CN101307108B - Anti-phosphorylation Tau protein antibody use for detecting AD sign abnormal phosphation Tau protein level - Google Patents
Anti-phosphorylation Tau protein antibody use for detecting AD sign abnormal phosphation Tau protein level Download PDFInfo
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Abstract
The invention provides an anti-phosphorylation Tau protein antibody and an application of the antibody in Alzheimer disease abnormal phosphorylation Tau protein level determination. The antibody comprises rabbit polyclonal antibodies which are respectively anti phosphorylation Tau protein at Thr181 site, anti phosphorylation Tau protein at Thr205 site, anti phosphorylation Tau protein at Thr231site, anti phosphorylation Tau protein at Ser262 site, anti phosphorylation Tau protein at Ser396 site and anti phosphorylation Tau protein at Ser404 site. The Tau protein comprises Tau protein from human beings, Tau protein from a rat and Tau protein from a mouse. The application of the antibody in the Alzheimer disease abnormal phosphorylation Tau protein level determination comprises the following steps of: preparing a brain tissue paraffin section for each group through establishing an Alzheimer disease rat model; adopting the antibodies which respectively resist the phosphorylation Tau protein of each site as a first antibody; and detecting the phosphorylation Tau protein in nerve cells of the rat brain tissue of each group so as to detect the phosphorylation Tau protein of each site. The six antibodies can be developed into kits for diagnoses and evaluations of the Alzheimer disease for human and related animal models.
Description
Technical field
The invention belongs to immunobiology and medical diagnosis category; Be specifically related to a kind of anti-respectively Thr181; Thr205, Thr231, Ser262; Ser396, Ser404 site proteic antibody of amino acid phosphorylation Tau and the purposes of measuring at the unusual phosphorylation Tau protein level of AD disease (alzheimer's disease) thereof.
Background technology
Tau albumen is a kind of phosphoglycoprotein that contains that extensively is present in the neurone, in brain, mainly concentrate among the neurocyte aixs cylinder (with the bonding force of aixs cylinder than strong with the bonding force of cell space or dendron).Through with neural axon in microtubule combine, play that promote microtubule polymerization, prevent depolymerization and keep the microtubule function equivalent should.Isolating Protein tau matter has 5～6 kinds of isomer at least in the healthy adult human brain, all is to form by being positioned at the same single-gene coding of human No. 17 karyomit(e) on long-armed.The about 100kb of this full length gene comprises 16 exons, owing to its transcript mRNA product has produced different isomer proteins in the difference (to tau exon 2,3, the splicing of 10 selectivity) of transcribing in the splicing of back.Minimum 352 amino acid of Protein tau isomer; 441 maximum amino acid; All the other four kinds is that 381,383,410,412 amino-acid residues are formed, and the difference between them is that C-terminal has 0,1 or 2 insertion sequence that is made up of 29 amino-acid residues by 3 or 4 microtubule land and N-terminal of being made up of 31～32 amino-acid residues.In the fetus period cerebral tissue the shortest a kind of Protein tau isomer (352 amino acid) is only arranged, its C-terminal contains 3 series connection repetitive sequences, and N hold no insertion sequence, and Protein tau is (the 7moles pi/ molecule) that is in the hyperphosphorylation state at this moment.Along with the progress phosphorylation degree of growth course reduces gradually, the Protein tau phosphorylation site seldom on average has only 2～3 in the normal mature brain, and this moment, Protein tau combined with tubulin, and Stability Analysis of Structures is difficult by phosphorylation.
Senile dementia; Be called alzheimer's disease (Alzheimer disease again; AD) be a kind of carrying out property of the elderly's neural system, degenerated disease; Performances such as mainly contain cognition dysfunctions such as memory, understanding, judgement and orientation clinically, mental act is unusual, and intelligence reduces and viability goes down.Since Alois Alzheimer in 1907 delivered the 1st routine clinical pathology report, existing centenary research was historical, but pathogenesis still imperfectly understands so far.It is generally acknowledged, when this disease is multifactor acting in conjunction due to, its morbidity and heredity, environment, metabolism factors are relevant.Show that from the data of epidemiology survey the sickness rate of Alzheimer and age ageing degree have obvious dependency: 65～74 years old sickness rate be 3%～6%, 75～85 years old 18.7%,＞85 years old 47%.The incidence and the western countries of China are roughly the same, estimate existing patient about 3,600,000, and along with the increasing of population predicted life, the arrival of social senilization, its number of patients also can increase gradually.
