WO2007015509A1

WO2007015509A1 - Immunoassay reagent

Info

Publication number: WO2007015509A1
Application number: PCT/JP2006/315283
Authority: WO
Inventors: Aki Iwasaki; Koji Suzuki; Takayuki Ishiuchi; Hiroko Kogo
Original assignee: Japan Science And Technology Agency; Keio University
Priority date: 2005-08-03
Filing date: 2006-08-02
Publication date: 2007-02-08
Also published as: JP2007040834A

Abstract

A reagent for a rapid and convenient immunoassay method whereby the washing step generally required in immunoassay methods can be omitted. Use is made of an immunoassay reagent containing an antibody or an antigen-binding fragment thereof to be used in the immunoassay together with an antigen to be assayed which are respectively bound to fluorescent substances undergoing FRET with each other. By contacting the reagent with a specimen, the binding and washing steps generally required in immunoassay methods can be omitted. Further, the antibody or an antigen-binding fragment thereof is bound to the antigen via a linker and fluorescent proteins are employed as the fluorescent substances and all of the constituting elements are made into a fused protein as a single molecule. Thus, a foreign antigen can be visualized in a cell.

Description

Specification

Reagent for immunoassay

Technical field

[0001] The present invention relates to a competitive immunoassay reagent using fluorescence resonance energy transfer (FRET). Background art

[0002] In basic research and clinical examinations, as a method for detecting a protein, an immunoassay method using an antibody against the protein is widely used. For example, the ELISA method is widely used for basic research, clinical examination, environmental research, and the like as a method capable of quantifying a target protein with high sensitivity. The Western Plot method is a method that detects specific proteins using a combination of the excellent resolution of electrophoresis and the high and specificity of antigen-antibody reaction, and the protein mixture force, and is mainly used in basic research. . In addition, by allowing a labeled antibody to act on the tissue specimen, the localization of the target protein in the tissue or cells can be observed.

However, these immunochemical methods require complicated operations such as antibody binding and subsequent washing.

[0004] On the other hand, methods using various fluorescent proteins typified by green fluorescent protein (GFP) are known as methods for observing protein dynamics in vivo. Since these fluorescent proteins can be expressed in vivo by genetic manipulation, they are useful for observing the localization and dynamics of endogenous proteins in tissues and cells.

[0005] However, in the method using such a fluorescent protein, the target protein must be expressed in a form fused with the fluorescent protein in the living cell!

The foreign antigen in the cell cannot be visualized.

[0006] Non-Patent Document 1: Science. 1999 Oct 29; 286 (5441): 952-4., TCR-Mediated internalizatio n of peptide— MHC complexes acquired by T cells. Huang JF, Yang Y, Sepulveda H,

Shi W, Hwang I, Peterson PA, Jackson MR, Sprent J, Cai Z. Disclosure of the invention

Problems to be solved by the invention

[0007] An object of the present invention is to provide a reagent for a rapid and simple immunoassay that can omit a washing step generally required for an immunoassay. Furthermore, another object of the present invention is to provide an immunoassay reagent capable of visualizing a foreign antigen in a cell.

Means for solving the problem

[0008] As a result of intensive research, the inventors of the present application have made fluorescent substances that cause FRET bind to the antibody or antigen-binding fragment thereof used for immunoassay and the antigen to be measured, respectively. It was found that by using a coexisting reagent as an immunoassay reagent and bringing it into contact with a specimen, the binding and washing steps generally required for immunoassay methods can be omitted. In addition, the antibody or antigen-binding fragment thereof and the antigen are bound via a linker, and the entire component is prepared as a single molecule fusion protein by using a fluorescent protein as a fluorescent substance. The inventors found that it is possible to visualize foreign antigens and completed the present invention.

That is, the present invention relates to an antibody or antigen-binding fragment thereof, a first fluorescent substance bound to the antibody or antigen-binding fragment thereof, and an antigen-antibody reaction with the antibody or antigen-binding fragment thereof. An immunoassay reagent comprising an antigen and a second fluorescent substance bound to the antigen, wherein the antibody or antigen-binding fragment thereof and the antigen are linked to each other to form one molecule, and the antibody Alternatively, the present invention provides an immunoassay reagent in which fluorescence resonance energy transfer occurs between the first and second fluorescent substances when an antigen-antibody reaction occurs between the antigen-binding fragment thereof and the antigen. The present invention also includes a step of bringing the immunoassay reagent of the present invention into contact with a test sample that may contain a test substance that competes with the antigen, and fluorescence resonance energy transfer of the immunoassay reagent at that time A method for measuring the test substance in the test sample using the change in efficiency of the test as an index is provided. Furthermore, the present invention provides a method for imaging a test substance present in a cell, which comprises contacting the reagent for immunoassay of the present invention with a test substance that competes with the antigen contained in the cell. The invention's effect

