CN101427132A - Method for differentially detecting multimeric form from monomeric form of multimer-forming polypeptides through three-dimensional interactions - Google Patents

Abstract

The present invention relates to a method for differentially detecting a multimeric form from a monomeric form of a multimer-forming polypeptide in a biosample, which comprises the steps of: (a) preparing a carrier-capturing antibody conjugate by binding a capturing antibody to the surface of a solid phase carrier in a three dimensional manner, wherein the capturing antibody is capable of recognizing an epitope on the multimer-forming polypeptide; (b) preparing a detection antibody, wherein an epitope recognized by the detection antibody is present at a position in the multimer-forming polypeptide to cause a steric hindrance by the capturing antibody bound to its epitope to prevent the binding of the detection antibody to the multimer-forming polypeptide; (c) contacting simultaneously the carrier-capturing antibody conjugate and the detection antibody to the biosample; and (d) detecting the formation of a carrier-capturing antibody-multimeric form-detection antibody complex.

Description

Detect the method for polymer form by three-dimensional interactions differential from the monomeric form of polymer formation polypeptide

Technical field

The present invention relates to detect the method and the immunoassay kit thereof of polymer form by three-dimensional interactions differential from the monomeric form of polymer formation polypeptide.

Background technology

The multimerization of the polypeptide of common known composition protein is that protein function is needed.Yet the polymer form usually causes disease or disorder in some protein.Especially, protein exists as monomer under normal condition and become polymer (or aggregated forms) under abnormality (for example forming the misfolding form by changing).

Misfolding and the final protein of assembling (or gathering) have fully been determined, i.e. the potein deficiency normal biological activity of the relevant conformation of those non-functionals.Fail correctly folding or fail to keep correctly to fold to cause many dissimilar biological function obstacles, and therefore cause many multi-form diseases (Massimo Stefani, et al., J.Mol.Med.81:678-699 (2003); With Radford SE, et al., cell.97:291-298 (1999)).Numerous disease finally results from and have the protein molecule (protein molecule that promptly is different from the biosome of normal functionating) with incorrect structure in life system.

For example, assemble or misfolding diseases associated or disorder comprise Alzheimer's with protein is unusual, Creutzfeldt-Jakob disease, spongiform encephalopathy, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, the serpin deficiency disease, pulmonary emphysema, cirrhosis, type ii diabetes, lubarsch-Pick disease, secondary systemic amyloidosis, frontotemporal bone dementia (Fronto-temporal dementias), senile systemic amyloidosis, familial amyloid polyneuropathy, heredity cerebral amyloid angiopathy and the amyloidosis relevant with haemodialysis.

Furtherd investigate and the early diagnosis of assembling relevant disease.But, also do not propose from any method and the approach of monomer whose (normally) form differential detection polymer (gathering) form.

Because transmissible spongiform encephalopathy (TSE), Creutzfeldt-Jakob disease, kuru, familial fatal insomnia and Ge-Shi-Sha (Gerstmann-Straussler-Scheinker) syndrome of people's accidental, variation, iatrogenic and familial, the itch disease of sheep and goat, the spongiform encephalopathy of cat, the spongiform encephalopathy of ermine, chronic wasting disease of deer, elk and elk and the spongiform encephalopathy of ox are fatal neurodegenerative disease (Prusiner S.B.Proc.Natl.Acad.Sci.USA 95:13363-13383 (1998); With Hope J.Curr.Opin.Genet.Dev.10,568-57 (2000)).Think the prion protein (PrP of the sick type of unusual isoform or itch strongly ^Sc) be the main pathogenesis (Caughey B.Trends Biochem.Sci.26:235-42 (2001)) of TSE.

Normal type prion protein (PrP ^C) comprise that partly and as monomeric form there be (Zahn, R., et al., Proc.Natl.Acad.Sci.USA97:145-150 (2000)) in alpha-helix and flexible disordered, wherein, the sick type (PrP of itch ^Sc) have the height beta sheet conformation and as polymer (gathering) or at least dimeric forms have (Caughey, B., et al., J.Biol.Chem.273:32230-35 (1998)).Conformation change from alpha-helix conformation to the beta sheet conformation is the main cause of its neuropathology disease seemingly.

PrP ^CTo proteinase sensitivity (PrP ^Sen), PrP ^ScProteolysis had partial resistance (PrP ^Res) and be easy to form high molecular gathering thing (Bolton D.C.Lancet, 358:164-5 (2001)).Make it be difficult to analysis in the back feature and cause forming PrP ^ResOr show the conformation transition of its feature.

Only keep the sick type of itch by the vitellophag type and detect with ELISA, Proteinase K (PK) digestion method has been used to distinguish the various forms of resistances of PrP (the sick type of itch).But the PK digestion method enjoys query.PrP conformation, concentration, organize antibody, digestion time and damping fluid can influence the susceptibility of PK, this significantly reduces the reliability of PK digestion method.

Therefore, need develop and have more high reliability and easily from monomer whose form (for example, PrP ^c, the cellular type of PrP) and middle differential detection polymer form (for example, PrP ^Sc, the sick type of the itch of PrP) new method.

Run through the application,, and respectively quote and be included in the bracket with reference to many pieces of patents and publication.In order more fully to describe the state of the art in field under the present invention and the present invention, the full content of these patents and publication is incorporated among the application as a reference at this.

Summary of the invention

Therefore, an object of the present invention is to provide and a kind ofly form the method that differential the monomeric form of polypeptide detects the polymer form from polymer.

Another object of the present invention provides a kind of kit that is used for forming from polymer the monomeric form differential detection polymer form of polypeptide.

By following detailed description and appended claims and accompanying drawing, other purpose of the present invention and advantage will become obvious.

Description of drawings

Fig. 1 a schematically shows the embodiment of the single pearl (polymer detection system-three-dimensional-single pearl) of MDS-3D-of the present invention system.

Fig. 1 b schematically shows the embodiment of the two pearl systems of MDS-3D-of the present invention.

Fig. 1 c schematically shows the embodiment with two pearl systems of MDS-3D-of double-tagging of the present invention.

Show Fig. 2 according to the present invention (method simultaneously) and conventional method (method of fractional steps) detect the experimental result of polymer prion protein." N " represents normal plasma respectively and comprises PrP with " Sc " ^ScBlood plasma." RLU " represents relative light unit.

Fig. 3 a represents to select to be suitable for the experimental result of the buffer type of the single pearl of MDS-3D-system.

Fig. 3 b shows by PrP ^ScThe result's of Fig. 3 a that the signal of blood plasma and the ratio of the signal of normal plasma are represented another kind of appearance form.

Fig. 4 shows the experimental result of using 3E7 and MA1 750 antibody to detect the polymer prion protein in the blood plasma according to the two pearl systems of MDS-3D-." RFU " represents relative fluorescence unit.

Fig. 5 represents to select to be suitable for the experimental result of the buffer type of the two pearl systems of MDS-3D-.

Fig. 6 represents to select to be suitable for the experimental result of the plasma concentration of the two pearl systems of MDS-3D-.

Fig. 7 represents to select to be suitable for the experimental result of the plasma concentration of the single pearl of MDS-3D-system.

