CN106568969A - ELISA detection method of alpha-synuclein aggregate with phosphorylation on 129th position of serine - Google Patents

ELISA detection method of alpha-synuclein aggregate with phosphorylation on 129th position of serine Download PDF

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CN106568969A
CN106568969A CN201610913085.1A CN201610913085A CN106568969A CN 106568969 A CN106568969 A CN 106568969A CN 201610913085 A CN201610913085 A CN 201610913085A CN 106568969 A CN106568969 A CN 106568969A
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phosphorylation
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CN106568969B (en
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杨慧
段春礼
邢昊
高歌
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Capital Medical University
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Abstract

The invention relates to an ELISA detection method of alpha-synuclein aggregate with phosphorylation on 129th position of serine. According to the method, a monoclonal antibody C140S and a polyclonal antibody-SN16 antibody are used to carry out sandwich ELISA so as to measure the content of alpha-synuclein aggregate with phosphorylation on 129th position of serine in a human blood sample.

Description

A kind of ELISA inspections of 129 phosphorylation alpha- synuclein aggregation bodies of serine Survey method
Technical field
The present invention relates to the serine of aggregation 129 phosphorylation alpha- synapse nucleoproteins (α-syn) detection method Set up.
Background technology
Parkinson disease (Parkinson ' s Disease, PD) are used as one of modal neurodegenerative diseases, difference shadow 1% and 5% population in sound 65 years old and 85 years old age, alpha- synapse nucleoproteins (α-syn) are right as new revision in 2014 The core protein of PD definition, has caused people to pay much attention to realities of the α-syn in the detection of PD early diagnosiss, the course of disease and curative effect of medication Apply on border.Therefore, one or more sensitive and special detection method is badly in need of now, detects various forms of α-syn with PD Generation variation tendency of the development in human body fluid, this requires that this detection method is objective, feasible, and is easy to clinically Promoted.Used as PD main pathological hallmark thing, its main component is made up of Louis body (Lewy ' s Body, LB) α-syn, And α-the syn for having 90% in LB there occurs phosphorylation (pS129- α-syn) at 129.In order to study the progress of the PD courses of disease, people Make use of various methods monitoring α-syn variation tendencies in vivo, make a general survey of the document of detection α-syn in recent years, report PD α-syn contents in patient body fluid are not quite similar, but the content increase of phosphorylation α-syn and α-syn aggregations is basic one The report of cause.In above-mentioned significant research report, the phosphorylation α-syn of detection is the aggregation of monomeric form and α-syn Form, is phosphorylation form or unphosphorylated form without α-syn in clear and definite aggregation, more without detection of aggregation state Phosphorylation α-syn, because at present still without the method for detection of aggregation pS129- α-syn.And the phosphorylation α-syn for assembling is in PD Occur to play more crucial effect in evolution.And have document report, and active aldehydes --- including ONE (4-oxo-2- ), nonenal HNE (4-hydroxynonenal) --- the content in the cerebrospinal fluid of PD patient constantly rises with progression of disease Height, they can occur adduction (as follows) as the product of lipid peroxidation with the 50th hyte propylhomoserin of α-syn.And promote α- Syn is more easy to assemble.In view of the unicity for setting up the standard antigen used needed for standard curve, this patent uses HNE's Oxidized byproduct (Oxidative Counterpart) ONE promotes people source α-syn and pS129- α-syn to assemble, and sets up base In the detection method of sandwich method ELISA, to observe clinical cerebrospinal fluid, blood plasma, brain homogenates in the future in aggregation pS129- α-syn with the course of disease variation tendency.
The histidine of HNE and protein carries out post translational modification by 1,4-Michael adductions to albumen
Therefore, the detection method of 129 phosphorylation alpha-synapse nucleoproteins of aggregationization serine that we invent has important Meaning.129 phosphorylations α of aggregation serine of the PD models of blood and spinal fluid samples and basic research for clinical PD- The detection of synapse nucleoprotein provides detection meanss, it is also possible to set up the diagnostic kit of PD by the method.
The basis of ELISA is the immobilization and antigen or the enzyme labelling of antibody of antigen or antibody.With reference in solid phase carrier table The antigen or antibody in face still keeps the antigen or antibody of its immunologic competence, enzyme labelling both to retain its immunologic competence, retains again The activity of enzyme.Reacted with the antigen or antibody of surface of solid phase carriers by inspection specimen.Shape on solid phase carrier is made with the method for washing Into antigen antibody complex and other materials in liquid separate.The antigen or antibody of enzyme labelling are added, is also tied by reaction Close on solid phase carrier.After adding the substrate of enzyme reaction, substrate becomes color products by enzyme catalysiss, receives in the amount of product and specimen The amount of inspection material is directly related, and according to the depth of colour generation qualitative or quantitative analysis are carried out.The catalytic efficiency of enzyme is very high, puts indirectly The big result of immunoreation, makes assay method reach very high sensitivity.
Double antibody sandwich method is detection antigen most common method, and operating procedure is as follows:
1) specific antibody is coupled with solid phase carrier, forms insolubilized antibody, washing removes unconjugated antibody and impurity,
2) plus by inspection specimen, insulation reaction, the antigen in specimen is combined with insolubilized antibody, is formed solid phase antigen antibody and is combined Thing, washing removes other uncombined materials,
3) add enzyme labelled antibody, insulation reaction, the antigen on solid-phase immunity complex to be combined with enzyme labelled antibody, thoroughly wash not With reference to enzyme labelled antibody, the enzyme amount for now carrying on solid phase carrier in specimen by inspection antigen amount it is related,
4) substrate colour developing is added, the substrate for enzymatic activity in solid phase becomes color products, by colorimetric, predicts antigen in specimen Amount, this two-site sandwich method has very high specificity.
In Clinical Laboratory, double antibody sandwich method method is applied to the macromole antigens such as the various protein of inspection, for example HBsAg, HBeAg, AFP, as long as hCG etc. obtain for the heterogenetic antibody by inspection antigen, so that it may for being coated with solid phase carrier and system This method is set up for enzyme conjugates.Source such as antibody is that the antibody of antiserum, coating and enzyme mark is preferably taken respectively from difference The animal of kind.Monoclonal antibody is such as applied, two monoclonal antibodies for different determinants on antigen are typically chosen, bag is respectively used to Solid phase carrier and prepared enzyme conjugates.
