CN101692092B - Method for quantitatively detecting alpha-synuclein auto-antibodies in human sera - Google Patents
Method for quantitatively detecting alpha-synuclein auto-antibodies in human sera Download PDFInfo
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Abstract
The invention relates to a method for quantitatively detecting alpha-synuclein auto-antibodies in human sera, and prepares recombinant human alpha-synuclein as antigens and rabbit anti-human alpha-synuclein polyclonal antibodies as antibodies into a kit, wherein, the quantities of alpha-synuclein auto-antibodies in the sera of patients with Parkinson's disease (PD) and in the sera of normal healthy persons are detected by utilizing the antigen-antibody reaction principle and are compared; and the presence and quantity of alpha-synuclein auto-antibodies in human sera are judged by the kit on the basis of color reaction.
Description
Technical field:
The present invention relates to a kind of detection method of alpha-synuclein auto-antibodies in human sera, the method can be used for Parkinsonian diagnosis.
Background technology:
Parkinson's (Parkinsonps disease, PD) be the old nerve degenerative diseases that a kind of incidence of disease is only second to senile dementia, its main pathological characters with carrying out property of substantia nigra of midbrain compact part dopaminergic neuron lose with residual neuron plasma in the eosinophilic inclusion appears, claim Lewy body.Alpha-synapse nucleoprotein is the principal ingredient that consists of Lewy body, and mainly the form with aggregation exists.In Parkinsonian pathogenic process, high-caliber alpha-synapse nucleoprotein can form the virose allosteric form of dopaminergic neuron tool, and this allosteric form might can cause auto-antibody to produce in the local microenvironment formation of dopaminergic neuron death, and then occurs increasing from body alpha-synapse nucleoprotein antibody horizontal in patient's Parkinson serum.Up to the present, only had one piece of bibliographical information and increased in the familial Parkinson's disease alpha-synuclein auto-antibodies in human sera level of Greece, and existed the defective such as the few and quantivative approach imperfection of case.At present, Parkinsonian diagnosis mainly relies on motor symptoms and doctor's experience, and higher misdiagnosis rate is arranged.Clinical pathological study shows in sporadic Parkinsonian diagnosis, to have 24% misdiagnosis rate at least.In addition, because the dead feature of Parkinsonian's substantia nigra dopaminergic neuron shows as the death of carrying out property, only have when substantia nigra dopaminergic neuron death has reached 70% left and right sides, just begin to occur motor symptoms, and at this moment be the late period of substantia nigra dopaminergic neuron death.There is no radical treatment this moment except the heteropathy of replenishing dopamine, certainly will have a strong impact on patient's quality of life and increase white elephant to society and family.Therefore early detection, diagnosis Parkinson's for timely treatment, protect dopaminergic neuron, delay the state of an illness, improve the Quality of Life tool and be of great significance.
The present invention works out the method that detects alpha-synuclein auto-antibodies in human sera, can be used for Parkinsonian diagnosis, is screening Parkinson's people at highest risk and the Parkinsonian important means of clinical diagnosis.
Summary of the invention:
The invention provides a kind of Parkinsonian diagnostic method, comprising the detection method of alpha-synuclein auto-antibodies in human sera.
The present invention is prepared into kit with antigen (recombined human alpha-synapse nucleoprotein) and antibody (the anti-human alpha-synapse nucleoprotein polyclonal antibody of rabbit), with the antigen-antibody reaction principle detect in patient Parkinson and the normal health human serum from the having quantity and compare of body alpha-synapse nucleoprotein antibody, kit adopt chromogenic reaction judge in the human serum from the existence of body alpha-synapse nucleoprotein antibody whether and quantity.
Parkinsonian diagnostic method of the present invention may further comprise the steps:
The preparation of step 1, people's alpha-synapse nucleoprotein
The preparation of step 2, anti-alpha-synapse nucleoprotein polyclonal antibody
The preparation of step 3, patient Parkinson and normal health human serum
The detection of step 4, alpha-synuclein auto-antibodies in human sera
Methods for the diagnosis of diseases of the present invention comprises uses diagnostic reagent that alpha-synuclein auto-antibodies in human sera is detected.