Fig. 8 shows the experimental result of using T2 and MA1 750 antibody to detect the polymer prion protein in the blood plasma according to the two pearl systems of MDS-3D-.

Fig. 9 shows that the detergent that is used for preparing plasma sample that uses variable concentrations and type detects the analysis result of the polymer prion protein of blood plasma by the single pearl of MDS-3D-system.

Figure 10 represents that the lavation buffer solution that uses dissimilar being used for to wash the pearl of using plasma sample and antibody incubation detects the analysis result of the polymer prion protein of blood plasma by the single pearl of MDS-3D-system.

Figure 11 represents to detect in concentration is 30% and 100% blood plasma according to the single pearl of MDS-3D-system the analysis result of polymer prion protein.

Figure 12 represents to detect in concentration is 20% and 25% blood plasma according to the single pearl of MDS-3D-system the analysis result of polymer prion protein.

Figure 13 shows that PK digestion is to detecting the influence of the polymer prion protein in the blood plasma according to the single pearl of MDS-3D-system.

Figure 14 a represents to use the analysis result of the antibody of Different Weight ratio by the polymer prion protein in the single pearl of the MDS-3D-system detection blood plasma.

Figure 14 b represents to use the analysis result of the antibody of Different Weight ratio by the polymer prion protein in the two pearl systems of the MDS-3D-detection blood plasma.

Figure 15 a and 15b represent to use the capture antibodies potpourri to detect the analysis result of the polymer prion protein in the blood plasma by the single pearl of MDS-3D-system.

Figure 15 c represents to use the detection mixtures of antibodies to detect the analysis result of the polymer prion protein in the blood plasma by the single pearl of MDS-3D-system.Left side post and right side post correspond respectively to (i) 3E7-pearl as capture antibodies and T2-biotin and MA1-biotin as detect mixtures of antibodies and (ii) MA1-pearl as capture antibodies and T2-biotin and 3E7-biotin as the detection mixtures of antibodies.

Figure 16 shows the comparison of the detectability of MDS and the single pearl of MDS-3D system.

Embodiment

In a technical scheme of the present invention, provide a kind of and in biological specimen, formed the method that differential the monomeric form of polypeptide detects the polymer form from polymer, it may further comprise the steps: (a) by capture antibodies is combined preparation carrier-capture antibodies bond with the surface of solid phase carrier with three dimensional constitution, wherein, this capture antibodies can be discerned the epi-position on the polymer formation polypeptide; (b) preparation detects antibody, wherein, is arranged in polymer by the epi-position of this detections antibody recognition and forms position of polypeptide and combine with its epi-position by capture antibodies and to cause steric hindrance, combines with prevention detection antibody and polymer formation polypeptide; (c) carrier-capture antibodies bond and detection antibody are contacted simultaneously with biological specimen; And the formation that (d) detects carrier-capture antibodies-polymer form-detection antibody complex.

In another technical scheme of the present invention, provide a kind of and in biological specimen, formed the kit that differential the monomeric form of polypeptide detects the polymer form from polymer, it comprises: (a) capture antibodies, this capture antibodies identification polymer form the epi-position on polypeptide and combine with the surface three dimension of solid phase carrier; (b) detect antibody, this detections antibody recognition is present in the epi-position that polymer forms a position in the polypeptide, causes that steric hindrance prevention detection antibody and polymer formation polypeptide combine thereby combine with its epi-position by capture antibodies.

Target of the present invention is a kind ofly to form method that the monomeric form of polypeptide differential detect polymer form from polymer by the immunoassays that relate to antigen-antibody reaction in biological specimen.In addition, the present invention uses two antibody-likes, capture antibodies and detection antibody, and the two all forms the polypeptide competition with polymer and combines.The antibodies that has occurred this competition by steric restriction.Particularly, owing to form the competition of the binding site on the polypeptide, form capture antibodies that the epi-position on the polypeptide combines with polymer and suppress to detect antibody and combine with epi-position on its polymer formation polypeptide with polymer.One of feature of the present invention is to carry out immunoassays under the Three-dimension Contact situation.Among the present invention, guarantee that the antigen of catching and detecting in antibody and the biological specimen has the Three-dimension Contact chance.

The inventor has proposed a kind ofly to form the prototype method that differential the monomeric form of polypeptide detects the polymer form from polymer, is called " polymer detection system (MDS) ", and has submitted PCT patented claim (PCT/KR2005/004001) to.In view of sensitivity that shows among the embodiment XV and distinguishing ability, the present invention will improve MDS.The most outstanding feature of the present invention is the antigen Three-dimension Contact that makes capture antibodies and detect antibody and biological specimen.Therefore, method called after of the present invention " MDS-3D (three-dimensional) system ".

Term used herein " polymer formation polypeptide " refers to a peptide species, this polypeptide can form gathering (being polymer) form, particularly after conformational change, cause as Alzheimer's, Creutzfeldt-Jakob disease, spongiform encephalopathy, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, the serpin deficiency disease, pulmonary emphysema, cirrhosis, type ii diabetes, lubarsch-Pick disease, secondary systemic amyloidosis, frontotemporal bone dementia, senile systemic amyloidosis, familial amyloid polyneuropathy, the extensive multiple disease of heredity cerebral amyloid angiopathy and the amyloidosis relevant with haemodialysis.Therefore, term " polymer formation polypeptide " can be used alternatingly with term " aggregation formation polypeptide ".

The inventive method is used two antibody-likes, i.e. capture antibodies and detection antibody.Term used herein " capture antibodies " refers to and can form the antibody that polypeptide combines with the purpose polymer in the biological specimen.Term " detection antibody " refers to and can form the antibody that polypeptide combines with the polymer that capture antibodies is caught." antibody " refer to can conjugated antigen immunoglobulin (Ig) protein.Antibody used herein refers to comprise can the binding purpose epi-position, the complete antibody of antigen or antigen fragment and any antibody fragment (for example, F (ab ') 2, Fab ', Fab, Fv).

Among the present invention, by capture antibodies and the epi-position that detects antibody specificity identification polymer forms be located in the polypeptide will with antibody that epi-position combines between cause steric hindrance (competition combination) the position.Preferably, by the amino acid sequence of the epi-position of capture antibodies identification with identical, overlapping by the amino acid sequence of the epi-position of detection antibody recognition or adjoin.Should be appreciated that, by under the amino acid sequence of the epi-position of capture antibodies identification and the situation identical or overlapping by the amino acid sequence of the epi-position that detects antibody recognition, the capture antibodies that combine and detect antibody and cause steric hindrance or compete combination with its epi-position.

Term used herein " overlapping " refers to that the epi-position of capture antibodies and detection antibody comprises the epi-position with overlapping wholly or in part amino acid sequence.For example, the epi-position of T2 and 3E7 antibody has the 147th～152 and the amino acid sequence of the 140th～160 amino acids of bovine prion protein sequence respectively.This epi-position can be described as overlapping epi-position fully.In addition, the epi-position of ICSM35 and 1E4 antibody has the 104th～113 and the amino acid sequence of the 108th～119 amino acids of bovine prion protein sequence respectively.This epi-position can be described as partly overlapping epi-position.