From pathology; AD disease shows as two big characteristics: 1. senile plaque (Senile Plaques, SP): claim the axon spot again, by a kind of kind of starch appearance precursor protein (Amyloid Precussor Protein that is called as; APP) (molecular weight 4KD; Belong to the laminar structured albumen of β, so claim β A4 albumen again) be core, on every side by axon, dendron, kind of starch appearance fiber and the spongiocyte and the projection thereof etc. of sex change around forming.Around those around nervous process usually be dystrophic, (Paired HelicalFilaments, PHFs), and PHFs is made up of the Protein taus of a large amount of highly unusual phosphorylations to contain the double stranded helix fibril.2. (Neurofibrillary Tangles, NFTs): it is by the neurone inclusion body that the abnormal cells skeleton is formed, and its form is different with the different sorts cell for NFT.The structure of NFTs also is made up of PHFs.Show that through the protein antibodies affinity tag staple of proof PHFs is the Protein tau of highly unusual phosphorylation, the Tau albumen of these highly unusual phosphorylations forms thread appearance settling and is deposited on and finally causes neuronic infringement in the neurone just.
Up to now; It is found that nearly 31 of the site that relates to the unusual phosphorylation modification of Protein tau; Wherein have at least 10 to be that proline(Pro) instructs, i.e. the protein kinase of these phosphorylation sites and proline(Pro) guidance (Proline Directed Protein Kinase, PDPK) relevant.Other has 15 sites on 46,198,199,202,208,210,235,262,396,400,404,409,412,413 and 422 serine residues; Other 6 sites are on 181,205,212,217,231 and 403 threonine residues.Can the Protein tau in the AD disease brain be divided into 3 components with different biochemical stripping technique: the 1. Protein tau of the non-unusual modification of endochylema (c-tau); 2. unusual the modification is prone to dissolve type albumen (AD P-tau) and 3. unusual the modification and accumulative Protein tau (PHF-tau).According to the dissolving properties difference of PHF-tau in 2% sodium lauryl sulphate, can it be divided into two kinds in PHFI-tau and PHFII-tau albumen again.Notable attribute is that agent structure is Pro-Gly-Gly-Gly and " repeats series connection " and occur in the Protein tau structure; Protein tau combines with duplex filament (PHF) by " repeating series connection " district; Promote PHF that irreversible polymerization takes place, caused formation and the neurone generation sex change of NFT.Cambridge etc. disclosed through immunocytochemistry and Biochemical Research in 1988: the Protein tau of hyperphosphorylation is the main subunit of PHF.The unusual phosphorylation of AD patient's brain tau has following characteristics: all special-shaped tau that 1. are not only among the PHF all exist with unusual phosphorylation form, and free tau is also by phosphorylation in the endochylema; 2. unusual phosphorylation appears not only at the N-terminal of tau, and in its intramolecular multi-section position; Though the phosphoric acid that 3. every mol albumen contains is 4 times of normal tau molecule, the aggregate level of the phosphoric acid protein phosphatase in AD patient's brain homogenate is compared with normal person not have significantly and is changed.The unusual phosphorylation modification of Protein tau in the AD brain can further promote it that glycosylation takes place, saccharification, and racemization, the wrong modification in translation such as ubiquitinization back, these process conformities have quickened the formation of PHF/NFTs together greatly.
