WO2008029965A1 - Dosage de réaction simultané destiné à détecter de façon différentielle une forme multimère - Google Patents

Dosage de réaction simultané destiné à détecter de façon différentielle une forme multimère Download PDF

Info

Publication number
WO2008029965A1
WO2008029965A1 PCT/KR2006/003587 KR2006003587W WO2008029965A1 WO 2008029965 A1 WO2008029965 A1 WO 2008029965A1 KR 2006003587 W KR2006003587 W KR 2006003587W WO 2008029965 A1 WO2008029965 A1 WO 2008029965A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
entrapping
indicative
affinity molecule
multimer
Prior art date
Application number
PCT/KR2006/003587
Other languages
English (en)
Inventor
Seong Soo Alexander An
Kun Taek Lim
Hyun Jung Oh
Original Assignee
Peoplebio, Inc.
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 Peoplebio, Inc. filed Critical Peoplebio, Inc.
Priority to PCT/KR2006/003587 priority Critical patent/WO2008029965A1/fr
Publication of WO2008029965A1 publication Critical patent/WO2008029965A1/fr

Links

Classifications

    • 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
    • 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
    • G01N33/54306—Solid-phase reaction mechanisms

Definitions

  • the present invention relates to methods for differentially detecting a multimeric form from a monomeric form of a multimer-forming polypeptide and immunoassay kits therefor.
  • 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 associated with abnormal aggregation or misfolding of proteins include Alzheimer's disease, Creutzfeldt-Jakob disease, Spongiform encephalopathies, Parkinson's disease, Huntington' s disease, Amyotrophic lateral sclerosis, Serpin deficiency, emphysema, cirrhosis, Type II Diabetes, primary systemic amyloidosis, secondary systemic amyloidosis Fronto-temporal dementias, senile systemic amyloidosis, familial amyloid polyneuropathy, hereditary cerebral amyloid angiopathy and haemodialysis- related amyloidosis.
  • the normal form of the prion protein (PrP c ) contains both an ⁇ -helical and a flexibly disordered portion and exists as a monomeric form (Zahn, R., et al . , Proc. Natl. Acad. Sci. USA 97:145-150(2000)), where the scrapie form (PrP Sc ) has highly ⁇ - sheet conformations and exists as a multimeric (aggregating) or at least dimer forms (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 c is protease sensitive (PrP sen )
  • PrP Sc is partially resistant to proteolysis (PrP res ) and prone to form high- molecular-weight aggregates (Bolton D. C. Lancet, 358:164-5 (2001)). This latter feature makes it difficult to analyze the conformational transition that leads to the formation of prP res or to characterize it.
  • Fig. 1 schematically represents the process of a specific embodiment of this invention.
  • Fig. 2 represents the results of the present method depending on the amount of protease K and treatment time with protease K.
  • the experiments used the varying concentrations of protease inhibitors.
  • RLU denotes relative luminescence unit.
  • Fig. 3 represents the results of the present method depending on the amount of protease K.
  • the experiments used the constant concentration of protease inhibitors and the constant treatment time (1 hr) with protease K.
  • Fig. 4 represents the results of the present method depending on the amount of protease K and treatment time with protease K.
  • the experiments used the constant concentration of protease inhibitors.
  • Fig. 5 represents that the present method using protease inhibitors successfully generates differential signals between scrapie and normal PrP plasma samples.
  • Fig. 6 represents that the present method using protease inhibitors successfully generates differential signals between scrapie and normal PrP plasma samples.
  • Figs. 7a and 7b represent the effect of protease inhibitors on the binding of normal PrP with antibodies.
  • Fig. 8 demonstrates that protease inhibitors increase the signal intensity in a process for detecting the scrapie form of PrP using entrapping antibodies bound to a solid support, a magnetic bead.
  • a method for differentially detecting a multimeric form from a monomeric form of a multimer-forming polypeptide in a biological sample which comprises the steps of: (a) obtaining the biological sample to be analyzed; (b) preparing an entrapping antibody-affinity molecule conjugate by binding an affinity molecule to the surface of the entrapping antibody, wherein the entrapping antibody recognizes an epitope on the multimer-forming polypeptide and is not bound to a solid support; (c) preparing an indicative antibody, wherein an epitope recognized by the indicative antibody is present at a position in the multimer-forming polypeptide to cause a steric hindrance by the entrapping antibody bound to its epitope to prevent the binding of the indicative antibody to the multimer- forming polypeptide; (d) contacting the entrapping antibody- affinity molecule conjugate and the indicative antibody to the biological sample in a three dimensional manner, thereby forming an entrapping antibody-affinity