[0010] According to the present invention, while maintaining the high sensitivity of an immunoassay method using an antigen-antibody reaction, a washing step generally required in such an immunoassay method is eliminated. Thus, an immunoassay reagent that enables a rapid and simple immunoassay has been provided. Furthermore, the present invention provides for the first time an immunoassay reagent capable of visualizing a foreign antigen in a living cell using antibody specificity, which has been impossible until now.

Brief Description of Drawings

FIG. 1 is a schematic view of the structure of an immunoassay reagent constructed in an example of the present invention.

FIG. 2 is a gene map of a nucleic acid encoding an immunoassay reagent prepared in an example of the present invention.

FIG. 3 is a fluorescence spectrum of a reaction mixture prepared in Example of the present invention when reagent KCS-03 is brought into contact with FLAG peptide to cause competition.

BEST MODE FOR CARRYING OUT THE INVENTION

[0012] The immunoassay reagent of the present invention binds a fluorescent substance that causes FRET to each of an antibody or an antigen-binding fragment thereof and an antigen that reacts with the antibody or the antigen-binding fragment thereof. Including things. The antibody or antigen-binding fragment thereof and the antigen are linked together to form a molecule, and may preferably be constructed as a molecule by binding via an appropriate linker. ! / ヽ. In the state in which binding due to an antigen-antibody reaction occurs between the antibody or antigen-binding fragment thereof and the antigen, FRET occurs between the fluorescent substances bound to each antigen. On the other hand, in the state where a substance competing with the antigen (test substance) is further present, some of the antibodies or antigen-binding fragments thereof bind to the test substance by antigen-antibody reaction. Since the test substance is not fluorescently labeled, the antibody or antigen-binding fragment thereof bound to the test substance does not produce FRET. The proportion of the antibody or antigen-binding fragment thereof that binds to the test substance increases as the amount of the test substance in the test sample increases. For this reason, by observing how the FRET efficiency changes when the immunoassay reagent of the present invention is brought into contact with the test sample, the presence or absence of the test substance in the test sample and its presence are observed. The amount can be measured. “Measurement” includes both detection and quantification. [0013] The antibody used in the present invention or an antigen-binding fragment thereof (hereinafter sometimes referred to as “antibody etc.” t) may be a Fab fragment or F () having binding properties to the antibody molecule itself and the corresponding antigen. ab ')

2 includes antibody fragments (such as “antigen-binding fragments” t ぉ herein). Furthermore, antigen-binding fragments include artificial single-chain antibodies such as ScFV (single chain fragment of variable region) constructed by linking the variable regions of the L and H chains of the antibody, for example. . Single chain antibodies are preferred because they can be expressed in cells other than mammalian animals such as prokaryotic cells such as E. coli and plant cells. In addition, the production method of a single chain antibody such as ScFV itself is well known, and is also described in the following examples. The antibody or antigen-binding fragment thereof used in the present invention is not limited as long as it retains the binding property by the antigen-antibody reaction with the antigen described later. It is not limited to fragments. Since the reagent for immunoassay of the present invention performs immunoassay based on the competition method, the antibody used and the like reacts with the test substance by antigen-antibody reaction.

[0014] The antigen used in the present invention undergoes an antigen-antibody reaction with the above-described antibody or antigen-binding fragment thereof, and the antigen molecule itself that can be used as an immunogen for inducing the production of the antibody, an antigenic determinant. Includes retained antigen fragments and non-immunogenic haptens. Furthermore, the antigen used in the present invention is not limited to an immunogen or a fragment thereof that induces the production of the antibody as long as it retains the binding property of the corresponding antibody by the antigen-antibody reaction. It may cross-react with the antibody or antigen-binding fragment thereof.

