WO2009011500A2 - Procédés pour diminuer de faux signaux dans un essai immunologique pour détecter une forme multimère d'une forme monomère de polypeptides formant des multimères - Google Patents

Procédés pour diminuer de faux signaux dans un essai immunologique pour détecter une forme multimère d'une forme monomère de polypeptides formant des multimères Download PDF

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Publication number
WO2009011500A2
WO2009011500A2 PCT/KR2008/003532 KR2008003532W WO2009011500A2 WO 2009011500 A2 WO2009011500 A2 WO 2009011500A2 KR 2008003532 W KR2008003532 W KR 2008003532W WO 2009011500 A2 WO2009011500 A2 WO 2009011500A2
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antibody
detection antibody
multimer
biosample
capturing
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PCT/KR2008/003532
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English (en)
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WO2009011500A3 (fr
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Hyun-Jung Oh
Kun-Taek Lim
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Peoplebio, Inc.
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Publication of WO2009011500A2 publication Critical patent/WO2009011500A2/fr
Publication of WO2009011500A3 publication Critical patent/WO2009011500A3/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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2828—Prion diseases

Definitions

  • the present invention relates to a method for decreasing a false signal in immunoassay to detect a multimeric form of a multimer-forming polypeptide by an antibody and a method for differentially detecting a multimeric form from a monomeric form of a multimer-forming polypeptide in a biosample therewith.
  • a multimerization of polypeptides constituting proteins has been generally known to be required for the function of proteins.
  • the multimeric forms often cause diseases or disorders in some proteins.
  • a protein exists as a monomer in normal conditions and is converted to a multimer (or aggregate form) in abnormal conditions (e.g., by the conversion to a misfolding form).
  • the diseases or disorders are caused by abnormal aggregation via misfolding of proteins including A ⁇ peptide and tau protein implicated in Alzheimer's disease, prion implicated in Spongiform encephalopathy, ⁇ -synuclein relating to Parkinson's disease, Ig light chains to primary systemic amyloidosis, serum amyloid A to secondary systemic amyloidosis, tau protein to Fronto-temporal dementia, transthyretin to senile systemic amyloidosis, transthyretin to familial amyloid polyneuropathy, cystatin C to hereditary cerebral amyloid angiopathy and ⁇ 2 -microglobulin to hemodialysis-associated amyloidosis.
  • proteins including A ⁇ peptide and tau protein implicated in Alzheimer's disease, prion implicated in Spongiform encephalopathy, ⁇ -synuclein relating to Parkinson's disease, Ig light chains to primary systemic amyloidosis, serum amyloid
  • the multimer-forming polypeptides are considered a crucial pathological factor in various diseases or disorders; however there has not yet been developed any method to differentially detect the multimeric form from the monomeric form of multimeric-forming polypeptides.
  • the present inventors have developed the system for specifically detecting a multimeric form of multimeric-forming polypeptides of which the monomeric form was converted into the polymeric form in a particular disease state.
  • the present inventors have made intensive studies to remarkably decrease these false signals. As results, it was confirmed that these false signals were significantly decreased where the immunoassay had been carried out after pretreating a biosample with NADPH.
  • a method for decreasing false signals in immunoassay to detect a multimeric form of multimer-forming polypeptides with an antibody which comprises the step of contacting a biosample to NADPH (Nicotiamide adenine dinucleotide phosphate (reduced form)).
  • a method for differentially detecting a multimeric form from a monomeric form of multimer- forming polypeptides in a biosample which comprises the steps of:
  • the present inventors have developed the system for specifically detecting a multimeric form of multimeric-forming polypeptides of which the monomeric form was converted into the polymeric form in a particular disease state.
  • the present inventors have made intensive studies to remarkably decrease these false signals. As results, it was confirmed that these false signals were significantly decreased where the immunoassay had been carried out after pretreating a biosample with NADPH.
  • the present invention provides a method for differentially detecting a multimeric form from a monomeric form of multimer-forming polypeptides.