  • kits for differentially detecting a multimeric form from a monomeric form of a multimer-forming polypeptide in a biological sample which comprises: (a) an entrapping antibody- affinity molecule conjugate wherein the entrapping antibody recognizes an epitope on the multimer-forrning polypeptide and is not bound to a solid support; and (b) an indicative antibody recognizing an epitope present at a position in the multimer- forming polypeptide to cause a steric hindrance by the entrapping antibody bound to its epitope to prevent the binding of the indicative antibody to the multimer-forming polypeptide.
  • an entrapping antibody-affinity molecule conjugate for differentially detecting a multimeric form from a monomeric form of a multimer-forming polypeptide in a biological sample, wherein the entrapping antibody recognizes an epitope on the multimer-forming polypeptide and is not bound to a solid support; wherein the indicative antibody recognizes an epitope present at a position in the multimer-forming polypeptide to cause a steric hindrance by the entrapping antibody bound to its epitope to prevent the binding of the indicative antibody to the multimer-forming polypeptide.
  • the present invention is directed to a method for differentially detecting a multimeric form from a monomeric form of a multimer-forming polypeptide in a biological sample by immunoassay involving antigen-antibody reactions.
  • the present invention uses two types of antibodies, an entrapping antibody and an indicative antibody both of which are competitive in binding to a multimer-forming polypeptide. Such competitive antibody binding occurs through steric inhibition.
  • the entrapping antibody bound to an epitope on a multimer-forming polypeptide inhibits the indicative antibody from binding to its epitope on the multimer-forming polypeptide because of competition to binding sites on the multimer-forming polypeptide.
  • One of the features of the present invention is to perform the immunoassay under three-dimensional contacting circumstances. In the present invention, the entrapping and indicative antibodies are ensured to have three-dimensional contacting opportunities to antigens in biological samples.
  • the process of this invention is named "SRA (simultaneous reaction assay) system".
  • SRA simultaneous reaction assay
  • the term "simultaneous” used herein with referring to the reaction of antibodies with biological samples means that the entrapping and indicative antibodies are contacted to biological samples simultaneously. This term encompasses a situation in which the entrapping and indicative antibodies are contacted to biological samples in a short time interval so long as the two antibodies have a similar opportunity (chance) to bind to multimer-forming polypeptides in biological samples.
  • multimer-forming polypeptide refers to a polypeptide capable of forming an aggregation (i.e., multimer) form, particularly, following conformational change, causing a wide variety of diseases such as Alzheimer's disease, Creutzfeldt-Jakob disease, Spongiform encephalopathies, Parkinson's disease, Huntington' s disease, Amyotrophic lateral sclerosis, Serpin deficiency, emphysema, cirrhosis, Type II diabetes, primary systemic amyloidosis, secondary systemic amyloidosis Fronto-temporal dementias, senile systemic amyloidosis, familial amyloid polyneuropathy, hereditary cerebral amyloid angiopathy and haemodialysis-related amyloidosis. Therefore, the term “multimer-forming polypeptide” will be interchangeably used with the term “aggregate-forming polypeptide” .
  • entrapping antibody means an antibody capable of binding to the multimer-forming polypeptide of interest in biological samples and conjugated with affinity molecules for isolating an antigen-antibody complex.
  • affinity molecules for isolating an antigen-antibody complex.
  • antigens binding to the entrapping antibody-biotin conjugate may be entrapped by incubating with streptavidin-magnetic beads, after which entrapped antigens may be isolated by applying magnetic field.
  • indicator antibody means an antibody capable of binding to the multimer-forming polypeptide and capable of indicating the presence of the multimeric form of the multimer- forming polypeptide.
  • the indicative antibody bound to the multimer-forming polypeptide binding to the entrapping antibody is indicative of the presence of the multimeric form, which may be readily detected by use of a signal-generating label conjugated with the indicative 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., F(ab')2, Fab', Fab, Fv) capable of binding the epitope, antigen or antigenic fragment of interest.
  • the epitopes specifically recognized by the entrapping antibody and indicative antibody are located at positions in multimer-forming polypeptides to cause steric hindrance (competitive binding) between antibodies to be bound to the epitopes.