As for the epi-position of adjoining that causes steric hindrance, an epi-position (for example in polymer formation polypeptide, epi-position by capture antibodies identification) can be positioned at except that the position outside another epi-position (for example), as long as two kinds of antibody combine with the epi-position competition of adjoining by the epi-position that detects antibody recognition.

Another feature of the present invention is to make capture antibodies to combine with the surface three dimension of the solid phase carrier that is used to prepare carrier-capture antibodies bond.For example, the surface combination of capture antibodies and the three-dimensional pearl that is used to prepare carrier-capture antibodies bond.Therefore, the present invention has got rid of the combining of surface of capture antibodies and plate, is two-dimentional because consider this combination.Make antigen Three-dimension Contact in itself and the biological specimen with the three-dimensional capture antibodies that combines of carrier, guarantee with biological specimen in the more touch opportunity of antigen.

The solid phase carrier that combines with capture antibodies can be any material with three-dimensional structure, is preferably to carry by the separable material of gravity, electric charge or magnetic force.Most preferably, solid phase carrier is a magnetic bead.

According to preferred implementation, detect antibody and have mark or the affine material that produces detectable signal.This mark includes but not limited to: (for example, alkaline phosphatase, peroxidase, beta galactosidase and the beta-glucosidase) of enzyme, radioactive (for example, I ¹²⁵And C ¹⁴), (for example, fluorescein), luminous, chemiluminescent and FREET (FRET (fluorescence resonance energy transfer)) mark of fluorescence.Affine material comprises biotin.The various marks of labelled antibody and method be known in the art (Harlow and Lane, eds.Antibodies:A Laboratory Manual (1988) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

Using radioactive label to detect under the situation of antibody, the antigen-antibody complex that forms in the final step of the present invention can detect by the radioactivity of measurement markers.Under the situation of the enzyme labelling assay for determining antibody of using the catalysis chrominance response, antigen-antibody complex can detect by the substrate that uses enzyme.For example, detecting under the situation of antibody with alkali phosphatase enzyme mark, bromine chloro-indole base phosphate (BCIP), nitro blue tetrazolium (NBT), naphthols-AS-BI-phosphate and ECF (chemiluminescence of enhancing) can be used as the substrate of chromogenic reaction; Using under the situation of horseradish peroxidase-labeled; can use chloronaphthol, aminoethyl carbazole, diaminobenzidine, D-luciferin, lucigenin (two-N-methylacridine nitrate (bis-N-methylacridinium nitrate)), resorufin benzyl ether, luminol, Amplex Red reagent (10-acetyl group-3; 7-two Qiang phenoxazines), TMB (3; 3; 5; the 5-tetramethyl benzidine) and ABTS (2,2-azine-two [3-ethylbenzene thiazoline sulphonic acid ester]) as substrate.

Under the situation with affine material marker detection antibody, the antigen-antibody complex of formation can detect (for example, chrominance response-catalyzing enzyme) with the mark that produces detectable signal is connected in conjunction with adminicle by using it.For example, detecting under the situation of antibody, can use the Streptavidin of desmoenzyme to detect antigen-antibody complex with biotin labeling.

Above-mentioned mark makes in the step (d) qualitative feasible with detection by quantitative polymer antigen-antibody complex.Under the situation that detects the antibody linkage flag, the signal that produces by measurement markers carries out step (d).

The preferred antibody group of being made up of capture antibodies and detection antibody comprises other antibody of discerning the detection antibody of bovine prion protein sequence the 147th～152 amino acids epi-position as a kind of antibody and the conduct of the capture antibodies of discerning bovine prion protein sequence the 140th～160 amino acids epi-position.Another preferred antibody group comprises other antibody as the detection antibody of a kind of antibody of the capture antibodies of identification bovine prion protein sequence the 104th～113 amino acids epi-position and conduct identification bovine prion protein sequence the 108th～119 amino acids epi-position.

Notable feature of the present invention is to make carrier-capture antibodies bond to contact with simultaneous system with biological specimen with detection antibody.Contact with detecting antibody if biological specimen begins to contact with capture antibodies then, then the most of capture antibodies epi-positions in the polypeptide of carrier-capture antibodies bond and polymer form combine, thereby cause the combination inhibition that detects antibody.Therefore, carrying out step-by-step program but not simultaneously under the situation of scheme, the signal that detects antibodies very a little less than.On the contrary, under making capture antibodies and detecting antibody and situation that biological specimen contacts simultaneously, two antibody-likes are under the race condition and it depends on their concentration with combining of antigen.

" contact simultaneously " about carrier-capture antibodies and the term that detects antibody and to refer to: each carrier-capture antibodies is contacted with simultaneous system with biological specimen with detection antibody; Or the potpourri that comprises carrier-capture antibodies and detection antibody is contacted with biological specimen.

According to preferred implementation, in step (c) with carrier-bound capture antibodies and detect antibody with the mol ratio of 5:1～1:5, more preferably 3:1～1:3 mol ratio, also more preferably the mol ratio of 2:1～1:2, most preferably from about the mol ratio of 1:1 (capture antibodies with detect antibody) is used.

Carrying out under the situation of the inventive method according to the two pearl systems of MDS-3D, in step (c) with carrier-bound capture antibodies and with carrier-bound detection antibody with the mol ratio of 5:1～1:5, more preferably 3:1～1:3 mol ratio, most preferably from about the mol ratio of 2:1 (capture antibodies with detect antibody) is used.

The present invention makes and forms the monomeric form of polypeptide differential from any polymer and detect the polymer form and become possibility.According to preferred implementation, polymer forms polypeptide and comprises: A β peptide relevant with Alzheimer's and Tau albumen, the prion relevant with Creutzfeldt-Jakob disease and spongiform encephalopathy, the alpha-synapse nucleoprotein relevant with Parkinson's disease, the Ig light chain relevant with lubarsch-Pick disease, the serum amyloid A protein relevant with secondary systemic amyloidosis, the Tau albumen relevant with frontotemporal bone dementia, the transthyretin relevant with senile systemic amyloidosis, the transthyretin relevant with familial amyloid polyneuropathy, the cysteine proteinase inhibitor C relevant with the heredity cerebral amyloid angiopathy, the β that the amyloidosis relevant with haemodialysis is relevant ₂-microglobulin, the Huntington protein relevant, the superoxide dismutase of being correlated with, the serpin of being correlated with and the dextrin relevant with type ii diabetes with serpin deficiency disease, pulmonary emphysema and cirrhosis with amyotrophic lateral sclerosis with Huntington's disease.

Most preferably, polymer formation polypeptide is the prion protein that causes Creutzfeldt-Jakob disease and spongiform encephalopathy.

The present invention gives prominence to and is used to detect polymer prion, the i.e. PrP that is formed by the prion protein conformational change ^Sc

When the inventive method was used for prion protein (PrP), monomeric form was PrP ^c(cell of prion or normal type) and the polymer form is PrP ^Sc(the sick or type that is infectious of the itch of prion).

One of feature of the present invention is to adopt the antibody that combines with the epi-position that has non repetitive sequence in antigen molecule.Unless the epi-position by antibody recognition has non repetitive sequence, otherwise the present invention can not effectively detect the polymer form from the monomeric form of polymer formation polypeptide.