This patent provides N's section albumen (amino acid/11-60) of the specificity capture α-syn that can be used for ELISA experiments The mouse monoclonal antibody C140S of rabbit polyclonal antibody-SN16 and specific detection pS129- α-syn, and explore antibody use In the optimum concentration of detection, the SN16 capture antigens of respectively 1ug/ml, the C140S testing goal antigens of 0.02ug/ml.This is specially It is sharp clearly to propose, for the double-antibody sandwich elisa of detection of aggregation pS129- α-syn, to verify detection antibody C140S first Reactive standard, i.e. purification for dry powder C140S antibody after the protein solution of 4mg/ml concentration is formulated as, for Positive antigen OP (the oligomerized 129-serine phosphorylated human alpha- of 1000ng/ml Synuclein, the people source alpha type synapse nucleoproteins of 129 serine phosphorylations of aggregationization) and Negative antigens OW (Oligomerized wild-type human alpha-synuclein, the wild type human source alpha type synapse cores of aggregationization Albumen), using the C140S of 0.125ug/ml, and 1:The m-HRP of 10000 dilutions are when doing indirect ELISA, in background signal and Negative control group 490nm wavelength absorption photometric value signal is respectively less than in the case of 0.2, the 490nm wavelength absorption photometrics of positive group Value will be reached between 2.0-2.5, and the C140S antibody of the Batch purification can be used for double-antibody sandwich elisa.
The content of the invention
The present invention provides a kind of sandwich ELISA detection of 129 phosphorylation alpha- synuclein aggregation bodies of serine Method, methods described includes using monoclonal antibody C140S, and polyclonal antibody SN16 to carry out sandwich method ELISA, to determine people The content of 129 phosphorylation alpha- synuclein aggregation bodies of the serine in blood sample.
The present invention also provides a kind of cell strain, and deposit number is CGMCC12993.
The present invention also provides a kind of monoclonal antibody C140S, is prepared by above-mentioned cell strain.
The present invention also provides a kind of rabbit polyclonal antibody of the capture alpha- synapse nucleoprotein antigens for ELISA method SN16。
The present invention also provides a kind of antigen, is to be marked by the α-syn of hPLK3 kinase catalytic 129 phosphorylations of serine Quasi- albumen.
The present invention also provides a kind of sandwich ELISA inspection of 129 phosphorylation alpha- synuclein aggregation bodies of serine The method for building up of survey method, comprises the steps：
1) people source α-syn are prepared；
2) by the serine phosphorylation of people source α-syn129 positions；
3) using ONE by step 1) in people source α-syn and step 2) in 129 phosphorylations people source α-syn (pS129- α-syn) aggregationization；
4) monoclonal antibody C140S of recognizable people source pS129- α-syn is prepared；
5) the rabbit polyclonal antibody SN16 that specificity captures α-syn is prepared；
6) sandwich method ELISA is done using monoclonal antibody C140S and rabbit polyclonal antibody SN16；
Preferably, described method for building up, comprises the steps：
1) people source α-syn are prepared, and using adsorbed onto glutathione agarose pearl -4B Purification of Human source α-syn；
2) using hPLK3 catalytic steps 1) in the people source α-syn that obtain make it that phosphorylations occur in 129 serines；
3) make step 1 using ONE) in people source α-syn and step 2) in 129 phosphorylations people source α-syn aggregationizations；
4) ascites C140S of the immune Balb/c mices of people source pS129- α-syn is can recognize that with dot-blot pilot experiments screening, Monoclonal antibody C140S is obtained, using protein G Sepharose beads FF affinity column monoclonal antibody purification C140S；
5) the rabbit polyclonal antibody SN16 that specificity captures α-syn is prepared；
6) concentration of detection antibody is determined；
7) sandwich assay is done using the rabbit polyclonal antibody SN16 of monoclonal antibody C140S and specificity capture α-syn ELISA。
The present invention also provides a kind of test kit, including following reagent：Mice resource monoclonal antibody C140S, and rabbit source Polyclonal antibody SN16.
The test kit of the present invention, also including following reagent：Phosphorylation agent, and reagent ONE.
The test kit of the present invention, also including following reagent：ELISA ELISA Plate, PBS, NaHCO3Buffer, confining liquid, PBST etc., the test kit, each reagent is packed respectively, and the amount of reagent is loaded in each packaging with enough sample usage amounts as base This amount, can expand 10 to, 100, the usage amount of 1000 samples.
ELISA detection method of the present invention, needs to use standard curve to determine the content about material, the mark The manufacture method of directrix curve is as follows：
Standard curve is set up using OP, uses OW as negative control in each concentration point, coloration method is AP (alkaline phosphorus Sour enzyme) catalysis pNPP (p-nitrophenyl phosphate, p- Nitrophenyl Phosphate, SurModics, article No. PNPS- 1000-01) postpone colour developing, i.e., from 30 minutes to scan for the first time, every 10 minutes run-downs reactivity and sensitivity are chosen The time point of scope satisfaction is used as the foundation of final standard curve.Each concentration OP that standard curve uses and OW will be set up During A405 (absorbance at 405nm) is input to Excel forms, the data for setting up standard curve are chosen, click insertion Menu, selects scatterplot, and in the scatterplot for generating, right button clicks an arbitrary data point, selects " to add in the menu for ejecting Plus Trendline ", select Trendline option to be " linear " in the window, and choose " display formula " and " display R-squared value on chart ".
Using the ELISA detection method of the present invention, absorbance data can be obtained, by the absorbance data for obtaining with above-mentioned Standard curve is compared, and can calculate the OP contents in sample, and the embodiment of the present invention is shown in the concrete operations of detection method.
It is below the explanation of the title term in the inventive method：
Sample is to set up the pure protein sample that standard curve is used.
People source α-syn people source α type synapse nucleoproteins, are distributed widely in central nervous system nerve tip, now think this egg It is relevant with neuron plasticity is maintained in vain.
Adsorbed onto glutathione agarose pearl -4B is to be coated with glutathion on sepharose 4B, in purification escherichia coli expression GST- It is in connection using GST (glutathione transferase) label on fusion protein N-terminal during α-syn fusion protein, reach purification Purpose, human thrombin is reused afterwards by the excision of this label, that is, obtain the people source α-syn pure proteins of purification.
HPLK3 is a hypotype in people source Polo sample kinase families (Polo-Like Kinase Family), in vitro Show in experiment, hPLK3 can be also true by whole people source α-syn catalytic phosphatases, Mass spectrometry experiments part in this patent The site of phosphorylation of accepting occurs over just the 129th serine.
ONE：4-oxo-2-nonenal, 4- oxo nonenyl aldehyde people's body lipid peroxidating (lipid peroxide) product One of, it is the oxidized byproduct (oxidative of HNE also including HNE (4-hydro-2-nonenal, 4- hydroxyl is for nonenyl aldehyde) counterpart)。
People source α-syn aggregations, english abbreviation OW (Oligomerized Wild-Type h α-syn) is by ONE to people source The hyte propylhomoserin post translational modifications of α-syn the 50th, formed ONE- protein adducts (ONE-protein adduct), when people source α- Syn is received after this post translational modification, HPLC results in aggregation, and document can be more readily formed and shows, its molecular weight is concentrated In 2000kD.
