CN101336374A - Method for identifying the genotype in position 171 of the ovine prion protein as well as kits for implementing said method - Google Patents

Abstract

The invention concerns a method for identifying the genotype in position 171 of the ovine PrP as well as a method for selecting an ovine for reproduction. The invention also concerns kits for implementing said methods. The inventive identifying method includes steps which consist in treating an ovine body fluid sample to be tested containing the PrP with a denaturing and reducing solution, immobilizing, optionally by a ligand, the denatured and reduced PrP on a solid phase, contacting the denatured, reduced and immobilized PrP with at least one detecting antibody, and detecting the possible presence of said at least one detecting antibody, one of the ligand or of said at least one detecting antibody specifically binding to the PrP having a particular allelic form in position 171. The invention is applicable in particular in the medical field, more particularly veterinary and sanitary medicine.

Description

Identify the 171st genotypic method of sheep prion protein and the kit of implementing described method

Invention field

The present invention relates to a kind of evaluation or analyze the 171st genotypic method of sheep prion protein (PrP), and the kit of implementing this method.

The invention still further relates to and a kind ofly allow according to the resistance of infectiousness SSE (TSSE) is carried out method for screening to the sheep population.

The invention still further relates to and a kind ofly allow according to the susceptibility of TSSE is carried out method for screening to the sheep population.

Prior art

The itch disease is a kind of disease that infects sheep, and is known before twoth century in Europe.Its special symptom is the social behavior in animal obstacle, have stay alone, eating disorder and dyskinetic tendency, such as trembling and stiff generation of its health rear portion.Other symptom generally include itch and fall hair.

The beginning in 1938 that works in about the experimental infection of scrapie is carried out on Britain sheep, helps very much to show the participation of inherent cause in this disease propagation.

Though also there is not clearly definite definite character that participates in the medium of itch disease as other infectiousness SSEs (TSSE), basis experimental evidence widely can provide near whole science common recognitions today.The infection medium that causes these diseases is a kind of protein of host seemingly, PrP (prion protein), and it is in anomaly pattern, and part is resisted proteolytic digestion, so-called PrPres, i.e. " prion protein resistance ".Verified relevant with these abnormal prion proteins infectiousness is verified and is had extreme resistance in the known ablation method that is used for deactivation " routine " pathogenic microorganisms (bacterium, virus, yeast).

The gene of sheep PrP is positioned at chromosome 13,256 the amino acid whose polypeptide of encoding.

The natural occurrence of having known itch disease in the sheep for a long time is relevant with the polymorphism of PrP gene, particularly this protein code 136,154 and 171 polymorphism.

Put down in writing several allele (perhaps allograft) of PrP, that main is allele ARQ, VRQ, ARR, AHQ and ARH.Two combination can produce a lot of different genotype in these allele.According to the sick plan of Britain country itch (British National Scrapie Plan, NSP), most of sheep population is carried a kind of in following 15 kinds of genotype on codon 136,154 and 171:

-A ₁₃₆R ₁₅₄R ₁₇₁/ A ₁₃₆R ₁₅₄R ₁₇₁, be labeled as ARR/ARR

-A ₁₃₆R ₁₅₄R ₁₇₁/ A ₁₃₆H ₁₅₄Q ₁₇₁, be labeled as ARR/AHQ

-A ₁₃₆R ₁₅₄R ₁₇₁/ A ₁₃₆R ₁₅₄H ₁₇₁, be labeled as ARR/ARH

-A ₁₃₆R ₁₅₄R ₁₇₁/ A ₁₃₆R ₁₅₄Q ₁₇₁, be labeled as ARR/ARQ

-A ₁₃₆H ₁₅₄Q ₁₇₁/ A ₁₃₆H ₁₅₄Q ₁₇₁, be labeled as AHQ/AHQ

-A ₁₃₆H ₁₅₄Q ₁₇₁/ A ₁₃₆R ₁₅₄H ₁₇₁, be labeled as AHQ/ARH

-A ₁₃₆H ₁₅₄Q ₁₇₁/ A ₁₃₆R ₁₅₄Q ₁₇₁, be labeled as AHQ/ARQ

-A ₁₃₆R ₁₅₄H ₁₇₁/ A ₁₃₆R ₁₅₄H ₁₇₁, be labeled as ARH/ARH

-A ₁₃₆R ₁₅₄H ₁₇₁/ A ₁₃₆R ₁₅₄Q ₁₇₁, be labeled as ARH/ARQ

-A ₁₃₆R ₁₅₄Q ₁₇₁/ A ₁₃₆R ₁₅₄Q ₁₇₁, be labeled as ARQ/ARQ

-A ₁₃₆R ₁₅₄R ₁₇₁/ V ₁₃₆R ₁₅₄Q ₁₇₁, be labeled as ARR/VRQ

-A ₁₃₆H ₁₅₄Q ₁₇₁/ V ₁₃₆R ₁₅₄Q ₁₇₁, be labeled as AHQ/VRQ

-A ₁₃₆R ₁₅₄H ₁₇₁/ V ₁₃₆R ₁₅₄Q ₁₇₁, be labeled as ARH/VRQ

-A ₁₃₆R ₁₅₄Q ₁₇₁/ V ₁₃₆R ₁₅₄Q ₁₇₁, be labeled as ARQ/VRQ

-V ₁₃₆R ₁₅₄Q ₁₇₁/ V ₁₃₆R ₁₅₄Q ₁₇₁, be labeled as VRQ/VRQ.

More briefly, according to genotype and they resistances to TSSE of sheep, sheep can be divided into 3 groups:

-resistance animal, even they are in the disease environment occurred frequently, it is infected also (perhaps not have only few), perhaps shows very high resistance under experimental infection.These are the animals with genotype ARR/ARR.But be noted that latest find that the itch disease also infects the genotypic case that is not true to type of ARR/ARR partly causes the query to the intimate absolute resistance conventional relevant with this genotype.

-hypersensitivity animal, they show the occurred frequently and short delitescence of disease.These be have genotype VRQ/VRQ (being considered as the most responsive genotype), ARQ/VRQ and ARQ/ARQ animal and

The animal of-demonstration various disease the incidence of disease and long latency.These are to have other genotypic animals.

For a long time, the plan of resistance itch disease is based on and slaughters all infected droves fully.But, determined that those animals of introducing again usually can be infected afterwards.This situation may with infect medium in the environment of letting animals feed strong persistence with and relevant to the resistance of the technology of depolluting.In the U.S., removed motion from nineteen fifty-two, see also from the overall point of view and do not succeed.This respect be it seems the result that the policy of having only Iceland leader produces.But this is an of a high price and enforceable policy.It specifically based on infecting district and health district in the territory is divided into, is slaughtered infected drove comprehensively and destroys all breeding apparatus, removes and raises district's soil on every side, and forbid in 5 years introducing animal again in described zone.Obviously, the animal that is used for breeding again " purification " zone is from infected zone not.

Carry A ₁₃₆R ₁₅₄R ₁₇₁The selection of the allelic animal of isozygotying, even it can not think to eradicate the absolute scheme of infectiousness spongiform encephalopathy (TSE) in the sheep drove, but still be the optimum strategy that the control major part comprises the TSE system breeding of bovine spongiform encephalopathy (BSE) medium, and has tangible economic benefit.

In order to resist the itch disease, for example Britain, France and Holland have come into effect the plan that enrichment has the sheep population of resistance animal.

The plan of France specifically comprises removing carries and the relevant V of hypersensitivity ₁₃₆R ₁₅₄Q ₁₇₁Allelic animal.Its high-frequency in population is the main hazard factor of transmission.Therefore, carry this allelic sheep and be considered to " unallowed " animal.Therefore, it can not be used to breed purpose by sale.Mark " itch sick sensitivity genes type " is to provide by promoting on the certificate that animal varieties alliance (Union forpromoting animal races) (Union Pour la Promotion des Races Animales-UPRA) or any other mechanism issue.

Described target need be identified the PrP genotype of sheep.Some have been proposed for this purpose based on molecular biological method.Can quote the several methods of using PCR, a kind of method of pyrophosphoric acid order-checking (pyrosequencing) and method of by PCR the PrP gene being carried out allowing after list increases simple colorimetric estimation of being called.

But all these relate to the method for pcr amplification must be undertaken by specialized laboratory, and cost is very high, and this has represented the real obstacle of its widespread adoption.

Known A ₁₃₆R ₁₅₄R ₁₇₁/ A ₁₃₆R ₁₅₄R ₁₇₁Genotype is to wish to be elected to be the genotype of the animal that is used to breed.

It is uniquely in 15 kinds of known types to have 2 amino acid whose genotype of R the 171st of PrP.This genotype is labeled as R/R.

We have understood manage to avoid V in breeding animals ₁₃₆R ₁₅₄Q ₁₇₁/ V ₁₃₆R ₁₅₄Q ₁₇₁Genotype.