According to preferred implementation, form in the polypeptide at polymer by the epi-position of the special specific recognition of capture antibodies and/or by the epi-position that detects antibody specificity identification and not repeat.

The used antibody of the present invention can prepare according to routine techniques, fusion method (Kohler andMilstein for example, European Journal of Immunology, 6:511-519 (1976)), (USP 4 for recombinant DNA method, 816,56) or phage antibody library (Clackson et al, Nature, 352:624-628 (1991); With Marks et al, J.Mol.Biol., 222:58,1-597 (1991)).The general procedure of Antibody Preparation is as described in the following document: Harlow, E.and Lane, D., Antibodies:A Laboratory Manual, Cold Spring Harbor Press, New York, 1988; Zola, H., MonoclonalAntibodies:A Manual of Techniques, CRCPress, Inc., Boca Raton, Florida, 1984; And Coligan, CURRENTPROTOCOLS IN IMMUNOLOGY, Wiley/Greene, NY, 1991.Be used for the preparation of the hybridoma cell line of monoclonal antibody goods by immortal cell line and the lymphocytic fusion that produces antibody.This can be undertaken by technology known in the art.Polyclonal antibody can by inject above-mentioned antigen to suitable animal, collect and comprise the antiserum of antibody in the animal and prepare from specific antibody via any known affine technical point.

Only react with the epi-position identical or overlapping with the epi-position that detects antibody or capture antibodies respectively as if capture antibodies of mixing or detection antibody capable, the present invention comprises that also the use mixtures of antibodies is as capture antibodies or detection antibody.Described in embodiment XIII and XIV, the present invention uses the capture antibodies of mixing or detects antibody can be from PrP ^cMiddle differential detects PrP ^Sc

Term used herein " biological specimen " is the sample from biosome of detected materials.Biological specimen refers to biogenic any cell, tissue or fluid, and perhaps can be according to the present invention convenient any other medium of estimating comprises sample, the sample of taking from animal of taking from the people, takes from the assigned foods sample for people and animals consuming.Preferably, biological specimen to be measured is a body fluid sample, comprises the extract of blood, serum, blood plasma, lymph, breast, urine, excrement, intraocular liquid, saliva, seminal fluid, brain extract (for example, brain homogenate), spinal fluid (SCF), appendix, spleen and tonsillotome tissue.More preferably biological specimen is brain homogenate or blood plasma, most preferably is blood plasma.

Using under the situation of brain homogenate as biological specimen, the inventive method comprises that further this is advantageously with the step of Proteinase K (PK) or trypsase pre-service biological specimen.

Using under blood or the situation of blood plasma as biological specimen, preferred biological sample is without proteinase (for example, PK) pre-service.Protease Treatment causes the inventive method to polymer form, especially PrP ^ScDetection and distinguishing ability significantly descend.Surprisingly, the PrP of the inventive method in detecting blood or plasma sample ^Sc(for example, PK) Xiao Hua requirement is described in embodiment X to have got rid of proteinase in the process fully.

Using under blood or the situation of blood plasma as biological specimen; advantageously the inventive method further comprises the step with flesh aminoacyl or Triton system (for example, Triton X-100) detergent (preferred Triton system, most preferably Triton X-100) pre-service biological specimen.With regard to the preparation of biological specimen (preferred blood, most preferably blood plasma), preferred damping fluid comprises TBST (the Tris-buffer saline that contains polysorbas20) and three (methylol) methylglycine (Tricine).Preferably, the concentration range of biological specimen in the step (c) (preferred blood, most preferably blood plasma) is 10v/v%～70v/v%, more preferably 20v/v%～40v/v%, most preferably 23v/v%～30v/v%.

In an also technical scheme of the present invention, provide a kind of and in biological specimen, form the method that differential the monomeric form of polypeptide detects the polymer form from polymer, it may further comprise the steps: (a) by making capture antibodies combine preparation magnetic bead-capture antibodies bond with the surface of magnetic bead with three dimensional constitution, wherein, this capture antibodies can be discerned the epi-position bond on the polymer formation polypeptide; (b) preparation detects antibody, wherein, is arranged in described polymer by the epi-position of this detections antibody recognition and forms position of polypeptide and combine with its epi-position by capture antibodies and to cause steric hindrance, to stop combining of described detection antibody and polymer formation polypeptide; (c) described magnetic bead-capture antibodies bond and detection antibody are contacted simultaneously with biological specimen; (d) utilize the product of step (c) to separate magnetic bead-capture antibodies-polymer form-detection antibody complex; (e) formation of detection magnetic bead-capture antibodies-polymer form-detection antibody complex.

Preferably, method of the present invention further comprises the step of capture antibodies-polymer form-detection antibody complex that washing has separated between step (d) and step (e).Can use various lavation buffer solutions to carry out washing step as PBS (phosphate buffered saline(PBS)), PBST (phosphate buffered saline(PBS) that contains polysorbas20), PBSX (phosphate buffered saline(PBS) that contains Triton X-100), TBSX (the Tris-buffer saline that contains Triton X-100) and TBST (the Tris-buffer saline that contains polysorbas20).Most preferably, use TBST to carry out washing step.

Fig. 1 a schematically shows the embodiment of use magnetic bead of the present invention as carrier.Describe the present invention more in detail with reference to Fig. 1 a.Make capture antibodies and HRP-on the magnetic bead detect antibody and comprise PrP ^cAnd PrP ^ScBiological specimen contact simultaneously, the detection antibody of HRP-combination can not with the PrP of magnetic bead-capture antibodies-combine ^cIn conjunction with, but only with the PrP of magnetic bead-capture antibodies-combine ^ScIn conjunction with.In addition, the PrP of magnetic bead-capture antibodies detection antibody-combination that can not combine with HRP- ^cIn conjunction with.The epi-position of capture antibodies and detection antibody has non repetitive sequence in prion protein.By the amino acid sequence of the epi-position of capture antibodies identification with identical by the amino acid sequence of the epi-position of detection antibody recognition, repeat or adjoin.Among Fig. 1 a, epi-position is expressed as triangle.Because occupy by the epi-position that the detects antibody recognition antibody that is captured, thus detect antibody can not with have the only PrP of an epi-position ^cIn conjunction with.Yet, since the polymer prion protein, PrP ^Sc, comprise a plurality of defined epitopes, thus detect antibody can with the PrP of capture antibodies-combine ^ScIn conjunction with.After antigen-antibody reaction, apply magnetic field to collect magnetic bead to resultant of reaction, then the pearl of washing collection.Use HRP substrate for induction color-, fluorescence-or chromogenic reaction and measure its result provides the qualitative and quantitative analysis data whether to form PrP with checking ^Sc-antibody complex.

The preferred implementation of kit according to the present invention, with carrier-bound capture antibodies and detect antibody with the mol ratio of 5:1～1:5, more preferably 3:1～1:3 mol ratio, also more preferably the mol ratio of 2:1～1:2, most preferably from about the mol ratio of 1:1 (capture antibodies with detect antibody) is included in the form of mixtures.This kit can further comprise magnetic sheet, damping fluid, colour developing enzyme and substrate.

MDS-3D of the present invention system is divided into single pearl system of MDS-3D and the two pearl systems of MDS-3D.