Tau protein level in the enzyme-linked immunosorbent assay of employing AD disease patient blood plasma, the cerebrospinal fluid (CSF) is arranged at present clinically; The result finds that the Tau protein level all significantly increases than normal and non-sacred disease patient group of the same age among the AD disease patient CSF; Be used for AD diagnosis and observation of curative effect with this; Its susceptibility is 82%, and it is 70% that specificity reaches.Reduce if also detect β-AP42 level simultaneously, the specificity that senile dementia is diagnosed can reach 70%～90%.So many in the world companies and research institution are all actively developing the research of this respect, but fewer based on the immunohistochemistry detection and the dependent diagnostic method of phosphorylation site specificity T au protein antibodies at present, major part is to stay in research category.So six kinds of anti-respectively Thr181 of this type provided by the invention; Thr205, Thr231, Ser262; Ser396, Ser404 site proteic antibody of amino acid phosphorylation Tau and the purposes on the unusual phosphorylation Tau protein level mensuration of AD disease thereof have important use and are worth.
Reference
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Summary of the invention
One of the object of the invention provides the proteic antibody of a kind of anti-phosphorylation Tau.This antibody-like is to the proteic phosphorylation site specific antibody of same Tau, has specifically comprised six kinds of anti-respectively Thr181, Thr205, Thr231, Ser262, Ser396, the proteic rabbit polyclonal antibody of Ser404 site phosphorylation modification Tau.
Another object of the present invention provides the purposes that a kind of such anti-phosphorylation Tau protein antibodies is measured at the unusual phosphorylation Tau protein level of alzheimer's disease cerebral tissue; That is, these six kinds of antibody are used for the method for use that the unusual phosphorylation Tau protein level of brain tissue slice AD disease is measured.These six kinds of antibody detect the method for phosphorylation Tau protein level in the alzheimer's disease rat model brain tissue slice respectively with routine immunization histochemical method, and further promote the diagnosis in human alzheimer's disease and estimate application.
The technical scheme of accomplishing the foregoing invention task is: the proteic antibody of a kind of anti-phosphorylation Tau, it is characterized in that,
Six kinds of anti-respectively Thr181, Thr205, Thr231; Ser262, Ser396, the proteic rabbit polyclonal antibody of Ser404 site phosphorylation modification Tau; Antigenic peptide below having adopted respectively during preparation; Its concrete sequence is respectively (annotate: italic and the amino acid as phosphorylation that uses double underline to mark in the sequence, N end halfcystine is artificial the interpolation, is used for the carrier connection):
The antigenic peptide CTPPAPK in Thr181 site
PPSSGE
The antigenic peptide CSPGSPG in Thr205 site
PGSRSR
The antigenic peptide CKVAVVR in Thr231 site
PPKSPS
The antigenic peptide CVKSKIG in Ser262 site
TENLKH
The antigenic peptide CAEIVYK in Ser396 site
PVVSGD
The antigenic peptide CVVSGDT in Ser404 site
PRHLSN
The preparation method of described six kinds of anti-phosphorylation Tau protein antibodies is: select above these six antigenic peptides respectively for use; Through adopting special-shaped difunctional coupling agent sulfosuccinimide 4-[N-methyl-maleic acid]-1-carboxylic hexanaphthene (SMCC) to be coupled to polypeptide on the carrier proteins KLH after the chemosynthesis, form the KLH-polypeptide antigen system that is fit to animal immune.Then through conventional steps such as rabbit immunity (every kind of antibody correspondence is selected the KLH-polypeptide antigen in this site for use), Antiserum Preparation and how anti-separation and purification; Final six kinds of anti-respectively Thr181 of this type that obtain; Thr205, Thr231, Ser262; Ser396, the proteic rabbit polyclonal antibody of Ser404 site phosphorylation modification Tau.
ELISA test shows: six kinds of anti-respectively Thr181 that the present invention is above-mentioned; Thr205, Thr231, Ser262; Ser396; The proteic rabbit polyclonal antibody of Ser404 site phosphorylation modification Tau, all to the binding specificity of specific site amino acid phosphorylation Tau albumen height, and the unphosphorylated Tau protein binding in site power is extremely low therewith.