  • the present method fundamentally uses antigen-antibody reaction and two types of antibodies, i.e., the capturing antibody and the detection antibody to cause a steric hindrance or be competitive in binding between antibodies to be bound to their epitopes.
  • the feature of the present method is that the capturing antibody and the detection antibody are contacted to samples of interest under conditions in which the contacting events occur in a three-dimensional manner.
  • the present inventors had already developed a prototype process for differentially detecting a multimeric form from a monomeric form of a multimer- forming polypeptide, called "Multimer Detection System (MDS)" and filed a patent application under PCT (PCT/KR2005/004001 with claiming priorities based on Korean Pat. Appln. No. 2005-0013877 (filed on February 19, 2005) and PCT/KR2005/000733 (filed on March 11, 2005)).
  • MDS Multimer Detection System
  • multimeric-forming polypeptide means a polypeptide capable of forming an aggregation form, particularly, following conformational change.
  • the aggregate-forming polypeptide cause the various diseases, for example, Alzheimer's disease, Spongiform encephalopathies, Parkinson's disease, primary systemic amyloidosis, secondary systemic amyloidosis, Fronto-temporal dementia, senile systemic amyloidosis, familial amyloid polyneuropathy, hereditary cerebral amyloid angiopathy, hemodialysis-associated amyloidosis, Creutzfeldt-Jakob Syndrome, huntington's disease and type II diabetes. Therefore, the term "multimeric-forming polypeptide" will be interchangeably used with the aggregate-forming polypeptide.
  • the present method uses two types of antibodies, i.e., the capturing antibody and the detection antibody.
  • the term "capturing antibody” means an antibody capable of binding to the multimer-forming polypeptide of interest in biosamples.
  • detection antibody means an antibody capable of binding to the multimer-forming polypeptide captured by the capturing antibody.
  • antibody is meant an immunoglobulin protein which is capable of binding an antigen.
  • Antibody as used herein is meant to include the entire antibody as well as any antibody fragments ⁇ e.g., ⁇ idb 1 )!, Fab', Fab, Fv) capable of binding the epitope, antigen or antigenic fragment of interest.
  • the epitopes of the capturing antibody and the detection antibody are present at the position to cause steric hindrance by the antibodies bound to their epitopes.
  • the amino acid sequence of the epitope recognized by the capturing antibody is identical to, overlapped with or adjacent to that of the epitope recognized by the detection antibody. It would be readily understood that the capturing antibody and detection antibody to be bound to their epitopes induce steric hindrance or are competitive in binding where the amino acid sequence of the epitope recognized by the capturing antibody is identical to or overlapped with that of the epitope recognized by the detection antibody.
  • the adjacent epitopes mean that one epitope in the multimer-forming polypeptide is located at a position apart from the other epitope so long as two antibodies are competitively bound to the adjacent epitopes.
  • Another feature of this invention is to bind capturing antibodies three- dimensionally to the surface of a solid phase carrier for preparing a carrier-capturing antibody conjugate. For instance, capturing antibodies are bound to the surface of three-dimensional beads for preparing a carrier-capturing antibody conjugate. Therefore, the present invention excludes capturing antibodies bound to the surface of plates because such binding is considered to be two-dimensional.
  • the capturing antibodies bound three-dimensionally to carriers allows for contacting sterically to antigens in biosamples, ensuring much more opportunities to contact to antigens in biosamples.
  • Solid phase carriers conjugated with capturing antibodies may be any materials having a steric structure, preferably, materials easily isolated or retrieved by gravity, charge or magnetic force. Most preferably, the solid phase carrier is a magnetic bead.
  • the detection antibody has a label generating a detectable signal or an affinity substance.
  • the label includes, but not limited to, an enzymatic ⁇ e.g., alkaline phosphatase, ⁇ -galactosidase, horseradish peroxidase, ⁇ -glucosidase and cytochrome P 450 ), a radioactive ⁇ e.g., C 14 , 1 125 , P 32 and S 35 ), a fluorescent ⁇ e.g., fluorescein), a luminescent, a chemiluminescent, OLED (organic light emitting diode) and a FRET (fluorescence resonance energy transfer) label.
