which the labeled unnatural amino acid is bound to the protein synthesis reaction solution, it is possible to synthesize a protein in which the labeled amino acid is introduced at the site substituted with the 4-base codon. The different color double label in the present invention uses a cell-free translation system and is co-expressed by combining the amber suppression method and the 4-base codon method, whereby a polypeptide containing a light chain variable region and a polypeptide containing a heavy chain variable region A complex can be formed by labeling with different fluorescent dyes. 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, a method of introducing a fluorescent dye in a site-specific manner by genetic recombination technology using E. coli or animal cells as a host can be used. 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. In addition, using an animal cell introduced with archaeal 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.

The antigen concentration measurement or detection kit of the present invention is intended for antigen concentration measurement or antigen detection. Antigen concentration measurement refers to quantifying the antigen concentration, and antigen detection refers to qualitative measurement of the antigen, including visualization with a fluorescent dye. Here, visualization means that the presence of an antigen can be confirmed by fluorescence by binding an antigen-binding protein labeled with a fluorescent dye to the antigen. In the present invention, for example, an antigen can be visualized by utilizing a decrease in fluorescence intensity by administering a labeled antigen-binding protein to a living body and binding it to the antigen.

The antigen is not particularly limited as long as it is an antigen specifically recognized by the antibody heavy chain variable region polypeptide and the antibody light chain variable region polypeptide. For example, proteins, peptides, carbohydrates, lipids, glycolipids And low molecular weight compounds. That is, in the method of the present invention, the antigen to be examined 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 endophite-producing toxins; 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 not limited, but biological body fluid samples such as blood, serum, plasma, urine, saliva, spinal fluid, culture supernatant, cell extract, fungus body extract, waste water, and 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. In addition, substances containing narcotic drugs such as cannabis components and stimulants can be mentioned. Compress sap from Asa plant parts such as leaves, stems, roots, seeds and petals, or plant fragments, or Asa plant parts such as leaves, stems, roots, seeds and petals, as a substance containing cannabis components And cannabis resin, which is a processed cannabis product made into a solid resin. Usually, a plant part or its plant piece is used as dry cannabis in a dry state. In the present invention, dry cannabis, particularly dry cannabis plant pieces, is used as a test object that is a plant part or a plant piece thereof.

The antibody light chain variable region polypeptide and antibody heavy chain variable region polypeptide constituting the antigen-binding protein of the present invention may be those derived from monoclonal antibodies. In other words, a hybridoma that produces a monoclonal antibody by a conventional method using an antigen to be tested as an immunogen, and a polypeptide containing an antibody light chain variable region of the monoclonal antibody produced by the hybridoma and an antibody heavy chain variable region Polypeptides can be utilized. Further, a DNA encoding the antibody light chain variable region and a DNA encoding the antibody heavy chain variable region are obtained from the hybridoma, and the polypeptide containing the antibody light chain variable region and the antibody heavy as a recombinant protein using the DNA are obtained. An antigen-binding protein comprising a polypeptide containing a chain variable region can also be produced.

Examples of hybridomas include hybridomas that produce antibodies against anti-tetrahydrocannabinol (THC) or its derivatives. Since THC, THC-A and CBN have similar structures and immunologically cross-react, THC-A and CBN can be detected by using an anti-THC antibody. An example of such a hybridoma is the hybridoma A-04, which was issued on November 20, 2014, on the basis of the National Institute for Product Evaluation Technology (NITE) Patent Microorganisms (NITE （Patent Microorganisms Depository). ) (National Institute of Technology 292-0818, Kisarazu City, Kazusa, Kazusa, 2-5-8 122) is deposited internationally under the accession number NITE BP-01970 (“Identification Display” is “A-04”) .

The antigen concentration measurement or detection kit of the present invention comprises a polypeptide containing an antibody light chain variable region and a polypeptide containing an antibody heavy chain variable region, and the polypeptide containing the antibody light chain variable region and the antibody heavy chain variable region Including antigen binding protein labeled with a fluorescent dye in either or both of the polypeptides containing, and antigens that can be used as standard substances, and reagents such as diluents usually used in this type of immunoassay kit Etc., may be equipped with instruments such as plates, instruction manuals, etc.
2. Antigen Detection Method Using Antigen Concentration Measurement or Detection Kit of the Present Invention The principle of antigen detection using the antigen concentration measurement or detection kit of the present invention is as follows.
(i) a polypeptide comprising an antibody light chain variable region and a polypeptide comprising an antibody heavy chain variable region in the antigen concentration measurement or detection kit of the present invention, the polypeptide comprising the antibody light chain variable region and the antibody heavy chain An antigen-binding protein in which one or both of the polypeptides including the variable region are labeled with a fluorescent dye is mixed with a test sample that may contain the antigen to be examined.