People source α-syn (pS129- α-syn) aggregation of 129 phosphorylations, english abbreviation OP (Oligomerized129- Serine-phosphorylated h α-syn) after hyte propylhomoserin translations of people source α-syn the 50th by ONE to 129 phosphorylations Modification, forms ONE- protein adducts (ONE-protein adduct), and promotion forms aggregation.
Nitrocellulose filter (NC films) used in dot-blot pilot experiments, puts thereon 1 μ l albumen samples, and using antibody with Its reaction, for the specificity and susceptiveness of detection antibody reaction.
Immune Balb/c mices Balb/c mices are the most frequently used laboratory animal strains for producing antibody, by purpose peptide The immune this mice of section, is to extract spleen the later stage and do fused cell to prepare.
Ascites C140S is tentatively filtering out one of clone number of antibody using indirect ELISA, according to residing orifice plate Position be named as C140S, due to being ascites, also without purification, therefore be named as ascites C140S and antibody phase region after purification Point.
Protein G Sepharose beads FF from the sepharose 4B that uses of antibody purification in ascites C140S and other clones number, thereon G-protein is coated with, so as to pass through to combine, wash the composition that the flow processs such as post remove non-antibody in ascites, can obtain pure with binding antibody Change antibody.
To set up ELISA detection method of the present invention, spy be prepared for it is a kind of be directed to 129 phosphorylations people source α- The mice resource monoclonal antibody C140S of syn, the preparation method of the monoclonal antibody is as follows：
Using P1 (M18631-1hz-1-1, Ac-CEAYEMP (pS) EGG-NH2,129, people source serine phosphorylation α- The 123-131 peptide fragments of syn) peptide fragment immunity balb-c mices, take spleen and prepare fusion myeloma cell line, indirect ELISA screening Go out cell line of the clone number for C140S, using the cell line 4 balb-c/nu mouse peritoneals are injected into, indirect ELISA is tested Antibody purification after card ascites atopic, after the reactivity of the antibody for reusing indirect ELISA checking purification (i.e. The positive group 490nm wavelength absorption photometric value of antibody recognition 1000ng/ml of 0.125ug/ml is higher than negative at least 30 times of group, and 0.2) background and negative group number-reading are less than, the detection antibody that can be used as in this method, and ultimate density is 4mg/ml.
(cell strain has been deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and numbering is CGMCC12993, preservation date September in 2016 14 days, Classification And Nomenclature：Hybridoma cell strain, address Chaoyang District, Beijing City North Star west The institute 3 of road 1).
To set up ELISA detection method of the present invention, a kind of special standby N-terminal (1-60 positions ammonia for people source α-syn Base acid) rabbit source polyclonal antibody SN16；The preparation method of the polyclonal antibody is as follows：
Using N-terminal (1-60 amino acids) peptide fragment 2 New Zealand white rabbit of immunity of α-syn, serum is collected in carotid artery blood-letting Antibody purification afterwards, ultimate density is 192ug/ml.
Due to the superiority of effect of the present invention, the present inventor is prepared for a kind of detection kit according to said method, described Test kit includes following reagent：
Mice resource monoclonal antibody C140S
Rabbit source polyclonal antibody SN16
Test kit of the present invention, may also include if necessary following reagent：
Phosphorylation agent (including hPLK3 kinases and the buffer of catalytic phosphataseization reaction)；
Reagent ONE
Test kit of the present invention, as needed, the test kit may also include any suitable the inventive method Auxiliary reagent：Such as, ELISA ELISA Plate, PBS (phosphate buffer), NaHCO3Buffer, confining liquid, PBST (is added in PBS 0.2% Tween-20) etc..
The present invention mentioned reagent box use be using in blood plasma recombined human alpha-synapse nucleoprotein polymer as standard egg In vain, using the relation curve of the aggregation bulk concentration and absorbance as standard curve, by the blood plasma-recombined human in measure sample The polymeric concentration of alpha-synapse nucleoprotein and formation rate reach the Parkinsonian purpose of diagnosis.
The test kit of the present invention, each reagent is packed respectively, preferably uses packing tube, and the amount of reagent is loaded in each packing tube With enough sample usage amounts as fundamental quantity, 10 can be expanded to, 100, the usage amount of 1000 samples.
The use of the test kit of the present invention is the side according to recombined human alpha-synuclein aggregation body in above-mentioned detection blood plasma What method was carried out, the reagent in test kit is configured to into as needed corresponding concentration when using, determine according to methods described.
For the research present invention, replica test, precision test, screening of detection method etc. are also carried out and have tested to guarantee The application of the present invention.
The specificity of mass spectral analyses PLK3 catalysis people source α-syn phosphorylation sites
Martial K.Mbefo in 2010 et al. send out when observing PLK3 phosphorylation α-syn using isotope-labelled protein It is existing, reach 2.5 hours 30 DEG C of response time, all of α-syn there occurs phosphorylation, and this patent will be made using this method Standby people source P α-syn do mass spectral analyses discovery, and PLK3 makes α-syn only there occurs phosphorylation (Fig. 3) in 129 serines, and There is no phosphorylation (Fig. 4) in S87 positions and Y125 positions, it was demonstrated that PLK3 has specificity for the phosphorylation site of α-syn.
The sensitivity experiment of the present invention：
Prepare the immune peptide fragment that C140S antibody is used：P1 (M18631-1hz-1-1, Ac-CEAYEMP (pS) EGG-NH2, The 123-131 peptide fragments of the 129 serine phosphorylation α-syn in people source)
Control includes：P2 (M18631-1hz-2-1, Ac-EMP (pS) EEGYQDC-NH2,129, people source serine phosphorylation Change the 126-135 peptide fragments of α-syn), W (Z18631-1hz-3-1, Ac-CEAYEMPSEEGYQD-NH2, the 123- of people source α-syn 135 peptide fragments), ASWT (h α-syn-FL, FL represent total length), SE (h α-syn/S129E are simulation phosphorylation mutant)
Experimental result such as following table：
Antibody Designation Volume (ml) Theoretical sensitivity (ng) Actual dilution ratio Using volume (μ l)
C140S 0.1 0.01 1:500 10
Show that C140S antibody can be with the P1 peptide fragments (Fig. 1) of specific recognition 100ng.
Indirect ELISA (Enzyme-Linked Immunosorbent Assay, enzyme-linked immunosorbent assay) screening is anti- Body recognizes phosphorylation site specificity
In S87, Y125, S129 site is three phosphorylation post translational modification sites to α-syn, and C140S antibody only can be known The peptide fragment of other S129 positions phosphorylation, for the peptide fragment and the equal nonrecognition of wild type peptide fragment (Fig. 2) of S87, Y125 phosphorylation.