More optimize with in particular, the proteic antibody of anti-phosphorylation Tau of the present invention is characterized in that, is meant the antibody that adopts following method to obtain:
(1). the antigenic peptide design is with synthetic:
According to the proteic sequence information of human Tau in the Protein Data Bank; The well-designed Thr181 that goes out six keys in six corresponding Tau albumen of difference, Thr205, Thr231; Ser262; Ser396, both sides, Ser404 phosphorylated amino acid site successive 14AA (comprising phosphoamino acid) polypeptide (N end artificial add a halfcystine), its concrete sequence is (annotating: italic and the amino acid as phosphorylation that uses double underline to mark in the sequence):
Thr181 CTPPAPK
PPSSGE
Thr205 CSPGSPG
PGSRSR
Thr231 CKVAVVR
PPKSPS
Ser262 CVKSKIG
TENLKH
Ser396 CAEIVYK
PVVSGD
Ser404 CVVSGDT
PRHLSN
(2). prepare above six antigenic peptide (P peptide) and other six unphosphorylated polypeptide of corresponding site amino acid (N peptide) that comprise corresponding site amino acid phosphorylation with chemical synthesis;
(3). be coupled to the P peptide on the carrier proteins KLH through the difunctional coupling agent sulfosuccinimide of abnormal shape 4-[N-methyl-maleic acid]-1-carboxylic hexanaphthene (SMCC), form the polypeptide-K LH antigen systems that is fit to animal immune;
Be coupled to P peptide, N peptide respectively on the carrier proteins BSA (bovine serum albumin) envelope antigen when being used as the ELISA detection;
(4). rabbit immunity and positive serum collection:
Dosage according to every rabbit 300ug polypeptide-K LH carries out fundamental immunity to rabbit; During fundamental immunity, adopt the polypeptide-K LH antigen emulsion that contains 50% Fu Shi Freund's complete adjuvant; Immunization route is subcutaneous multiple spot; Every later on booster immunization that carried out a time at a distance from 20 days; What adopt is the polypeptide-K LH antigen emulsion that contains 50% freund 's incomplete adjuvant; Take a blood sample during this time and do the ELISA test with the BSA-polypeptide, collection reached the positive serum of blood sampling standard according to ELISA result in 7 days behind the 4th booster immunization of completion;
(5). antibody separation and purification and evaluation:
Adopting chemical connection method to link GEL to polypeptide antigen (P peptide, N peptide) idol goes up to prepare the antigen post that affinity chromatography is used; The positive serum of gathering is behind the filtration of 0.45um filter, pH regulator; It is mixed 4 ℃ of slow stirred overnight with the even affinity chromatography GEL that has connected the phosphorylation polypeptide antigen; Second day with in its chromatography column of packing into, washes post with the glycine solution of pH2.8 after washing post with 10mM PBS, collects OD
280>=0.15 elutriant is also regulated pH neutrality rapidly; Corresponding site amino acid phosphorylation Tau protein antibodies (P-antibody) of anti-this P peptide and the corresponding site amino acid non-phosphorylating Tau protein antibodies (Pan-antibody) of anti-this P peptide have been comprised in this elutriant; Further this elutriant and the even affinity column that has connected corresponding non-phosphorylating polypeptide antigen are combined to remove the Pan-antibody that wherein is mixed with; Collect it and penetrate liquid, finally obtained the proteic rabbit polyclonal antibody finished product of the corresponding site amino acid phosphorylation modification Tau of anti-this P peptide and (both selected for use Thr181 site antigenic peptide finally can produce the proteic antibody finished product of anti-Thr181 site phosphorylation modification Tau, all the other Thr205 through dialysis, concentrated, interpolation glycerin for protecting agent; Thr231; Ser262, Ser396, the preparation process of Ser404 site antibody is similar);
The enzyme plate that encapsulates with the BSA-polypeptide antigen is done the ELISA test, data presentation its height tire and specify the specificity of site phosphorylation Tau albumen height.