Goal of the invention

The objective of the invention is to solve following new technical matters, the 171st the genotypic method of sheep prion protein (PrP) of identifying promptly is provided.

Purpose of the present invention also comprises the following new technical matters of solution, promptly provides and identifies the 171st genotypic kit of sheep PrP.

Specific purposes of the present invention are to solve following new technical matters, promptly provide and identify the 171st genotypic method and/or at this genotypic identification kit, allowing sheep breeder screening, select or to identify resistance animal or susceptibility animal, particularly the resistance animal is distinguished from the susceptibility animal or on the contrary.

Specific purposes of the present invention are to solve these technical matterss under the breeding plan, to remove or to reduce existing of prion in the sheep at least.

Specific purposes of the present invention are to solve this technical problem, to resist the sheep disease of scratching where it itches.

Specific purposes of the present invention are to utilize the method except that PCR or Western trace type to solve these technical matterss, PCR or Western trace type method be costliness and need specialized laboratory's equipment.

Therefore, another object of the present invention is to utilize fast detecting to solve these technical matterss, it should be noted that described fast detecting does not need any professional equipment except that the conventional analysis laboratory is used.

Specific purposes of the present invention be with can reproduction, industrialization, reliably, mode and minimum cost solve these technical matterss rapidly.

Summary of the invention

Have been found that now and might identify the amino acid that is positioned at the 171st of sheep PrP by at least a antibody that utilizes some allelic form of specific recognition PrP.

This method can be applicable to the biology body fluid of sheep, such as blood plasma, blood, serum, milk, saliva and urine.

In addition, this method is easy to implement, and quick and economical.

For this purpose, the present invention proposes a kind of evaluation or analyzes the 171st genotypic method of sheep PrP, may further comprise the steps:

A) comprise the sheep biology body fluid to be measured of PrP with the solution-treated that causes sex change and have a reductibility,

B) PrP after sex change, the reduction is fixed on the solid phase, chooses wantonly and fix by part,

C) sex change, reduction, the PrP after fixing are contacted with antibody with at least a detection, and

D) detect described at least a detection with may the existing of antibody,

And

Wherein said part and described at least a detection can combine (l ' un du ligand et du au moins un anticorps dedetection se lie sp é cifiquement à la PrP ayant une forme all é lique particuli é re enposition 171) with the 171st the PrP specificity with specific allelic form with one of antibody.

Preferably, the described solution that causes sex change and have a reductibility comprises:

A) at least a denaturant, it is selected from:

-surfactant, it is selected from:

Anionic surfactant is such as SDS (lauryl sodium sulfate), sarcosyl (Hamposyl L), sodium taurocholate, sodium glycocholate, NaTDC, natrium taurocholicum, Sodium Caprylate, 1-decane sodium sulfonate, NaLS and lauryl sulfate lithium;

Zwitterionic surfactant is such as SB 3-10 (the sweet Lay alkali of decyl-sulfo group), SB 3-12 (the sweet Lay alkali of dodecyl-sulfo group), SB 3-14 (the sweet Lay alkali of myristyl-sulfo group), SB 3-16 (the sweet Lay alkali of cetyl-sulfo group), SB 3-18 (the sweet Lay alkali of octadecyl-sulfo group), CHAPS and CHAPSO and deoxidation CHAPS;

Non-ionic surfactant, such as Triton X-100, Triton X-114, Tween 20, Tween 80, Brij 35 (polyoxyethylene 23 lauryl ethers), nonidet P-40, just-decyl-β-D-glucopyranoside, just-dodecyl-β-D-glucopyranoside, just-octyl-β-D-glucopyranoside and just-octyl-α-D-glucopyranoside;

Their potpourri, and/or

-chaotropic agent, and

B) at least a reductive agent.

Described chaotropic agent is selected from one of urea, guanidine, guanidine hydrochloride, guanidine thiocyanate and their potpourri.

More preferably, the described solution that causes sex change and have a reductibility comprises the potpourri, particularly anionic surfactant of ionic surface active agent, more particularly SDS and sarcosyl.

More precisely, the described solution that causes sex change and have a reductibility comprises with respect to by pending sample and cause sex change and have potpourri cumulative volume that the solution of reductibility the constitutes surfactant of 0.5wt.% at least, particularly is equal to, or greater than the 2wt.% with respect to the potpourri cumulative volume.

Preferably, at least a reductive agent is selected from: one of DTT (dithiothreitol (DTT)), TCEP (three (2-carboxy ethyl) phosphine) hydrochloride, DTE (dithioerythritol), beta-mercaptoethanol, 2-mercaptoethylmaine and their potpourri.

In this case, preferably, by pending sample and cause sex change and have the reductant concentration that comprises in the potpourri that the solution of reductibility constitutes between 2.5mM and 100mM, particularly between 5mM and the 50mM, more particularly between 5mM and the 30mM.

Most preferably, the described solution that causes sex change and have a reductibility comprises the potpourri of sarcosyl, SDS and DTT.

If the solution that causes sex change and have a reductibility comprises chaotropic agent, the concentration of so described chaotropic agent preferably is equal to, or greater than 1M, more particularly is equal to, or greater than 3M.If described chaotropic agent is a urea, concentration 8M preferably so.

Identify in first embodiment of the 171st genotype method of sheep PrP in the present invention, by part fixedly sex change and the reduction after PrP, described part is a kind of seizure antibody that can keep PrP by affine combination, and described detection antibody is a kind of antibody that can combine with the 171st the PrP specificity with specific allelic form, and this position is different from the epi-position site that described seizure is discerned with antibody.

In second embodiment of the inventive method, by part fixedly sex change and the reduction after PrP, described part is the seizure antibody that can combine with the 171st the PrP specificity with specific allelic form, and described detection antibody be a kind of can be by the antibody of affine combination in conjunction with PrP, described combination occurs in and is different from the described seizure epi-position site with the antibody recognition site.

In the 3rd embodiment of the inventive method, part by being selected from following molecule is the PrP after sex change and the reduction fixedly: plasminogen, affinity element, strepto-affinity element, monose aminoglycan, hesperidine, porphyrin, streptomysin and tetracycline, and described detection antibody is a kind of antibody that can combine with the 171st the PrP specificity with specific allelic form.

In the 4th embodiment of the inventive method, the PrP after sex change and the reduction directly is fixed on the solid phase, and described detection antibody is a kind of antibody that can combine with the 171st the PrP specificity with specific allelic form.

In all embodiments of the inventive method, preferably, described solid phase is selected from microtiter plate, pearl, pipe, and it is made by polymkeric substance, it should be noted that polystyrene, tygon or latex.Preferably, described solid phase is the microtiter plate that polystyrene is made.

In addition, in all embodiments of the inventive method, preferably, described biology humoral sample is selected from blood, blood plasma, serum and milk.

In first embodiment of the inventive method, described seizure antibody be a kind of can be in conjunction with the antibody of PrP, with described detection with combining between antibody and the PrP without any competition, described detection is specific with antibody to the 171st allelic form.Particularly, this antibody can be selected from antibody SAF-15, SAF-31, SAF-32, SAF-33, SAF-34, SAF-35, SAF-37,3B5, SAF-84, SHA-31, BAR-222, BAR-224, BAR-233 and 8G8.

Use antibody as catching, the antibody of preferred target Prp eight (peptide) repeat region, that is: SAF-15, SAF-31, SAF-32, SAF-33, SAF-34, SAF-35, SAF-37 and 3B5.

In this case, described detection is preferably selected from the 2A11 antibody through mark, the 11C6 antibody through mark, the BAR226 antibody of process mark and the antibody 12F10 of process mark with antibody.

But preferably, in first embodiment of the inventive method, described seizure antibody is SAF-34 antibody or 3B5 antibody, and described detection antibody is the 2A11 antibody through mark.

Advantageously, in second embodiment, described part is the seizure antibody that is selected from 2A11,11C6, BAR-226 and 12F10.

Advantageously, described detection is selected from SAF-15, SAF-31, SAF-32, SAF-33, SAF-34, SAF-35, SAF-37,3B5, SAF-84, SHA-31, BAR-222, BAR-224, BAR-233 and 8G8 with antibody.

Preferably, in second embodiment of the inventive method, described seizure is selected from antibody 2A11,12F10, BAR226 and 11C6 with antibody, and described detection is selected from antibody SAF-15, SAF-31, SAF-32, SAF-33, SAF-34, SAF-35, SAF-37,3B5, BAR-224, SHA-31 and the 8G8 of process mark with antibody.

The present invention also provides a kind of basis that the resistance of infectiousness SSE is carried out method for screening to the sheep population, it is characterized in that it comprises the embodiment of previous described method.

The present invention also provides a kind of basis that the susceptibility of infectiousness SSE is carried out method for screening to the sheep population, it is characterized in that it comprises the embodiment of previous described method.

This special permission breeder tells the susceptibility animal or only keeps the resistance animal.These screening methods especially can be used under the breeding plan.