The antigen list for using：
Coding Full name Sequence Concentration Detection antibody
P① P1 CEAYEMP(pS)EEG 4mg/ml C140S&C54S
P② P2 EMP(pS)EEGYQDC 4mg/ml C48S
S 22667-1-1 KTVEGAGS(p)IAAATG 8mg/ml
Y 22667-1-2 VDPDNEAY(p)EMPSEE 8mg/ml
W Z18631-1hz-3-1 CEAYEMPSEEGYQD 4mg/ml
It is an advantage of the current invention that：
The present invention uses capture antibody SN16 and detection antibody C140S, can go out aggregate form with specific detection 129 The people source α-syn of position serine phosphorylation, and nonrecognition monomer form, also nonrecognition wild type α-syn monomers and aggregation； The signal of identification is significantly higher than unphosphorylated aggregation, phosphorylated monomeric, wild syn monomers.
The present invention can voluntarily adjust the developing time of pNPP, to control background, and obtain the sensitivity model needed for detecting Enclose, for the people source α-syn of 129 serine phosphorylations of aggregate form, peak response is up to 10ng/ml.
Description of the drawings
Fig. 1 is Dot blot results prompting 1:The C140S ascites of 500 dilutions is 100ng for the sensitivity of P1 peptide fragments, right In P2 peptide fragments, wild type peptide fragment W, wild type α-syn full length proteins (ASWT), α-syn-S129E mutants (SE) then without anti- Ying Xing.
Fig. 2 represents C140S, the strain antibody of function identical three that C48S, C54S are filtered out for Dot-Blot, indirect method ELISA points out three strain antibodies for immune peptide fragment P has reactivity, for 87 phosphated peptide sections (S), 125 Phosphorylated Peptides , then without reactivity, the 3D5 antibody used in indirect ELISA result is Xuan Wu hospital in suitable for section (Y), wild type peptide fragment (W) The anti-h α-syn115-121 antibody of mouse monoclonal prepared by professor, WAKO is that the mouse monoclonal of Japan's manufacture resists 129 phosphoric acid Change h α-syn antibody (pSyn#64), CWRA is the rabbit source Anti-TNF-α h α-syn-FL antibody of this room manufacture, three after as a result pointing out Person is for the equal anergy of four kinds of peptide fragments.
Fig. 3 shows that one of the peptide fragment of Glu-C cuttings α-syn124-131 mass spectrums show that 129 serines there occurs phosphoric acid Change, and there is no phosphorylation in 125 L-Tyrosine.
Fig. 4 shows that one of the peptide fragment of Trypsin cuttings α-syn84-104 mass spectrums show that 87 serines do not occur phosphoric acid Change.
Fig. 5 is that C140S ascites is for 129 phosphorylation α-syn full-length proteins in indirect method experimentReactivity There is no a significant difference with WAKO antibody, and reactivity is significantly higher than C48S and C54S ascites, 3D5 antibody forWild type α-syn total lengths (W), S129E (simulation phosphorylation α-syn protein mutants, E), S129A (blocking phosphorylation α-syn protein mutation Body, A) there is reactivity, and there was no significant difference, 3A5 antibody is the anti-phosphorylation α-syn of this room early stage self-control mice resource monoclonal Antibody, can 129 simulations phosphorylation α-syn protein mutants (E) of specific recognition in ELISA.
Fig. 6 is in same pvdf membrane (polyvinylidene difluoride, polyvinylidene fluoride) successively two kinds of incubation Antibody is observed, and left side C140S antibody only recognizes 129 phosphorylations h α-syn (P) of 17kD, washes incubation BD antibody after film visible 129 phosphorylations h α-syn (P) are consistent with wild type h α-syn (W) applied sample amounts.
Fig. 7 is filtered out for indirect ELISA and met using the antibody purification batch of standard.
Fig. 8 is anti-for capture using Anti-TNF-α phosphorylation α-syn antibody (Santa Cruz, sc-135638) of Santa Body, biotinylation 3D5 monoclonal anti-humans α-syn115-121 is detection antibody, find nonrecognition phosphorylation (P) and wild type α- Syn monomers (W), and α-syn aggregations (OW), only recognize aggregationization P α-syn (OP).
Fig. 9 does capture antibody for this room self-control SN16 for the anti-human α-syn-N ends antibody of polyclone, and C140S antibody is detected Antibody, finds nonrecognition phosphorylation (P) and wild type α-syn monomers (W), and α-syn aggregations (OW), only recognizes aggregationization Pα-syn(OP)。
Figure 10 is the standard curve that OP is set up using SN16/C140S antibody, and antigen concentration includes 64000,32000, The A405 readings of 16000,8000,4000,2000,1000,500pg/ml OP and OW, AP-pNPP developing times 60 minutes.
Figure 11 is that indirect ELISA verifies that the N-terminal for α-syn of SN16 antibody can be with specific recognition.
Specific embodiment
The present invention is further illustrated by the following examples.
Embodiment 1
The expression of α-syn and purification：
Experiment material：
Plasmid：pGEX-4T-1-hαsyn-WT(4969bp+423bp)
AMP (Ampicillin, ammonia benzyl mycin) resistance, expresses GST-h α syn-WT fusion protein, molecular weight 26kD+ 16kD；
LB (lysogeny broth or Luria-Bertani, bacteriolyze meat soup) solids and fluid medium, AMP+； 100mM IPTG (Isopropyl β-D-1-Thiogalactopyranoside, isopropyl-beta D-thio galactopyranoside), 0.1mM PBS (phosphate buffer saline, phosphate buffered saline(PBS)), Thrombin (people source thrombase 1ku Sigma, article No. T1063), Glutathione Sepharose-4B (Glutathione Sepharose 10ml GE, article No. 17- 0756-01), 20% ethanol solution, 10mM reduced glutathions, 50mM Tris-HCl pH8.0 (Tris, Aminomethylidinetrimethanol, trimethylol aminomethane), 6mol guanidine hydrochloride (Guanidine Hydrochloride) solution；
Ultrasonic Cell Disruptor, centrifuge, little shake pipe, 1L conical flasks, 500mL large beakers, 50mL centrifuge tubes, the flat burnings of 50mL Bottle, 5mL droppers, Polypropylene Column (polypropylene post QIAGEN, article No. 34924).