  • the amino acid sequence of the epitope recognized by the entrapping antibody is identical to, overlapped with or adjacent to that of the epitope recognized by the indicative antibody. It would be readily understood that the entrapping antibody and indicative 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 entrapping antibody is identical to or overlapped with that of the epitope recognized by the indicative antibody.
  • overlapped with used herein with referring to epitopes to entrapping and indicative antibodies encompasses epitopes having completely or partially overlapped amino acid sequences.
  • the epitopes to 3E7 and T2 antibodies have amino acid sequences spanning amino acid 129-148 and 135- 140, respectively of a human prion sequence.
  • Such eptiopes can also be described as completely overlapped epitopes.
  • the epitopes to 3F4 and 308 have amino acid sequences spanning amino acid 109-112 and 111-118, respectively, of a human prion sequence.
  • Such epitopes can be described as partially overlapped epitopes.
  • one epitope (e.g., epitope recognized by the entrapping antibody) in the multimer-forming polypeptide may be located at a position apart from the other epitope (e.g., epitope recognized by the indicative antibody) so long as two antibodies are competitively bound to the adjacent epitopes.
  • the amino acid sequence of the epitope recognized by the entrapping antibody is identical to or overlapped with that of the epitope recognized by the indicative antibody.
  • the amino acid sequence of the epitope recognized by the entrapping antibody is identical to or completely overlapped with that of the epitope recognized by the indicative antibody.
  • the cocktail form of several types of entrapping antibodies may be used in this invention.
  • the cocktail form of several types of indicative antibodies may be used in this invention.
  • Another feature of this invention is to utilize an entrapping antibody-affinity molecule rather than an entrapping antibody per se.
  • the affinity molecule bound to entrapping antibodies is intended to isolate an entrapping antibody- affinity molecule-multimeric form-indicative antibody complex finally formed through binding to its affinity counterpart molecule.
  • biotin bound to entrapping antibodies binds to its affinity counterpart molecule, streptavidin to form a biotin-conjugated entrapping antibodies-streptavidin complex followed by isolating the complex.
  • the affinity molecule bound to the entrapping antibody and the counterpart affinity molecule are selected from the group consisting of biotin, avidin, streptavidin, antigens, haptens, antibodies, hormones, vitamins, receptors, carbohydrates, lectins, metals, chelators, polynucleotides, cofactor or prosthetic groups, apoproteins, effecter molecules, one member of a hydrophobic interactive pair, enzyme cofactors, enzymes, polymeric acids, polymeric bases, dyes, protein binders, peptides, protein binders and enzyme inhibitors, provided that the affinity molecule and the counterpart affinity molecule are different.
  • the affinity pair includes biotin/avidin (or streptavidin) , antigens (or haptens) /antibodies, hormones (or vitamins) /receptors, carbohydrates/lectins, metals /chelators, cofactor (or prosthetic groups) /apoproteins, and enzyme cofactors/enzymes .
  • the affinity molecule bound to the entrapping antibody is biotin, avidin or streptavidin
  • the counterpart affinity molecule is biotin, avidin or streptavidin.
  • the counterpart affinity molecule is avidin or streptavidin.
  • the counterpart affinity molecule is bound to a solid support.
  • Solid support conjugated with the counterpart affinity molecule may be any materials isolatable by gravity, charge or magnetic force.
  • the solid phase carrier is a magnetic bead.
  • the step (e) for isolating an entrapping antibody- affinity molecule-multimeric form-indicative antibody complex may be carried out by applying a magnetic field to the resultant of the step (d) .
  • the isolation of antigen-antibody complexes by use of a pair of affinity molecule/counterpart affinity molecule induces concentration effect of antigen-antibody complexes to be detected.
  • the entrapping antibody and indicative antibody used in this invention are not bound to a solid support such as plates and beads .
  • the entrapping antibody and indicative antibody in a free form allows for contacting three- dimensionally to antigens in biological samples, ensuring much more opportunities to contact to antigens in biological samples.
  • the indicative 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, peroxidase, ⁇ - galactosidase and ⁇ -glucosidase) , a radioactive (e.g., I 125 and
  • a fluorescent (e.g., fluorescein), a luminescent, a chemiluminescent, an electrical and a FRET (fluorescence resonance energy transfer) label includes biotin.
  • FRET fluorescence resonance energy transfer