Advantageously, described screening technique comprises a step that the signal that records in the sample to be analyzed and resistance and/or susceptibility sample are compared.

The present invention also provides a kind of evaluation sheep PrP the 171st genotypic kit, comprising:

-being fixed with the solid phase of at least a seizure with antibody, described seizure particularly is selected from antibody SAF-15, SAF-31, SAF-32, SAF-33, SAF-34, SAF-35, SAF-37,3B5, SAF-84, SHA-31, BAR-222, BAR-224, BAR-233 and 8G8 with antibody;

-cause sex change and have reductibility solution and

-at least a detection antibody particularly is selected from antibody 12F10, BAR226,11C6 and 2A11 through mark, more particularly passes through the 2A11 of mark.

The present invention also provides a kind of the 171st genotypic kit of sheep PrP that be used to identify, it is characterized in that it comprises:

-being fixed with the solid phase of at least a seizure with antibody, described seizure particularly is selected from antibody 12F10, BAR226,11C6 and 2A11 with antibody,

-cause sex change and have reductibility solution and

-at least a detection antibody particularly is selected from antibody SAF-15, SAF-31, SAF-32, SAF-33, SAF-34, SAF-35, SAF-37,3B5, SAF-84, SHA-31, BAR-222, BAR-224, BAR-233 and 8G8 through mark.

The accompanying drawing summary

Illustrative below reading will better be understood the present invention after describing also with reference to the accompanying drawings, and it is clearer that other features and advantages of the present invention will become:

-Fig. 1 show that the blood plasma with ARR/ARR sheep (promptly having the R/R genotype the 171st of PrP) or ARR/VRQ sheep (promptly having the R/Q genotype the 171st of PrP) or VRQ/VRQ sheep (promptly having the Q/Q genotype the 171st of PrP) obtains with respect to the standardized relative absorbance of genotype Q/Q.The test of the sandwich form of these ELISA is carried out carrying out after sex change and the reduction to blood plasma by embodiment 1 is described.All sandwich methods relate to SAF-34 antibody (anti-eight (peptides) repeat), all allografts that its indiscriminate identification is the 171st.Depend on the circumstances, SAF-34 antibody is used as catches with antibody (SAF-34), perhaps is used as to detect with antibody (SAF-34-AChE is with the SAF-34 of exposed (gymnote) acetylcholinesterase mark).

-Fig. 2 shows that having the optical density (OD) that the blood plasma of the sheep of amino acid R/R or R/H or R/Q or H/Q or Q/Q obtains with the 171st of PrP distributes.These tests utilize SAF-34 antibody to carry out with antibody as detecting as catching with antibody and biotin labeled 2A11 antibody.

-Fig. 3 shows that the optical density (OD) that obtains with the 171st blood plasma with sheep of amino acid Q/Q or amino acid R/Q or amino acid R/R distributes.These tests are to utilize SAF-34 to carry out with antibody as detecting as the 2A11 antibody of catching with antibody and AChE mark.

-Fig. 4 is presented at the influence of reductive agent (being dithiothreitol (DTT)) existence in the composition of denaturing soln or disappearance and denaturant (being urea) disappearance.These tests utilize biotin labeled 2A11 antibody to carry out with antibody as catching as detecting with antibody and SAF-34.

-Fig. 5 shows that having the optical density (OD) that the serum of the sheep of amino acid R/R or R/H or R/Q or H/Q or H/H or Q/Q obtains with the 171st of PrP distributes.These tests are to utilize antibody 3B5 to carry out with the DTT concentration of antibody and 20mM as detecting as catching with antibody, biotin labeled 2A11.

-Fig. 6 shows that DTT concentration is to the influence of plasma sample in the denaturing soln.These tests utilize 3B5 to carry out with antibody as detecting as catching with antibody and biotin labeled 2A11.

-Fig. 7 shows that DTT concentration is to the influence of blood serum sample in the denaturing soln.These tests utilize antibody 3B5 to carry out with antibody as detecting as catching with antibody and biotin labeled 2A11.

-Fig. 8 shows that the optical density that obtains with the different blood serum samples of taking from sheep distributes, and proves the different genotype corresponding to PrP.These tests utilize antibody 3B5 to carry out with antibody as detecting as catching with antibody and biotin labeled 2A11.

Detailed Description Of The Invention

The objective of the invention is a kind of for the identification of the 171st genotypic method of sheep PrP, wherein with leading The solution-treated that causes sex change and have a reproducibility comprises the sheep biology body fluid to be measured of PrP, fixedly sex change With the PrP after the reduction, the PrP after making sex change and reducing contacts described antibody with at least a following antibody Can with the 171st the sheep PrP specific binding with specific allelic form, and detect PrP antibody May exist.

Within the scope of the present invention, " biology body fluid " can specifically be blood, blood plasma, serum, urine, Cerebrospinal fluid, saliva, milk etc.

According to a preferred embodiment, this biology body fluid is selected from blood, serum, blood plasma and milk. This biology body fluid comprises PrP.

Term " antibody " refer to comprise or by at least a antigen binding site form any complete anti-The functional fragment of body or antibody, described antigen binding site make described antibody capable and antigenicity chemical combination At least a antigenic determinant combination of thing. The example of mentioned antibody fragment can comprise fragment Fab, Fab ', F (ab ')₂With scFv chain (single chain variable fragment), dsFv chain (double-stranded variable fragment), Etc.. These functional fragments specifically can obtain by genetic engineering.

The preparation of the monoclonal antibody that can use in the present invention or monospecific polyclonal serum belongs to In routine techniques, hereinafter will describe in detail.

Term " specific " is when relating to the spy of antibody to the 171st specific allelic form of sheep PrP In the time of opposite sex identification or specific binding, expression is compared with other possible allelic forms, should Antibody preferably interacts with specific allelic form, makes it to distinguish and to distinguish possible etc. Position gene form. Greater than 10⁸L.mol ^-1Binding constant be preferred.

The antibody that the present invention uses be specificity for the antibody of prion protein, so they are preferably Monoclonal antibody or monospecific polyclonal antibody, i.e. other protein of their nonrecognition.

Can according toAnd the lymphocyte of Milstein (1975) record merges and hybridoma is cultivated Conventional method obtains monoclonal antibody. Other methods that prepare monoclonal antibody also are known (people such as Harlow, 1988). Can pass through immune mammal (for example mouse, rat, rabbit even people, Etc.) and the lymphocyte integration technology that utilize to produce hybridoma prepare monoclonal antibody (

With Milstein, 1975).

The substitute technology that has this common method. For example might be by expressing from hybridoma clone's nuclear Acid prepares monoclonal antibody. Also might utilize " phage display " technology to come Dispersal risk, namely add Add antibody cDNA to carrier, described carrier normally filobactivirus (for example is used for colibacillary FUSE5) (people such as Scott, 1990). The latter consists of the storehouse, has the scFv fragment on their surface. Marks Put down in writing the constructing plan of these antibody libraries Deng people (1991).

Follow mode of operation commonly used, can obtain from the serum for the animal of the antigen immune of peptide type Polyclonal antibody.

Usually for example might utilize polypeptide as immunogene, particularly recombinant polypeptide or oligopeptides. Follow Conventional scheme, rabbit is followed the people such as Benoit (1982) record with the immunogenic equivalent immunity of 1mg peptide Rules carry out. Behind 4 weekly intervals, animals received is injected the processing of 200 μ g antigens, gets blood after 10 to 14 days. After the injection, antagonistic Serum is estimated profit with the ability that the radiolabeled peptide antigen of iodine is combined for the third time Carry out with chloramine-t method. On carboxymethyl cellulose (CMC) post, undertaken pure by ion-exchange chromatography then Change. To well known to a person skilled in the art that by the antibody molecule utilization that wash-out is collected method is adjusted to then Desired concn for example utilizes DEAE Sephadex to obtain the IgG fraction.

In order to improve the specificity of polyclonal serum, utilize to be used for immunity and to be fixed on titre peptide on the solid phase (titer peptide), antibody can carry out purifying by affine by immunity (perhaps immunity absorption) chromatography. Make Antiserum contacts enough time and exempts to cause peptide and antibody molecule with the described peptide that is fixed on the solid phase Epidemic disease is reacted and formation solid-phase immunity compound.

As with the suitable antibodies of the specific allelic form specific binding of the 171st of PrP, can carry To antibody 2A11,11C6, BAR226,12F10, V5 and V61.

But, as with the 171st antibody with PrP specific binding of particular form, preferred antibody 2A11, antibody 11C6, antibody BAR226 or antibody 12F10.