In the unbound state before the antigen-binding protein binds to the antigen, the fluorescent dye that labels the polypeptide containing the antibody light chain variable region and / or the polypeptide containing the antibody heavy chain variable region is quenched. No or weakly quenched. The weak quench occurs when the fluorescent dye is located in the vicinity of the tryptophan residue in the heavy chain variable region of the antigen-binding protein and interacts with the tryptophan residue, whereby the antigen-binding protein acts as a quencher. On the other hand, if the fluorescent dye is not located near the tryptophan residue in the heavy chain variable region of the fluorescent binding protein, the fluorescent dye is not quenched.

When the antigen to be examined is present in the test sample, the antigen and the antigen binding protein bind to form a complex. When the antigen and the antigen binding protein are bound, the antigen-antigen binding protein complex or antigen acts as a quencher for the labeled fluorescent dye, and the fluorescent dye is further quenched. That is, the fluorescent dye interacts with the antigen-antigen-binding protein complex or antigen with hydrophobic interaction or electrostatic interaction, enters the antigen-binding pocket of the antigen-binding protein, and remains in the tryptophan residue of the antibody heavy chain variable region. The interaction with the group becomes stronger and the degree of quenching becomes stronger. Alternatively, the degree of quenching increases because the interaction between the antigen and the fluorescent dye increases.

In addition, the antigen-binding protein of the present invention in which the same color fluorescent dye is labeled on each of the polypeptides provides a quenching effect (H-dimer) between the fluorescent dyes. In addition, in the fluorescent labeling complex of the present invention in which a different color fluorescent dye is labeled on each of the polypeptides, in addition to quenching by the tryptophan residue and quenching between fluorescent dyes, the fluorescence resonance energy transfer (FRET) effect is used. Quenching effect is obtained and quenching is increased.
(ii) The presence of an antigen can be detected or quantified by measuring the fluorescence emitted by the fluorescent dye and detecting the decrease in fluorescence intensity.

At this time, it is preferable to prepare a calibration curve in advance by mixing and contacting a test sample containing an antigen-binding protein and a known amount of antigen, 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. The measured fluorescence intensity and the amount of antigen in the test sample have a negative correlation, and the amount of antigen to be detected can be measured using the fluorescence intensity as an index.

FIG. 1 shows the principle and the structure of the antigen-binding protein. FIG. 1D shows a polypeptide comprising the antibody light chain variable region of the present invention and a polypeptide comprising the antibody heavy chain variable region, and the polypeptide comprising the antibody light chain variable region and the polypeptide comprising the antibody heavy chain variable region. FIG. 1 is a schematic diagram showing the structure of an antigen-binding protein in which either or both are labeled with a fluorescent dye, wherein 1 is an scFv antibody, 2 is a Fab antibody, 3 is an F (ab ′) ₂ antibody, and 4 is a complete body. Antibody is shown. In the figure, the cylinders labeled VL1 and VL2 (black and diagonal cylinders, respectively) indicate the polypeptide containing the antibody light chain variable region, and the cylinders labeled VH1 and VH2 (white and horizontal cylinders, respectively) ) Indicates a polypeptide containing an antibody heavy chain variable region. In addition, the vertical cylinders labeled C indicate antibody constant regions, and the circles with S indicate disulfide bonds. In the example of FIG. 1D, in 1 and 2, only one polypeptide containing the antibody light chain variable region is labeled, and in 3 and 4, two polypeptides containing the antibody light chain variable region are labeled. 1A, B and C show the principle of the method of the invention using labeled Fab antibodies. FIG. 1A is an example of a single-label antigen-binding protein in which only a polypeptide containing an antibody light chain variable region is labeled. FIG. 1B shows a polypeptide containing an antibody light chain variable region and a polypeptide containing an antibody heavy chain variable region. Is an example of the same color double-label antigen-binding protein labeled with the same fluorescent dye, and FIG. 1C shows fluorescent dyes (fluorescent dye 1 and fluorescent dye 1 and polypeptide having antibody heavy chain variable regions and polypeptides containing antibody heavy chain variable regions). It is an example of a different color double-label antigen binding protein labeled with a fluorescent dye 2). In FIGS. 1A, B and C, a and b are states in which the antigen is not bound, a is a state in which the fluorescent dye is not interacted with the tryptophan of the antigen binding protein and is not quenched, and b is a fluorescent dye. Interacts with the antigen binding protein tryptophan, indicating that the antigen binding protein acts as a quencher and is weakly quenched. c indicates a state in which the antigen is bound, and the fluorescent dye interacts with the complex of the antigen and the antigen binding protein, and the complex of the antigen and the antigen binding protein acts as a quencher and is strongly quenched.