The technical scheme of accomplishing the 2nd invention of the present invention task is: the purposes that the proteic antibody of described anti-phosphorylation Tau is measured at the unusual phosphorylation Tau protein level of alzheimer's disease (AD disease) is characterized in that step is following:
At first set up AD disease rat model (setting up castration group, estrogen receptor agonist intervention group simultaneously) through intracerebral injection A β 25-35 condensed state, model success back sacrificed by decapitation rat is respectively organized the cerebral tissue paraffin section with the ordinary method preparation.Adopt six kinds of anti-respectively Thr181 among the present invention then; Thr205, Thr231, Ser262; Ser396; When Ser404 site amino acid phosphorylation Tau protein antibodies is done the immunohistochemical methods detection one resists, and detects with routine immunization histochemical method and respectively organizes the phosphorylation Tau albumen in rat cerebral tissue's neurocyte, and the result respectively organizes and detected phosphorylation Tau albumen on the brain section; Show as that neurocyte endochylema and nervous process position present pale brown look (DAB colour developing) in the cerebral tissue specific region, and have certain difference on the intensity between each group.In conjunction with the pathological report of biochemical molecular, six kinds of anti-phosphorylation Tau protein antibodies of this invention can be developed as the immunohistochemical methods test kit, and the diagnosis and the treatment that are used for human alzheimer's disease are estimated.
In view of the proteic antibody of the anti-phosphorylation Tau of this type provided by the invention can be good at discerning the Tau albumen that comprises people, rat, mouse species; So it can be used in the diagnosis of the unusual phosphorylation Tau protein level of human alzheimer's disease for further promoting in the purposes that rat cerebral tissue's unusual phosphorylation Tau protein level of section AD disease is measured.
Description of drawings
Fig. 1 is that six kinds of anti-phosphorylation Tau albumen rabbits resist the immunohistochemical methods in the section of AD disease rat cerebral tissue to detect effect more.
Specific embodiment
1, one type of proteic Antibody Preparation of anti-phosphorylation Tau of embodiment:
(1). antigenic peptide is synthetic to be connected with the carrier idol: according to the proteic sequence information of human Tau in the Protein Data Bank; Designed the Thr181 of six keys in six difference corresponding Tau albumen; Thr205; Thr231, Ser262, Ser396; Both sides, Ser404 phosphorylated amino acid site successive, comprise the 14AA polypeptide (N end artificial add a halfcystine) of phosphoamino acid, the concrete sequence of these six antigenic peptides is (annotating: italic and the amino acid as phosphorylation that uses double underline to mark in the sequence):
Thr181 CTPPAPK
PPSSGE
Thr205 CSPGSPG
PGSRSR
Thr231 CKVAVVR
PPKSPS
Ser262 CVKSKIG
TENLKH
Ser396 CAEIVYK
PVVSGD
Ser404 CVVSGDT
PRHLSN
(2). prepare above six antigenic peptide (P peptide) and other six unphosphorylated polypeptide of corresponding site amino acid (N peptide) that comprise corresponding site amino acid phosphorylation with chemical synthesis.
(3). do coupling agent with sulfosuccinimide 4-[N-methyl-maleic acid]-1-carboxylic hexanaphthene (SMCC), be coupled to the P peptide on the carrier proteins KLH, form the polypeptide-K LH antigen that is fit to animal immune; Be coupled to P peptide, N peptide on the carrier proteins BSA (bovine serum albumin) respectively with quadrat method, be used for the envelope antigen that ELISA detects.Idol connects good antigen and is stored in-70 ℃ of refrigerators.
(4). rabbit immunity and positive serum collection: the dosage according to every rabbit 300ug polypeptide-K LH is implemented fundamental immunity to rabbit.During fundamental immunity, adopt the Fu Shi Freund's complete adjuvant: the emulsion that polypeptide-K LH antigen is processed with volume ratio at 1: 1, immunization route are subcutaneous multiple spot.Every later on booster immunization that carried out a time at a distance from 20 days; The dosage of every rabbit 200ug polypeptide-K LH during reinforcement; Adopt freund 's incomplete adjuvant: the emulsion that polypeptide-K LH antigen is processed with volume ratio at 1: 1; The separation of serum of taking a blood sample is during this time done the ELISA test with the BSA-polypeptide, and collection reached the positive serum of blood sampling standard according to ELISA result in 7 days behind the 4th booster immunization of completion.Generally according to the method described above, after four times booster immunization was accomplished, all more than 1: 64000, immune effect was splendid for the serum titer of rabbit.