[0015] The first and second fluorescent dyes used in the present invention are fluorescent dyes that cause mutual FRET. FRET is a phenomenon in which the excitation energy moves between two fluorescent dyes (donor and acceptor) with different excitation wavelengths. When light with the excitation wavelength of the donor is irradiated, FRET occurs when the donor and the acceptor are at a short distance, and the donor fluorescence decreases and the acceptor fluorescence increases, but when both are separated, FRET occurs. The donor fluorescence increases and the acceptor fluorescence decreases. Therefore, the distance between the donor and the acceptor can be known by observing the fluorescence generated when the light having the excitation wavelength of the donor is irradiated. FRET itself is well known, and donors and acceptors for FRET Are commercially available. As a donor and an acceptor, any combination can be used as long as FRET occurs between the donor and acceptor, and the donor and acceptor can be freely selected on the basis of information such as literature or commercially available products. When used in living cells, it is hopeful for low cytotoxicity \, CFP, cyan fluorescent protein) ^ YFP, yellow fluorescent protein) ^ GFP, BFP (blue fluorescent protein), and their variants A suitable fluorescent protein can be preferably used. Note that FRET between CFP-YFP, GFP-BFP, and FRET between these variants are widely known, and these fluorescent proteins are also commercially available. If a fluorescent protein is used, it can be produced as a single molecule fusion protein by linking with a labeling antibody or the like, and when used in a living cell, a gene encoding the fusion protein may be introduced. Fluorescent proteins are convenient when used in living cells. Nucleic acids encoding these fluorescent proteins are well known, and various vectors including them are also available for sale. Therefore, fluorescently labeled proteins in which fluorescent proteins are fused to desired polypeptides can be obtained from commercially available vectors. It can be easily prepared by using. In addition, you may couple | bond a donor with either an antibody etc. and an antigen.

[0016] The method of labeling a fluorescent dye with an antibody or the like or an antigen is appropriately selected depending on the type of fluorescent dye used. When the fluorescent dye is a non-peptidic compound, it can be labeled by a known method such as chemical modification by attaching a reducing group such as maleimide to the thiol-amino group of the antibody. When the fluorescent dye is a peptide compound such as a fluorescent protein, it can be produced as an antibody or a fusion protein with an antigen as described above. The method for producing the fusion protein is also described in the following examples, but is not limited thereto, and can be produced using any known method. The first and second fluorescent dyes bind to the position where FRET occurs when the antibody or the like and the antigen are bound by the antigen-antibody reaction. The fluorescent dye may be bound directly to an antibody or an antigen or an antigen via a linker (spacer). By using such a linker (spacer), it is possible to appropriately adjust the position of the fluorescent dye so that FRET occurs when the antibody and the antigen are bound by an antigen-antibody reaction. .

[0017] In the immunoassay reagent of the present invention, the antibody or the like and the antigen are linked to each other to form one molecule, and preferably, the antibody or the like and the antigen are combined via a linker to form one molecule To do. If a fluorescent protein is used as the fluorescent dye, all the components of the immunoassay reagent of the present invention can be produced as a single molecule protein. By using a single-molecule immunoassay reagent, it is not necessary to mix multiple reagent components at the time of use, and by expressing the nucleic acid encoding this in living cells, donors and acceptors can be expressed in equal amounts. Thus, the knock ground can be suppressed, which is very advantageous when the immunoassay reagent of the present invention is used in living cells.

[0018] The structure of the linker is not limited as long as the antibody or the like and the antigen have a distance sufficient to form a bond by an antigen-antibody reaction in the molecule by folding the linker part. It is not a thing. In general, if the polypeptide chain has an amino acid strength of about 3 to 200 residues, preferably about 7 to 30 residues, the distance between the antibody and the antigen is appropriately maintained, Allows antigen-antibody reaction. It also prevents non-specific binding within the linker or between the linker and other sites in the molecule, and does not affect the antigen-antibody reaction between the antibody and antigen and has a low amino acid strength. Among them, a linker having an amino acid having no side chain as a main component (ie, more than 50% in terms of the number of amino acids) such as glycine is preferable.