Preferred antibody 2A11 more particularly. This antibody is to follow the people such as J.Bru; Neuroscience Research 48,2004, the scheme preparation of 75-83 record, namely use recombinant bovine Prp immunity PrP^0/0Mouse. 171-179 sequence (QVYYRPVDQ) combination of this antibody and ox PrP, with have identical epi-position mostly The PrP of number mammalian species (sheep, goat, cat, rabbit, mouse, pig and hamster) shows strong the friendship The fork reaction, but do not show strong cross reaction with the people PrP with sequence (QVYYRPMDE). Also show When PrP be reduced with sex change after, its affinity raises strongly, maximum possible be since epi-position with participate in PrP Approaching of first serine residue of disulphide bridges. In addition, immunohistochemical analysis is presented at and uses Proteinase K Immunoreactivity raises greatly after processing.

In addition, proved that 2A11 antibody and the 171st carry glutamine (Q) or histidine (H) is residual The allelic form specific binding of the sheep PrP of base, and ARR allele is not observed immunity instead Answering property.

In other words, 2A11 antibody is not in conjunction with the R residue. Therefore, between 2A11 antibody and the PrP albumen not In conjunction with (described 2A11 antibody is not only as detecting with antibody but also as seizure antibody, in conjunction with not being converted into this Optical density in the invention identification method equal zero or close to zero), prove survey in the biology body fluid and comprise The genotype of the 171st of PrP be the R/R genotype, i.e. genotype A₁₃₆R ₁₅₄R ₁₇₁/A ₁₃₆R ₁₅₄R ₁₇₁。

In addition, because compare with the H residue, the 171st Q residue of 2A11 antibody and PrP is with quantitatively upper different And preferred mode combination, if detect any combination between 2A11 antibody and the PrP, have so May distinguish the isozygoty biology of sheep derived from Q/Q according to the signal strength signal intensity (being converted into OD) that obtains Body fluid and derived from the isozygoty biology body fluid of sheep of Q/H heterozygosis sheep or H/H.

Similarly, the 171st is carried the allelic heterozygosis animal of single R (R/Q or R/H) and will produce Even more weak signal, because half of the PrP that comprises in the sample do not identified by 2A11 antibody.

Therefore, the PrP and the specificity that are actually in the sample that detect in the authentication method of the present invention are anti-Between the body may in conjunction with existence.

Therefore, might design a simple immunological type test, come according to expressed genotype Show the different differences between the animal. According to first embodiment, this test can be used for identifying The ARR/ARR animal. According to second embodiment, this test can be used for other genotype (Q/Q, Q/H, H/H) compare, identify heterozygosis animal (R/ (Q, H)).

Method of the present invention comprises causing sex change and have also biology humoral sample and suitable quantity The solution contact of originality.

Advantageously, described solution comprises:

A) at least a denaturant, it is selected from:

-surfactant, it is selected from:

Anion surfactant is such as SDS (lauryl sodium sulfate), lauryl creatine acid Sodium (Hamposyl L), sodium taurocholate, sodium glycocholate, NaTDC, natrium taurocholicum, Sodium Caprylate, 1-decane sodium sulfonate, NaLS and lauryl sulfate lithium;

Zwitterionic surfactant is such as SB 3-10 (the sweet Lay alkali of decyl-sulfo group), SB 3-12 (ten The sweet Lay alkali of dialkyl group-sulfo group), SB 3-14 (the sweet Lay alkali of myristyl-sulfo group), SB 3-16 (ten The sweet Lay alkali of six alkyl-sulfo group), SB 3-18 (the sweet Lay alkali of octadecyl-sulfo group), CHAPS and CHAPSO and deoxidation CHAPS;

Non-ionic surface active agent, such as Triton X-100, Triton X-114, Tween 20, Tween 80, Brij 35 (polyoxyethylene 23 lauryl ethers), nonidet P-40, just-decyl-β-D-Glucopyranoside, just-dodecyl-β-D-glucopyranoside, just-octyl-β-D-pyrans Glucoside and just-octyl-α-D-glucopyranoside;

The mixture of these surfactants, and/or

-chaotropic agent, and

B) at least a reducing agent.

Reducing agent refers to cut the reagent of PrP albumen disulphide bridges.

In can be used for reducing agent of the present invention, can mention especially DTT (dithiothreitol (DTT)), TCEP (three (2-carboxy ethyl) phosphine) hydrochloride, DTE (dithioerythritol), beta-mercaptoethanol, 2-sulfydryl Ethamine or their mixture.

By pending sample and cause sex change and have in the mixture that the solution of reproducibility consists of existing The content of reducing agent usually between 2.5mM and 100mM, particularly between 5mM and the 50mM, more Specifically between 5mM and the 30mM.

Described chaotropic agent is selected from one of urea, guanidine, guanidine hydrochloride, guanidine thiocyanate and their mixture.

If there is chaotropic agent, their concentration is generally equal to or greater than 1M, is preferably greater than 3M so.

Preferred chaotropic agent is urea, and preferred concentration is 8M.

By pending sample and cause sex change and have usually quilt of mixture that the solution of reproducibility consists of Be heated to the temperature between 37 ℃ and 100 ℃, particularly 50 ℃ to 70 ℃, more particularly about 60 ℃, Reach 5 minutes to 1 hour, particularly 7 to 20 minutes, more particularly about 10 minutes.

Select these conditions to satisfy the requirement of following contradiction, the enough sex change of PrP are so that the 171st on the one hand Amino acids formed epi-position is not blocked, on the other hand PrP too sex change so that epi-position still can be resisted Body identification. In addition, these processing must not affect follow-up antibody and fix.

According to a specific embodiment, described solution comprises the mixture of surfactant, is worth annotating What anticipate is the mixture of ionic surface active agent, particularly anion surfactant, more particularly The mixture of SDS and sarcosyl.

By pending sample and cause sex change and have surface in the mixture that the solution of reproducibility consists of The concentration of activating agent is generally equal to or greater than the 0.5wt.% with respect to the mixture cumulative volume, particularly etc. In or greater than the 2wt.% with respect to the mixture cumulative volume.

Therefore, the described solution that causes sex change and have a reproducibility comprises at least a denaturant and at least one Plant reducing agent, described denaturant can be surfactant and/or chaotropic agent.

As shown in hereinafter embodiment 4, utilize this be sex change be again solution the locating PrP of reproducibility Reason is that the embodiment institute of the inventive method is indispensable.

Usually, allow and comprise the solution of the specific antibody of the 171st amino acids and the solid phase that is fixed with PrP Contact at least 10 minutes, particularly about 1 hour, temperature range can be 4 to 50 ℃, particularly 37 ℃.

Obviously, the method for mark depends on employed label on the detection solid phase. These are this area skills The known method of art personnel. For example can mention biotin labeling. For example, in this case, support May existing of biotin fixing on the thing can followingly manifest, and namely adds streptavidin-peroxide The solution of enzyme conjugates allows described solution in about 37 ℃ thermotonus about 10 to 30 minutes. At last, The existence that is bonded to the peroxidase activity of holder can followingly manifest, and for example namely adds chromogenic substrate Tetramethyl benzidine allows described chromogenic substrate about 30 minutes of 20 ℃ of reactions. At last, 450 and 620 Nm measures absorbance.

Consider the fixing of prion protein after sex change, the reduction, this may be on solid phase directly Fixing or indirectly fixing.

Described solid phase can be the trace that polymer, particularly polystyrene, polyethylene or latex are made Titer plate, pearl, pipe.

Preferred solid phase is the microtiter plate that polystyrene is made.

For direct fixing on solid phase of PrP, be fixed to the prion protein on the plate, at fixing PrP egg Before the Bai Zishen, as all proteins, must carry out purifying (sex change) from sample, to remove to the greatest extent May many unwanted protein.

In addition, prion protein must reduce.

Secondly, the detection of direct or indirect labelling is reacted with antibody.

For PrP indirectly fixing on solid phase, can utilize antibody or another kind is not the spy of antibody Opposite sex part is fixed. So, can (it can be by the parent by being called the antibody that catches with antibody With in conjunction with keeping PrP), perhaps by with the 171st allelic particular form specific binding Antibody, perhaps by not being that the part of antibody is fixed indirectly, described seizure can be by the parent with antibody Keep PrP with combination. This part can be such as plasminogen, affinity element, streptavidin, monose The molecule of aminoglycan, hesperidine, porphyrine, streptomysin or tetracycline.

If PrP is by means of can being fixed by affine part or antibody in conjunction with keeping PrP, that The antibody of the 171st the allele particular form of PrP albumen and energy specific binding after fixing is sent out Give birth to reaction, described antibody is by direct or indirect labelling.

On the other hand, if PrP passes through the anti-of the 171st allelic particular form of energy specific binding Body is fixed, make so after fixing PrP albumen with can send out in conjunction with the antibody that keeps it by affine Give birth to reaction.

In this case, be that this antibody is by direct or indirect labelling.

Then, in all situations, detect by the antibody through mark and undertaken.

Advantageously, method of the present invention utilizes the form of sandwich immunoassay to implement, particularly The ELISA type. In described situation, suitable is, use can be simultaneously with purifying, reduction after PrP Protein bound so-called the seizure with antibody and so-called the detection used antibody.

