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WO2016104549A1 - Méthode d'analyse d'immunomarquage par fluorescence à l'aide de protéines se liant à l'antigène, comprenant un polypeptide comprenant un domaine anticorps variable à marquage fluorescent - Google Patents

Méthode d'analyse d'immunomarquage par fluorescence à l'aide de protéines se liant à l'antigène, comprenant un polypeptide comprenant un domaine anticorps variable à marquage fluorescent Download PDF

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Publication number
WO2016104549A1
WO2016104549A1 PCT/JP2015/085909 JP2015085909W WO2016104549A1 WO 2016104549 A1 WO2016104549 A1 WO 2016104549A1 JP 2015085909 W JP2015085909 W JP 2015085909W WO 2016104549 A1 WO2016104549 A1 WO 2016104549A1
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antigen
chain variable
variable region
antibody
polypeptide
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Japanese (ja)
Inventor
亮二 阿部
富士男 斎木
典裕 小林
亨介 山根
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Ushio Denki KK
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Ushio Denki KK
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens

Definitions

  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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,
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 method according to [7] wherein the antigen concentration is selected from the group consisting of 2 antibodies and complete antibodies.
  • 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 a derivative thereof deposited internationally under the deposit number NITE BP-01970.
  • 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.
  • 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.
  • 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
  • an antigen-binding protein both of which are labeled with a fluorescent dye
  • 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.
  • 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.
  • 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.
  • CBN cannabinol
  • mold antigen binding protein It is a figure which shows the cannabinol (CBN) measurement result using different color double label Fab type
  • THC tetrahydrocannabinol
  • THC-A tetrahydrocannabinolic acid
  • CBN cannabinol
  • 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
  • VL antibody light chain variable region
  • VH antibody heavy chain variable region
  • the present invention is also a method for detecting an antigen using the above-described antigen concentration measurement or detection kit.
  • 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.
  • 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 (C ⁇ ) or a polypeptide further having a hinge portion. Among them, a polypeptide having an antibody light chain variable region with C ⁇ is preferred.
  • 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.
  • 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.
  • a polypeptide comprising an antibody heavy chain variable region capable of recognizing the antigen can be appropriately prepared.
  • 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.
  • the peptide containing the sequence is bound thereto.
  • a peptide that forms a complex in addition to the antibody constant region (CH1 and C ⁇ , 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • scFv antibody single chain antibody: single chain variable fragment
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • any of a polypeptide comprising an antibody light chain variable region and a polypeptide comprising an antibody heavy chain variable region are labeled with a fluorescent dye.
  • a fluorescent dye for example, a single-label Fab antibody.
  • the same kind of fluorescent dye may be sufficient and another kind of fluorescent dye may be sufficient.
  • the same color double-label antigen binding protein For example, it is called the same color double-label Fab antibody
  • the different case is called a different color double-label antigen binding protein (for example, a different color double-label Fab antibody).
  • 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.
  • 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).
  • an antigen-binding protein labeled with a fluorescent dye 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • FRET fluorescence resonance energy transfer
  • a polypeptide comprising an antibody light chain variable region and a polypeptide comprising an antibody heavy chain variable region 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
  • FRET fluorescence resonance energy transfer
  • 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.
  • the antigen-binding protein 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.
  • 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.
  • the antigen concentration can be appropriately selected based on the principle that the fluorescence intensity of the fluorescent dye is negatively correlated.
  • fluorescent dyes used for fluorescent labeling include rhodamine, coumarin, Cy, EvoBlue, oxazine, Carbopyronin, naphthalene, biphenyl, anthracene, phenenthrene, pyrene, carbazole, etc.
  • 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 558/5
  • 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).
  • IANBD, CNBD, Acrylodan, 5-IAF and the like can be mentioned.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a protein in which the labeled amino acid is introduced at the site substituted for the amber codon can be synthesized.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • antigen detection refers to qualitative measurement of the antigen, including visualization with a fluorescent dye.
  • 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.
  • 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.
  • proteins, peptides, carbohydrates, lipids, glycolipids And low molecular weight compounds are examples of proteins, peptides, carbohydrates, lipids, glycolipids And low molecular weight compounds.
  • 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.
  • chemical substances including low-molecular compounds such as narcotics, explosives, agricultural chemicals, fragrances, and pollutants can also be measured.
  • 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
  • Tetrahydrocannabinol has double bond regioisomers, ⁇ 8 -THC and ⁇ 9 -THC.
  • Reference to THC includes ⁇ 8 -THC and ⁇ 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.
  • 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
  • cannabis resin which is a processed cannabis product made into a solid resin.