A preferred embodiment of the immunoassay reagent of the present invention is shown in FIG. The reagent in Fig. 1 consists of two types of fluorescent proteins (YFP and CFP in the figure) that cause FRET in each other, as well as the antibody site (ScFv in the figure) and antigen site (in the figure, "ScFv"). It is a single molecule fusion protein consisting of an antigen "). In FIG. 1, the fluorescent protein linked to the antigen site is the FRET donor, and the fluorescent protein linked to the antibody site is the FRET acceptor. In the normal state, the fusion protein is folded at the partial position of the linker, and binding between the antibody site and the antigen site is caused by the antigen-antibody reaction. As a result, the two fluorescent proteins are present in the vicinity. Then, FRET occurs and acceptor fluorescence is observed (top of Fig. 1). However, when a free antigen (test substance) is present in the system (lower part of Fig. 1), the test substance binds to the antibody site competitively with the antigen site, so that the antibody site and the antigen depend on the amount of antigen. Antigen-antibody reaction with the site is eliminated. Thus, FRET is eliminated, and the fluorescence of the acceptor decreases and the fluorescence of the donor increases. This change in fluorescence intensity enables quantitative analysis of the binding between the antibody and the antigen. [0020] In the measurement using the immunoassay reagent of the present invention, the immunoassay reagent is brought into contact with the test sample, and then irradiated with light having the excitation wavelength of the FRET donor, and the wavelength of the resulting fluorescence is measured. It is done by setting. The contact time between the immunoassay reagent and the test sample is not particularly limited, but it is usually about 5 to 120 minutes. The concentration of the reagent to be used is a force that can be appropriately set according to the expected concentration of the test substance, etc. Usually, it is about lfM to: LmM

[0021] As described above, the test substance measured by immunoassay using the immunoassay reagent of the present invention reacts with the antibody or the like in an antigen-antibody reaction. That is, as the above-described antibody or the like, an antibody that undergoes an antigen-antibody reaction with a desired test substance is employed. Further, the antigen may be the same substance as the test substance, or a substance different from the test substance as long as it has an antigen-antibody reaction with the antibody or the like (that is, has the same or similar epitope). It may be. When a test substance is present in the test sample, the test substance competes with the antigen in the immunoassay reagent of the present invention, and among the antibodies and the like in the immunoassay reagent of the present invention, Some of them bind by reacting with the original antibody. The ratio of the antibody or the like that binds to the test substance by reacting with the antigen antibody increases as the amount of the test substance in the test sample increases. On the other hand, when an antibody or the like binds to a test substance, the antigen in the reagent cannot bind to the antibody or the like, and since no fluorescent dye is bound to the test substance, FRET does not occur. Donor fluorescence increases. Therefore, the test substance can be detected or quantified by measuring the fluorescence of the test sample. When quantification is performed, fluorescence is measured as described above for a plurality of standard samples containing antigens of known concentrations, and the measurement results are plotted with the horizontal axis representing the antigen concentration in the standard sample and the vertical axis representing the measured fluorescence intensity. A calibration curve is created by plotting, and the concentration of antigen in the test sample can be quantified by applying the measured fluorescence intensity to the test sample of unknown concentration. .

[0022] Further, by expressing the immunoassay reagent of the present invention in living cells, irradiating the cells with light having an excitation wavelength of FRET donor, and observing with a fluorescence microscope equipped with an appropriate filter, The localization of the target substance in living cells can be observed. That is, the present invention also provides a method for imaging a test substance present in a cell, which comprises contacting the immunoassay reagent of the present invention with a test substance that competes with the antigen contained in the cell. Is. In this case, it is necessary to express a reagent constructed as a fusion protein with a fluorescent protein in living cells, but all the components of the immunoassay reagent can be expressed in one gene. It is possible to express the donor and acceptor in the same amount, and the knock ground can be suppressed, which is preferable. When the immunoassay reagent of the present invention expressed in a living cell encounters a test substance in the cell, the test substance and the antigen compete with each other, and an antibody or the like that binds to the test substance is also generated. When bound to the test substance, the distance between the FRET donor and the acceptor increases. Therefore, when irradiated with light of the excitation wavelength of the donor and observed, the fluorescence of the acceptor is observed at the site where there is almost no test substance, but the fluorescence of the donor is observed mainly at the site where the target antigen is present. . For example, when CFP is used as the donor and YFP is used as the acceptor, yellow to yellow-green fluorescence is observed at the site where there is almost no test substance, and blue fluorescence from CFP is observed at the site where there is a lot of test substance. Is observed.

Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to these.

Example 1

[0024] Probe construction and competition test

A probe having the structure shown in FIG. 1 was constructed. The antigen is a FLAG peptide (MDK YDDDDK (SEQ ID NO: 33), described as “antigen” in FIG. 1), and the antibody is a single chain fragment of variable region (ScFv), Using cyan fluorescent protein (CFP) as the donor and yellow fluorescent protein (YFP) as the acceptor as the photochromic dye, FLAG peptide and acceptor, ScFv and donor, and ScFv

The FLAG peptide was conjugated with a linker. Details will be described below.