  • the antigen-antibody complex formed may be detected by measuring radioactivity from label.
  • the indicative antibody is labeled with enzymes catalyzing colorimetric reactions
  • the antigen-antibody complex formed may be detected by use of substrates for enzymes.
  • the indicative 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; in the case of labeled with horseradish peroxidase, chloronaphtol, aminoethylcarbazol, diaminobenzidine, D-luciferin, lucigenin (bis-IV-methylacridinium nitrate) , resorufin benzyl ether, luminol, Amplex Red reagent (10-acetyl- 3, 7-dihydroxyphenoxazine) , TMB (3, 3, 5, 5-tetramethylbenzidine) and ABTS (2, 2-Azine-di [3-ethylbenzthiazoline sulfonate]) may be used as
  • 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
  • enzyme-conjugated streptavidin may be used for detecting the antigen-antibody complex; however, in this case, a biotin/streptavidin pair should not been used as affinity molecules bound to entrapping antibodies and counterpart affinity molecules.
  • the step (f) may be carried out by measuring a signal generated from the label.
  • the step (d) is carried out by contacting the entrapping antibody-affinity molecule conjugate and the indicative antibody to the biological sample in a three dimensional manner.
  • the term "three dimensional manner" used herein with describing the contacting event of two types of antibodies means that both the entrapping antibody-affinity molecule conjugate and the indicative antibody are not bound to solid supports such as plates and beads, i.e., is in the free form, and therefore both of them are able to survey three- dimensionally their epitopes on antigens for specific binding. If either the entrapping antibody-affinity molecule conjugate or the indicative antibody is bound to plates, the three- dimensional scrutiny is not longer possible. The three- dimensional contact allows for enabling the antibodies to have much more opportunities to contact to antigens in biological samples .
  • the step (d) is carried out by contacting the entrapping antibody-affinity molecule conjugate and the indicative antibody simultaneously or in a short time interval to the biological sample. More preferably, the step (d) is carried out by contacting simultaneously the entrapping antibody-affinity molecule conjugate and the indicative antibody. In such simultaneous reactions, either the entrapping antibody or indicative antibody can be firstly bound to epitopes on multimer-forming polypeptides.
  • the entrapping antibody-affinity molecule conjugate is initially incubated with biological samples for a long period of time (e.g., 30 min) and then the indicative antibody is contacted to biological samples, the entrapping antibody- affinity molecule conjugate are bound to most of epitopes to the entrapping antibody in the multimeric form, giving rise to the substantially complete loss of the binding opportunity of the indicative antibody to epitopes.
  • the entrapping antibody and indicative antibody are contacted to biological samples simultaneously or in a short time interval, two types of antibodies is rendered to be under competition circumstances and their binding to epitopes depends on their concentration.
  • the time interval of the contacting of the entrapping antibody-affinity molecule conjugate and the indicative antibody is more than 30 min, the signal from labeled indicative antibodies becomes negligible.
  • the time interval of the contacting is 0-10 min, more preferably 0-8 min, still more preferably 0-5 min, most preferably 0-3 min.
  • the order of the contacting of the entrapping antibody-affinity molecule conjugate and the indicative antibody becomes meaningless.
  • either the entrapping antibody-affinity molecule conjugate or the indicative antibody can be firstly contacted to the biological sample.
  • the entrapping antibody-affinity molecule conjugate and the indicative antibody in the step (c) are used at 5:1-1:5 mole ratio of the entrapping antibody to the indicative antibody, more preferably, 3:1-1:3, still more preferably, 2:1-1:2, most preferably about 1:1.
  • the present invention makes it possible to differentially detect a multimeric form from a monomeric form of any multimer- forming polypeptide.
  • the multimer-forming polypeptide includes A ⁇ peptide and tau protein related to Alzheimer's disease, prion related to Creutzfeldt-Jakob disease and Spongiform encephalopathies, ⁇ - synuclein related to Parkinson' s disease Ig light chains related to primary systemic amyloidosis, serum amyloid A related to secondary systemic amyloidosis, tau related to Fronto-temporal dementias, transthyretin related to senile systemic amyloidosis, transthyretin related to familial amyloid polyneuropathy, cystatin C related to hereditary cerebral amyloid angiopathy, ⁇ 2 -microglobulin related to haemodialysis- related amyloidosis, huntingtin related to Huntington' s disease, superoxide dismutase related to Amyotrophic lateral