Experimental procedure：
Experiment one：The expression of destination protein
By plasmid convert to BL21 (DE3) (A.Roca (University of Wisconsin) build BL21recA spread out Raw bacterium, can stablize some genes of interest with repetitive sequence) in cell, after 37 DEG C of 200rpm incubation 50min activation strains, 10 μ l bacterium solutions are taken to ammonia benzyl resistance LB solid medium, 12-16 hours are incubated in 37 DEG C of environment；
1 monoclonal bacterium colony of picking, is infected filling the little of 6ml AMP+LB fluid mediums and shake in pipe, 37 DEG C 220rpm is incubated；
After 12 hours, make the little pipe that shakes depart from former incubation environment, take 2.5ml bacterium solutions, (can consider to protect bacterium herein) is added to In filling the 1L conical flasks of 250ml ammonia benzyl resistance LB fluid mediums, 37 DEG C of 260rpm are incubated 3 hours；
Take the IPTG Induction of bacterial expressing protein of final concentration of 0.1mM 4 hours；
After the completion of induction 4 hours, allow flask to depart from incubation environment in time, carry out bacteria breaking experiment.
Experiment two：Bacteria breaking is tested
Pre-cooling centrifuge rotor is to 4 DEG C when can carry out testing one；
Contain from ice machine and take a box trash ice, be compacted standby；
50mL specialty centrifuge tubes are taken, 40mL bacterium solutions are poured into every time, 10000g continues 4 minutes collection bacterium；
After the completion of collection bacterium, abandoning supernatant is put into centrifuge tube in ice, adds 10ml 0.1M PBS to fill using 5ml droppers Divide resuspended；
After the completion of resuspended, bacterium solution poured into or is moved in 50ml centrifuge tubes with dropper, bacterium solution is placed in ice-water bath (can fit When adding water, to ensure low temperature), according to 2 seconds every time ultrasound, 2 seconds interval, amount to 150 times condition implement ultrasonication, ultrasound visit At head immersed in liquid level 1/3, it is careful not to get foam；
Equivalent bacterium solution refunds two professional centrifuge tubes, using 15000g centrifugal force 45min, supernatant is taken, on ice Next step experiment is carried out, or albumen supernatant is preserved in -20 DEG C.
Experiment three：Affinity chromatography purification destination protein
Take 1ml Glutathione Sepharose-4B (GE, article No. 17-0756-01) to be placed directly within In Polypropylene Column (QIAGEN, article No. 34924), balanced three times with 10ml 0.1mMPBS altogether.Note liquid Face is controlled in the middle part of pillar, if too high, because liquid is excessive to the pressure of post bed support can be caused to be attached to post bed Tension, is unfavorable for liquid smoothly lower drop；
It is with the supernatant prepared in previous step experiment that the Sepharose-4B in post is resuspended, exhaust, be put into a 50ml After centrifuge tube, pillar is placed in into room temperature standby；
Centrifuge tube is sealed, vertical shaking 1h, should not heat under room temperature, and speed should not be too fast；
Mixed liquor in small beaker is poured in the clean amateur centrifuge tubes of 50ml, using 4 DEG C of centrifuge 3000rpm Centrifugation 10min；
Take out centrifuge tube, supernatant discarded, with 1ml 0.1M PBS it is resuspended be sunken to centrifugation bottom of the tube Sepharose-4B, and Pillar is moved back to, with the PBS of 3 bed volumes post is washed, the PBS volumes for finally staying remain 1 with Sepharose-4B volume ratios: 3；
20U (40 μ l) thrombin (Sigma, article No. T1063) is added in 1ml 0.1M PBS and dilutes, and suctions out 1ml dilutions Liquid seals pillar to resuspended in pillar with sealed membrane；
At room temperature 16h is incubated on shaking table, shaking speed should not be too fast；
Column flow exports switch is opened, after reclaiming effluent, 0.1M PBS eluting four times, each 1ml is added；Collect Eluent.
Experiment four：Glutathione Sepharose-4B and void column are reclaimed
The solution of 2-3 pillar volume is now prepared, wherein containing 10mM reductive glutathiones and 50mM pH8.0Tris- HCl, with the washed pillar of this solution after, 20% ethanol solution is added into post；
It is resuspended fully after, solution in post is moved to into one individually has in spiral cover container, is shown to be the Glutathione of recovery Sepharose-4B；
After solution takes only in post, add guanidine hydrochloride solution of 1mL 6mol that whole residual protein removings in pillar is dry to it Only, with distilled water by pillar wash clean after, that is, reclaim void column finish.
Embodiment 2
129 phosphorylations of α-syn：
Part I：Required reagent
HEPES：N-2-Hydroxyethylpiperazine-N'-2'-ethanesulfonic Acid, 4- ethoxy piperazine Piperazine ethyl sulfonic acid；N- (2- ethoxys) piperazine-N'-2- ethane sulfonic acids, pH7.4
MgCl2：Magnesium Chloride, magnesium chloride
DTT：Dithiothreitol, dithiothreitol, DTT, 1M
HCl：Hydrochloride, hydrochloric acid
hPLK3：Homo sapiens Polo-Like Kinase 3, people source Polo samples kinases 3,10ug (0.42mg/ Ml), there are the liquid of 23.8 μ l, -80 DEG C of preservations in main pipe
hα-syn：Homo sapiens alpha synuclein, people source α type synapse nucleoproteins, 4mg/ml
ATP：Adenosine triphosphate, adenosine triphosphate, 100mM
EDTA Na2：Ethylenediaminetetraacetic Acid-2Na, disodium EDTA
Tris：Aminomethylidinetrimethanol, trimethylol aminomethane, 50mM pH7.5
NaCl：Sodium Chloride, Sodium Chloride 150mM
Triton X-100：Polyethylene Glycol Octylphenol Ether, Triton X-100
Glycerol：Glycerol
Part II：Buffer
Storage buffer solution (Storage buffer)：
1.1 purpose：For storing hPLK3Kinases, and reaction system it is too small when, diluted using this buffer
1.2 constituents and compound method：
With cumulative volume 100ml calculating
Prepare the 50mM Tris-HCl pH7.5 solution of 100ml：
The Tris alkali for taking 0.606g is added in the ultra-pure water that 90ml height is pressed through, and pH is adjusted to 7.5 using HCl after dissolving, Constant volume to 100ml, after preparing, this solution room temperature preservation；
A. 50ml Tris-HCl solution is taken, 0.88g NaCl are added thereto to, is mixed；
B. the 1M DTT of 200 μ l are added；
C. 18mg EDTA Na are weighed2Add and mix；
D. the Triton X-100 of 20 μ l are added；
E. it is eventually adding the mixing of 50ml glycerol；
1.3 storage method：- 20 DEG C of preservations
2. reaction buffer (Working buffer)：
2.1 purpose：Kinases, ATP and reaction environment are provided for α-syn phosphorylation reactions
2.2 constituents and compound method：
With cumulative volume 100ml calculating：
Take 0.477g HEPES to be added in the ultra-pure water that 90ml height is pressed through, HCl adjusts pH to 7.4；
Add 95.2mg MgCl2；
Add the 1M DTT of 200 μ l；
Ultra-pure water constant volume is to 100ml；
2.3 storage method：- 20 DEG C of preservations
3. substrate solution：
3.1 purpose：Reaction environment is provided for α-syn phosphorylation reactions
3.2 constituents and compound method：
(MWCO is Molecular in the 5MWCO super filter tubes of the α-syn to 5ml for A. taking a small amount of 0.1M PBS dissolvings Weight Cut Off, represent molecular cut off)；
B. in super filter tube top half container, with reaction buffer 5ml is settled to；
C. 10min is centrifuged using refrigerated centrifuge 10000g；
D. residual liquid in container is drawn, it is 1269ug/ml that BCA determines concentration
4. terminating reaction：
It is placed in -80 DEG C
Part III：Phosphorylation reaction
Reaction volume is set as 50 μ l；
The working buffer solution that 11.3 μ l contain α-syn (144ug) is added in 37 μ l working buffer solutions, hPLK3 is added 1.2 μ l, add the μ l of ATP 0.5,30 DEG C react 3 hours, be put into -80 DEG C of terminating reaction α-syn aggregation and 129 phosphorylations The aggregation of α-syn (P α-syn)：
Thomas et al. 2011 using ONE (4-oxo-2-nonenal, 4- oxo nonenyl aldehyde) prepare aggregationization α- Syn, HPLC (Chromatography High Pressure Liquid, high-pressure liquid phase) result shows that α-syn receive ONE's The soluble aggregate of 2000kD can be formed after post translational modification.