  • a plant part or its plant piece is used as dry cannabis in a dry state.
  • 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.
  • 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.
  • 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.
  • hybridomas examples 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.
  • 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.
  • 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.
  • the antigen and the antigen binding protein bind to form a complex.
  • 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.
  • 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.
  • 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.
  • FRET fluorescence resonance energy transfer
  • 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.
  • 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.
  • the cylinders labeled VL1 and VL2 (black and diagonal cylinders, respectively) indicate the polypeptide containing the antibody light chain variable region
  • the cylinders labeled VH1 and VH2 (white and horizontal cylinders, respectively)
  • the vertical cylinders labeled C indicate antibody constant regions
  • the circles with S indicate disulfide bonds.
  • 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).
  • FIGS. 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a 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.
  • MF20 / FluoroPoint-Light (manufactured by Olympus) III manufactured by Hitachi Software Engineering.
  • the fluorescence detection may be a fluorescence spectrum detection or a fluorescence intensity detection at a specific wavelength.
  • 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.
  • Example 1 Production of anti-tetrahydrocannabinol (THC) hybridoma Hybridoma production
  • a mouse strain BALB / c
  • BSA-conjugated THC antigen Genway Biotech
  • 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%).
  • the solution was removed by suction, and the plate was washed 3 times with PBS.
  • PBS containing 2% skim milk was dispensed (300 ⁇ L / well) and allowed to stand at 37 ° C. for 1 hour.
  • 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.
  • 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.
  • a ProX tag amber
  • a His tag is added to the C-terminus.
  • 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 ⁇ L) 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 ⁇ L. The fluorescence intensity of these solutions was measured using a fluorescence plate reader (SpectraMax Paradigm; manufactured by Molecular Devices).
  • the excitation wavelength (Ex) was set to 530 nm, and the fluorescence intensity at the fluorescence wavelength (Em) of 580 nm was measured.
  • the excitation wavelength (Ex) was set to 630 nm, and the fluorescence intensity at a fluorescence wavelength (Em) of 680 nm was measured.
  • Example 3 Measurement using Fab type antigen binding protein Fab type antigen binding protein (construction of expression vector)
  • the base sequence corresponding to the ninth amino acid is TTT.
  • the DNA sequence of MSKQIEVNFSNET SEQ ID NO: 6
  • 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).
  • 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.
  • MSKQIEVNXSNET 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).
  • a ProX tag VH is labeled when translated and VL is unlabeled
  • a His tag or FLAG tag is added to the C-terminus. It is designed as follows.
  • the reaction solution (60 ⁇ L) consists of 3 ⁇ L Enzyme Mix, 0.6 ⁇ L Methionine, 30 ⁇ L 2 ⁇ Reaction Mix, 20 ⁇ L E-coli Lysate, 2 ⁇ L of two types of plasmid DNA (200 ng each), 3 ⁇ L of fluorescently labeled aminoacyl-tRNAamber (480 pmol), 1.4 ⁇ L 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 TM 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.
  • the above reaction solution (60 ⁇ L) 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 ⁇ L of Elute buffer (20 mM Phosphate buffer (pH 7.4) /0.5 M NaCl / 100 ⁇ g FLAG peptide / 0.1% Polyoxyethylene (23) Lauryl Ether). Next, the eluate was applied to a His-Spin Trap Column.
  • the resulting fluorescently labeled Fab type antigen binding proteins were the following four types. The first four alphabets are abbreviations for each.
  • 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)
  • 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)
  • 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)
  • Antigen-binding protein consisting of a polypeptide containing an antibody heavy chain variable region labeled with
  • the excitation wavelength (Ex) was set to 480 nm, and the fluorescence intensity at the fluorescence wavelength (Em) of 530 nm was measured.
  • 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.
  • the excitation wavelength (Ex) was set to 630 nm, and the fluorescence intensity at the fluorescence wavelength (Em) of 680 nm was measured.
  • THC tetrahydrocannabinol
  • HALT tetrahydrocannabinol
  • HTLC HTLC type
  • HCLT HCLT type antigen binding proteins
  • Fab type antigen binding protein (construction of expression vector)
  • 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).
  • 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.
  • His-tagged DNA sequences were incorporated into a pIVEX2.3d vector (Roche Diagnostics).
  • 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.
  • the reaction solution (60 ⁇ L) consists of 3 ⁇ L Enzyme Mix, 0.6 ⁇ L Methionine, 30 ⁇ L 2 ⁇ Reaction Mix, 20 ⁇ L E-coli Lysate, 2 ⁇ L of two types of plasmid DNA (200 ng each), 3 ⁇ L of fluorescently labeled aminoacyl-tRNAamber (480 pmol), 1.4 ⁇ L 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.
  • TAMRA-X-AF-tRNAamber fluorescently labeled aminoacyl-tRNA
  • 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 ⁇ L of the reaction solution, and protein expression was observed with a fluorescence image analyzer (FMBIO-III; manufactured by Hitachi Software Engineering).
  • the above reaction solution (60 ⁇ L) 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 ⁇ L of Elute buffer (20 mM Phosphate buffer (pH 7.4) /0.5 M NaCl / 100 ⁇ g FLAG peptide / 0.1% Polyoxyethylene (23) Lauryl Ether). Next, the eluate was applied to a His-Spin Trap Column.
  • the resulting fluorescently labeled Fab-type antigen binding protein was as follows. The first four alphabets are abbreviations for each.
  • 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.
  • THC tetrahydrocannabinol
  • THC-A tetrahydrocannabinolic acid
  • CBN cannabinol
  • 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
  • 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.