  • the multimer-forming polypeptide is the prion protein causing Creutzfeldt-Jakob disease and Spongiform encephalopathies .
  • 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 entrapping antibody and/or the epitope specifically recognized by the indicative 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 described in Harlow, E.
  • biological sample used herein is an organism- originated sample of material to be tested.
  • the biological sample refers to any cell, tissue, or fluid from a biological source, or any other medium that can advantageously be evaluated according to this invention, including a sample drawn from human, a sample drawn from an animal, a sample drawn from food designed for human or animal consumption.
  • the biological sample to be tested is a body fluid sample including blood, serum, plasma, lymph, milk, urine, feces, ocular fluid, saliva, semen, brain extracts (e.g., brain homogenates) , cerebral spinal fluid (CSF) , appendix, spleen and tonsillar tissue extracts.
  • CSF cerebral spinal fluid
  • the biological sample is a brain homogenate, blood, serum or plasma, still more preferably blood or plasma, most preferably, plasma.
  • the present method further comprises the step of (a-2) treating the biological sample with a protease inhibitor.
  • protease inhibitors enhances the binding of a multimeric form (e.g. PrP Sc ) with antibodies in biological samples (e.g. plasma) .
  • Protease inhibitors interfere with the binding of a normal monomer form (e.g. PrP c ) with antibodies in biological samples. Therefore, protease inhibitors increases both signal intensity and differentiation that indicate the existence of a multimeric form (e.g. PrP Sc ) .
  • the protease K treatment conventionally used for detecting PrP Sc may contribute to signal differentiation, signal intensity is decreased. In this respect, the protease K treatment is very likely to induce false negative data.
  • the present invention overcomes the shortcomings of conventional techniques described above.
  • the present method can differentiate a multimeric form (e.g. PrP Sc ) from monomer form (e.g. PrP c ) in biological samples (e.g. plasma) with no help of protease K.
  • a multimeric form e.g. PrP Sc
  • monomer form e.g. PrP c
  • biological samples e.g. plasma
  • protease inhibitor refer to any molecule or collection of molecules that are capable of interfering with the proteolytic activity of one or more proteases.
  • the protease inhibitors may be capable of inhibiting serine proteases, cysteine proteases, metalloproteases and aspartic proteases.
  • the protease inhibitor useful in this invention is a serine protease inhibitor, more preferably, 4- (2-aminoethyl) -benzenesulfonyl fluoride, leupeptin, phenylmethylsulfonyl fluoride, aprotinin, chymostatin, antithrombin III, 3, 4-dichloroisocoumarin, tosyl-
  • L-lysine chloromethyl ketone tosyl-L-phenylalanine chloromethyl ketone, diisopropyl fluorophosphates, antipain or ⁇ 2-macroglobulin, most preferably, 4- (2-aminoethyl) - benzenesulfonyl fluoride.
  • the concentration of protease inhibitors is in the range of 0.05-1.0 mM, more preferably 0.1-0.8 mM and most preferably 0.3-0.7 mM.
  • the biological sample is not treated with protease K or trypsin.
  • the present method further comprises the step of pretreating the biological sample with sarkosyl or Triton series (e.g., Triton X-100) detergent.
  • sarkosyl or Triton series e.g., Triton X-100
  • a method for differentially detecting a multimeric form from a monomeric form of a multimer-forming polypeptide in a biological sample which comprises the steps of: (a-1) obtaining the biological sample to be analyzed; (a-2) treating the biological sample with a protease inhibitor; (b) preparing an entrapping antibody-affinity molecule conjugate by binding an affinity molecule to the surface of the entrapping antibody, wherein the entrapping antibody recognizes an epitope on the multimer-forming polypeptide and is not bound to a solid support; (c) preparing an indicative antibody, wherein an epitope recognized by the indicative antibody is present at a position in the multimer-forming polypeptide to cause a steric hindrance by the entrapping antibody bound to its epitope to prevent the binding of the indicative antibody to the multimer- forming polypeptide and wherein the indicative antibody has a label generating a detectable signal; (d) contacting the entrapping
  • Fig. 1 schematically represents an example of this invention using the entrapping antibody-biotin conjugates, streptavidin-magnetic beads and HRP-conjugated indicative antibodies.
  • the entrapping antibody-biotin conjugates and the HRP-conjugated indicative antibodies are simultaneously contacted to biological samples containing PrP c and PrP Sc and both of them have opportunity to bind to their epitopes on PrP c or PrP Sc .