[0025] 1. Production of ScFv

1) Preparation of anti-FLAG antibody cDNA

Total RNA was extracted from ⁶ cells of 4E11 cells (ATCC Cat. No. H B9259) 1.2 X 10 ⁶ which are B cell hyperpridoma producing anti-FLAG antibody using SV Total Isolation System (Promega). Immediately after extraction, TAKARA RNA PCR Kit (AMV) (Takara Shuzo) and oligo_dT primer One was used to obtain cDNA from total RNA by reverse transcription.

[0026] 2) Amplification of Fab region

A gene encoding the Fab region was specifically amplified from the obtained cDNA by PCR. Primers 1 and 2 in Table 1 were used for amplification of antibody heavy chains, and primers 3 and 4 of Table 1 were used for amplification of antibody light chains. The reaction conditions are as follows: 95 ° C for 5 minutes, 95 ° C for 1 minute, 63 ° C for 1 minute, 72 ° C for 2 minutes, 95 ° C for 1 minute, 55 ° C for 1 minute, 72 ° C for 2 minutes, then 72 ° C for 10 minutes . After electrophoresis, a target fragment of about 700 Kb was recovered from the gel by a conventional method, and cloned using pCR2.1-TOPO (Invitrogen) according to the attached manual to confirm the base sequence.

[0027] 3) Variable region amplification

In order to obtain only the variable region of the PCR product power, primers for H chain and L chain were designed and amplified as follows. Sense primer for H chain; 5'-GGC T ^ C TAG "AGC GTA ATA GGT CCG ATT TCT GGC-3 '(SEQ ID NO: 23) (Primer 5, added to Xbal site. 5) -A CGT A ^ AG CTT ATG CTT AAG GCC CAA CCG GCC 3 '(SEQ ID NO: 24) (Primer 6, Hindlll site addition), L chain sense primer; 5 ACG TA ^ A GOT TTA TTT CCA GCT TGG T CC CCC-3 '(SEQ ID NO: 25) (Primer 7, Hindlll site addition), L chain anti Sense primer; 5 ~ T ~ CTA G'AG GGT GGC GGT GGC TCG GGC GGT GGT GGG T CG GGT GGC G GC GGA TCT TCC GCT AGC GGG GAC ATT GTG-3 '(SEQ ID NO: 26) (Primer 8, Xbal site And a linker of (GGGGS) (SEQ ID NO: 34)

3 Add one). Amplification was performed by gradient PCR. The reaction conditions are as follows: 98 ° C for 10 seconds 60-70 ° C for 30 seconds — 72 ° C for 1 minute for 30 cycles, then 72 ° C for 10 minutes. The temperature of each column of gradient PCR is 60.0 ° C, 60.3 ° C, 60.8 ° C, 61.7 ° C, 62.9 ° C, 64.3 ° C, 66.0 ° C, 67.5 ° C, 68.5 ° C, 69.3 ° C , 69.8 ° C, 70.0 ° C. As a result of agarose gel electrophoresis, an H chain variable region fragment was observed near 350 Kb, and an L chain variable region fragment was observed near 400 Kb. Each fragment was also recovered for gel strength.

[0028] 4) Ligation of H chain variable region fragment and L chain variable region fragment

Each fragment was reacted with Xbal at 37 ° C for 1 hour, and each treated fragment was electrophoresed and recovered from the gel. In the system of 20 1 lye the heavy chain variable region fragment 0.78 pmol and the light chain variable region fragment 0.69 pmol. -Reacted. The reaction was performed at 16 ° C for a whole day and night. Immediately after the reaction, PCR was performed using primers 6 and 7 with the ligation sample as a saddle type (98 ° C 10 seconds 60 ° C 30 seconds 72 ° C 1 minute after 30 cycles, 72 ° C 10 minutes). Immediately after the completion of PCR, gel electrophoresis and fragment collection were performed, and TA cloning was performed by a conventional method. The cloung product was transferred to a competent cell (One shot TOP10 (Invitrogen)) by the heat shock method (42 ° C for 30 seconds) and pre-cultured according to the manual attached to the competent cell. After incubation, apply culture medium 50 1 to the surface of ampicillin-containing LB agar medium coated with 40 μl of 40 mg / ml X-gal and 40 1 of 100 mM IPTG. From this, a white single colony was selected and further cultured to recover the plasmid. The sequence of the inserted fragment is determined, and the presence of the target fragment, that is, the heavy chain variable region is upstream and the light chain variable region is downstream, and the (GGGGS) linker is