Seizure refers to the part of following antibody or antibody with antibody, and it is preferably by simple affine knot Close or by means of the affine combination by the identification of defined epitope site, be fixed on the solid phase, described solid Thereby can keep the antigen that exists in the biological sample mutually.

When antigen was captured with antibody fixedly, detection must be able to make self and still can approach with antibody And be different from the epi-position site combination that catches with the antibody recognition site.

Term " (process) mark " refers to that direct mark is (by enzyme, radio isotope, fluorescence Dyestuff, luminophor, etc.) and indirect labelling (for example the antibody by self direct mark or Utilize the reagent through " affine to " of mark, such as, but not limited to the affinity of biotin-process mark Plain right, etc.).

Therefore, a concrete theme of the present invention is to be used to identify the 171st genotypic method of PrP, may further comprise the steps:

-handle biology humoral sample to be measured, make PrP sex change and the reduction that comprises in the described sample,

-sample after the described processing is contacted with being fixed with so-called solid phase of catching with antibody,

The solid phase that obtains in-cleaning the abovementioned steps,

-solid phase after the described cleaning is contacted with being called the solution that detects with the antibody that passes through mark of antibody,

-clean the solid phase that obtains in the abovementioned steps, then

-detect may existing of mark on the solid phase,

A kind of antibody specificity identification PrP, regardless of the allele variant of the 171st of PrP, another kind of antibody specificity is in conjunction with the 171st the sheep PrP with specific allele variant.

As no matter the 171st allele variant of PrP and the antibody of specific recognition PrP, can mention especially can specific recognition octapeptide repetitive structure antibody, such as those of record among the patented claim WO 01/35104.In these antibody, can mention the antibody that is selected from down group especially: SAF-15, the SAF-31, SAF-32, SAF-33, SAF-34, SAF-35 and the SAF-37 that put down in writing among the application WO01/35104.

Can also mention people Krasemann such as Krasemann, S., Groschup, M.H., Harmeyer, S., Hunsmann, G. and Bodemer, W. (1996a) Generation of monoclonal antibodiesagainst human prion proteins in PrP ^0/0Mice.Molecular Medecine, 2, the antibody 3B5 of 725-734 record.

In addition, can quote people's such as F é raudet paper (Screening of 145Anti-PrPMonoclonal Antibodies for Their Capacity to Inhibit PrPSc Replication inInfected Cells, the Journal of Bio1ogical Chemistry, Vol.280, No.12, Issue ofMarch 25, pp.11247-11258,2005).Special preferred antibody SAF-34 or antibody 3B5.

As the antibody of the 171st specific allele variant of specific recognition PrP, quote antibody 2A11.Also quote Krasemann, S., Jurgens T. and Bodemer, W. (1999) Generation ofmonoclonal antibodies against prion proteins, an unconventional nucleic acidbased immunization strategy.Journal of Biotechnology, 73, the antibody 12F 10 of 119-129 record, and Krasemann, S., Grosehup, M.H., Hunsmann, G. and Bodemer, W. (1996b) Induction of antibodies against human prion proteins (PrP) by DNA-mediatedimmunization of PrP ^0/0Mice.J.Immunol.Methods, 199, the antibody 11C6 of 109-118 record.

Also mention people such as Moudjou, Journal of Virology, Sept.2004, the V5 and the V61 antibody of 9270-9276 record.

Preparation or acquisition and above-mentioned antibody have the specific monoclonal antibody of similar or identical epi-position obviously in those skilled in the art's scope in power, and described monoclonal antibody is suitable for embodiment of the present invention.

Following drawings and Examples are for the present invention being described rather than limiting its scope.

Embodiment

Material and method

Obtain and characterize the specific monoclonal antibody of octapeptide repetitive structure (SAF)

Press patented claim WO 01/35104 described preparation to the specific antibody SAF-15 of octapeptide repetitive structure, 31,32,33,34,35 and 37.

Synthetic and the mark of people PrP 79-92 peptide

(Milligen 9050, Waters, Milford, MI) the synthetic peptide of representing PrP octapeptide repetitive structure, for example structure G-G-W-G-Q-P-H-G-G-G-W-G-Q-G-(NH to utilize automatic synthesizer ₂), corresponding to the sequence 79-92 of people PrP.As the previously described (people such as McLaughlin who is used for other peptides or protein, 1987, people such as Grassi, 1989), by heterobifunctional agent succinimido 4-(N-maleimide methyl) cyclohexane-1-carboxylate (SMCC, Calbiochem France), makes peptide and gymnote acetylcholinesterase (AChE) covalent coupling.This method relates to mercapto that is added to peptide and the reaction that is fixed to the maleimide amine function group of AChE by the reaction with SMCC.According to previous description (people such as McLaughlin, 1987), by with the reaction of N-succinimido S-acetyl thio acetic acid esters (SATA), make mercapto be added to peptide.By making AChE-SMCC and excessive mercaptan reactive polypeptide, realize coupling.

-immunity and MONOCLONAL ANTIBODIES SPECIFIC FOR

According to previous description people such as (, 1997) Lasmezas, obtain the goods of " the sick relevant fibrillation of itch " (SAF, PrPre goods) from the hamster brain (the itch disease is 263K) that infects.Before immune mouse by handle these goods of deactivation with formic acid.With these SAF goods immunity PrP knock out mice (PrP ^0/0Mouse), and according to previous description people such as (, 1988,1989) Grassi preparation hybridoma.According to the screening of hereinafter described carrying out culture supernatants.57 hybridomas can be identified and stablize: they are known as SAF-1 to SAF-90.All these antibody prove that all identification is fixed on SAF on the microtiter plate, and wherein minority shows the ability of identification polypeptide-AChE conjugate.In the latter, obviously discern octapeptide repetitive structure (reacting) for 7 with the 79-92 peptide that is coupled to AChE; They are antibody SAF-15, SAF-31, SAF-32, SAF-33, SAF-34, SAF-35 and SAF-37.After ascites liquid form clone and amplification, on albumin A Sepharose post by the affinitive layer purification monoclonal antibody ,-20 ℃ of preservations up to use.Follow the isotype that the OuchterlonyShi technology is determined antibody by immunodiffusion radially.

-screening hybridoma culture supernatants

Prove the existence of PrP specific antibody in the hybridoma culture supernatants in two ways, test the ability that they combine with peptide-AChE conjugate (particularly peptide 79-92-AChE) or hamster SAF.In first kind of situation, in the enterprising row filter of plate that comprises as described previously fixing goat anti-mouse IgG antibody (people such as Cr é minont, 1993, people such as Frobert, 1991).Briefly, allow 100 μ, 1 culture supernatants and 100 μ l peptide-AChE conjugates in the plate that comprises immobilization goat anti-mouse IgG antibody, spend the night 4 ℃ of reactions.After washing plate, Xiang Kongzhong adds 200 μ l E1lman reagent (people such as Ellman, 1961) and is fixed to existing of AChE on the solid phase with detection.In second kind of situation, by at the 0.05M phosphate buffer, allow 50 μ l, 2 μ g/ml solution spend the night among the pH 7.4 in the environment temperature reaction, prepare the plate that comprises immobilization SAF goods.After the cleaning, plate uses EIA damping fluid (100mM phosphate buffer, ph7.4 comprises 150mMNaCl, 0.1% bovine serum albumin (BSA) and 0.01% sodium azide) 4 ℃ of saturated spending the night, and keeps this temperature up to use.According to previous description (people such as Negroni, 1998), utilize the anti-mouse IgG antibody of AChE labelled goat to prove combining of monoclonal antibody and immobilization SAF.

Use the AChE labeled monoclonal antibody

According to previous description (people such as Grassi, 1989), utilizing heterobifunctional agent is succinimido 4-(N-maleimide methyl) cyclohexane-1-carboxylate (SMCC, Calbiochem, France), by the Fab ' fragment of reduction and the tetramer form coupling of enzyme are prepared the monoclonal antibody of using the AChE mark.Mercapto and the reaction that is fixed to the maleimide amine function group of AChE by reaction that Fab ' fragment that this method need be reduced is entrained with SMCC.

Obtain monoclonal antibody 3B5,11C6 and 12F10

With recombined human PrP immunity PrP knock-out mice and after following the different technologies screening of people's descriptions such as Krasemann, obtain monoclonal antibody 3B5,11C6 and 12F10.

3B5 is a kind of antibody and identification polypeptide 79-92 at recombined human PrP.

11C6 is the antibody that comformational epitope is not identified in a kind of identification.

12F10 is a kind of antibody and identification polypeptide 142-160 at recombined human PrP.

Obtain monoclonal antibody Bar226

As C.Feraudet, N.Morel, S.Simon, H.Volland, Y.Frobert, C.Creminon, D.Vilette, S.Lehmann, J.Grassi (2005) Screening of 145anti PrP Monoclonalantibodies for their capacity to inhibit PrPSc Replication in Infected Cells J.Biol.Chem., 280,11247-11258 is described, by with the immune PrP knock out mice of reorganization PrP (ARQ allograft), obtained antibody Bar-226.