PBS is 20mM, ONE subpackages, using being immediately placed in -80 DEG C of preservations after complete.
Reagent name Pα-syn α-syn ONE
Original concentration 2500μg/ml 2314.6μg/ml 5mg/ml
Initial volume 30μl 1000μl 200μl
Original quality 75μg 2.3mg 1mg
Target response volume 20μl 100μl 100μl
Target response concentration 140μM 140μM 1mM
Target response quality 39.2μg 202.44μg 15.42μg
Volume aspirated 15.68μl 87.46μl 3.09μl
PBS supplies volume 1.32μl 9.45μl -
Concentration after reaction 1960μg/ml 2024.4μg/ml
Reaction condition is 37 DEG C of o/n (18-24hrs)
Embodiment 3
The anti-human P α-syn antibody cell strain C140S of Dot Blot screening Mus resource monoclonals
Using Dot-Blot (Dot blot) experimental technique screen the standby anti-human P α of Mus resource monoclonal of Abmart company systems- Syn antibody cell strains.There are two rows five to arrange each 1 μ l albumen samples, upper behavior 100ng on each NC film (nitrocellulose filter) Peptide fragment, lower behavior 500ng peptide fragments, the immune peptide fragment of the C140S antibody for filtering out is P1 (M18631-1hz-1-1, Ac- The 123-131 peptide fragments of the 129 serine phosphorylation α-syn in CEAYEMP (pS) EGG-NH2 people source), control includes：
P2 be (129, M18631-1hz-2-1, Ac-EMP (pS) EEGYQDC-NH2 people source serine phosphorylation α-syn's 126-135 peptide fragments)；
W (the 123-135 peptide fragments of Z18631-1hz-3-1, Ac-CEAYEMPSEEGYQD-NH2 people source α-syn)；
ASWT (h α-syn-FL, Full Length, total length)；
SE (- syn/S129E, Ser129Glu, 129 mutant serine of h α is glutamic acid)
Antibody Designation Volume (ml) Theoretical sensitivity (ng) Actual dilution ratio Using volume (μ l)
C140S 0.1 0.01 1:500 10
Protein G-Sephrose FF affinitive layer purification C140S IgG antibody
Material：
Protein G-Sephrose-FF
Mouse immune ascites 10ml
Method：
1.Protein G-sephrose FF (G-protein agarose gel FF) affinity columns are balanced with PBS, are stored in 4 DEG C and are treated With.Ascites is mixed with isopyknic PBS carries out doubling dilution, after low-temperature and high-speed centrifugation, then Jing 0.22um filtering with microporous membranes.
2., by upper Protein G sephrose FF posts after ascites-PBS solution mix homogeneously, slow post of crossing is conducive to IgG Combined with Protein G.Repeat loading 5 times.
3. ascites solution is crossed after post finishes, and fully rinsing pillar with PBS makes absorbance value be down to baseline (at least 10 post beds Volume, it is therefore an objective to which thorough eluting is clean from post by the foreign protein of non-specific binding).
4. the IgG on post is disintegrated down and is collected protein eluate (about 5 with 0.1M Glycine-HCl pH 2.7 Individual bed volume).
5. the 1M Tris-HCl pH 8.0 and 0.1M NaHCO3,0.5M of 1/10 volume are added in the eluent collected The solution of NaCl pH 8.3, (reaching the purpose of neutralization).
6. by the IgG solution desalinations of collection, concentrate, do protein quantification, subpackage, lyophilization.
7.-20 DEG C preservation.
Product subpackage：
Purification obtains 13mg IgG, takes 6mg and does biotin labeling, a remaining 7mg (dry powder)
Embodiment 4
The reactivity of the C140S antibody of indirect ELISA checking purification
First, reagent configuration：
1. coating buffer, washing are identical with 1.7 with diluent, confining liquid,
2. substrate solution：OPD (o-phenylenediamine)-H2O2Preparing method, phosphate-citrate buffer
0.1M citric acid 19.2g add H2O to 1000ml takes 48.6ml
0.2M Na2HPO2·12H2O 71.7g add H2O to 1000ml takes 51.4ml
Developer：Take out 10ml+4mgOPD+5 μ l H2O2Matching while using
3. terminate liquid：100ml
10%2N H2SO4 10ml+H2O 90ml
2nd, operating process：
1. wrapper sheet：Antigen OP and OW plus CB dilutions are taken, is added in 96 orifice plates per the μ l of hole 100,4 DEG C overnight；
2. with 200 μ l cleaning mixture board-washing 5 minutes, wash 3 times, pat dry；
3. the μ l/ holes of confining liquid shrouding 200 are used, and 37 DEG C are closed 2 hours；
4. board-washing 3 times；
5. the μ l of C140S antibody 100 of diluted are added；
After 6.37 DEG C are incubated 2 hours, board-washing 3 times；
7. 37 DEG C of anti-mouse IgG for adding 100 μ l HRP labellings is incubated 1 hour；
8. board-washing 4 times；
9. substrate developer is added, and 37 DEG C are developed the color 20 minutes；
10. terminate liquid is added；
11. determine 490nm wavelength absorption photometric values with ELISA detectors；
Embodiment 5
Indirect ELISA verifies the sensitivity of C140S antibody and specificity before purification
Using antigen：
Using antibody and dilution ratio：C140S(1:1000)、C54S(1:1000)、C48S(1:1000)、3D5(1: 2000)、WAKO(1:4000)、3A5(1:4000) (Fig. 5)
WB (Western Blot, Western blotting) verifies the specificity of C140S antibody
To P α-syn and α-syn monomers, successively using the total α-syn monoclonal antibodies of C140S and BD (BD, article No. 610787) Observation, determines that C140S can be used for WB experiments, and can recognize P α-syn monomers (Fig. 6).Indirect ELISA is verified after purification The reactivity (HRP catalytic substrates develop the color, A490) of C140S antibody use Protein G- in C140S mouse ascites Sephrose-FF affinity columns after purification, need whether the C140S antibody using indirect ELISA checking after purification has Satisfied reactive and specificity.In an experiment, it is coated on orifice plate using the positive antigen OP and Negative antigens OW of 1000ng/ml Bottom, the C140S antibody tests of the purification of 0.125ug/ml, 1:The anti-mouse IgG antibody incubation of the HRP labellings of 10000 dilutions Afterwards, catalyzed coloration.Then background reading and negative control 490nm wavelength absorption photometric value are being less than in the case of 0.2, positive group 490nm wavelength absorption photometric value must be between 2.0-2.5, and the antibody of the Batch purification can be used for double-antibody sandwich elisa Experimental technique sets up the examination criteria curve (Fig. 7) of 129 phosphorylated human source α-syn for aggregationization, and this step is important Step.