The single pearl of MDS-3D system uses that capture antibodies-(Fig. 1 a) and the two pearl systems of MDS-3D use capture antibodies-in conjunction with pearl and detect antibody-in conjunction with pearl (Fig. 1 b and 1c) in conjunction with pearl.With reference to Fig. 1 a the single pearl of MDS-3D system is described hereinbefore.

In the two pearl systems of MDS-3D, detect antibody and be connected with the surface three dimension of solid phase carrier.Contact with three dimensional constitution with the polymer polypeptide with carrier-bound detection antibody.

Can be any material with the solid phase carrier that detects antibodies with three-dimensional structure, preferred, by the separable material of gravity, electric charge or magnetic force.Most preferably, solid phase carrier is a latex beads.Carrier can be labeled.Have at carrier under the situation of mark (for example, carrier is the latex beads that comprises fluorescent material), the detectable signal that carrier produces (existence of indication polymer polypeptide) can need not marker detection antibody and obtain.

The two pearl systems of MDS-3D are further divided into " the two pearl systems of MDS-3D with single mark " (Fig. 1 b) and " the two pearl systems of MDS-3D with double-tagging " (Fig. 1 c).

In the two pearl systems of the MDS-3D with single mark, detect antibody or carrier and have the mark that produces detectable signal.In the two pearl systems of the MDS-3D with double-tagging, detect antibody and carrier and all have the mark that produces detectable signal.This double-tagging strategy can double check the signal that exists of indication polymer polypeptide.

With reference to Fig. 1 b in detail, the two pearl systems of the MDS-3D with single mark will be described more.Make on the magnetic bead capture antibodies and fluorescence (Fluor)-detection antibody (the detection antibody that is connected with the latex beads that comprises fluorescent material) with comprise PrP ^cAnd PrP ^ScBiological specimen contact simultaneously, and fluorescence-detection antibody can not with the PrP of magnetic bead-capture antibodies-combine ^cIn conjunction with, and only can with the PrP of magnetic bead-capture antibodies-combine ^ScIn conjunction with.The epi-position of capture antibodies and detection antibody has non repetitive sequence in prion protein.By the amino acid sequence of the epi-position of capture antibodies identification with identical, overlapping by the amino acid sequence of the epi-position of detection antibody recognition or adjoin.Among Fig. 1 b, epi-position is expressed as triangle.Because occupy by the epi-position that the detects antibody recognition antibody that is captured, thus detect antibody can not with have the only PrP of an epi-position ^cIn conjunction with.Yet, since the polymer prion protein, PrP ^ScComprise a plurality of defined epitopes, thus detect antibody can with the PrP of capture antibodies-combine ^ScIn conjunction with.After antigen-antibody reaction, apply magnetic field to collect magnetic bead to resultant of reaction, then the pearl of washing collection.Measure with analysis of fluorescence intensity, whether checking forms PrP ^Sc-antibody complex.

To describe the two pearl systems of the MDS-3D with double-tagging more in detail with reference to Fig. 1 c.Make the capture antibodies on the magnetic bead detect antibody (the detection antibody of the HRP-combination that is connected with the latex beads that comprises fluorescent material) and comprise PrP with fluorescence-HRP- ^cAnd PrP ^ScBiological specimen contact simultaneously, fluorescence-detection antibody can not with the PrP of magnetic bead-capture antibodies-combine ^cIn conjunction with, and only can with the PrP of magnetic bead-capture antibodies-combine ^ScIn conjunction with.The epi-position of capture antibodies and detection antibody has non repetitive sequence in prion protein.By the amino acid sequence of the epi-position of capture antibodies identification with identical, overlapping by the amino acid sequence of the epi-position of detection antibody recognition or adjoin.Among Fig. 1 c, epi-position is expressed as triangle.Because occupy by the epi-position that the detects antibody recognition antibody that is captured, thus detect antibody can not with have the only PrP of an epi-position ^cIn conjunction with.Yet, since the polymer prion protein, PrP ^ScComprise a plurality of defined epitopes, thus detect antibody can with the PrP of capture antibodies-combine ^ScIn conjunction with.After antigen-antibody reaction, apply magnetic field to collect magnetic bead to resultant of reaction, then the pearl of washing collection.Measure with analysis of fluorescence intensity and HRP reaction, whether checking forms PrP ^Sc-antibody complex.

Following specific embodiment is used for illustrating the present invention, but can not be used for restriction as the defined scope of the present invention of appended claims.

Embodiment

Example I: use the polymer PrP in the single pearl of the MDS-3D-system detection blood plasma

The 2.5 x detergents (comprising 3% Triton X-100,1.5% NaTDC and 0.25% flesh aminoacyl) that mix 2705 μ l sheep blood plasmas, 22.5 μ l reorganization polymer sheep PrP (genotype ARQ, 120 times of dilutions) and 3605 μ l are with the preparation sample.Also preparation comprises the negative control that 22.5 μ l PBS substitute reorganization polymer sheep PrP.The magnetic bead of preparation capture antibodies-combination, wherein 1 μ g capture antibodies combines with 2.5 μ l magnetic beads.The capture antibodies that combines with magnetic bead is 3E7 or MA1-750 monoclonal antibody.Epi-position at the 3E7 monoclonal antibody is PrP ^cThe 140th～160 amino acids sequence (with reference to the bovine prion protein sequence) or the 132nd～152 amino acids sequence (with reference to sheep prion sequence) find that it is a non repetitive sequence in prion.MA1-750 antibody specificity identification PrP ^cOn epi-position, Ser-Arg-Pro-Leu-Ile-His-Phe-Gly-Ser-Asp-Tyr-Glu-Asp-Arg finds that it is a non repetitive sequence in prion.

With the magnetic bead of capture antibodies-combination with detect antibody and the mode incubation of plasma sample with the while.Then, determine whether the reorganization polymer sheep PrP in the plasma sample is detected by the single pearl of MDS-of the present invention system.Use 3B8/D5-HRP or T2-HRP as detecting antibody.As HirokoHayashi, et al., J.Vet.Med.Sci., 66 (6): described in 515 (2004), T2 antibody recognition PrP _147～152Epi-position (with reference to the bovine prion protein sequence) or PrP _140～145Epi-position (with reference to sheep prion sequence).Epi-position at the 3B8/D5 monoclonal antibody is the 132nd～152 amino acids sequence (with reference to sheep prion sequence) and the 140th～160 amino acids sequence (with reference to the bovine prion protein sequence).

In plasma sample, add the magnetic bead of capture antibodies-combination simultaneously and detect antibody and 37 ℃ of incubations 1 hour.Then, apply magnetic field to separate magnetic bead, then wash this pearl three times with TBST to reaction mixture.Carrying out ECL (chemiluminescence of enhancing) detects.

Table 1

For relatively, with the magnetic bead of capture antibodies-combination begin with the plasma sample incubation then with detect the antibody incubation.

The preparation of plasma sample is as indicated above.In plasma sample, add the magnetic bead of capture antibodies-combination and 37 ℃ of incubations 1 hour.Then, apply magnetic field to separate magnetic bead, then wash this pearl three times with TBST to reaction mixture.Then with the pearl that separates with detect antibody 37 ℃ of incubations 1 hour.Apply magnetic field to separate magnetic bead to reaction mixture, then wash this pearl three times with TBST.At last, carrying out ECL (chemiluminescence of enhancing) detects.