  • an enzymatic ⁇ e.g., alkaline phosphatase, ⁇ -galactosidase, horseradish peroxidase, ⁇ -glucosidase and cytochrome P 450
  • an affinity substance bound to the detection antibody includes a biotin.
  • Various labels and methods for labeling antibodies are described in Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1999), which is incorporated herein by reference.
  • the antigen- antibody complex formed in the final step of this invention may be detected by measuring radioactivity from label.
  • the detection antibody is labeled with enzymes catalyzing colorimetric reactions
  • the antigen-antibody complex formed may be detected by use of substrates for enzymes.
  • the detection antibody is labeled with alkaline phosphatase, bromochloroindolylphosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-Bl-phosphate and ECF (enhanced chemifluorescence) may be used as a substrate for color developing reactions.
  • BCIP bromochloroindolylphosphate
  • NBT nitro blue tetrazolium
  • ECF enhanced chemifluorescence
  • the antigen-antibody complex formed may be detected by use of its binding partner linked to label generating a detectable signal (e.g., colorimetric reaction-catalyzing enzymes).
  • a detectable signal e.g., colorimetric reaction-catalyzing enzymes
  • biotin e.g., biotin
  • a streptavidin-conjugated colorimetric reaction-catalyzing enzyme e.g., horseradish peroxidase
  • the step (e) may be carried out by measuring a signal generated from the label of the detection antibody linked to multimeric form of polypeptide.
  • both the carrier-capturing antibody conjugate and detection antibody are contacted to biosamples in a simultaneous manner.
  • the capturing antibody is initially contacted and then the detection antibody is contacted to biosamples
  • free-form carrier-capturing antibody conjugates are bound to most of epitopes in the multimeric form of a polypeptide to the capturing antibody, giving rise to the steric hindrance in the binding of the detection antibody for the epitope (the epitope identical to, overlapped with or adjacent to the epitope for the capturing antibody). Therefore, where the step by step protocol is executed rather than the simultaneous protocol, the signal from the detection antibody binding becomes far poor.
  • the capturing antibody and detection antibody are simultaneously contacted to biosamples, two types of antibodies are rendered to be under competition circumstances and their binding to an antigen depends on their concentration.
  • carrier-capturing antibodies and detection antibodies refers to: (i) contacting each of the carrier- capturing antibody and detection antibody to biosamples in a simultaneous manner; or (ii) contacting a mixture containing both the carrier-capturing antibody and detection antibody to biosamples.
  • the capturing antibody bound to the carrier and the detection antibody in the step (c) are simultaneously used at 5:1-1:5 mole ratio of the capturing antibody to the detection antibody.
  • This invention may differentially detect a multimeric form from a monomeric form of any multimeric-forming polypeptides.
  • the multimeric-forming polypeptides detected by the present method are A ⁇ peptide and tau protein related to Alzheimer's disease, prion related to Spongiform encephalopathy, ⁇ -synuclein related to Parkinson's disease, Ig light chains related to primary systemic amyloidosis, serum amyloid A related to secondary systemic amyloidosis, tau protein related to Fronto-temporal dementia, transthyretin related to senile systemic amyloidosis, transthyretin related to familial amyloid polyneuropathy, cystatin C related to hereditary cerebral amyloid angiopathy or ⁇ 2 -microglobulin related to hemodialysis-associated amyloidosis.
  • the multimeric-forming polypeptide of interest is the prion protein.
  • Sporadic and familial Creutzfeldt-Jakob diseases, kuru and Gerstmann- Straussler-Scheinker syndrome in humans, scrapie in sheep and goats, feline spongiform encephalopathy in cat are the fatal neurodegenerative diseases, due to transmissible spongiform encephalopathies (TSE) (Prusiner S.B. Proc. Natl. Acad. ScL USA, 95:13363-13383(1998); and Hope J. Curr. Opin. Genet Dev., 10:568- 57(2000)).