(HRP catalytic substrates develop the color the double-antibody sandwich elisa of specific recognition aggregationization P α-syn, observe 490nm extinctions Shading value)
The use of Anti-TNF-α phosphorylation α-syn antibody (Santa Cruz, sc-135638) of Santa is capture antibody, it is raw Thing elementization 3D5 monoclonal anti-humans α-syn115-121 is detection antibody, finds nonrecognition phosphorylation (P) and wild type α-syn lists Body (W), and α-syn aggregations (OW), only recognize aggregationization P α-syn (OP) (Fig. 8).
This room self-control SN16 does capture antibody for the anti-human α-syn-N ends antibody of polyclone, and C140S antibody does detection antibody, It was found that nonrecognition phosphorylation (P) and wild type α-syn monomers (W), and α-syn aggregations (OW), only identification aggregationization P α- Syn (OP) is (Fig. 9).
Embodiment 6
The preparation of SN16：
1. pre- blood-letting
Being placed on rabbit in special rabbit box gently, the rabbit blood sampling in relaxation state can be easier to.Pressing rabbit ear Then root inserts the needle into the middle and upper part of ear's blood vessel up to blood vessel enhancement, it was observed that careful piston of releasing is collected after inserting needle Blood 1ml-5ml.After terminating to collect, exit syringe needle and press injury to prevent blood flow, then use ethanol disinfection.Take the blood of collection 30 minutes are placed in 37 DEG C of calorstats preventing activating complement system, then test tube is stood overnight at 4 DEG C is made blood coagulation.With Blood clot is dialled by medicine shovel from tube wall, blood is transferred in plastic centrifuge tube, 4 DEG C, 10,000g centrifugations 10 minutes, collects Supernatant is in 4 DEG C of preservations.
2. injections of antigens
(1) prepare two adult rabbits, 100 μ g antigens/rabbit is dissolved in stand-by in 1ml phosphate buffer solutions.In 1ml Fu Shi not Add mycobacteria to make Freund's complete adjuvant in Freund's complete adjuvant, and add 1ml antigenic solutions (N-terminal of the α-syn of source containing someone), acutely Concussion is allowed to fully emulsified, and with 3ml syringes the emulsion is extracted, and connects 25G syringe needles, the bubble in Inside Syringe.From cage Middle taking-up rabbit is placed on flat place, and at 4 different positions subcutaneous injection is carried out, and two are in back, and two are at thigh.Comfort The rabbit hair at injection and with the exposed skin of ethanol disinfection.Skin is pinched out, by syringe needle with the angle inserting needle of 15 degree of Relative Skin, is entered Pin depth is 1cm-2cm, carefully should not be pierced in muscle, in 4 different parts difference about 500 μ l antigenic solutions of each injection.Note After penetrating end, gently extract again after pin is placed into several seconds at injection, and sterilized at injection with ethanol.In 4 position weights Multiple aforesaid operations.With same procedure immunity another rabbit.
(2) the week injections of antigens per 4-6, and -10 days 7 days after injection are according to step 2 collect blood.The blood that will be collected It is compared with the blood collected before injection, checks whether there is antibody generation.After generation antibody to be determined can collect blood in a large number, But every rabbit collect blood can not be more than 40ml to prevent shock.
3. collect blood
(1) rabbit is gently put on fixed mount, dimethylbenzene is applied to the upper middle part of ear's blood vessel, with 45 ° of blade tilt at this Place cuts out the otch of 0.23cm-0.3cm enables blood freely to flow out.The blood for oozing is collected with the pipe after sterilization, if in knot Occur the available warm water of solidification before beam and dab incision, be further continued for collecting.The gauze collected after appropriate blood after available sterilization is light Affected part is wiped, flicking affected part determines that blood flow can terminate after stopping for -20 seconds 10 seconds.
(2) blood is placed 30 minutes in 37 DEG C of calorstats, then stood overnight at 4 DEG C.Medication is shoveled blood clot from pipe Dial on wall, blood be transferred in plastic centrifuge tube, 4 DEG C, 10,000g centrifugations 10 minutes are collected supernatant and are antiserum, The several years can be preserved at -20 DEG C.
4. using the method purification part antibody mentioned in patent.