Antigen detection using the antigen concentration measurement or detection kit of the present invention can be performed in the following steps.
(A) a step of bringing an antigen-binding protein into contact with an antigen to be examined in a liquid phase,
The antigen-binding protein comprises a polypeptide comprising an antibody light chain variable region and a polypeptide comprising an antibody heavy chain variable region, and any one of the polypeptide comprising the antibody light chain variable region and the polypeptide comprising the antibody heavy chain variable region One or both are labeled with a fluorescent dye that is quenched in a state labeled with a polypeptide containing an antibody heavy chain variable region or a polypeptide containing an antibody light chain variable region, and the antigen binding protein and the antigen to be tested are combined with the antigen. A process in which a complex of an antigen to be tested and an antigen-binding protein acts as a quencher of a fluorescent dye when forming a complex of an antigen-binding protein;
(B) measuring or detecting fluorescence of the fluorescent dye; and (c) measuring antigen concentration or detecting antigen,
When the antigen binding protein consisting of the polypeptide containing the antibody heavy chain variable region and the polypeptide containing the antibody light chain variable region binds to the antigen to form a complex of the antigen and the antigen binding protein, quenching of the fluorescent dye is eliminated. Based on the measured or detected fluorescence, using as an indicator that the antigen concentration in the liquid phase and the fluorescence intensity of the fluorescent dye have a negative correlation due to a decrease in fluorescence intensity due to stronger quenching. A step of calculating the amount of the antigen to be examined or detecting the antigen to be examined.

According to the method of the present invention, the presence of the antigen can be detected by the decrease in the fluorescence intensity, and the antigen can be quantified by measuring the fluorescence intensity.

For the measurement of the fluorescence in the antigen concentration measurement or detection method of the present invention using the detection kit of the present invention, a light source or a measurement device usually used for fluorescence detection can be 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) 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.

In the antigen detection method using the antigen concentration measurement or detection kit 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. That's fine. 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 present invention will be specifically described by the following examples, but the present invention is not limited to these examples.
[Example 1] Production of anti-tetrahydrocannabinol (THC) hybridoma Hybridoma production A mouse strain (BALB / c) was immunized with adjuvant with BSA-conjugated THC antigen (Genway Biotech), and after the serum titer increased, the spleen was removed and myeloma cell NS-1 strain (P3.NS- Cell fusion was carried out with 1 / 1.Ag4.1) by the PEG method (40%). Further, selection by HAT medium and selection by ELISA (details are shown in 2) yielded an anti-THC antibody (IgG1, kappa) -producing hybridoma. The resulting hybridoma A-04, dated November 20, 2014, NITE Patent Microorganisms Depository (Kazusa, Kisarazu, Chiba, 292-0818, Japan) 2-5-8, Kamashita Room 122) was deposited internationally under the deposit number NITE BP-01970 (“Identification” is “A-04”).
2. Assay of monoclonal antibody produced by hybridoma Anti-THC antibody reactivity was measured by competitive ELISA (Enzyme-Linked ImmunoSorbent Assay).
(i) A 0.10 M carbonate buffer solution (pH 8.6) (100 ng / mL) of THC-BSA was dispensed (100 μL / well) into a 96-well ELISA plate and left overnight at room temperature.
(ii) The solution was removed by suction, and the plate was washed 3 times with PBS. PBS containing 2% skim milk was dispensed (300 μL / well) and allowed to stand at 37 ° C. for 1 hour.
(iii) The solution was removed by aspiration, and the plate was washed 3 times with PBS containing 0.05% (v / v) Tween 20 to obtain an antigen-immobilized plate.
(iv) On the plate of (iii), a 50% (v / v) ethanol solution of tetrahydrocannabinol (THC) or a similar compound (tetrahydrocannabinolic acid (THC-A) or cannabinol (CBN)) (25 μL / Then, the culture supernatant (100 μL / well) of hybridoma A-04 appropriately diluted with PBS containing 0.10% gelatin was mixed and incubated at 37 ° C. for 1 hour.
(v) Aspirate the solution and wash the plate 3 times with T-PBS, add peroxidase-labeled goat anti-mouse IgG (Fc specific) antibody (1.6 μg / 100 μL / well) and incubate at 37 ° C. for 30 minutes did.
(vi) The plate is again washed three times with PBS containing 0.05% (v / v) Tween 20 and contains the substrate solution {o-PD (0.04%) and H ₂ O ₂ [0.06% (v / v)] Citrate / phosphate buffer (pH 5.0)} (100 μL / well) was added and incubated at room temperature for 30 minutes.
(vii) An enzyme reaction stop solution (1.0 MH ₂ SO ₄ ) (100 μL / well) was added and mixed, and then the absorbance at 490 nm was measured with a microplate reader (Bio-Rad).