  • TSE transmissible spongiform encephalopathies
  • Abnormal isoform or the scrapie form of prion protein (PrP ⁇ ) has been strongly suggested to the main culprit of TSE (Caughey B. Trends Biochem. ScL, 26:235-42(2001)).
  • PrP c The normal form of the prion protein (PrP c ), contains both an ⁇ -helical and a flexibly disordered portion (Zahn, R., et al., Proc. Natl. Acad. ScL USA, 97:145- 150(2000)), where the scrapie form (PrP 50 ) has highly ⁇ -sheet conformation (Caughey, B., et al., J. Biol. Chem., 273:32230-35(1998)). The conformational change from ⁇ -helical to ⁇ -sheet conformations is the central event of the disease that seems to be responsible for its neuropathology. PrP 50 is prone to form high— molecular-weight aggregates ⁇ i.e., multimer) (Bolton D. C. Lancet, 358:164-5 (2001)).
  • the present invention is significantly useful in detecting a multimeric prion, i.e., PrP Sc formed by conformational change of prion proteins.
  • the monomeric form is PrP c (cellular or normal form of prion) and the multimeric form is PrP Sc (scrapie or infectious form of prion).
  • One of the features of this invention is to employ antibodies which are bound to epitopes having non-repeated sequence in an antigen molecule. Unless epitopes recognized by antibodies have a non-repeated sequence, the present invention may not effectively detect a multimeric form from a monomeric form of a multimer- forming polypeptide. According to a preferred embodiment, the epitope specifically recognized by the capturing antibody and/or the epitope specifically recognized by the detection antibody are not repeated in the multimer-forming polypeptide.
  • the antibodies used in this invention could be prepared according to conventional techniques such as a fusion method (Kohler and Milstein, European Journal of Immunology, 6:511-519(1976)), a recombinant DNA method (USP 4,816,56) or a phage antibody library (Clackson et al, Nature, 352:624-628(1991); and Marks et al, J. MoI. Biol., 222:58, 1-597(1991)).
  • a fusion method Kelham and Milstein, European Journal of Immunology, 6:511-519(1976)
  • USP 4,816,56 recombinant DNA method
  • a phage antibody library a phage antibody library
  • the general procedures for antibody production are minutely described in Harlow, E.
  • Biosample as used herein means an organism-originated sample of material to be tested.
  • the biosample refers to any cell, tissue, or fluid from a biological source.
  • the biosample is a body fluid sample including blood, serum, plasma, lymph, milk, urine, feces, saliva, brain extracts (e.g., brain homogenates) and spinal cord fluid (SCF).
  • the biosample is a brain homogenate, blood and plasma, most preferably, plasma.
  • PK protease K
  • trypsin ase K
  • the present method further comprises the step of pretreating the biosample with sarkosyl or Triton series ⁇ e.g., Triton X-100) detergent.
  • a method for differentially detecting a multimeric form from a monomeric form of multimer- forming polypeptides in a biosample which comprises the steps of: (a) preparing a magnetic bead-capturing antibody conjugate by binding a capturing antibody to the surface of a magnetic bead in a three dimensional manner, wherein the capturing antibody is capable of recognizing an epitope on the multimer-forming polypeptide;
  • the present invention will be described in more detail as follows:
  • the capturing antibodies on magnetic beads and the HRP-detection antibodies are simultaneously contacted to biosamples containing PrP c and PrP Sc , and HRP- conjugated detection antibodies cannot be bound to magnetic bead-capturing antibody-bound PrP c but bound only to magnetic bead-capturing antibody-bound
  • PrP Sc In addition, magnetic bead-capturing antibodies cannot be also bound to HRP- conjugated detection antibody-bound PrP c .
  • the epitopes to the capturing antibody and detection antibody have a non-repeated sequence in the prion protein.
  • the amino acid sequence of the epitope recognized by the capturing antibody is identical to, overlapped with or adjacent to that of the epitope recognized by the detection antibody. Since the epitope recognized by the detection antibody is occupied by the capturing antibody, the detection antibody cannot be bound to PrP c having only one epitope. However, since the multimeric prion protein, PrP 50 contains a plurality of certain epitope, the detection antibody can be bound to capturing antibody-bound PrP Sc .