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Abstract

L'invention concerne un kit de mesure d'antigène ou de détection d'antigène, grâce auquel il est possible de mesurer une concentration d'antigène en utilisant comme indicateur de cette dernière la corrélation négative existant entre l'intensité de fluorescence d'un marqueur fluorescent et la concentration de l'antigène. L'invention concerne également une méthode de mesure ou de détection d'antigène qui utilise le kit. L'invention concerne un kit de mesure de la concentration d'antigène ou de détection d'antigène comprenant une protéine de liaison à l'antigène comprenant un polypeptide comprenant un domaine variable de chaîne légère d'anticorps et un polypeptide comprenant un domaine variable de chaîne lourde d'anticorps, l'un ou les deux, du polypeptide comprenant un domaine variable de chaîne légère d'anticorps et du polypeptide comprenant un domaine variable de chaîne lourde d'anticorps, étant marqués par un marqueur fluorescent, et le kit de mesure de la concentration d'antigène ou de détection d'antigène étant caractérisé en ce que lorsque la protéine de liaison à l'antigène se lie à l'antigène à tester et forme un complexe, le complexe formé par l'antigène et la protéine se liant à l'antigène devient un inhibiteur de fluorescence (quencher) du marqueur fluorescent, la concentration de l'antigène dans la phase liquide et l'intensité de fluorescence du marqueur fluorescent étant corrélées négativement, et le marqueur fluorescent étant plus fortement inhibé lorsque le complexe formé par l'antigène et la protéine se liant à l'antigène est formé. L'intensité de fluorescence étant en conséquence diminuée, il est possible de mesurer la concentration d'antigène ou de visualiser l'antigène sur la base de la fluorescence mesurée ou détectée, en utilisant comme indicateur de ceux-ci la corrélation négative existant entre la concentration d'antigène dans la phase liquide et l'intensité de fluorescence du marqueur fluorescent.
PCT/JP2015/085909 2014-12-24 2015-12-24 Méthode d'analyse d'immunomarquage par fluorescence à l'aide de protéines se liant à l'antigène, comprenant un polypeptide comprenant un domaine anticorps variable à marquage fluorescent Ceased WO2016104549A1 (fr)

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CN110455765A (zh) * 2019-08-29 2019-11-15 中国科学院深圳先进技术研究院 一种应用于细胞体的多色荧光蛋白浓度的检测方法及设备
CN110455765B (zh) * 2019-08-29 2021-11-19 中国科学院深圳先进技术研究院 一种应用于细胞体的多色荧光蛋白浓度的检测方法及设备

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