3 The presence of the fragments was confirmed through binding.

[0029] 5) Evaluation of binding ability

Confirmation of the binding ability was confirmed by ELISA. Recovered 501, which had been solvent-substituted with 50 mM bicarbonate buffer (pH 9.6), was bound overnight to a 96-well plate for ELISA. At this time, four types of concentrations were obtained: concentrated stock solution, 10-fold dilution, 100-fold dilution, and 1000-fold dilution. The next day, each tool was washed 3 times with 200 µ 1 Tween20-containing phosphate buffer (PBS-T), blocked with 1% urine serum albumin (BSA) -containing PBS for 2 hours at room temperature, and then 100 A 1: 100 diluted biotinylated FLAG was added to the wells and incubated for 2 hours at 4 ° C. Each well is then washed 3 times with 200 µl PBS-T, 50 1 of 1000-fold diluted alkaline phosphatase-labeled avidin is added, and incubated at 4 ° C for 30 minutes. Finally, ρ-nitrophenol phosphate is added. The plate was incubated for 1 hour at room temperature, and the absorbance was measured with a plate reader. As a result, it was confirmed that it has sufficient binding ability.

[0030] 2. Intramolecular competition FRET probe preparation

1) Intramolecular competition FRET probe gene construction (KCS-03)

ScFv was obtained by treating the vector of 1.4) above with Hindlll. The ScFv was ligated to the Hindlll site of the pECFP-Nl vector (Clontech). After ligation, the plasmid vector is transferred to a competent cell according to the description in 1.4), and a LB agar medium containing kanamycin is used for blue / white determination, followed by cultivation and extraction to obtain the desired plasmid. Obtained . The presence of ScFv in the plasmid was confirmed by sequencing.

[0031] The YFP gene was obtained by PCR using the pEYFP-Nl vector (Clontech) as a saddle and using primers 9 and 10 in Table 1 (98 ° C for 10 seconds 66 ° C for 30 seconds 72 ° C for 1 minute) After 30 cycles, 72 ° C for 10 minutes). The target band around 700 bp was collected from the electrophoresis gel, treated with BglII and Sad, and ligated to the ScFv-introduced pECFP-Nl vector according to the above.

[0032] The FLAG peptide was prepared as follows: 5 -gat ccg atg gac as DNA corresponding to the FLAG peptide fragment so that the sticky end complementary to BamH I was exposed in the sequence encoding the FLAG peptide. tac aag gat gac gat gac aag gga tec egg- 3 (Self number 29) and 5 — g ate ccg gga tec ctt gtc ate gtc ate ctt gta gtc cat eg— 3 (¾ 歹 iO) Each 10 1 (lOOpmol / 1) was reacted at 30 ° C. for 30 minutes to form a duplex. The fragment was ligated to the BamHI site of the above-mentioned ScFvZYFP-introduced pECFP-Nl vector plasmid to construct an intramolecular competitive FRET probe gene (Fig. 2, KCS-01) _{o In} Fig. 2, "MCS" It is a vector cloning site and functions as a linker. The MCS Shiomi-Kami line is as follows. g cta gcg cta ccg gac tea gat etc gag etc aa g ctt cga att ctg cag tcg acg gta ccg egg gcc egg gat cca ccg gtc gcc acc atg gtg (IS column number 35)

[0033] Next, the MCS of KCS-01 obtained in this way was cut out with Hindlll and BamHI, and during this time, A ATT CTG ATG GGT GGC GGT GGC TCG GGC GGT GGT GGG TCG GGT GGC GGC GGA TCT G A linker having the sequence -3 ′ (SEQ ID NO: 31) was inserted. In addition, KSC-01 is cut with Sad, and the sequence 5'- AA TTC AGA TCC GCC GCC ACC CGA CCC ACC ACC GCC CGA GCC ACC GCC ACC CAT CAG-5 '(SEQ ID NO: 32) between YFP and ScFv region (Fig. 2, KCS-03) _o

[0034] The primers used in this experiment are shown in Table 1 below.