  • a magnetic field is applied to the reaction resultant to collect magnetic beads, followed by washing the collected beads.
  • the color-, fluorescence- or luminescence-developing reactions are induced using HRP substrates and their results are measured, providing qualitative and quantitative analysis data to verify whether the PrP Sc -antibody complex is formed.
  • the capturing antibody bound to the carrier and the detection antibody are contained at 5:1-1:5 mole ratio of the capturing antibody to the detection antibody in the form of cocktail.
  • the kit may further comprise magnetic plate, buffer, color-developing enzymes and substrates.
  • the MDS-3D System of this invention is classified into MDS-3D Single Bead System and MDS-3D Dual Bead System.
  • the MDS-3D Single Bead System as described above uses capturing antibody- conjugated beads and the MDS-3D Dual Bead System uses both capturing antibody- conjugated beads and detection antibody-conjugated beads.
  • the MDS-3D Dual Bead System is more advantageous than the MDS-3D Single Bead System in view of intensity of the signals.
  • the detection antibody is three- dimensionally ⁇ i.e., steric) linked to the surface of a solid phase carrier.
  • the detection antibodies bound to carriers permit to contact to multimeric polypeptides in a three-dimensional and concentrated manner, contributing to increase in contacting opportunities of the antibodies to multimeric polypeptides.
  • Solid phase carriers conjugated with detection antibodies may be any material having three-dimensional structure, preferably, materials isolatable by gravity, charge or magnetic force. Most preferably, the solid phase carrier is a latex bead.
  • the carrier may be labeled. Where the carrier has a label ⁇ e.g., the carrier is a latex bead containing fluorescent substance), a detectable signal generated from the carrier (indicative of the presence of multimeric polypeptides) may be obtained without labeling ⁇ e.g., HRP) detection antibodies.
  • the MDS-3D Dual Bead System is classified into "MDS-3D Dual Bead System with Single Label” and "MDS-3D Dual Bead System with Double Label”.
  • either detection antibody or carrier has a label generating a detectable signal. Both detection antibody and carrier have a label generating a detectable signal in the MDS-3D Dual Bead System with Double Label.
  • Double labeling strategy enables to dually check a signal generated from carrier and a signal generated from the detection antibody.
  • the MDS-3D Dual Bead System with Single Label will be described in more detail as follows.
  • the capturing antibodies on magnetic beads and Fluor-detection antibodies (detection antibodies linked to latex beads containing fluorescent substance) are simultaneously contacted to biosamples containing PrP c and PrP 50 , and Fluor-detection antibodies cannot be bound to magnetic bead-capturing antibody-bound PrP c but bound only to magnetic bead-capturing antibody-bound PrP 50 .
  • the epitopes to the capturing antibody and detection antibody have a non- repeated sequence in the prion protein.
  • the amino acid sequence of the epitope recognized by the capturing antibody is identical to, overlapped with or adjacent to that of the epitope recognized by the detection antibody.
  • the detection antibody Since the epitope recognized by the detection antibody is occupied by the capturing antibody, the detection antibody cannot be bound to PrP c having only one epitope. However, since the multimeric prion protein, PrP 50 contains a plurality of certain epitope, the detection antibody can be bound to capturing antibody-bound PrP 50 .
  • a magnetic field is applied to the reaction resultant to collect magnetic beads, followed by washing the collected beads. Measurements are carried out to analyze fluorescence intensities, verifying whether the PrP ⁇ -antibody complex is formed.
  • the MDS-3D Dual Bead System with Double Label will be described in more detail as follows.
  • the capturing antibodies on magnetic beads and Fluor-HRP- detection antibodies (HRP-conjugated detection antibodies linked to latex beads containing fluorescent substance) are simultaneously contacted to biosamples containing PrP c and PrP ⁇ , and Fluor-detection antibodies cannot be bound to magnetic bead-capturing antibody-bound PrP c but bound only to magnetic bead- capturing antibody-bound PrP 50 .
  • the epitopes to the capturing antibody and detection antibody have a non-repeated sequence in the prion protein.
  • the amino acid sequence of the epitope recognized by the capturing antibody is identical to, overlapped with or adjacent to that of the epitope recognized by the detection antibody.
  • the detection antibody Since the epitope recognized by the detection antibody is occupied by the capturing antibody, the detection antibody cannot be bound to PrP c having only one epitope. However, since the multimeric prion protein, PrP 50 contains a plurality of certain epitope, the detection antibody can be bound to capturing antibody-bound PrP Sc . After the antigen-antibody reaction, a magnetic field is applied to the reaction resultant to collect magnetic beads, followed by washing the collected beads. Measurements are carried out to analyze fluorescence intensities and HRP reactions, verifying whether the PrP ⁇ -antibody complex is formed.
  • the biosample in the step (c) contacts with (i) NADPH and (ii) a metal ion selected from copper, znic, nickel and manganese. More preferably, the metal ion is copper.
  • NADPH is used in a concentration of 0.4-2 mM, more preferably 0.8-1.2 mM.
  • copper is used in a concentration of 30-100 ⁇ M, more preferably 70-90 ⁇ M.
  • the present invention provides a method for decreasing false signals in immunoassay to detect a multimeric form of multimer- forming polypeptides with an antibody and a method for differentially detecting a multimeric form from a monomeric form of multimer-forming polypeptides in a biosample.
  • the present invention may differentially detect a multimeric form from a monomeric form of multimer-forming polypeptides easily in a high-throughput manner.
  • the present invention may detect a multimeric form of multimer-forming polypeptides associated with various diseases in a high-throughput manner and particularly is significantly useful in detecting a scrapie form of prion.
  • Fig. Ia represents a schematic diagram for an operation principle of a MDS- 3D-Single Bead (Multimer Detection System-3 Dimensional-Single Bead) system of this invention.
  • Rg. Ib represents a schematic diagram for an operation principle of a MDS- 3D-Single Bead (Multimer Detection System-3 Dimensional-Single Bead) system of this invention.
  • Rg. Ib represents a schematic diagram for an operation principle of a MDS- 3D-Single Bead (Multimer Detection System-3 Dimensional-Single Bead) system of this invention.
  • Rg. Ib represents a schematic diagram for an operation principle of a MDS-
  • Fig. Ic represents a schematic diagram for an operation principle of a MDS- 3D-Dual Bead system with Double Label of this invention.
  • Fig. 2 represents the results to detect PrP Sc according to the MDS-3D-Single Bead system after pretreating a plasma sample with CuCI 2 and NADPH.
  • Fig. 3 is a graph representing effects of NADPH in the pretreatment of the plasma sample.
  • CuCI 2 solution (8 mM) was prepared by dissolving CuCI 2 (Sigma) in TBST buffer.
  • NADPH solution 100 mM was prepared by dissolving NADPH (Sigma) in
  • TBST buffer Prion sample was prepared by mixing a plasma sample containing the multimeric form of PrP with 8 mM CuCI 2 (in TBST) and 100 mM NADPH (in TBST).
  • the sample was vortexed briefly, incubated for 1 hr at 37°C, centrifuged at 7,000 xg for 1 min, and added with 160 ⁇ l Triton X-100. Finally, the reaction solution was vortexed briefly and then centrifuged at 7,000 xg for 1 min.
  • Capturing antibody-bound magnetic beads were prepared in which 1 ⁇ g of capturing antibodies was incubated with 2.5 ⁇ l of magnetic beads.
  • the magnetic bead-bound capturing antibody is 3E7 monoclonal antibody. The epitope against the
  • 3E7 monoclonal antibody is an amino acid sequence spanning amino acid 140-160 (with reference to a bovine prion sequence) which is not repeated in PrP c .
  • the sample was incubated with the magnetic bead-capturing antibody conjugate and the detection antibody simultaneously to determine whether the multimeric sheep PrP might be detected.
  • T2-HRP was used as detecting antibodies.
  • the T2 monoclonal antibody is described in Hiroko Hayashi, et al., J. Vet. Med. Sci., 66(5): 515-520(2004), specifically recognizing PrPi 35 - I40 epitope.
  • the capturing antibody-conjugated magnetic bead and the detection antibody were simultaneously added to the sample and incubated for 1 hr at 37°C.
  • the magnetic field was then applied to the reaction mixture to separate magnetic beads, followed by washing the beads three times with TBST.
  • the ECL (enhanced chemiluminescence) detection was carried out. The results are shown in Fig. 2 and Fig. 3.
  • Fig. 3 is a graph representing the effect of NADPH in the pretreatment of the plasma sample. As shown in Fig. 3, NADPH has no effect on the signals for PrP 50 in the plasma but significantly contributes to reduction in the signals from the normal plasma.

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Abstract

La présente invention propose un procédé pour diminuer de faux signaux dans un essai immunologique pour détecter une forme multimère de polypeptides formant des multimères avec un anticorps, qui consiste à mettre en contact un bio-échantillon avec du NADPH (nicotinamide adénine dinucléotide phosphate (forme réduite)), et un procédé pour détecter de manière différentielle une forme multimère d'une forme monomère de polypeptides formant des multimères dans un bio-échantillon. La présente invention permet de détecter de façon différentielle une forme multimère d'une forme monomère de polypeptides formant des multimères facilement et à haut débit. La présente invention permet de détecter à haut débit une forme multimère de polypeptides formant des multimères associés à diverses maladies et ladite invention est en particulier très efficace pour détecter une forme polio-encéphalomyélite de prion.
PCT/KR2008/003532 2007-07-16 2008-06-20 Procédés pour diminuer de faux signaux dans un essai immunologique pour détecter une forme multimère d'une forme monomère de polypeptides formant des multimères WO2009011500A2 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017138497A1 (fr) * 2016-02-08 2017-08-17 シスメックス株式会社 Méthode de détection d'analyte et kit de réactifs permettant de détecter un analyte

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6846640B2 (en) * 2002-04-30 2005-01-25 Pharmacia & Upjohn Company Time-resolved fluorescence assay for the detection of multimeric forms of A-beta 1-40

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6846640B2 (en) * 2002-04-30 2005-01-25 Pharmacia & Upjohn Company Time-resolved fluorescence assay for the detection of multimeric forms of A-beta 1-40

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
A.BELLON ET AL.: 'Improved conformation-dependent immunoassay:suitability for human prion detection with enhanced sensitivity' J.GEN.VIOL. vol. 84, 2003, pages 1921 - 1925, XP1921 *
E.BIRKMANN,ET AL.: 'Counting of single prion particles bound to a capture-antibody surface(surface-FIDA).' VET.MICROBIOL. vol. 123, no. 4, 07 April 2007, pages 294 - 304 *
J.-I.KIM, ET AL.: 'Simple and specific detection of abnormal prion protein by a magnetic bead-base immunoassay coupled with laser-induced fluorescence spectrofluorometry' JOURNAL OF NEUROIMMUNOLOGY vol. 158, no. 1, 2005, pages 112 - 119 *
V.C.SERBEC, ET AL.: 'Monoclonal antibody against a peptide of human prion protein discriminates between Creutzfeldt-Jacob's disease-affected and normal brain tissue.' THE JOURNAL OF BIOLOGICAL CHEMISTRY vol. 279, no. 5, 30 January 2004, pages 3694 - 3698 *

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WO2017138497A1 (fr) * 2016-02-08 2017-08-17 シスメックス株式会社 Méthode de détection d'analyte et kit de réactifs permettant de détecter un analyte
JPWO2017138497A1 (ja) * 2016-02-08 2018-02-22 シスメックス株式会社 被検物質の検出方法および被検物質の検出用試薬キット

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