As shown in Figure 2, under with magnetic bead-capture antibodies (3E7 antibody) and detection antibody (T2-HRP) and the situation of sample with mode incubation simultaneously, detection signal at polymer PrP demonstrates the detection signal more much higher than substep scheme, and this explanation scheme has simultaneously greatly strengthened the sensitivity that the polymer prion detects.In addition, much higher in the step-by-step program frequently of the polymer prion signal intensity in the scheme and normal prion signal intensity simultaneously drawn the reason that the present invention shows fabulous distinguishing ability to the polymer prion.

Example II: the determining of buffer type that is suitable for the single pearl of MDS-3D-system

For the buffer type of determining to be suitable in the single pearl of the MDS-3D-of the present invention system, use the capture antibodies (3E7-pearl) of magnetic-combination and detect antibody (T2-HRP) and finish MDS-3D-list pearl system with the weight ratio of 1:1 (0.8 μ g:0.8 μ g is equivalent to the mol ratio of 1:1 substantially).Plasma sample is prepared as follows: mix 120 μ l d-H ₂10% Triton X-100 among the O, 580 μ l damping fluids (pH 8.0 for TAPS, TBST or three (methylol) methylglycine) and 300 μ l show to have PrP ^ScThe sheep blood plasma of clinical symptom comprises the plasma sample of 1.2% Triton X-100 and 30% blood plasma so that cumulative volume 1ml to be provided.Mix 2 μ l (0.8 μ g) as the magnetic bead of the 3E7-combination of capture antibodies and 200 μ l (0.8 μ g) as the T2-HRP (4 μ g/ml among the TBST) that detects antibody with the preparation mixed antibody.In the 1ml plasma sample, add 200 μ l mixed antibodies and 37 ℃ of incubations 1 hour.Then, apply magnetic field to separate magnetic bead, then wash this pearl three times with TBST to reaction mixture.Carry out ECL and detect (Fig. 3 a and 3b).Among Fig. 3 a and the 3b, " N " and " Sc " represents normal plasma respectively and comprises PrP ^ScBlood plasma.As shown in Fig. 3 a and 3b, three (methylol) methylglycine damping fluid is to comprising PrP ^ScBlood plasma demonstrate peak signal and normal plasma demonstrated weak signal.

EXAMPLE III: use the two pearl systems of MDS-3D-to detect polymer PrP in the blood plasma

Mix 120 μ l, 10% Triton X-100,730 μ l TBST damping fluids (pH 8.0) and 150 μ l sheep blood plasmas and comprise the plasma sample of 1.2% Triton X-100 and 15% blood plasma so that 1ml to be provided.Mix 4 μ l as the magnetic bead of the 3E7-combination of capture antibodies, 2 μ l as the fluorescent latex pearl of the MA1750-combination that detects antibody and 200 μ l TBST damping fluids with the preparation mixed antibody (the 3E7-pearl: MA1 750-fluorescent bead, 2:1).In the 1ml plasma sample, add 200 μ l mixed antibodies and 37 ℃ of incubations 1 hour.Then, apply magnetic field to separate magnetic bead, then wash this pearl three times with TBST to reaction mixture.At last, detect fluorescence signal.As shown in Figure 4, the two pearl systems of MDS-3D-can differential detect the PrP in the blood plasma ^Sc

EXAMPLE IV: the determining of buffer type that is suitable for the two pearl systems of MDS-3D-

Mix 4 μ l as the magnetic bead of the 3E7-combination of capture antibodies and 1 μ l as the fluorescent latex pearl of the MA1 750-combination that detects antibody with the preparation mixed antibody (the 3E7-pearl: MA1 750-fluorescent bead, 4:1).Mix 120 μ l, 10% Triton X-100,730 μ l damping fluids (TBST or three (methylol) methylglycine, pH 8.0) and 150 μ l sheep blood plasmas to make plasma sample.Other programs are identical with EXAMPLE III.As shown in Figure 5, use the two pearl systems of MDS-3D-, in plasma sample, detect PrP ^Sc, TBST demonstrates than higher signal and the distinguishing ability of three (methylol) methylglycine damping fluid.

In addition, use T2 and MA1 antibody to make the two pearl systems of MDS-3D-as capture antibodies and detection antibody respectively, carry out said procedure then.As shown in Figure 5, the two pearl systems of the MDS-3D-that uses another antibody to make up can also detect the PrP in the blood plasma ^Sc

EXAMPLE V: the determining of plasma concentration that is suitable for the two pearl systems of MDS-3D-

Mix 2 μ l as the magnetic bead of the 3E7-combination of capture antibodies and 1 μ l as the fluorescent latex pearl of the MA1 750-combination that detects antibody with the preparation mixed antibody (the 3E7-pearl: MA1 750-fluorescent bead, 2:1).Preparation concentration is 30%, 15% and 7.5% plasma sample.Use three (methylol) methylglycine (pH 8.0) damping fluid.Other programs are identical with EXAMPLE III.

As shown in Figure 6, the two pearl systems of MDS-3D-of the present invention can the differential detectable concentration be the PrP in whole plasma samples of 30%, 15% and 7.5% ^ScYet the ratio of Sc/N reduces when reducing plasma concentration, and when reducing plasma concentration, signal intensity increases.Therefore, be appreciated that suitable plasma concentration is about 15% in the two pearl systems of MDS-3D-.

Example VI: the determining of plasma concentration that is suitable for the single pearl of MDS-3D-system

In order to determine suitable plasma concentration in the single pearl of the MDS-3D-system, use the capture antibodies (3E7-pearl) of magnetic-combination and detect antibody (T2-HRP) to finish MDS-3D-list pearl system with the ratio (0.8 μ g:0.8 μ g) of 1:1.Preparation concentration is 30%, 15% and 7.5% plasma sample.Use three (methylol) methylglycine (pH 8.0) damping fluid.Other programs are identical with example II.

As shown in Figure 7, the single pearl of MDS-3D-of the present invention system can the differential detectable concentration be the PrP in whole plasma samples of 30%, 15% and 7.5% ^ScWhen plasma concentration reduces, at normal plasma and PrP ^ScThe ratio of the Sc/N of blood plasma, signal intensity all reduce.Therefore, be appreciated that the plasma concentration that is suitable for the single pearl of MDS-3D-system is about 30%.

In addition, using 3E7-pearl antibody and T2-HRP antibody and use three (methylol) methylglycine (pH 8.0) buffer preparation concentration with the ratio of 1:2 is 30% and 100% plasma sample.Other programs are identical with example II.As shown in Figure 11, observe 100% plasma concentration and produce much lower signal intensity and discriminating than 30% plasma concentration.

In addition, using 3E7-pearl antibody and T2-HRP antibody and use three (methylol) methylglycine (pH 8.0) buffer preparation concentration with the ratio of 1:1 is 20% and 25% plasma sample.The reaction volume that comprises 20% and 25% plasma sample is respectively 300 μ l and 400 μ l.Other programs are identical with example II.As shown in Figure 12, observe 25% plasma concentration and produce much higher signal intensity and discriminating than 20% plasma concentration.