Obtain monoclonal antibody 2A11

Follow people such as Brun; J.Neuroscience Research 48,2004, the scheme that 75-83 describes, preparation monoclonal antibody 2A11.

Conjugate: monoclonal antibody 2A11-biotin

Follow Greg T.Hermanson, 1996 schemes of describing prepare the conjugate with biotin labeled monoclonal antibody 2A11.

Conjugate: with the strepto-affinity element of peroxidase labelling

Follow Greg T.Hermanson, 1996 schemes of describing, the preparation conjugate of the strepto-affinity element of peroxidase labelling.

Biology body fluid

Employed biology body fluid is serum or the blood plasma from the sheep of Cheviot, Romanov, Casserarde and Manech kind.

Environment temperature

Environment temperature is defined as the temperature between 18 ℃ and 30 ℃, comprises 8 ℃ and 30 ℃.

Embodiment 1: with different antibody to testing

Plasma sample is taken from the 171st sheep with genotype R/R or R/Q or Q/Q of PrP.

The solution that causes sex change and have a reductibility that each sheep plasma sample of 65 μ l and 65 μ l is comprised 8M urea and 10mM DTT mixes.Then potpourri was heated 30 minutes at 50 ℃.

Comprise catch with the hole of the microtiter plate of antibody in interpolation 100 μ potpourris that l obtains, described seizure antibody is antibody 12F10 or antibody BAR233 or antibody BAR226 or antibody SAF-34 or antibody 11C6.

Then with comprising 10 ^-2The M phosphate buffer, the cleaning fluid of pH 7.4 and 0.05%Tween 20 cleans the hole 3 times.

After the cleaning, 100 μ l detection antibody is added in every hole, the antibody SAF-34 that described detection antibody is the AChE mark (is antibody 12F10 or BAR233 or BAR226 or 11C6 if catch with antibody), perhaps use the antibody BAR224 of AChE mark, perhaps use biotin labeled antibody 2A11, perhaps using biotin labeled antibody BAR208, is every liter 3 μ g antibody with respect to the final mixture that obtains like this.

With the hole 37 ℃ of incubations 1 hour.

Then, carry out 5 times with cleaning fluid and clean, described cleaning fluid comprises 10 ^-2The M phosphate buffer, pH7.4 and with respect to the Tween 20 of cleaning fluid gross weight 0.05wt%.

If the antibody through mark is biotin labeled antibody 2A11 and Bar208, then the existence of labelled antibody on the solid phase is developed the color by adding strepto-affinity element-superoxide enzyme conjugates, described conjugate is 0.2 μ g/ml final mixture.In this case, with them 37 ℃ of incubations 30 minutes.Then, carry out 5 times with cleaning fluid and clean, described cleaning fluid comprises 10 ^-2The M phosphate buffer, pH 7.4 and with respect to the Tween 20 of cleaning fluid gross weight 0.05wt%.In each hole, add 100 μ l peroxidase substrate and as the 2ml tetramethyl biphenyl amine aqueous solution (TMB) of chromogen.With them in the dark environment temperature incubation 30 minutes.In each hole, add 100 μ l 1N sulfuric acid solutions as stop buffer.450 and 620nm measure absorbance.

For the antibody of AChE mark, in the hole, add 200 μ l Ellman reagent people such as (, 1961) Ellman after, detect the existence that is fixed to the AChE on the solid phase by the absorbance of measuring 414nm.

Fig. 1 shows the gained result.

Can know from Fig. 1 and to find out, catch the animal that the 171st animal with genotype R/R and this position can be had the animal of genotype R/Q with antibody to 12F10/SAF-34-AChE, BAR226/SAF-34-AChE, 11C6/SAF-34-AChE, SAF-34/2A11-biotin and have a genotype Q/Q and obviously make a distinction with antibody/detection.

Determine that also all the other catch with antibody/detection with antibody not making discriminating between the different genotype of the 171st of prion protein PrP.

Can also know from Fig. 1 and to find out that not in conjunction with the antibody of the 171st specific allelic form, SAF-34 antibody both can also can be used antibody as detecting as catching with antibody as a kind of.When using antibody as detection, it uses the AChE mark.

Can also know and to find out with the 171st animal with genotype R/R and animal and to have right that the best difference of animal of genotype Q/Q identifies to being biotin labeled SAF-34/2A11 with genotype R/Q from Fig. 1.

In fact, antibody 2A11 is not in conjunction with the R allelic form of the 171st of PrP.

Embodiment 2: plasma sample is tested

Plasma sample is taken from the sheep that the 171st of PrP has genotype R/R or R/H or R/Q or H/Q or Q/Q.

The solution that each sheep plasma sample of 75 μ l is caused sex change with 75 μ l and have a reductibility mixes, the described solution that causes sex change and have a reductibility comprises with respect to causing sex change and having the sarcosyl of the overall solution volume 2wt.% of reductibility, with respect to causing sex change and having the lauryl sulfate of overall solution volume 2wt.% of reductibility and the dithiothreitol (DTT) of 10mM.

Then potpourri was heated 10 minutes at 60 ℃.

Comprise catch with the hole of the microtiter plate of antibody in interpolation 100 μ potpourris that l obtains, described seizure antibody is SAF-34 antibody.

Then, with cleaning fluid the hole is carried out 3 times and clean, described cleaning fluid comprises 0.01M Tris, 0.3MNaCl, pH 7.4 and with respect to the Tween 20 of cleansing solution gross weight 0.1wt.%.

Cleaning fluid is buffered to neutral pH or alkalescent, and comprises the neutrality/non-ionic detergent (0.01%Tween 20) of low concentration.

After the cleaning, every hole is added 100 μ l and is detected that to use antibody, described antibody be the 2A11 antibody of biotin coupling, being that the concentration of every liter 3 μ g antibody exists with respect to obtained final mixture gross weight.

With the hole 37 ℃ of incubations 1 hour.

Carrying out 3 times with previously described cleaning fluid then cleans.

By adding the described strepto-affinity element of " material " chapters and sections-superoxide enzyme conjugates the existence of labelled antibody on the solid phase is developed the color, described conjugate is 0.2 μ g/ml final mixture.

With the hole 37 ℃ of incubations 30 minutes.

Carrying out 5 times with previously described cleaning fluid then cleans.

In each hole, add 100 μ l peroxidase substrate and as the 2ml tetramethyl biphenyl amine aqueous solution (TMB) of chromogen.

With the hole in the dark environment temperature incubation 30 minutes.

In each hole, add 100 μ l 1N sulfuric acid solutions as stop buffer.

450 and 620nm measure absorbance (optical density).

Fig. 2 provides the gained result.

Fig. 2 shows that the optical density that obtains from the genotypic blood plasma of all kinds of sheep distributes.

From Fig. 2 as seen, SAF-34 antibody/biotin labeled 2A11 is to realizing fabulous discriminating between different sheep genotype.

Embodiment 3: under the condition different with embodiment 2 plasma sample is tested

In this embodiment, denaturant is a chaotropic agent, more precisely is 8M urea.

Plasma sample is taken from the 171st sheep with genotype R/R or R/Q or Q/Q of PrP.

The solution that causes sex change and have a reductibility that each sheep plasma sample of 65 μ l and 65 μ l is comprised 8M urea and 10mM dithiothreitol (DTT) mixes.

Then potpourri was heated 10 minutes at 60 ℃.

Then, add 130 μ l EIA damping fluids (Enzyme Immuno Assay), described EIA damping fluid comprises the 1M kaliumphosphate buffer, and pH 7.4,0.15M NaCl and with respect to the bovine serum albumin (BSA) of EIA damping fluid cumulative volume 0.1wt.%.

Comprise catch with the hole of the microtiter plate of antibody in interpolation 100 μ potpourris that l obtains, described seizure antibody is SAF-34 antibody.

With potpourri 20 ℃ of incubations 1 hour.

Then, with cleaning fluid the hole is carried out 3 times and clean, described cleaning fluid comprises 0.01M Tris, 0.3MNaCl, pH 7.4 and with respect to the Tween 20 of cleaning fluid cumulative volume 0.1wt.%.

After the cleaning, every hole is added 100 μ l and is detected that to use antibody, described antibody be biotin labeled 2A11, and the concentration of 2A11 antibody-biotin conjugates is obtained final mixture by 0.5 μ g/ml.

With the hole 4 ℃ of incubations 1 hour.

Carrying out 3 times with previously described cleaning fluid then cleans.

After the cleaning, described according to " material " chapters and sections, 100 μ l strepto-affinity element-superoxide enzyme conjugates are added in every hole, and concentration is 0.1 μ g/ml final solution.

With the hole 20 ℃ of incubations 30 minutes.

Carrying out 5 times with previously described cleaning fluid then cleans.