(5). antibody separation and purification and evaluation: adopt chemical connection method to link GEL to polypeptide antigen (P peptide, N peptide) idol and go up to prepare the antigen post that affinity chromatography is used.The positive serum of gathering filters through the 0.45um filter, and adds 10% pH7.6Tris-HCl damping fluid (Tutofusin tris-hydrochloric acid) adjusting pH.Then serum is mixed 4 ℃ of slow stirred overnight with the affinity chromatography GEL that idol has connected the phosphorylation polypeptide antigen, second day with in its chromatography column of packing into, washes post with the glycine solution of pH2.8, collection OD after washing post with 10mM PBS
280>=0.15 elutriant is also regulated pH neutrality rapidly; Corresponding proteic antibody of site amino acid phosphorylation Tau (P-antibody) of anti-this P peptide and the corresponding proteic antibody of site amino acid non-phosphorylating Tau (Pan-antibody) of anti-this P peptide have been comprised in this elutriant; Further elutriant and the even affinity column that has connected corresponding N peptide are combined to remove the Pan-antibody that wherein is mixed with; Collect it and penetrate liquid, finally obtained the proteic rabbit polyclonal antibody finished product of the corresponding site amino acid phosphorylation modification Tau of anti-this P peptide and (both selected for use Thr181 site antigenic peptide finally can produce the proteic antibody finished product of anti-Thr181 site phosphorylation modification Tau, all the other Thr205 through dialysis, concentrated, interpolation glycerin for protecting agent; Thr231; Ser262, Ser396, the preparation process of Ser404 site antibody is similar);
The enzyme plate that encapsulates with the BSA-polypeptide antigen is done the ELISA test, and data (seeing table 1) have shown that the height of these six kinds of antibody tires (all greater than 1: 64000) and specify the specificity (under 1: 64000 weaker concn antibody to the OD value of P peptide far above the OD value to corresponding N peptide) of site phosphorylation Tau albumen height.
The ELISA test result that six kinds of anti-phosphorylation Tau albumen rabbits of table 1 resist more
Embodiment 2, anti-phosphorylation Tau protein antibodies detect phosphorylation Tau albumen in the AD disease rat animal model cerebral tissue with immunohistochemical method:
1, AD disease rat model is set up in A β 25-35 injection:
1mgA β 25-35 is dissolved in the 200 μ L sterile salines, processes 5nmol/ μ L concentration, sealing is placed on 96 hours formation A β 25-35 condensed states in 37 ℃ of cell culture incubators.Be divided into three groups of administration modelings:
AD disease model group rat: behind the 4% vetanarcol intraperitoneal injection of anesthesia; With the stereotaxic apparatus fixedly rat of cranium head position of making even; Confirm 3.8mm behind the bregma of right side, the other 2.5mm of center line; 3.0mm is a right side hippocampus drosal part under the skull surface, with slowly injecting A β 25-35 μ L in the 1 μ L microsyringe 5min, and the 10min that gives that let the acupuncture needle remain at a certain point.Postoperative gives the intramuscular injection of penicillin sodium salt 50,000 units, every day 1 time, for three days on end, puts to death rat to the 14th day.
AD castration group rat: (3-4mL/kg ip) undergos surgery under the anesthesia, and the about 3-4cm of aseptic abdomen center longitudinal incision extracts the bilateral ovaries tissue, puts uterine tube and goes into the abdominal cavity, intestinal tube and stomach return at 10% Chloral Hydrate.Before sewing up abdominal muscle, inject 100,000 unit penicillium mould, skin suture to the abdominal cavity.The capable continuously vaginal smear of postoperative.The same AD model of making in 2 week of castration art back.(the modeling time is totally 4 weeks)
The estrogen receptor agonist intervention group: castration art AD rat skin lower injection 17 beta estradiols after 4 weeks, the 200ug/Kg body weight 2 times weekly, continues two weeks (the modeling time is totally 6 weeks).