Example VII A: use the polymer PrP in the two pearl systems of the MDS-3D-detection blood plasma

With the ratio of 2:1 or 4:1 use as the magnetic bead of the T2-combination of capture antibodies and as the fluorescent latex pearl of the MA1 750-combination that detects antibody to finish the two pearl systems of MDS-3D-.Use 15% plasma sample and TBST (pH 8.0) damping fluid.Other programs are identical with EXAMPLE III.As shown in Figure 8, find that T2-pearl and MA1750-pearl are with the ratio of the Sc/N of the ratio of 4:1 and the signal intensity ratio height with the ratio of 2:1.

Example VII A I: the detergent that uses variable concentrations and type is by the polymer PrP in the single pearl of the MDS-3D-system detection blood plasma

Except the detergent condition, the detergent that uses variable concentrations and type in the mode identical with example II detects to demonstrate by the single pearl of MDS-3D-system has PrP ^ScPolymer PrP in the sheep blood plasma sample of clinical symptom.As shown in Figure 9, the concentration of Triton X-100 (1%, 1.5% and 2%) demonstrates detection and the differential result similar to polymer PrP.In addition, although result error is higher relatively, (USB Corp. USA) has also shown suitable detection and differential result to Np-40.Np-40 is a kind of nonionic detergent and expression [Octylphenoxy] polyethoxy ethanol.

Example I X: use dissimilar lavation buffer solutions by the polymer PrP in the single pearl of the MDS-3D-system detection blood plasma

Except antibody weight ratio (3E7-pearl: T2-HRP, 1:2) and wash conditions outside, in the mode identical, use dissimilar being used to wash lavation buffer solution with the pearl of plasma sample and antibody incubation and detect by the single pearl of MDS-3D-system and contain PrP with example II ^ScThe sheep blood plasma sample in polymer PrP.As shown in Figure 10, with other damping fluids, PBST (phosphate buffered saline(PBS) that contains polysorbas20), PBSX (phosphate buffered saline(PBS) that contains Triton X-100) compare with TBSX (the Tris-buffer saline that contains Triton X-100), and TBST (the Tris-buffer saline that contains polysorbas20) lavation buffer solution demonstrates detection and the differential result of the most excellent polymer PrP.

Embodiment X:PK digestion detects the influence of the polymer PrP in the blood plasma to using the single pearl of MDS-3D-system

Except the antibody weight ratio (3E7-pearl: T2-HRP, 1:4) outside, in the mode identical, carry out or do not have PK (Proteinase K) digestion with example II, detect by the single pearl of MDS-3D-system and contain PrP ^ScThe sheep blood plasma sample in polymer PrP.As shown in figure 13, PK digestion has greatly reduced at the PrP in the plasma sample ^ScSignal intensity.In this point, can confirm that the single pearl of MDS-3D-of the present invention system can be at the PrP in detecting blood or plasma sample ^ScThe time get rid of the needs of PK digestion fully.

Embodiment XI: the antibody that uses the Different Weight ratio is by the polymer PrP in the single pearl of the MDS-3D-system detection blood plasma

In the mode identical, use the antibody of Different Weight ratio to contain PrP by the detection of the single pearl of MDS-3D-system with example II ^ScThe sheep blood plasma sample in polymer PrP.The weight ratio that 3E7-pearl capture antibodies and T2-HRP detect antibody is 4:1,2:1,1.33:1 and 1:1.As shown in Figure 14 a, using with identical amount under the situation of two kinds of antibody, the single pearl of MDS-3D-of the present invention system demonstrates the most excellent in the PrP in the plasma sample ^ScDetection and distinguishing ability.

Embodiment XII: the antibody that uses the Different Weight ratio is by the polymer PrP in the two pearl systems of the MDS-3D-detection blood plasma

In the mode identical, use the antibody of Different Weight ratio to contain PrP by the detection of the two pearl systems of MDS-3D-with EXAMPLE III ^ScThe sheep blood plasma sample in polymer PrP.The weight ratio that 3E7-pearl capture antibodies and MA1 750 fluorescent beads detect antibody is 4:1 and 2:1.As shown in Figure 14 b, using with the ratio of 2:1 under capture antibodies and the situation that detects antibody, the two pearl systems of MDS-3D-of the present invention demonstrate the most excellent in the PrP in the plasma sample ^ScDetection and distinguishing ability.

Embodiment XIII: use the capture antibodies potpourri by the polymer PrP in the single pearl of the MDS-3D-system detection blood plasma

In the mode identical, use the capture antibodies potpourri to detect and contain PrP by the single pearl of MDS-3D-system with example II ^ScThe sheep blood plasma sample in polymer PrP.Use by 3E7-pearl and T2-pearl antibody (Figure 15 a) or the capture antibodies potpourri formed of 3E7-pearl and MA1-pearl antibody (Figure 15 b) as capture antibodies.The capture antibodies potpourri is 1:1 with the weight ratio that detects antibody T2-HRP.Shown in Figure 15 a and 15b, the capture antibodies potpourri demonstrates excellent in the PrP in the plasma sample equally ^ScDetection and distinguishing ability.

Embodiment XIV: use and detect mixtures of antibodies by the polymer PrP in the single pearl of the MDS-3D-system detection blood plasma

In the mode identical, use the detection mixtures of antibodies to detect and contain PrP by the single pearl of MDS-3D-system with example II ^ScThe sheep blood plasma sample in polymer PrP.Used antibody group be (i) 3E7-pearl as capture antibodies and T2-biotin and MA1-biotin as detect mixtures of antibodies and (ii) the MA1-pearl as capture antibodies and T2-biotin and 3E7-biotin as detecting mixtures of antibodies.As shown in Figure 15 c, detect mixtures of antibodies and demonstrate excellent equally the PrP in the plasma sample ^ScDetection and distinguishing ability.

Embodiment XV: the comparison of the detectability of the single pearl of MDS and MDS-3D-system under the same conditions

The inventor has proposed the prototype method of differential detection polymer form from the monomeric form of polymer formation polypeptide, is called " polymer detection system (MDS) ", and has submitted PCT patented claim (PCT/KR2005/004001) to.

In order to compare the PrP of MDS and the single pearl of MDS-3D system with reliable fashion ^ScDetect and distinguishing ability, under same experimental conditions, contain PrP according to each program detection ^ScThe sheep blood plasma sample in polymer PrP.Use 3E7 and T7 antibody as capture antibodies and detection antibody respectively.

As shown in Figure 16, find out obviously, compare that the single pearl of MDS-3D of the present invention system demonstrates higher to the PrP in the sheep blood plasma sample with MDS ^ScSensitivity and distinguishing ability.