In each hole, add 100 μ l peroxidase substrate and as the 2ml tetramethyl biphenyl amine aqueous solution (TMB) of chromogen.

With the hole in the dark environment temperature incubation 30 minutes.

In each hole, add 100 μ l 1N sulfuric acid solutions as stop buffer.

450 and 620nm measure absorbance.

Fig. 3 provides the gained result.

By comparison diagram 2 and 3, find that antibody can realize fabulous discriminating to SAF-34/2A11 between different sheep genotype, regardless of causing sex change and having the composition of the solution of reductibility.

Embodiment 4: the research of denaturant/influence that the reductive agent combination exists

The purpose of this embodiment is to be evaluated at and employedly causes sex change and have the importance that comprises reductive agent and denaturant in the solution of reductibility.

For this purpose, the scheme of following embodiment 3 is to testing from the 171st blood plasma with sheep of genotype Q/Q or R/R of PrP, promptly uses to comprise 8M urea as denaturant and the 10mMDTT solution as reductive agent.

Use that only to comprise denaturant be that the solution of 8M urea is tested identical sheep plasma.

These identical samples are that the solution of 10mM DTT is handled with only comprising reductive agent also.

Measure in these blood plasma every part optical density at 450-620nm.

Institute obtains and the results are shown in Fig. 4, the wherein white post representative optical density that blood plasma obtained of the solution-treated that comprises denaturant and reductive agent, grid posts representative is with only containing denaturant and do not contain the density that the biology humoral sample of the solution-treated of any reductive agent is obtained, and the column with slant lines representative is with only containing reductive agent and not containing the optical density that sample obtained of the solution-treated of any denaturant.

As can be seen from Figure 4, the combination of reductive agent and denaturant (surfactant and/or chaotropic agent) exists differentiating that different genotype is indispensable.

Should be noted that because the 171st R allelic form of antibody 2A11 and PrP do not combine the result that obtains of institute does not show any difference between the test carried out of use denaturing soln, whether comprise reductive agent regardless of denaturing soln.

Sheep serum is carried out the test identical with embodiment 1 to 4.Obtain similar result.

In case the genotype that protein is the 171st identified, might select to expect the sheep that is used to breed.

Preferably, selected sheep is those sheep with genotype R/R, the antipruritic disease of described genotype.

Therefore, might select to have this genotypic ram and ewe, make the sheep that only can breed genotype R/R, perhaps only select the ram of genotype R/R, and allow they and the ewe of range gene type breed according to plan.

Advantageously, method of the present invention can utilize kit to implement.

In first embodiment, kit of the present invention comprises:

-a kind of solid phase that is fixed with at least a seizure with antibody, described seizure is selected from antibody SAF-15, SAF-31, SAF-32, SAF-33, SAF-34, SAF-35, SAF-37,3B5, SAF-84, SHA-31, BAR-222, BAR-224, BAR-233 and 8G8 with antibody

-cause sex change and have reductibility solution and

-at least a detection antibody particularly is selected from antibody 12F10, BAR226,11C6 and 2A11 through mark, more particularly passes through the 2A11 of mark.

In second embodiment, kit of the present invention comprises:

-a kind of solid phase that is fixed with at least a seizure with antibody, described seizure is selected from antibody 12F10, BAR226,11C6 and 2A11 with antibody,

-cause sex change and have reductibility solution and

-at least a detection antibody is selected from antibody SAF-15, SAF-31, SAF-32, SAF-33, SAF-34, SAF-35, SAF-37,3B5, SAF-84, SHA-31, BAR-222, BAR-224, BAR-233 and 8G8, and described antibody is through mark.

Preferably, in kit of the present invention, described solid phase is a microtiter plate, is preferably made by polystyrene.

Preferably, in first embodiment of kit of the present invention, described seizure antibody is SAF-34 antibody or 3B5 antibody, and described detection antibody is 2A11 antibody.

And preferably, in kit of the present invention, described denaturing soln comprises lauryl sodium sulfate, dithiothreitol (DTT) and sarcosyl.

Embodiment 5: serum is tested

Research has amino acid R/R from the 171st of PrP, perhaps R/H, perhaps R/Q, perhaps H/Q, the optical density that the sheep serum of H/H and Q/Q obtains (OD) distributes, wherein spectrodensitometry is followed and similar scheme described in the embodiment 2, just the sample of being studied is a serum, DTT concentration is 20mM, catching with antibody is 3B5, detecting with antibody is that 2A11 and consumption are with respect to obtaining final mixture gross weight 2.2 μ g/L antibody, and the existence of labelled antibody is 0.37 μ g/L final mixture by adding concentration on the solid phase, the described strepto-affinity element of " material " chapters and sections/superoxide enzyme conjugates develops the color.

Fig. 5 shows the gained result.

Between different sheep genotype, observe good discriminating.

Embodiment 6: reductant concentration is to the research of plasma sample influence

Plasma sample is taken from the 171st sheep with genotype R/R or R/Q or Q/Q of PrP.

The solution that each sheep plasma sample of 75 μ l is caused sex change with 75 μ l and have a reductibility mixes, the described solution that causes sex change and have a reductibility comprises with respect to causing sex change and having the sarcosyl of the overall solution volume 2wt.% of reductibility, with respect to causing sex change and having the lauryl sulfate of overall solution volume 2wt.% of reductibility and the dithiothreitol (DTT) of 10mM, 20mM or 30mM.

Then potpourri was heated 10 minutes at 60 ℃.

Comprise catch with the hole of the microtiter plate of antibody in interpolation 100 μ potpourris that l obtains, described seizure antibody is 3B5 antibody.

Then, with cleaning fluid the hole is carried out 3 times and clean, described cleaning fluid comprises 0.01M Tris, 0.3MNaCl, pH 7.4 and with respect to the Tween 20 of cleaning fluid gross weight 0.1wt.%.

Cleaning fluid is buffered to neutral pH or alkalescent, and comprises the neutrality/non-ionic detergent (0.01%Tween 20) of low concentration.

After the cleaning, every hole is added 100 μ l and is detected and to use antibody, i.e. conjugate biotin 2A11 antibody, and it is being that the concentration of every liter 3 μ g antibody exists with respect to obtained final mixture gross weight.

With the hole 37 ℃ of incubations 1 hour.

Carrying out 3 times with previously described cleaning fluid then cleans.

By adding strepto-affinity element-superoxide enzyme conjugates of describing in " material " chapters and sections the existence of labelled antibody on the solid phase is developed the color, described conjugate is 0.4 μ g/ml final mixture.

37 ℃ of incubations 30 minutes.

Then, carrying out 5 times with previously described cleaning fluid cleans.

In each hole, add 100 μ l peroxidase substrate and as the 2ml tetramethyl biphenyl amine aqueous solution (TMB) of chromogen.

With the hole in the dark with environment temperature incubation 30 minutes.

In each hole, add 100 μ l 1N sulfuric acid solutions as stop buffer.

450 and 620nm measure absorbance (optical density).

Fig. 6 shows the gained result.

Observe, the variation of reductant concentration has no significant effect the discriminating between the population.Embodiment 7: reductant concentration is to the research of serum influence

Blood serum sample is taken from the 171st sheep with genotype R/R or R/Q or Q/Q of PrP.

The solution that each sheep sample of 75 μ l is caused sex change with 75 μ l and have a reductibility mixes, the described solution that causes sex change and have a reductibility comprises with respect to causing sex change and having the sarcosyl of the overall solution volume 2wt.% of reductibility, with respect to causing sex change and having the lauryl sulfate of overall solution volume 2wt.% of reductibility and the dithiothreitol (DTT) of 20mM or 30mM.

Then potpourri was heated 10 minutes at 60 ℃.

Comprise catch with the hole of the microtiter plate of antibody in interpolation 100 μ potpourris that l obtains, described seizure antibody is 3B5 antibody.

Then, with cleaning fluid the hole is carried out 3 times and clean, described cleaning fluid comprises 0.01M Tris, 0.3MNaCl, pH 7.4 and with respect to the Tween 20 of cleaning fluid gross weight 0.1wt.%.

Cleaning fluid is buffered to neutral pH or alkalescent, and comprises the neutrality/non-ionic detergent (0.01%Tween 20) of low concentration.

After the cleaning, every hole is added 100 μ l and is detected and to use antibody, i.e. biotin coupling 2A11 antibody, and it is being that the concentration of every liter 2 μ g antibody exists with respect to obtained final mixture gross weight.

With the hole 37 ℃ of incubations 1 hour.

Carrying out 3 times with previously described cleaning fluid then cleans.

By adding strepto-affinity element-superoxide enzyme conjugates of describing in " material " chapters and sections the existence of labelled antibody on the solid phase is developed the color, described conjugate is 0.33 μ g/ml final mixture.

Then 37 ℃ of incubations 30 minutes.

Then, carrying out 5 times with previously described cleaning fluid cleans.

In each hole, add 100 μ l peroxidase substrate and as the 2ml tetramethyl biphenyl amine aqueous solution (TMB) of chromogen.

With the hole in the dark environment temperature incubation 30 minutes.

In each hole, add 100 μ l 1N sulfuric acid solutions as stop buffer.

450 and 620nm measure absorbance (optical density).

Fig. 7 shows the gained result.

Observe, the variation of reductant concentration has no significant effect the discriminating between the population.

Embodiment 8: blind test

195 parts of blood serum samples are taken from different sheep.

These 195 parts of samples of being gathered are carried out the scheme of description among the embodiment 5.

Also use the serum (contrast) of the known R/R of being of its genotype, R/Q and Q/Q to carry out the scheme of describing among the embodiment 5.

Fig. 8 shows the gained result.

Observe, can bestly identify the genotype of institute's test sample product.

Claims (26)

1. identify the 171st genotypic method of sheep PrP, it is characterized in that it may further comprise the steps:

A) comprise the sheep biology humoral sample to be measured of PrP with the solution-treated that causes sex change and have a reductibility,

B) PrP after sex change and the reduction is fixed on the solid phase, optional fixes by part,

C) make sex change, reduction and fixing after PrP contact with antibody with at least a detection, and

D) detect described at least a detection with may the existing of antibody,

And

Wherein said part and described at least a detection are with combining with the 171st the PrP specificity with specific allelic form one of in the antibody.

2. the method for claim 1 is characterized in that the described solution that causes sex change and have a reductibility comprises:

A) at least a denaturant, it is selected from:

-surfactant, it is selected from:

● anionic surfactant, such as SDS (lauryl sodium sulfate), sarcosyl (Hamposyl L), sodium taurocholate, sodium glycocholate, NaTDC, natrium taurocholicum, Sodium Caprylate, 1-decane sodium sulfonate, NaLS and lauryl sulfate lithium;

● zwitterionic surfactant, such as SB 3-10 (the sweet Lay alkali of decyl-sulfo group), SB 3-12 (the sweet Lay alkali of dodecyl-sulfo group), SB 3-14 (the sweet Lay alkali of myristyl-sulfo group), SB 3-16 (the sweet Lay alkali of cetyl-sulfo group), SB 3-18 (the sweet Lay alkali of octadecyl-sulfo group), CHAPS and CHAPSO and deoxidation CHAPS;

● non-ionic surfactant, such as Triton X-100, Triton X-114, Tween 20, Tween 80, Brij 35 (polyoxyethylene 23 lauryl ethers), nonidet P-40 ,-just-decyl-β-D-glucopyranoside, just-dodecyl-β-D-glucopyranoside, just-octyl-β-D-glucopyranoside and just-octyl-α-D-glucopyranoside;

● their potpourri, and/or

-chaotropic agent, and

B) at least a reductive agent.

3. method as claimed in claim 2 is characterized in that described chaotropic agent is selected from one of urea, guanidine, guanidine hydrochloride, guanidine thiocyanate or their potpourri.

4. any described method in the claim as described above is characterized in that the described solution that causes sex change and have a reductibility comprises the potpourri of ionic surface active agent.

5. any described method in the claim as described above, the wherein said solution that causes sex change and have a reductibility comprise with respect to being the surfactant of 0.5wt.% at least by pending sample and the cumulative volume that causes sex change and have a potpourri that the solution of reductibility constitutes.

6. as any described method in the claim 2 to 5, it is characterized in that described at least a reductive agent is selected from: one of DTT (dithiothreitol (DTT)), TCEP (three (2-carboxy ethyl) phosphine) hydrochloride, DTE (dithioerythritol), beta-mercaptoethanol, 2-mercaptoethylmaine and their potpourri.

7. as any described method in the claim 2 to 6, wherein by pending sample and cause sex change and have the reductant concentration that comprises in the potpourri that the solution of reductibility constitutes between 2.5mM and 100mM.

8. any described method in the claim as described above is characterized in that the described solution that causes sex change and have a reductibility comprises the potpourri of sarcosyl, lauryl sodium sulfate and dithiothreitol (DTT).

9. any described method in the claim as described above is characterized in that describedly causing sex change and having that chaotropic agent concentration is equal to, or greater than 1M in the solution of reductibility.

10. as any described method of claim, it is characterized in that described chaotropic agent is a urea.

11. any described method in the claim as described above, wherein in step b), PrP after sex change and the reduction fixes by part, described part is the seizure antibody that can keep PrP by affine combination, and wherein in step c), described detection antibody is the antibody that can combine with the 171st the PrP specificity with specific allelic form, and this position is different from the epi-position site that described seizure is discerned with antibody.

12. as any described method in the claim 1 to 10, wherein in step b), PrP after sex change and the reduction fixes by part, described part is the seizure antibody that can combine with the 171st the PrP specificity with specific allelic form, and wherein in step c), described detection antibody is the antibody that can combine with PrP by affine combination, and described combination occurs in and is different from the epi-position site of described seizure with the antibody recognition site.

13. as any described method in the claim 1 to 10, wherein in step b), PrP after sex change and the reduction fixes by part, described part is selected from plasminogen, affinity element, strepto-affinity element, monose aminoglycan, hesperidine, porphyrin, streptomysin and tetracycline, and wherein in step c), described detection antibody is the antibody that can combine with the 171st the PrP specificity with specific allelic form.

14. as any described method in the claim 1 to 10, wherein in step b), PrP after sex change and the reduction directly is fixed on the solid phase, and wherein in step c), and described detection antibody is the antibody that can combine with the 171st the PrP specificity with specific allelic form.

15. any described method in the claim as described above, that wherein said solid phase is selected from is that polymkeric substance is made, polystyrene is that make, that tygon is made or latex is made, the microtiter plate that microtiter plate, pearl, pipe and polystyrene are made.

16. any described method in the claim as described above, the sample of wherein said biology body fluid is blood, blood plasma, serum or milk.

17. as any described method in the claim 1 to 11,13 to 16, it is characterized in that described part is to catch to use antibody, it is selected from antibody SAF-15, SAF-31, SAF-32, SAF-33, SAF-34, SAF-35, SAF-37,3B5, SAF-84, SHA-31, BAR-222, BAR-224, BAR-233 and 8G8.

18., it is characterized in that described detection is selected from the 2A11 antibody through mark, the 11C6 antibody through mark, the BAR226 antibody of process mark and the antibody 12F10 of process mark with antibody as any described method in the claim 1 to 11,13 to 17.

19. method as claimed in claim 11 is characterized in that described seizure antibody is SAF-34 antibody or 3B5 antibody, and described detection antibody is the 2A11 antibody through mark.

20. as any described method in the claim 1 to 10,12 and 15 to 16, it is characterized in that described part is to catch to use antibody, it is selected from antibody 2A11,11C6, BAR-226 and 12F10.

21., it is characterized in that described detection is selected from SAF-15, SAF-31, SAF-32, SAF-33, SAF-34, SAF-35, SAF-37,3B5, SAF-84, SHA-31, BAR-222, BAR-224, BAR-233 and 8G8 with antibody as any described method in the claim 1 to 10,12,15 to 16 and 20.

22. method as claimed in claim 12, it is characterized in that described seizure is selected from antibody 2A11,12F10, BAR226 and 11C6 with antibody, and described detection is selected from antibody SAF-15, SAF-31, SAF-32, SAF-33, SAF-34, SAF-35, SAF-37,3B5, SAF-84, SHA-31, BAR-222, BAR-224, BAR-233 and the 8G8 of process mark with antibody.

23. according to resistance the sheep population is carried out method for screening, it is characterized in that it comprises the embodiment of any described method in the claim as described above to the infectiousness SSE.

24. according to susceptibility the sheep population is carried out method for screening, it is characterized in that it comprises the embodiment of any described method in the claim as described above to the infectiousness SSE.

25. identify the 171st genotypic kit of sheep PrP, comprising:

-solid phase, fixed at least a seizure antibody on the described solid phase, described seizure particularly is selected from antibody SAF-15, SAF-31, SAF-32, SAF-33, SAF-34, SAF-35, SAF-37,3B5, SAF-84, SHA-31, BAR-222, BAR-224, BAR-233 and 8G8 with antibody

-cause sex change and have reductibility solution and

-at least a detection antibody particularly is selected from antibody 12F10, BAR226,11C6 and 2A11 through mark, more particularly passes through the antibody 2A11 of mark.

26. identify the 171st genotypic kit of sheep PrP, it is characterized in that it comprises:

-solid phase has been fixed at least a seizure antibody on the described solid phase, described seizure particularly is selected from antibody 12F10, BAR226,11C6 and 2A11 with antibody,

-cause sex change and have reductibility solution and

-at least a detection antibody particularly is selected from antibody SAF-15, SAF-31, SAF-32, SAF-33, SAF-34, SAF-35, SAF-37,3B5, SAF-84, SHA-31, BAR-222, BAR-224, BAR-233 and 8G8 through mark.

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