The results are shown in FIG. As shown in FIG. 2, the obtained antibody showed affinity for THCA, THC and CBN.
[Example 2] Measurement using scFv-type antigen-binding protein A method for producing an antigen protein is described in WO2011 / 061944, and can be produced based on the description in the publication.
1. Production of scFv type antigen binding protein (construction of expression vector)
The DNA sequence encoding the light chain variable region (VL; SEQ ID NO: 1) of the antibody against human osteocalcin (BGP) and the heavy chain variable region (VH; SEQ ID NO: 2) of the antibody against BGP of SEQ ID NO: 3 A gene in which a DNA sequence of a ProX tag (MSKQIEVNXSNET (SEQ ID NO: 3) when translated) containing an amber codon at the N-terminus is added to the DNA sequence linked by a linker, and a His-tag DNA sequence is added at the C-terminus Was incorporated into the pIVEX2.3d vector. The constructed expression vector is designed such that a ProX tag (amber) is added to the N-terminus of scFv and a His tag is added to the C-terminus.
(Synthesis of scFv antibody)
Introduction of fluorescently labeled amino acid into antibody variable region-containing peptide and / or N-terminal region of antibody variable region-containing peptide by cell-free translation system using RYTS (trade name) E. coli cell-free synthesis kit (Protein Express) Went.

The fluorescent dyes used for labeling were Cy3 and EvoBlue10.

The resulting fluorescently labeled scFv type antigen binding protein has the structure shown in D-1 of FIG.
2. Antigen measurement Fluorescently-labeled scFv-type antigen-binding protein (320 nM, 1.25 μL) obtained in 1) and BGP (0, 0.11, 1.1, 11, 110, 1100, 11000 nM) as an antigen were added to PBS containing 1% BSA (+0. (05% Tween20) to a total of 50 μL. The fluorescence intensity of these solutions was measured using a fluorescence plate reader (SpectraMax Paradigm; manufactured by Molecular Devices). When Cy3-labeled scFv was used, the excitation wavelength (Ex) was set to 530 nm, and the fluorescence intensity at the fluorescence wavelength (Em) of 580 nm was measured. When EvoBlue10-labeled scFv was used, the excitation wavelength (Ex) was set to 630 nm, and the fluorescence intensity at a fluorescence wavelength (Em) of 680 nm was measured.