Embodiment XVI: the determining of plasma concentration that is suitable for using the single pearl of the MDS-3D-system of other antibody groups

Except the type and plasma concentration of antibody group, contain PrP by the detection of the single pearl of MDS-3D-system in the mode identical with example II ^ScThe sheep blood plasma sample in polymer PrP.Use ICSM35-biotin Streptavidin pearl as capture antibodies and 1E4-HRP as detecting antibody.The concentration of sheep blood plasma is 25%.(D-Gen, Inc.) identification is corresponding to the epi-position of the 96th～105 amino acids sequence (with reference to sheep prion sequence) or the 104th～113 amino acids sequence (with reference to the bovine prion protein sequence) for ICSM35 antibody.(Sanquin Reagents, Inc.) identification is corresponding to the epi-position of the 100th～111 amino acids sequence (with reference to sheep prion sequence) or the 108th～119 amino acids sequence (with reference to the bovine prion protein sequence) for 1E4 antibody.

As shown in Figure 17, another antibody group of the overlapping epi-position of identification division demonstrates excellent in the PrP in the plasma sample equally ^ScSensitivity and distinguishing ability.

Described preferred implementation of the present invention, be appreciated that the distortion and the modification that fall into essential scope of the present invention for a person skilled in the art are significantly, and scope of the present invention has been determined by described claims and equivalent thereof.

Claims (38)

1, a kind ofly form the method that differential the monomeric form of polypeptide detects the polymer form from polymer in biological specimen, this method may further comprise the steps:

(a) by capture antibodies is combined preparation carrier-capture antibodies bond with the surface of solid phase carrier with three dimensional constitution, wherein, described capture antibodies can be discerned the epi-position on the described polymer formation polypeptide;

(b) preparation detects antibody, wherein, is arranged in described polymer by the epi-position of described detection antibody recognition and forms position of polypeptide and combine with its epi-position by capture antibodies and to cause steric hindrance, to stop combining of described detection antibody and polymer formation polypeptide;

(c) described carrier-capture antibodies bond and detection antibody are contacted simultaneously with biological specimen;

(d) formation of detection carrier-capture antibodies-polymer form-detection antibody complex.

2, method according to claim 1, wherein, described detection antibody combines with three dimensional constitution with the surface of described solid phase carrier.

3, method according to claim 1, wherein, described polymer formation polypeptide is selected from by A β peptide, amyloid beta, Tau albumen, prion, alpha-synapse nucleoprotein, Ig light chain, serum amyloid A protein, transthyretin, cysteine proteinase inhibitor C, β ₂In the group that-microglobulin, Huntington protein, superoxide dismutase, serpin and dextrin are formed.

4, method according to claim 3, wherein, it is prion that described polymer forms polypeptide.

5, method according to claim 4, wherein, described monomeric form is PrPc and described polymer form is PrP ^Sc

6, method according to claim 1, wherein, by the amino acid sequence of the epi-position of described capture antibodies identification with identical, overlapping by the amino acid sequence of the epi-position of described detection antibody recognition or adjoin.

7, method according to claim 1 wherein, is formed in the polypeptide at described polymer by the epi-position of described capture antibodies identification and not to repeat.

8, method according to claim 1 wherein, is formed in the polypeptide at described polymer by the epi-position of described detection antibody recognition and not to repeat.

9, method according to claim 1, wherein, the solid phase carrier that combines with described capture antibodies is a magnetic bead.

10, method according to claim 2, wherein, with the solid phase carrier of described detection antibodies be latex beads.

11, method according to claim 1, wherein, described detection antibody has the mark that produces detectable signal.

12, method according to claim 2, wherein, the solid phase carrier that combines with described capture antibodies and/or have the mark that produces detectable signal with the solid phase carrier of described detection antibodies.

13, method according to claim 11 wherein, is carried out described step (d) by the signal that detects the mark generation that is connected with described detection antibody.

14, method according to claim 12, wherein, the signal that produces by the mark that detects with described carrier-the capture antibodies bond is connected carries out described step (d).

15, according to claim 11 or 12 described methods, wherein, described with detect chemical, enzyme, radioactive, fluorescence, luminous, the chemiluminescent and FREET mark of being labeled as that antibody is connected.

16, method according to claim 1, wherein, described biological specimen is brain homogenate or blood.

17, method according to claim 16, wherein, described biological specimen is a blood.

18, method according to claim 17, wherein, described biological specimen is a blood plasma.

19, method according to claim 16, wherein, when using brain homogenate as described biological specimen, described method further comprises with Proteinase K or the described biological specimen of trypsase pre-service.

20, method according to claim 1, wherein, capture antibodies and detection antibody with 2: 1～1: 2 mol ratio in described step (c) use and carrier-bound capture antibodies and detection antibody.

21, a kind of kit that is used for detecting the polymer form in biological specimen differential from the monomeric form of polymer formation polypeptide, it comprises:

(a) capture antibodies, this capture antibodies are discerned described polymer and are formed the epi-position on the polypeptide and combine with the surface three dimension of solid phase carrier; And

(b) detect antibody, this detections antibody recognition is present in the epi-position that described polymer forms a position in the polypeptide, causes that steric hindrance stops described detection antibody and polymer formation polypeptide to combine thereby combine with its epi-position by described capture antibodies.

22, kit according to claim 21, wherein, described detection antibody combines with three dimensional constitution with the surface of solid phase carrier.

23, kit according to claim 21, wherein, described polymer formation polypeptide is selected from by A β peptide, amyloid beta, Tau albumen, prion, alpha-synapse nucleoprotein, Ig light chain, serum amyloid A protein, transthyretin, cysteine proteinase inhibitor C, β ₂In the group that-microglobulin, Huntington protein, superoxide dismutase, serpin and dextrin are formed.

24, kit according to claim 23, wherein, it is prion that described polymer forms polypeptide.

25, kit according to claim 24, wherein, described monomeric form is PrP ^cAnd described polymer form is PrP ^Sc

26, kit according to claim 21, wherein, by the amino acid sequence of the epi-position of described capture antibodies identification with identical, overlapping by the amino acid sequence of the epi-position of described detection antibody recognition or adjoin.

27, kit according to claim 21 wherein, is formed in the polypeptide at described polymer by the epi-position of described capture antibodies identification and not to repeat.

28, kit according to claim 21 wherein, is formed in the polypeptide at described polymer by the epi-position of described detection antibody recognition and not to repeat.

29, kit according to claim 21, wherein, the solid phase carrier that combines with described capture antibodies is a magnetic bead.

30, kit according to claim 22, wherein, with the solid phase carrier of described detection antibodies be latex beads.

31, kit according to claim 21, wherein, described detection antibody has the mark that produces detectable signal.

32, kit according to claim 22, wherein, the solid phase carrier that combines with described capture antibodies and/or have the mark that produces detectable signal with the solid phase carrier of described detection antibodies.

33, according to claim 31 or 32 described kits, wherein, described with detect chemical, enzyme, radioactive, fluorescence, luminous, the chemiluminescent and FREET mark of being labeled as that antibody is connected.

34, kit according to claim 21, wherein, described biological specimen is brain homogenate or blood.

35, kit according to claim 34, wherein, described biological specimen is a blood.

36, kit according to claim 35, wherein, described biological specimen is a blood plasma.

37, kit according to claim 34, wherein, when using brain homogenate as described biological specimen, described kit further comprises Proteinase K or trypsase.

38, kit according to claim 21 wherein, comprises and carrier-bound capture antibodies and detection antibody with the described capture antibodies of 2:1～1:2 mol ratio and the form of mixtures of detection antibody.

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