Embodiment 7
SN16/C140S sandwich method ELISAs detection of aggregation P α-syn (OP)
First, reagent configuration：
1. coating buffer：CB(Coating Buffer,pH 9.6)500ml
2. cleaning mixture and diluent：0.01M PBS+0.2%Tween-20 (Polysorbate 20, polysorbate 20), pH 7.4
0.1M PBS, pH 7.4 are prepared first
NaH2PO4·2H2O 2.964g
Na2HPO4·12H2O 28.998g
NaCl 9.0g
ddH2O (ultra-pure water) 1.0L
It is made into the working solution of 500ml again every time
Take 0.1M PBS pH7.4 50ml, plus ddH2O 450ml, plus 1ml Tween-20 make its final concentration of 0.2% (v/v)。
3. confining liquid：5%BSA (Bovine Serum Albumin, bovine serum albumin, 25ml), this is 96 holes The amount of plate, matching while using
BSA 1.25g+25ml diluents
4. nitrite ion：PNPP (p-nitrophenyl phosphate, p- Nitrophenyl Phosphate, SurModics, article No. PNPS-1000-01)
2nd, experiment flow：
1. coating captures antibody：
SN16 (the anti-human α-syn-N ends antibody of the self-produced rabbit source polyclone in this room, concentration 192ug/ml) is diluted with coating buffer For 1 μ g/ml, 100 μ l are added per hole, 4 DEG C of coatings are overnight (about 16-20 hours)；
2. board-washing：
Coating buffer is discarded, the cleaning mixture of 200 μ l is added per hole, be stored at room temperature 5min every time, wash three times, hereinafter referred to as " washing × 3 "
3. orifice plate is closed：
The confining liquid of 200 μ l is added per hole, 37 DEG C are closed 2 hours；
4. board-washing × 3；
5. detection antibody is added：
Using diluent by C140S (the self-produced mice in this room original monoclonal anti-human p α-syn antibody, concentration 4mg/ml, batch 6.4) 0.02 μ g/ml are diluted to, 100 μ l is added per hole, 37 DEG C are incubated 2 hours；
6. board-washing × 3；
7. two anti-m-AP (anti-mouse IgG of alkali phosphatase enzyme mark) are added：
Using diluent by the anti-mouse IgG antibody of AP (alkaline phosphatase, alkali phosphatase) labelling with 1:10000 dilutions, 37 DEG C are incubated 1 hour；
8. board-washing × 4；
9. substrate solution is prepared, adds 100 μ lpNPP continuously to be developed the color per hole, the time is with 10min to be spaced from 20min Start to 80min, standard curve is set up using the reading of 80min according to the response rate in patent；
10. 405nm wavelength absorption photometric values are read using PerkinElmer Victor3
Embodiment 8
SN16/C140S makes ELISA standard curves (AP catalysis pNPP colour developings)
Set up standard curve and do capture antibody using the SN16 of 1 μ g/ml, the C140S of 0.02 μ g/ml is detection antibody, OP For purpose albumen, OW is reference protein, establishes standard curve (Figure 10).
Embodiment 9
Real patient samples are detected using the inventive method, and calculate OP contents in sample,
Step 1, using the anticoagulant tube containing EDTA 5ml ulnar vein blood is extracted,
Step 2,2000g is centrifuged 20 minutes separated plasmas and erythrocyte under 4 DEG C of environment, immediately in -80 DEG C of preservations
Step 3, using the method in embodiment 7 ELISA standard curves are done, while to patients blood plasma respectively according to different Dilution ratio dilutes, and makes the concentration for measuring OP in the range of linearity of standard curve, carries out multiple holes, and the value then measured is multiplied by again Dilution ratio, the content of OP in as actual every milliliter of blood plasma.
Embodiment 10
The preparation of test kit of the present invention
Test kit contains coating buffer, captures antibody SN16, washing/diluent, standard antigen OP, detection antibody C140S, anti-mouse IgG of alkali phosphatase enzyme mark, pNPP substrate nitrite ions
The content of this test kit each component is respectively：
1.10x is coated with buffer 50ml
2. capture antibody SN16 and be coated on 96 orifice plates that (orifice plate is detachable, -20 DEG C of guarantors of good seal with the concentration of 1ug/ml Deposit)
3.10x washing/diluent 100ml
4. in standard antigen OP 44.8ng/0.7ml cleaning mixture (curve concentration range 64000pg/ml to 500pg/ is set up ml)
5. detection antibody C140S concentration is 0.2 μ g/ml, containing 1ml, 10ml is diluted to when using
6. anti-mouse IgG of alkali phosphatase enzyme mark is with 1:The common 1ml of 1000 dilutions, with diluted to 1 when using: 10000
7.pNPP substrate nitrite ion 10ml.

Claims (10)

1. a kind of sandwich ELISA detection method of 129 phosphorylation alpha- synuclein aggregation bodies of serine, its feature exists In methods described includes using monoclonal antibody C140S, and polyclonal antibody-SN16 to carry out sandwich method ELISA, to determine people The content of 129 phosphorylation alpha- synuclein aggregation bodies of the serine in blood sample.
2. a kind of cell strain, it is characterised in that deposit number is CGMCC12993.
3. a kind of monoclonal antibody C140S, it is characterised in that prepared by the cell strain of claim 2.
4. a kind of rabbit polyclonal antibody SN16 of the capture alpha- synapse nucleoprotein antigens for ELISA method.
5. a kind of antigen, it is characterised in that by the α-syn standard eggs of hPLK3 kinase catalytic 129 phosphorylations of serine In vain.
6. the foundation side of the sandwich ELISA detection method of 129 phosphorylation alpha- synuclein aggregation bodies of a kind of serine Method, it is characterised in that comprise the steps：
1) people source α-syn are prepared；
2) by the serine phosphorylation of people source α-syn129 positions；
3) using ONE by step 1) in people source α-syn and step 2) in 129 phosphorylations people source α-syn (pS129- α- Syn) aggregationization；
4) monoclonal antibody C140S of recognizable people source pS129- α-syn is prepared；
5) the rabbit polyclonal antibody SN16 that specificity captures α-syn is prepared；
6) sandwich method ELISA is done using monoclonal antibody C140S and rabbit polyclonal antibody SN16.
7. method for building up according to claim 6, it is characterised in that comprise the steps：
1) people source α-syn are prepared, and using adsorbed onto glutathione agarose pearl -4B Purification of Human source α-syn；
2) using hPLK3 catalytic steps 1) in the people source α-syn that obtain make it that phosphorylations occur in 129 serines；
3) make step 1 using ONE) in people source α-syn and step 2) in 129 phosphorylations people source α-syn aggregationizations；
4) ascites C140S of the immune Balb/c mices of people source pS129- α-syn is can recognize that with dot-blot pilot experiments screening, is obtained Monoclonal antibody C140S, using protein G Sepharose beads FF affinity column monoclonal antibody purification C140S；
5) the rabbit polyclonal antibody SN16 that specificity captures α-syn is prepared；
6) concentration of detection antibody is determined；
7) sandwich method ELISA is done using the rabbit polyclonal antibody SN16 of monoclonal antibody C140S and specificity capture α-syn.
8. a kind of test kit, it is characterised in that including following reagent：Mice resource monoclonal antibody C140S, and many grams of rabbit source Grand antibody SN16.
9. test kit according to claim 8, it is characterised in that also including following reagent：Phosphorylation agent, and reagent ONE。
10. test kit according to claim 8, it is characterised in that also including following reagent：ELISA ELISA Plate, PBS, NaHCO3Buffer, confining liquid, PBST etc., the test kit, each reagent is packed respectively, each packaging in load reagent amount with Enough sample usage amounts are fundamental quantity, can expand 10 to, 100, the usage amount of 1000 samples.
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