  • the reaction resultants are incubated with streptavidin-magnetic beads and then placed under magnetic field for isolation of an entrapping antibody-affinity molecule-multimeric form (PrP Sc ) -indicative antibody complex.
  • the process of this invention produces four types of results:
  • the present invention is directed to providing a kit for differentially detecting a multimeric form from a monomeric form of a multimer-forming polypeptide in a biological sample.
  • the kit may further comprise a counterpart affinity molecule, a counterpart affinity molecule-solid support conjugate, buffer, protease inhibitors and/or detergent.
  • the SRA SYSTEM requires no multimer-specific antibodies. For example, it is not dependent on PrP Sc -specific antibodies. Antibodies having cross-reactivity between PrP S ⁇ and PrP 0 can be successfully applied to the present invention for differentially detecting PrP Sc in biological samples;
  • the present invention does not need proteinase K (PK) treatment having been conventionally used for PrP Sc detection.
  • PK proteinase K
  • the present SRA method per se exhibits a sufficient potential to discriminate PrP So from PrP c without PK treatment;
  • the present invention enables aggregating forms (particularly, PrP Sc ) in plasma samples to be detected by immunoassay. Little has been suggested about successful detection of PrP Sc in plasma; and,
  • the present invention can carried out in a convenient and speedy manner, which enables the automation of the SRA SYSTEM.
  • the plasma was centrifuged at 1500 x g for 5 min to be used in the ELISA.
  • the samples were prepared by diluting plasma (400 ⁇ l) with 10%
  • Triton X-100 160 ⁇ l, Samchun Inc., Korea
  • dH 2 O and TBST Triton X-100
  • Proteinase K (Sigma) at final concentrations (120, 60, 30, 15 and 0 ⁇ g/ml) was added to the plasma samples and incubated at 37°C for 0.5, 1 and 2 hr.
  • protease inhibitor 4- (2-aminoethyl) -benzenesulfonyl fluoride (2.0, 1.0, 0.5 and 0 I* in dH 2 O) were added to the diluted plasma samples and incubated for 15 min at room temperature. The samples were centrifuged at 1500 x g for 5 min.
  • the SRA SYSTEM set was prepared by mixing the entrapping antibody (3E7-biotin, 0.4 ⁇ g, Roboscreen Inc. Germany) and the indicative antibody (3E7-HRP, 0.4 ⁇ g, Roboscreen Inc. Germany) in TBST (200 ⁇ l) .
  • the SRA SYSTEM set and the samples were mixed and incubated for 1 hr at
  • the antibody and antigen complex with biotin and HRP were concentrated by adding streptavidin-conjugated magnetic beads (5 ⁇ l of 1 mg/ml, Invitrogen Inc.) and incubating at room temperature for 30 min. The magnetic beads were separated from the mixture using magnet (Invitrogen Inc.). Unbound complexes were washed away with TBST and the washing procedure was repeated three times. After transferring the magnetic beads to the microtiter plate, the HRP chemiluminescent substrate (150 ⁇ l, Pico HRP substrate, Pierce Inc.) was added to detect the signal and the siqnal was measured in the Perkin Elmer Vector 3 luminomitor. As represented in Fig.
  • protease K shows the highest signal intensity and signal differentiation.
  • Fig. 3 30 ⁇ g/ml of protease K
  • UMD pooled scrapie sheep plasma (Grade A, University of Maryland, US) and the commercial sheep normal plasma (Innovative Research Inc.) were used.
  • the plasma was centrifuged at 1500 x g for 5 min to be used in the ELISA.
  • the samples were prepared by diluting plasma (400 ⁇ l) with 10% Triton X-IOO (160 ⁇ l, Samchun Inc., Korea) in dH 2 O and TBST
  • the 3E7 antibody specifically reacts with the epitope of the sequence 132-152 of sheep PrP.
  • the antibody and antigen complex with biotin and HRP were concentrated by adding streptavidin-conjugated magnetic beads (5 ⁇ l of 1 mg/ml, Invitrogen Inc.) and incubating at room temperature for 30 min. The magnetic beads were separated from the mixture using magnet (Invitrogen Inc.) . Unbound complexes were washed away with TBST and the washing procedure was repeated three times. After transferring the magnetic beads to the microtiter plate, the HRP chemiluminescent substrate (150 ⁇ l, Pico HRP substrate, Pierce Inc.) was added to detect the signal and the signal was measured in the Perkin Elmer Vector 3 luminomitor.
  • protease inhibitors [4- (2-aminoethyl) -benzenesulfonyl fluoride] could perfectly differentiate PrP Sc from PrP c in plasma as represented in Fig. 5.
  • the treatment of protease inhibitors increases both signal intensity and differentiation.
  • the SRA system of this invention using protease inhibitors successfully generates differential signals between scrapie and normal plasma samples.
  • the concentrations of protease inhibitors higher than 0.5 mM interfered with the detection of PrP Sc .
  • the protease inhibitor, 4- (2-aminoethyl) -benzenesulfonyl fluoride enhances the differentiation of PrP Sc from the normal PrP in sheep plasma.
  • Increasing concentrations of 4- (2-aminoethyl) -benzenesulfonyl fluoride further enhance the binding of PrP Sc with antibodies in sheep scrapie plasma.
  • increasing concentrations of 4- (2-aminoethyl) -benzenesulfonyl fluoride interfered with binding of normal PrP in normal sheep plasma, resulting in decreasing signal to the normal PrP.
  • EXAMPLE III Detecting PrP c in Normal Sheep Plasma in the Presence of Protease Inhibitor
  • the Control antibody set was prepared by mixing the entrapping antibody (ICSM-35-biotin, 0.1 ⁇ g, D-Gen Inc., UK) and the indicative antibody (T2-HRP, 0.1 ⁇ g, NIAH, Japan) in TBST (200 ⁇ l) .
  • the Control antibody set and the samples were mixed and incubated for 1 hr at 37 ° C .
  • the ICSM-35 antibody specifically reacts with human PrP c and PrP Sc in both native and denatured conformations.
  • the antibody also reacts with sheep, mouse, hamster and bovine PrP c and PrP Sc .
  • the epitope is defined within the sequence 93-102 of human PrP.
  • the T2 antibody (Hiroko Hayashi, et al., J. Vet. Med. Sci. r 66 (6) : 515 (2004) ) specifically reacts with the epitope of the sequence 135-140 of human PrP or the sequence of 139-144 of sheep PrP.
  • the antibody and antigen complex with biotin and HRP were concentrated by adding streptavidin-conjugated magnetic beads (5 ⁇ l of 1 mg/ml, Invitrogen Inc.) and incubating at room temperature for 30 min. The magnetic beads were separated from the mixture using magnet (Invitrogen Inc.) . Unbound complexes were washed away with TBST and the washing procedure was repeated three times. After transferring the magnetic beads to the microtiter plate, the HRP chemiluminescent substrate (150 ⁇ l, Pico HRP substrate, Pierce Inc.) was added to detect the signal and the signal was measured in the Perkin Elmer Vector 3 luminomitor.
  • the plasma was centrifuged at 1500 x g for 5 min to be used in the ELISA.
  • the samples were prepared by diluting plasma (400 ⁇ l) with 10%
  • Triton X-100 160 ⁇ l, Samchun Inc., Korea
  • dH-O and TBST Triton X-100
  • the SRA SYSTEM set was prepared by mixing the capturing antibody (3E7- biotin, 2.4, 1.6, 0.8 and 0.4 ⁇ g, Roboscreen Inc. Germany) and the indicative antibody (T2-HRP, 0.4 ⁇ g) in TBST (200 ⁇ l) .
  • the SRA SYSTEM set and the samples were mixed and incubated for 1 hr at 37 ° C.
  • the 3E7 antibody specifically reacts with the epitope of the sequence 132-152 of sheep PrP.
  • the T2 antibody specifically reacts with the epitope of the sequence 139-144 of sheep PrP.
  • the antibody and antigen complex with biotin and HRP were concentrated by adding streptavidin-conjugated magnetic beads (8- ⁇ l of 1 mg/ml, Invitrogen Inc.) and incubating at room temperature for 30 min. The magnetic beads were separated from the mixture using magnet (Invitrogen Inc.). Unbound complexes were washed away with TBST and the washing procedure was repeated three times. After transferring the magnetic beads to the microtiter plate, the HRP chemiluminescent substrate (150 ⁇ l, Pico HRP substrate, Pierce Inc.) was added to detect the signal and the signal was measured at Perkin Elmer Vector 3 luminomitor.
  • EXAMPLE V Detecting Differentially PrP Sc from PrP c in the Scrapie and Normal Sheep Plasmas Using Combinations of Entrapping and Indicative Antibodies UMD pooled scrapie sheep plasma (Grade A, University of
  • the plasma was centrifuged at 1500 x g for 5 min to be used in the ELISA.
  • the samples were prepared by diluting plasma (400 ⁇ l) with 10% Triton X-100 (160 ⁇ l, Samchun Inc., Korea) in dH 2 O and TBST (840 ⁇ l, Sigma) , followed by voltex and centrifugation.
  • Protease inhibitor, 4- (2-aminoethyl) -benzenesulfonyl fluoride at 0.5 mM in dHoO was added to the diluted plasma samples and voltexed.
  • the SRA SYSTEM set was prepared by mixing the entrapping antibody (3E7-biotin, 0.2 ⁇ g, Roboscreen Inc. Germany; and MAl-750-biotin, 0.2 ⁇ g, Bioreagents Inc. US) and the indicative antibody (T2-HRP, 0.2 ⁇ g, NIAH, Japan; 3E7-HRP, 0.2 ⁇ g, Roboscreen Inc. Germany) in TBST (200 ⁇ l) .
  • the SRA SYSTEM set and the samples were mixed and incubated for 1 hr at 37 ° C.
  • the 3E7 and T2 antibody specifically reacts with the epitope of the sequence 132-152 and 139-144 of sheep PrP, respectively.
  • the MAl-750 antibody specifically reacts with the epitope of the sequence 139-152 of sheep PrP.
  • the antibody and antigen complex with biotin and HRP were concentrated by adding streptavidin-conjugated magnetic beads (5 ⁇ l of 1 mg/ml, Invitrogen Inc.) and incubating at room temperature for 30 min. The magnetic beads were separated from the mixture using magnet (Invitrogen Inc.) . Unbound complexes were washed away with TBST and the washing procedure was repeated three times. After transferring the magnetic beads to the microtiter plate, the HRP chemiluminescent substrate (150 ⁇ l, Pico HRP substrate, Pierce Inc.) was added to detect the signal and the signal was measured in the Perkin Elmer Vector 3 luminomitor.
  • the SRA system of this invention successfully using cocktail forms of antibodies generates differential signals between scrapie and normal plasma samples.