[0035] [Table 1]

[0036] 3. Intracellular expression

The plasmid encoding KCS-03 constructed in 2 above was transferred to 293FT cells (Invitrogen) using Lipofectamine 2000 (Invitrogen). Each FRET probe and control protein was expressed in 293FT cells using 24 well plates by incubating each well with 0.8 g of plasmid and 293FT cells. The fluorescence image of the cells was observed using an inverted fluorescence microscope. As a result, it was confirmed that CFP and YFP were expressed in all cells.

[0037] 4. Competition test

Twenty-four hours after transfection, cells were treated with trypsin EDTA and planted in three 10 cm dishes. After another 24 hours, the cells were disrupted with M-PER (Piece), and 3 ml of protein solution was obtained according to the protocol. Amicon ultra (100,000 MWCO, Millipore) was centrifuged at 400 Orpm for 40 minutes, and the protein solution was concentrated about 15 times.

[0038] Each concentrated protein solution is divided into 20 1 cells, and a fluorescent plate reader (Molecular Devices SPECTRA max GEMIN lx) is used with a 384-well plate in a 40 1 volume system. sK / R). FLAG peptide (custom synthesis by SIGMA Genosys) to LxlO ⁴ or al 1x10- ³ M added 30 minutes followed by culturing, and Do include peptides! /ヽwas measured sample (blank). The results are shown in Figure 3.

[0039] From FIG. 3, the blank fluorescence YFP when (FLAG peptide was not added) was added FLAG peptide 1X10- ³ M compared to (529 nm) has decreased, the FRET is eliminated I understood. Therefore, it was clarified that immunoassay can be performed by a competitive method using this.

Industrial applicability

[0040] The immunoassay reagent of the present invention enables visualization of foreign proteins, and is particularly useful for basic research such as protein functional analysis. In addition, the competitive FRET probe of the present invention does not require the binding and washing operations that are generally required in the assembly using conventional antigen-antibody reactions such as ELISA, so that rapid measurement is possible. It is useful for clinical examinations and environmental measurements.

Claims

The scope of the claims

[1] An antibody or an antigen-binding fragment thereof, a first fluorescent substance bound to the antibody or the antigen-binding fragment thereof, an antigen that undergoes an antigen-antibody reaction with the antibody or the antigen-binding fragment thereof, and the binding to the antigen An immunoassay reagent comprising the second fluorescent substance, wherein the antibody or antigen-binding fragment thereof and the antigen are linked to each other to form one molecule, and the antibody or antigen-binding fragment thereof. Reagent for immunoassay in which fluorescence resonance energy transfer occurs between the first and second fluorescent substances when an antigen-antibody reaction occurs between the antigen and the antigen

[2] The reagent for immunoassay according to claim 1, wherein the reagent is constituted as a molecule by binding to the antibody or antigen-binding fragment thereof via the antigen carrier.

[3] The immunoassay reagent according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is a single-chain antibody.

[4] The immunoassay reagent according to any one of claims 1 to 3, wherein the first and Z or second fluorescent substances are fluorescent proteins.

[5] The immunoassay reagent according to claim 4, wherein the first and Z or second fluorescent proteins are in the form of a fusion protein with the antibody or antigen-binding fragment thereof and Z or the antigen.

[6] The antibody or the antigen-binding fragment thereof is bound via the antigen handler to form a molecule, and the first and second fluorescent materials are the first and second fluorescent substances. The immunoassay reagent according to claim 5, wherein the reagent is a fluorescent protein, and the antibody or antigen-binding fragment thereof, the first fluorescent protein, the linker, the antigen, and the second fluorescent protein are in the form of a fusion protein. .

[7] A nucleic acid encoding the fusion protein according to claim 5 or 6.

[8] A vector comprising the nucleic acid according to claim 7 and capable of expressing the nucleic acid in a host cell.

[9] The method includes a step of contacting the immunoassay reagent according to claim 1 with a test sample that may contain a test substance that competes with the antigen. A method for measuring the test substance in the test sample, using the change in the efficiency of fluorescence resonance energy transfer of the immunoassay reagent as an index.

[10] A test that exists in a cell, comprising contacting the immunoassay reagent according to any one of claims 1 to 8 with a test substance that competes with the antigen contained in the cell. Substance imaging method.

[11] The nucleic acid according to claim 7 is expressed in a cell into which the vector according to claim 8 is introduced, and the generated immunoassay reagent is brought into contact with a test substance that competes with the antigen contained in the cell. The method of claim 10, comprising: