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WO2007015509A1 - Reactif pour dosage immunologique - Google Patents

Reactif pour dosage immunologique Download PDF

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Publication number
WO2007015509A1
WO2007015509A1 PCT/JP2006/315283 JP2006315283W WO2007015509A1 WO 2007015509 A1 WO2007015509 A1 WO 2007015509A1 JP 2006315283 W JP2006315283 W JP 2006315283W WO 2007015509 A1 WO2007015509 A1 WO 2007015509A1
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WO
WIPO (PCT)
Prior art keywords
antigen
antibody
immunoassay
fluorescent
binding fragment
Prior art date
Application number
PCT/JP2006/315283
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English (en)
Japanese (ja)
Inventor
Aki Iwasaki
Koji Suzuki
Takayuki Ishiuchi
Hiroko Kogo
Original Assignee
Japan Science And Technology Agency
Keio University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Japan Science And Technology Agency, Keio University filed Critical Japan Science And Technology Agency
Publication of WO2007015509A1 publication Critical patent/WO2007015509A1/fr

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    • 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

Definitions

  • the present invention relates to a competitive immunoassay reagent using fluorescence resonance energy transfer (FRET).
  • FRET fluorescence resonance energy transfer
  • an immunoassay method using an antibody against the protein is widely used.
  • 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. .
  • a labeled antibody to act on the tissue specimen, the localization of the target protein in the tissue or cells can be observed.
  • GFP green fluorescent protein
  • the target protein 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.
  • 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,
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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 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.
  • an immunoassay reagent that enables a rapid and simple immunoassay has been provided.
  • 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.
  • 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.
  • 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.
  • 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. ! / ⁇ . 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.
  • the antibody 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.
  • the antibody used in the present invention or an antigen-binding fragment thereof may be a Fab fragment or F () having binding properties to the antibody molecule itself and the corresponding antigen. ab ')
  • 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.
  • 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.
  • 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.
  • FRET itself is well known, and donors and acceptors for FRET Are commercially available.
  • 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.
  • CFP CFP
  • cyan fluorescent protein ⁇ YFP
  • yellow fluorescent protein ⁇ GFP
  • BFP blue fluorescent protein
  • 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.
  • a fluorescent protein 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
  • 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.
  • 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.
  • 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. .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • glycine is preferable.
  • FIG. 1 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 ").
  • the fluorescent protein linked to the antigen site is the FRET donor
  • the fluorescent protein linked to the antibody site is the FRET acceptor.
  • the fusion protein 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.
  • 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
  • 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.
  • 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.
  • 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. .
  • 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.
  • the donor and acceptor in the same amount, and the knock ground can be suppressed, which is preferable.
  • 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.
  • 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. .
  • the FLAG peptide was conjugated with a linker. Details will be described below.
  • 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.
  • AMV TAKARA RNA PCR Kit
  • AMV Takara Shuzo
  • oligo_dT primer One was used to obtain cDNA from total RNA by reverse transcription.
  • 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 .
  • 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.
  • 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.
  • each fragment was reacted with Xbal at 37 ° C for 1 hour, and each treated fragment was electrophoresed and recovered from the gel.
  • the reaction was performed at 16 ° C for a whole day and night.
  • 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).
  • 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 ⁇ l 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
  • each tool was washed 3 times with 200 ⁇ 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.
  • PBS-T Tween20-containing phosphate buffer
  • BSA urine serum albumin
  • 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.
  • 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.
  • 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 ctt gta gtc cat eg— 3 (3 ⁇ 4 ⁇ 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.
  • 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.
  • 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! / ⁇ was measured sample (blank). The results are shown in Figure 3.
  • the immunoassay reagent of the present invention enables visualization of foreign proteins, and is particularly useful for basic research such as protein functional analysis.
  • 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.

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Abstract

Réactif pour dosage immunologique, rapide et pratique, permettant de faire l'économie de l'opération de lavage généralement requise par ce type de dosage. A cette fin, on utilise un réactif pour essai immunologique contenant un anticorps ou un fragment de liaison à l'antigène de l'anticorps appelé à être utilisé dans le dosage conjointement avec un antigène à analyser qui sont liés respectivement là des substances fluorescentes en relation FRET (fluorescence de transfert de l'énergie de résonance) l'une par rapport à l'autre. Lorsque l'on met le réactif en contact avec un spécimen, on peut sauter les opérations de liaison et de lavage normalement requises. De plus, l'anticorps ou un fragment de liaison à l'antigène de cet anticorps est lié à l'antigène via un lieur, des protéines fluorescentes sont utilisées comme substances fluorescentes et la totalité des éléments constitutifs est généralement réunie sous la forme d'une protéine fusionnée en tant que molécule unique. Il est ainsi possible de visionner un antigène étranger dans une cellule.
PCT/JP2006/315283 2005-08-03 2006-08-02 Reactif pour dosage immunologique WO2007015509A1 (fr)

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JP2005225577A JP2007040834A (ja) 2005-08-03 2005-08-03 免疫測定用試薬

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Cited By (2)

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US8236575B2 (en) 2007-11-22 2012-08-07 Fujifilm Corporation Carrier for analysis of an analyte and process for producing the same
WO2015124690A1 (fr) * 2014-02-24 2015-08-27 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Composition à paire-fret de géométrie définie

Families Citing this family (3)

* Cited by examiner, † Cited by third party
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KR101429247B1 (ko) * 2011-06-23 2014-08-12 한국생명공학연구원 ｉＦＲＥＴ용 탐침 및 이의 용도
US20140231274A1 (en) * 2011-11-22 2014-08-21 Panasonic Corporation Single molecule detection method and single molecule detection apparatus for biological molecule, and disease marker testing apparatus
JP6551923B2 (ja) * 2014-05-12 2019-07-31 学校法人東日本学園 被検物質測定ｆｒｅｔ分子センサー

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JP2003042955A (ja) * 2001-07-30 2003-02-13 Sekisui Chem Co Ltd 分子認識蛍光体、それを用いた標的物質の測定方法
JP2003513271A (ja) * 1999-11-05 2003-04-08 アイシス・イノベーション・リミテッド 汎用蛍光センサー
JP2006505774A (ja) * 2002-11-07 2006-02-16 エラスムス　ユニフェルシテイト　ロッテルダム 腫瘍特異的融合蛋白質の検出のための方法およびプローブ
JP2006506634A (ja) * 2002-11-18 2006-02-23 ヴァルション テクニリネン ツッキムスケスクス 小さな分析物のための非競合的な免疫アッセイ法

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Publication number Priority date Publication date Assignee Title
JP2001124770A (ja) * 1999-08-18 2001-05-11 Japan Science & Technology Corp 物質の濃度測定方法
JP2003513271A (ja) * 1999-11-05 2003-04-08 アイシス・イノベーション・リミテッド 汎用蛍光センサー
JP2003042955A (ja) * 2001-07-30 2003-02-13 Sekisui Chem Co Ltd 分子認識蛍光体、それを用いた標的物質の測定方法
JP2006505774A (ja) * 2002-11-07 2006-02-16 エラスムス　ユニフェルシテイト　ロッテルダム 腫瘍特異的融合蛋白質の検出のための方法およびプローブ
JP2006506634A (ja) * 2002-11-18 2006-02-23 ヴァルション テクニリネン ツッキムスケスクス 小さな分析物のための非競合的な免疫アッセイ法

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8236575B2 (en) 2007-11-22 2012-08-07 Fujifilm Corporation Carrier for analysis of an analyte and process for producing the same
WO2015124690A1 (fr) * 2014-02-24 2015-08-27 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Composition à paire-fret de géométrie définie

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