Landscapes

  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne un procédé destiné à détecter de façon différentielle une forme multimère d'une forme monomère d'un polypeptide formant des multimères dans un échantillon biologique, qui comprend les étapes consistant: (a) à obtenir l'échantillon biologique à analyser; (b) à préparer un conjugué molécule d'affinité-anticorps de piégeage par liaison d'une molécule d'affinité à la surface de l'anticorps de piégeage, cet anticorps reconnaissant un épitope sur le polypeptide formant des multimères; (c) à préparer un anticorps indicateur, un épitope reconnu par cet anticorps étant présent dans le polypeptide formant des multimères afin d'entraîner un encombrement stérique par l'anticorps de piégeage lié à son épitope; (d) à mettre en contact de façon tridimensionnelle le conjugué molécule d'affinité-anticorps de piégeage et l'anticorps indicateur dans l'échantillon biologique; (e) à isoler le complexe anticorps indicateur-forme multimère-molécule d'affinité-anticorps de piégeage par utilisation d'une molécule d'affinité de contrepartie; et (f) à détecter la formation du complexe anticorps indicateur-forme multimère-molécule d'affinité-anticorps de piégeage.
PCT/KR2006/003587 2006-09-08 2006-09-08 Dosage de réaction simultané destiné à détecter de façon différentielle une forme multimère WO2008029965A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/KR2006/003587 WO2008029965A1 (fr) 2006-09-08 2006-09-08 Dosage de réaction simultané destiné à détecter de façon différentielle une forme multimère

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/KR2006/003587 WO2008029965A1 (fr) 2006-09-08 2006-09-08 Dosage de réaction simultané destiné à détecter de façon différentielle une forme multimère

Publications (1)

Publication Number Publication Date
WO2008029965A1 true WO2008029965A1 (fr) 2008-03-13

Family

ID=39157377

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2006/003587 WO2008029965A1 (fr) 2006-09-08 2006-09-08 Dosage de réaction simultané destiné à détecter de façon différentielle une forme multimère

Country Status (1)

Country Link
WO (1) WO2008029965A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2103938A1 (fr) * 2006-12-11 2009-09-23 Abbott Laboratories Procede de mesure immunologique pour precurseur peptidique de liberation de gastrine

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002097444A2 (fr) * 2001-05-31 2002-12-05 Arete Associates Procede de detection de proteines mal pliees
US6765088B1 (en) * 1997-02-21 2004-07-20 Universität Zürich Immunological detection of prions
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
US20060057671A1 (en) * 2004-09-10 2006-03-16 Orser Cindy S Immobilized probes and methods of detecting conformationally altered prion proteins

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6765088B1 (en) * 1997-02-21 2004-07-20 Universität Zürich Immunological detection of prions
WO2002097444A2 (fr) * 2001-05-31 2002-12-05 Arete Associates Procede de detection de proteines mal pliees
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
US20060057671A1 (en) * 2004-09-10 2006-03-16 Orser Cindy S Immobilized probes and methods of detecting conformationally altered prion proteins

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2103938A1 (fr) * 2006-12-11 2009-09-23 Abbott Laboratories Procede de mesure immunologique pour precurseur peptidique de liberation de gastrine
EP2103938A4 (fr) * 2006-12-11 2010-06-30 Abbott Lab Procede de mesure immunologique pour precurseur peptidique de liberation de gastrine
US9459248B2 (en) 2006-12-11 2016-10-04 Abbott Laboratories Immunoassay method for pro-gastrin-releasing peptide
US10067124B2 (en) 2006-12-11 2018-09-04 Abbott Laboratories Immunoassay method for pro-gastrin-releasing peptide

Similar Documents

Publication Publication Date Title
US8008026B2 (en) Methods for differentially detecting a multimeric form from a monomeric form of a multimer-forming polypeptide through three-dimensional interactions
US8026070B2 (en) Differential detection of multimeric and monomeric forms of multimer-forming polypeptides
KR100987639B1 (ko) 멀티머-형성 폴리펩타이드의 모노머로부터 멀티머를 분별검출하는 방법
US11802867B2 (en) Cardiac troponin assay method and assay reagent
JP2018503807A (ja) 凝集形−形成ポリペプチドの凝集形を検出する方法
WO2008029965A1 (fr) Dosage de réaction simultané destiné à détecter de façon différentielle une forme multimère
WO2009011500A2 (fr) Procédés pour diminuer de faux signaux dans un essai immunologique pour détecter une forme multimère d'une forme monomère de polypeptides formant des multimères
CN101427132A (zh) 通过三维相互作用从多聚体形成多肽的单体形式中示差检测多聚体形式的方法
JP6745351B2 (ja) 凝集型−形成ポリペプチドの凝集型を検出する方法
US20240003873A1 (en) Conjugate formation method, substance of interest measurement method, and kit
KR101363576B1 (ko) 알츠하이머 질환에 대한 신규 바이오마커 및 그의 용도
KR102086852B1 (ko) 응집형-형성 폴리펩타이드의 응집형을 검출하는 방법
US20090162943A1 (en) Reagents and Methods for the Detection of Transmissible Spongiform Encephalopathy
KR20060025296A (ko) 정상형 프라이온에 대한 항체 및 그의 용도

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 06783784

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 06783784

Country of ref document: EP

Kind code of ref document: A1