2, brain tissue slice is made
Each organizes the rat sacrificed by decapitation, takes out full brain and places 10% formaldehyde to soak after about 1～2 hour, and the crown cerebral tissue that cuts between optic chiasma and the mammillary body continued to place formaldehyde fixing 24 hours.Tissue block becomes wax stone, the crown section of row, every thickness 4um through routine dehydration, transparent, waxdip, embedding.
3, histochemical method detects phosphorylation Tau albumen
A, press standard immunoassay histochemical stain method, earlier with tissue slice in 60 ℃ of roasting sheet 1h, conventional YLENE soaks dewaxing, after the gradient ethanolic soln aquation, PBST flushing 3 times, each 5min.
B, antigen retrieval (boiling method): the citrate buffer (pH6.0) of 10mmol/L is put in section, and the heating timing is 3 minutes in microwave oven, and it is to be cooled to room temperature to take out container, section with PBST flushing 3 times, each 5min.
C, 3%H
2O
2-methanol solution block endogenous property px 15min, PBST flushing 3 times, each 5min;
D, section is performed mark, add above six kinds of anti-respectively Thr181, Thr205 respectively according to numbering with the immunohistochemical methods pen; Thr231, Ser262, Ser396; Amino acid phosphorylation Tau proteic rabbit in Ser404 site places 37 ℃ to hatch 1hr after resisting (extent of dilution 1: 50) more; PBST replaces an anti-negative control of doing.
E, PBST flushing 3 times, behind each 5min, every drips the instant ELivisionplus of new company s-generation group test kit reagent A advanced in years: one 50 μ L of Post blocking reagent, room temperature continues to hatch 30min in the wet box.
F, PBST flushing 3 times, each 5min, 50 μ L of every dropping group test kit reagent B (polymer two of HRP mark is anti-), room temperature was hatched 30 minutes.
G, PBST flushing 3 times, each 5min, the colour developing of DAB substrate, Hematorylin is redyed, dehydration, the neutral gum sealing of transparent back.
4, the proteic immunohistochemical methods interpretation of result of phosphorylation Tau
After each organized the immunohistochemical assay completion, section placed under the opticmicroscope and observes, and gets representational visual field photographic analysis.Result such as Fig. 2, visible six kinds of anti-Thr181, Thr205; Thr231, Ser262, Ser396; Ser404 site amino acid phosphorylation Tau albumen rabbit polyclonal antibody is in three groups of AD disease rat: all detected the positive in model group (A row), castration group (B row), the estrogen receptor agonist intervention group (C row); Coloration result shows as the neurocyte aixs cylinder and cell space partly presents pale brown look, and shows as certain trend between the group: AD castration group is that positive rate is the highest, and the AD group will be hanged down relatively; Estrogen intervention group is lower than AD castration group, shows as between AD castration group and AD group.
Claims (3)
1. the proteic antibody of anti-phosphorylation Tau is characterized in that, adopted respectively during described Antibody Preparation to Thr205 in the human Tau albumen, and Thr231, following three 14AA antigenic peptides of these three phosphorylation sites designs of Ser404, its concrete sequence is:
The antigenic peptide CSPGSPG in Thr205 site
PGSRSR
The antigenic peptide CKVAVVR in Thr231 site
PPKSPS
The antigenic peptide CVVSGDT in Ser404 site
PRHLSN
Italic and the amino acid as phosphorylation that uses double underline to mark in the above sequence, N end halfcystine is artificial the interpolation, is used for carrier and connects.
2. the proteic antibody of anti-phosphorylation Tau according to claim 1; It is characterized in that the proteic antibody of described anti-phosphorylation Tau is meant the antibody that adopts following method to obtain: select for use described respectively to Thr205 in the human Tau albumen; Thr231; Three 14AA antigenic peptides of these three phosphorylation site designs of Ser404 are coupled to polypeptide on the carrier proteins KLH through adopting special-shaped difunctional coupling agent sulfosuccinimide 4-[N-methyl-maleic acid]-1-carboxylic hexanaphthene after the chemosynthesis, form the KLH-polypeptide antigen system that is fit to animal immune; Through conventional rabbit immunity, every kind of antibody correspondence is selected the KLH-polypeptide antigen in this site for use then; Antiserum Preparation and how anti-separation and purification, final these three kinds anti-respectively Thr205 that obtain, Thr231, the proteic rabbit polyclonal antibody of Ser404 site phosphorylation modification Tau.
3. the proteic antibody of anti-phosphorylation Tau according to claim 1 and 2 is characterized in that, the proteic antibody of described anti-phosphorylation Tau is meant the antibody that adopts following method to obtain:
(1). the antigenic peptide design is with synthetic:
According to the proteic sequence information of human Tau in the Protein Data Bank, design three proteic Thr205 of the corresponding Tau of difference, Thr231, the one section successive in both sides, three phosphorylated amino acid sites of Ser404,14 amino acid whose polypeptide; Halfcystine of the artificial interpolation of N end of each polypeptide, its concrete sequence is:
Thr205?CSPGSPG
PGSRSR
Thr231?CKVAVVR
PPKSPS
Ser404?CVVSGDT
PRHLSN
(2). prepare above three antigenic peptides that comprise corresponding site amino acid phosphorylation as the phosphorylation polypeptide of P peptide with prepare the non-phosphorylating polypeptide of other three unphosphorylated polypeptide of corresponding site amino acid with chemical synthesis as the N peptide;
(3). be coupled to the P peptide on the carrier proteins KLH through the difunctional coupling agent sulfosuccinimide of abnormal shape 4-[N-methyl-maleic acid]-1-carboxylic hexanaphthene, form the polypeptide-K LH antigen systems that is fit to animal immune; Be coupled to P peptide, N peptide respectively on the carrier proteins BSA envelope antigen when being used as the ELISA detection;
(4). rabbit immunity and positive serum collection:
Dosage according to every rabbit 300ug polypeptide-K LH carries out fundamental immunity to rabbit; During fundamental immunity, adopt the polypeptide-K LH antigen emulsion that contains 50% Fu Shi Freund's complete adjuvant; Immunization route is subcutaneous multiple spot; Every later on booster immunization that carried out a time at a distance from 20 days; What adopt is the polypeptide-K LH antigen emulsion that contains 50% freund 's incomplete adjuvant; Take a blood sample during this time and do the ELISA test with the BSA-polypeptide, collection reached the positive serum of blood sampling standard according to ELISA result in 7 days behind the 4th booster immunization of completion;
(5). antibody separation and purification and evaluation:
Adopt chemical connection method to link the P peptide of polypeptide antigen, N peptide idol GEL and go up to prepare the antigen post that affinity chromatography is used; The positive serum of gathering is behind the filtration of 0.45um filter, pH regulator; It is connected the antigenic affinity chromatography GEL of P peptide with idol mixed 4 ℃ of slow stirred overnight; Second day with in its chromatography column of packing into, washes post with the glycine solution of pH2.8 after washing post with 10mM PBS, collects OD
280=0.15 elutriant is also regulated pH neutrality rapidly; Proteic antibody of the corresponding site amino acid phosphorylation Tau of anti-this P peptide and the corresponding site of anti-this P peptide amino acid non-phosphorylating Tau protein antibodies have been comprised in this elutriant; Further this elutriant and idol are connected the antigenic affinity column of corresponding N peptide and combined to remove the Pan-antibody that wherein is mixed with; Collect it and penetrate liquid, finally obtained the proteic rabbit polyclonal antibody finished product of the corresponding site amino acid phosphorylation Tau of anti-this P peptide through dialysis, concentrated, interpolation glycerin for protecting agent;
Enzyme plate so that the BSA-polypeptide antigen encapsulates is done the ELISA test.
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| CN106950378A (en) * | 2016-01-07 | 2017-07-14 | 牛远杰 | One kind diagnosis castration-resistant prostate cancer kit |
| CA3045294A1 (en) * | 2016-12-07 | 2018-06-14 | Genentech, Inc. | Anti-tau antibodies and methods of use |
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| CN112812187B (en) * | 2021-01-18 | 2021-12-17 | 南昌大学第一附属医院 | Phosphorylated EIF5B specific antibody and preparation method and application thereof |
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