The results when using Cy3-labeled scFv are shown in FIG. 3, and the results when using EvoBlue10-labeled scFv are shown in FIG. As shown in FIGS. 3 and 4, in the absence of antigen (BGP), the fluorescent dye was not quenched and emitted fluorescence, but as the antigen concentration increased, the fluorescent dye was quenched and the fluorescence intensity was increased. Fell. As shown in the figure, a negative correlation was observed between the antigen concentration and the fluorescence intensity.
[Example 3] Measurement using Fab type antigen binding protein Fab type antigen binding protein (construction of expression vector)
A DNA sequence encoding a polypeptide comprising a light chain variable region (VL; SEQ ID NO: 4) of an antibody against tetrahydrocannabinol (THC) and an antibody light chain constant region (Cκ; SEQ ID NO: 5), and a ProX tag (N-terminal) The base sequence corresponding to the ninth amino acid is TTT. When translated, the DNA sequence of MSKQIEVNFSNET (SEQ ID NO: 6) is added, and the linker (SEQ ID NO: 7) and FLAG tag DNA sequence are added to the C-terminus. The obtained gene was incorporated into a pIVEX2.3d vector (Roche Diagnostics). In addition, a ProX tag containing an amber codon at the N-terminus in a DNA sequence encoding a polypeptide containing an antibody heavy chain variable region (VH; SEQ ID NO: 8) and antibody heavy chain constant region (CH1; SEQ ID NO: 9) against THC ( The base sequence corresponding to the ninth amino acid is TAG. When translated, the DNA sequence of MSKQIEVNXSNET (X is a fluorescently labeled amino acid); SEQ ID NO: 10) is given, and further, a linker (SEQ ID NO: 7) and The gene to which the DNA sequence of the His tag was added was incorporated into a pIVEX2.3d vector (Roche Diagnostics). In these constructed expression vectors, a ProX tag (VH is labeled when translated and VL is unlabeled) is added to the N-terminus of the inserted VL or VH, and a His tag or FLAG tag is added to the C-terminus. It is designed as follows.
(Synthesis of Fab type antigen binding protein)
The reaction solution (60 μL) consists of 3 μL Enzyme Mix, 0.6 μL Methionine, 30 μL 2 × Reaction Mix, 20 μL E-coli Lysate, 2 μL of two types of plasmid DNA (200 ng each), 3 μL of fluorescently labeled aminoacyl-tRNAamber (480 pmol), 1.4 μL of Nuclease Free Water was added. Fluorescently labeled aminoacyl-tRNAs (TAMRA-X-AF-tRNAamber, CR110-X-AF-tRNAamber, and ATTO655-X-AF-tRNAamber) for producing fluorescently labeled proteins are CloverDirect ™ tRNA Reagents for Site -Directed Protein Functionalization (manufactured by Protein Express) was used. The reaction solution was allowed to stand at 20 ° C. for 2 hours for reaction to synthesize the protein, and then complex formation was completed by further reaction at 4 ° C. for 16 hours. After completion of the reaction, SDS-PAGE (15%) was performed using 0.5 μL of the reaction solution, and protein expression was observed with a fluorescence image analyzer (FMBIO-III; manufactured by Hitachi Software Engineering). Furthermore, Western blotting was performed using an anti-His tag antibody or an anti-FLAG tag antibody, and it was confirmed that the target fluorescently labeled antibody variable region-containing peptide was synthesized.
(Purification of fluorescently labeled Fab-type antigen binding protein)
The synthesized fluorescently labeled Fab type antigen binding protein was purified by an anti-FLAG M2 affinity gel (Sigma Aldrich) or His-Spin Trap Column (GE Healthcare). The above reaction solution (60 μL) was applied to a column containing anti-FLAG M2 affinity gel, incubated for 15 minutes at room temperature, and then washed with a wash buffer (20 mM Phosphate buffer (pH 7.4) /0.5 M NaCl / 0.1% Polyoxyethylene (23) Lauryl Ether) was washed 3 times. Next, elution was performed 3 times with 200 μL of Elute buffer (20 mM Phosphate buffer (pH 7.4) /0.5 M NaCl / 100 μg FLAG peptide / 0.1% Polyoxyethylene (23) Lauryl Ether). Next, the eluate was applied to a His-Spin Trap Column. After incubation at room temperature for 15 minutes, washing was performed 3 times with Wash buffer (20 mM Phosphate buffer (pH 7.4) /0.5 M NaCl / 60 mM imidazole / 0.1% Polyoxyethylene (23) Lauryl Ether). Next, elution was performed 3 times with 200 μL of Elute buffer (20 mM Phosphate buffer (pH 7.4) /0.5 M NaCl / 0.5 M imidazole / 0.1% Polyoxyethylene (23) Lauryl Ether). Furthermore, the eluate was Amicon Ultra-0.5 centrifugal filter 10 kDa (Millipore), buffer exchanged with PBS (+ 0.05% Tween20), and concentrated. The concentration of the purified sample was measured using a fluorescence image analyzer (FMBIO-III; manufactured by Hitachi Software Engineering).

The resulting fluorescently labeled Fab type antigen binding proteins were the following four types. The first four alphabets are abbreviations for each.
(i) Antigen-binding protein consisting of a polypeptide containing an antibody heavy chain variable region labeled with HTLA type TAMRA and a polypeptide containing an antibody light chain variable region labeled with ATTO655 (different color double label)
(ii) Antigen binding protein consisting of a polypeptide containing an antibody heavy chain variable region labeled with HALT type ATTO655 and a polypeptide containing an antibody light chain variable region labeled with TAMRA (different color double label)
(iii) Antigen-binding protein consisting of a polypeptide containing an antibody heavy chain variable region labeled with HTLC type TAMRA and a polypeptide containing an antibody light chain variable region labeled with CR110 (different color double label)
(iv) Antigen-binding protein consisting of a polypeptide containing an antibody heavy chain variable region labeled with HCLT type CR110 and a polypeptide containing an antibody light chain variable region labeled with TAMRA (different color double label)
2. Antigen measurement And the fluorescently labeled Fab-type antigen-binding protein (320 nM, 1.25 μL) obtained in the above, and tetrahydrocannabinol (THC), which is an antigen, or cannabinol (CBN), which is an analog thereof (0, 0.01, 0.1, 1, 10, 100 μg / mL) was prepared with PBS containing 1% BSA (+ 0.05% Tween 20) to a total of 50 μL. The fluorescence intensity of these solutions was measured using a fluorescence plate reader (SpectraMax Paradigm; manufactured by Molecular Devices). When CR110 fluorescently labeled antibody was used, the excitation wavelength (Ex) was set to 480 nm, and the fluorescence intensity at the fluorescence wavelength (Em) of 530 nm was measured. When TAMRA fluorescent dye-labeled antibody was used, the excitation wavelength (Ex) was set to 530 nm, and the fluorescence intensity at the fluorescence wavelength (Em) of 580 nm was measured. When the ATTO655 fluorescent dye-labeled antibody was used, the excitation wavelength (Ex) was set to 630 nm, and the fluorescence intensity at the fluorescence wavelength (Em) of 680 nm was measured.

The measurement results of tetrahydrocannabinol (THC) using HTLA type, HALT type, HTLC type and HCLT type antigen binding proteins are shown in FIG. 5, and the measurement result of cannabinol (CBN) is shown in FIG.

As shown in FIGS. 5 and 6, in the absence of antigen, the fluorescent dye was not quenched and emitted fluorescence, but as the antigen concentration increased, the fluorescent dye was quenched and the fluorescence intensity decreased. . As shown in the figure, a negative correlation was observed between the antigen concentration and the fluorescence intensity. Among HTLA, HALT, HTLC, and HCLT, HTLA and HCLT, which have a large difference between the fluorescence intensity in the absence of the antigen and the fluorescence intensity when the antigen is present at a high concentration, were good.
[Example 4] Measurement using Fab type antigen binding protein Fab type antigen binding protein (construction of expression vector)
A DNA sequence encoding a polypeptide comprising a light chain variable region (international deposit “A-04”) of an antibody against tetrahydrocannabinol (THC) and an antibody light chain constant region (Cκ; SEQ ID NO: 5); The tag (the base sequence corresponding to the 9th amino acid is TTT, and when translated, the DNA sequence of MSKQIEVNFSNET; SEQ ID NO: 6) is given, and the linker (SEQ ID NO: 7) and FLAG tag DNA sequence at the C-terminus The gene to which was added was incorporated into a pIVEX2.3d vector (Roche Diagnostics). In addition, a DNA sequence encoding a polypeptide containing an antibody heavy chain variable region against THC (International Deposit “A-04”) and an antibody heavy chain constant region (CH1; SEQ ID NO: 9), and ProX containing an amber codon at the N-terminus. The tag (base sequence corresponding to the 9th amino acid is TAG, and when translated, the DNA sequence of MSKQIEVNXSNET (X is a fluorescently labeled amino acid); SEQ ID NO: 10) is given, and a linker (SEQ ID NO: 7) is added to the C-terminus. ) And His-tagged DNA sequences were incorporated into a pIVEX2.3d vector (Roche Diagnostics). In these constructed expression vectors, a ProX tag (VH is labeled when translated and VL is unlabeled) is added to the N-terminus of the inserted VL or VH, and a His tag or FLAG tag is added to the C-terminus. It is designed as follows.
(Synthesis of Fab type antigen binding protein)
The reaction solution (60 μL) consists of 3 μL Enzyme Mix, 0.6 μL Methionine, 30 μL 2 × Reaction Mix, 20 μL E-coli Lysate, 2 μL of two types of plasmid DNA (200 ng each), 3 μL of fluorescently labeled aminoacyl-tRNAamber (480 pmol), 1.4 μL of Nuclease Free Water was added. CloverDirect (trade name) tRNA Reagents for Site-Directed Protein Functionalization (manufactured by Protein Express) was used as a fluorescently labeled aminoacyl-tRNA (TAMRA-X-AF-tRNAamber) for producing a fluorescently labeled protein. The reaction solution was allowed to stand at 20 ° C. for 2 hours for reaction to synthesize the protein, and then complex formation was completed by further reaction at 4 ° C. for 16 hours. After completion of the reaction, SDS-PAGE (15%) was performed using 0.5 μL of the reaction solution, and protein expression was observed with a fluorescence image analyzer (FMBIO-III; manufactured by Hitachi Software Engineering). Furthermore, Western blotting was performed using an anti-His tag antibody or an anti-FLAG tag antibody, and it was confirmed that the target fluorescently labeled antibody variable region-containing peptide was synthesized.
(Purification of fluorescently labeled Fab-type antigen binding protein)
The synthesized fluorescently labeled Fab type antigen binding protein was purified by anti-FLAG M2 affinity gel (Sigma Aldrich) or His-Spin Trap Column (GE Healthcare). The above reaction solution (60 μL) was applied to a column containing anti-FLAG M2 affinity gel, incubated for 15 minutes at room temperature, and then washed with a wash buffer (20 mM Phosphate buffer (pH 7.4) /0.5 M NaCl / 0.1% Polyoxyethylene (23) Lauryl Ether) was washed 3 times. Next, elution was performed 3 times with 200 μL of Elute buffer (20 mM Phosphate buffer (pH 7.4) /0.5 M NaCl / 100 μg FLAG peptide / 0.1% Polyoxyethylene (23) Lauryl Ether). Next, the eluate was applied to a His-Spin Trap Column. After 15 minutes of incubation at room temperature, washing was performed 3 times with Wash buffer (20 mM Phosphate buffer (pH 7.4) /0.5 M NaCl / 60 mM imidazole / 0.1% Polyoxyethylene (23) Lauryl Ether). Next, elution was performed 3 times with 200 μL of Elute buffer (20 mM Phosphate buffer (pH 7.4) /0.5 M NaCl / 0.5 M imidazole / 0.1% Polyoxyethylene (23) Lauryl Ether). Furthermore, the eluate was Amicon Ultra-0.5 centrifugal filter 10 kDa (Millipore), buffer exchanged with PBS (+ 0.05% Tween20), and concentrated. The concentration of the purified sample was measured using a fluorescence image analyzer (FMBIO-III; manufactured by Hitachi Software Engineering).

The resulting fluorescently labeled Fab-type antigen binding protein was as follows. The first four alphabets are abbreviations for each.
(v) HTLT type antigen-binding protein consisting of a polypeptide containing an antibody heavy chain variable region labeled with TAMRA and a polypeptide containing an antibody light chain variable region labeled with TAMRA (double label of the same color)
The resulting fluorescently labeled Fab type antigen binding protein has the structure shown in D-2 of FIG.

FIG. 7 shows the results of measuring tetrahydrocannabinol (THC), tetrahydrocannabinolic acid (THC-A) and cannabinol (CBN) using an HTLT type antigen binding protein.

As shown in FIG. 7, reactivity was observed with any antigen of tetrahydrocannabinol (THC), tetrahydrocannabinolic acid (THC-A) and cannabinol (CBN). In the absence of the antigen, the fluorescent dye was not quenched and emitted fluorescence. However, as the antigen concentration increased, the fluorescent dye was quenched and the fluorescence intensity decreased. As shown in the figure, a negative correlation was observed between the antigen concentration and the fluorescence intensity.

The polypeptide comprising an antibody light chain variable region of the present invention and a polypeptide comprising an antibody heavy chain variable region, either one of the polypeptide comprising the antibody light chain variable region and the polypeptide comprising the antibody heavy chain variable region, or By using an antigen concentration measurement or detection kit that contains an antigen-binding protein that is both labeled with a fluorescent dye, high sensitivity without requiring a solid phase or washing step Can be detected.

SEQ ID NOs: 1-10 Synthesis All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

A polypeptide comprising an antibody light chain variable region and a polypeptide comprising an antibody heavy chain variable region, and either one or both of the polypeptide comprising the antibody light chain variable region and the polypeptide comprising the antibody heavy chain variable region are fluorescent An antigen concentration measurement or detection kit comprising an antigen-binding protein labeled with a dye,
When the antigen binding protein binds to the antigen to be tested to form a complex, the complex of the antigen and the antigen binding protein becomes the quencher of the fluorescent dye,
There is a negative correlation between the concentration of the antigen in the liquid phase and the fluorescence intensity of the fluorescent dye. When the complex of antigen and antigen-binding protein is formed, the fluorescence intensity is decreased by quenching the fluorescent dye more strongly. And
It is possible to measure the antigen concentration or visualize the antigen based on the fluorescence measured or detected by using as an index that the antigen concentration in the liquid phase and the fluorescence intensity of the fluorescent dye have a negative correlation. An antigen concentration measurement or detection kit.
2. The antigen concentration measurement or detection kit according to claim 1, wherein the antigen-binding protein comprising a polypeptide containing an antibody light chain variable region and a polypeptide containing an antibody heavy chain variable region is an scFv antibody.
An antigen-binding protein comprising a polypeptide comprising an antibody light chain variable region and a polypeptide comprising an antibody heavy chain variable region, a Fab antibody, and two Fab antibodies bound by a disulfide bond via a hinge, F (ab ′) 2 antibody; And a kit for antigen concentration measurement or detection according to claim 1, wherein the kit is selected from the group consisting of intact antibodies.
The antigen concentration measurement or detection kit according to claim 3, wherein the polypeptide containing the antibody light chain variable region and the polypeptide containing the antibody heavy chain variable region are labeled with the same fluorescent dye.
The antigen concentration measurement or detection kit according to any one of claims 1 to 4, wherein the fluorescent dye is a rhodamine fluorescent dye.
6. The antigen concentration measurement or detection kit according to claim 5, wherein the fluorescent dye is carboxytetramethylrhodamine.
An antigen concentration measurement or detection method comprising sequentially performing the following steps (a) to (c):
(A) a step of bringing an antigen-binding protein into contact with an antigen to be examined in a liquid phase,
The antigen-binding protein comprises a polypeptide comprising an antibody light chain variable region and a polypeptide comprising an antibody heavy chain variable region, and any one of the polypeptide comprising the antibody light chain variable region and the polypeptide comprising the antibody heavy chain variable region One or both are labeled with a fluorescent dye that is quenched in a state labeled with a polypeptide containing an antibody heavy chain variable region or a polypeptide containing an antibody light chain variable region, and the antigen binding protein and the antigen to be tested are combined with the antigen. A process in which a complex of an antigen to be tested and an antigen-binding protein acts as a quencher of a fluorescent dye when forming a complex of an antigen-binding protein;
(B) measuring or detecting fluorescence of the fluorescent dye; and (c) measuring antigen concentration or detecting antigen,
When the antigen binding protein consisting of the polypeptide containing the antibody heavy chain variable region and the polypeptide containing the antibody light chain variable region binds to the antigen to form a complex of the antigen and the antigen binding protein, quenching of the fluorescent dye is eliminated. Based on the measured or detected fluorescence, using as an indicator that the antigen concentration in the liquid phase and the fluorescence intensity of the fluorescent dye have a negative correlation due to a decrease in fluorescence intensity due to stronger quenching. A step of calculating the amount of the antigen to be examined or detecting the antigen to be examined.
The method for measuring or detecting an antigen concentration according to claim 7, wherein the antigen-binding protein comprising a polypeptide comprising an antibody light chain variable region and a polypeptide comprising an antibody heavy chain variable region is an scFv antibody.
An antigen-binding protein comprising a polypeptide comprising an antibody light chain variable region and a polypeptide comprising an antibody heavy chain variable region, a Fab antibody, and two Fab antibodies bound by a disulfide bond via a hinge, F (ab ′) 2 antibody; And the antigen concentration measurement or detection method according to claim 7, wherein the antigen concentration measurement or detection method is selected from the group consisting of a whole antibody and a whole antibody.
10. The method for measuring or detecting an antigen concentration according to claim 9, wherein the polypeptide containing the antibody light chain variable region and the polypeptide containing the antibody heavy chain variable region are labeled with the same fluorescent dye.
The method for measuring or detecting an antigen concentration according to any one of claims 7 to 10, wherein the antigen is a low molecular compound.
The method for measuring or detecting an antigen concentration according to any one of claims 7 to 11, wherein the antigen is a cannabis component.
The method for measuring or detecting an antigen concentration according to claim 12, wherein the cannabis component is selected from the group consisting of tetrahydrocannabinol (THC), tetrahydrocannabinolic acid (THC-A) and cannabinol (CBN).
A hybridoma that produces an antibody that binds to tetrahydrocannabinol (THC) or its derivatives, deposited internationally under the deposit number NITE BP-01970.
A monoclonal antibody that binds to tetrahydrocannabinol (THC) or a derivative thereof produced by the hybridoma according to claim 14.
The antigen-binding protein comprising a polypeptide comprising an antibody light chain variable region and a polypeptide comprising an antibody heavy chain variable region, wherein the polypeptide comprising the antibody light chain variable region of the monoclonal antibody according to claim 15 and the antibody weight of the monoclonal antibody The antigen concentration measurement or detection kit according to any one of claims 1 to 6, which is an antigen-binding protein comprising a polypeptide containing a chain variable region.
The antigen-binding protein comprising a polypeptide comprising an antibody light chain variable region and a polypeptide comprising an antibody heavy chain variable region, wherein the polypeptide comprising the antibody light chain variable region of the monoclonal antibody according to claim 15 and the antibody weight of the monoclonal antibody The method for measuring or detecting an antigen concentration according to any one of claims 7 to 13, which is an antigen-binding protein comprising a polypeptide containing a chain variable region.