Therefore the present invention includes diagnostic kit.
The composition of diagnostic kit of the present invention comprises:
Antigen (recombined human alpha-synapse nucleoprotein)
Antibody (the anti-human alpha-synapse nucleoprotein polyclonal antibody of rabbit)
Kit of the present invention comprises that also following auxiliary constituent is used for the reaction assay of antigen-antibody.
Antigenic dilution (0.05M carbonic acid buffer, pH 9.6)
Antibody and serum dilution (10%BSA/PBS)
Wash plate liquid (0.1% (v/v) Tween-20/PBS)
Confining liquid (10%BSA/PBS)
Mark two anti-(AP-goat anti-rabbit iggs; AP-mountain goat anti-human igg)
Chromogenic substrate (p-Nitrophenyl Phosphate Liquid Substrate System, Sigma)
96 hole elisa plates.
Kit of the present invention, each reagent is packed respectively, preferably uses packing tube, and the amount of the reagent of packing in each packing tube can expand 10 to take enough-individual sample use amount as fundamental quantity, and 100, the use amount of 1000 samples.
The detection method of the alpha-synuclein auto-antibodies in human sera that the present invention also comprises, the method may further comprise the steps:
Antigen (recombined human alpha-synapse nucleoprotein) and antibody (the anti-human alpha-synapse nucleoprotein polyclonal antibody of rabbit) react, according in the diversity judgement human serum of reaction solution from the existence of body alpha-synapse nucleoprotein antibody whether and quantity.
The method that adopts kit of the present invention to detect is as follows:
1) wrapper sheet: people's alpha-synapse nucleoprotein (10 μ g/ml, carbonic acid buffer dissolving) coated 96 hole elisa plates in 100 μ l/ holes, 4 ℃, 24 hours.
2) wash plate: wash plate 3 times, each 5 minutes with PBS (PBS-T) the 200 μ l/ holes that contain 0.1% (v/v) Tween-20.
3) sealing: adopt to contain 10%BSA/PBS 200 μ l/ holes sealing 1 hour.
4) application of sample: 1～3 row at 96 orifice plates add from 1: 10000～1: 640000 the anti-human alpha-synapse nucleoprotein polyclonal antibody of different dilution rabbits 100 μ l/ holes (20% goat normal serum/PBS dilution), and 3 of each dilutabilitys are done 3 multiple holes.9～12 row add the human serum sample 100 μ l/ holes (1: 100,20% goat normal serum/PBS dilution) of dilution, and each sample is done 3 multiple holes.4 ℃, spend the night.
5) wash plate: wash plate 3 times, each 5 minutes with PBS (PBS-T) the 200 μ l/ holes that contain 0.1% (v/v) Tween-20.
6) adding mark two resists: the many resisting samples of rabbit at 1～3 row added alkali phosphatase enzyme mark goat anti-rabbit igg 100 μ l/ holes (1: 5000, the 10%BSA/PBS dilution), 9～12 row human serum samples added alkali phosphatase enzyme mark mountain goat anti-human igg 100 μ l/ holes (1: 5000, the 10%BSA/PBS dilution), incubated at room 1 hour.
7) wash plate: wash plate 3 times, each 5 minutes with PBS (PBS-T) the 200 μ l/ holes that contain 0.1% (v/v) Tween-20.
8) colour developing: add chromogenic substrate (p-Nitrophenyl Phosphate Liquid Substrate System, Sigma) 100 μ l/ holes, 37 ℃, 30 minutes.
9) color development stopping reaction: 3N NaOH 100 μ l/ hole color development stopping reactions.
10) detect absorbance: microplate reader 405nm and survey absorbance.
11) the antibody unit value is calculated
High dilution is set to contain 1 antibody unit at 1: 640000, and be 2 antibody units at 1: 320000 successively, and be 4 antibody units at 1: 160000, be 8 antibody units at 1: 80000, be 16 antibody units at 1: 40000, and be 32 antibody units at 1: 20000, and be 64 antibody units at 1: 10000.According to the mean light absorbency of each antibody unit, make typical curve.According to lineal relevant formula, can calculate serum sample and contain relative quantity from the anti-human alpha-synapse nucleoprotein antibody of body.As: the absorbance in 3 multiple holes of 80314 serum is respectively 0.84973,0.86223,0.69677.The absorbance average is 0.80291.Can calculate according to formula that the relative quantity from body people alpha-synapse nucleoprotein antibody is 37.27744 in this serum.
Kit of the present invention, wherein being prepared as follows of recombined human alpha-synapse nucleoprotein:
The conventional BL21 competent cell that transforms of pGEX-h α-SYN recombinant plasmid (by the present of U.S. Parkinson's disease research institute), (every liter contains the 16g peptone to get 1 bacterium colony implantation 10ml, 2 * YTA-amp fluid nutrient medium, the 10g yeast extract, 5g NaCl and 0.1g ampicillin) 37 ℃ of shaken overnight, be transferred to after the preculture in 1 liter of fresh 2 * YTA-amp nutrient culture media, adding IPTG (ultimate density 0.1mmol/L) inducible protein behind 37 ℃ of shaken cultivation 3 h expresses, gather in the crops bacterium after continuing to cultivate 4 h, with ice-cold PBS suspension bacterium, centrifugal behind the ultrasonication bacterium, supernatant obtains GST-h α-SYN fusion through glutathione-Sepharose affinitive layer purification, decomposes through fibrin ferment again, enzyme was cut the post purifying and was obtained the recombined human alpha-synapse nucleoprotein.Freeze drying ,-80 ℃ of preservations are stand-by.BCA standard measure alpha-synapse nucleoprotein.Western Blot identifies restructuring alpha-synapse nucleoprotein (Fig. 1).Being prepared as follows of the anti-human alpha-synapse nucleoprotein polyclonal antibody of rabbit:
4 of rabbit, blood sampling before the immunity, preparation serum is for subsequent use.The Fu Shi Freund's complete adjuvant that the warp of 2ml alpha-synapse nucleoprotein solution (1mg/ml) and equivalent fully shakes is fully emulsified.Antigenic solution hypodermic injection after every animals received 0.8ml emulsification, initial immunity are after 14 days, and blood sampling preparation antiserum detects immune effect.Initial immunity is after 4 weeks, the animals received reinforced immunological, and after 2 weeks, blood sampling preparation antiserum.Take ammonium sulfate salting-out process separation and purification antibody, Western Blot detects specificity and the susceptibility (Fig. 2) of antibody.
When using kit of the present invention, need to gather patient Parkinson and normal healthy people control serum, the preparation method of described control serum is as follows:
Collect venous blood in heparin tube (BD company), avoid haemolysis.Vibrate up and down lentamente heparin tube 5 times makes the coagula mixing in the blood.Room temperature (25 ℃) was vertically placed 1 hour, made blood clotting.(blood must accurately condense 1 hour, otherwise sample difference setting time may cause difference).Under the room temperature, usefulness hydro-extractor centrifugal 10 minutes with 2000g.Draw supernatant, packing (10 μ l/ pipe) is in the mark sample hose of correspondence.Frozen blood serum sample is in-80 ℃ of refrigerators immediately.Avoid multigelation.
In test of the present invention, use therein reagent and starting material comprise: pGEX-h α-SYN recombinant plasmid all can be bought from the market and obtain, and belongs to known technology.
Description of drawings:
Fig. 1 is 96 hole elisa plate synoptic diagram.
Fig. 2 is canonical plotting
Fig. 3 is the evaluation of recombined human alpha-synapse nucleoprotein
Fig. 4 is the evaluation of the anti-human alpha-synapse nucleoprotein polyclonal antibody of rabbit
It is simple to operate, highly sensitive that Fig. 5 is that Parkinson's alpha-synuclein auto-antibodies in human sera content contrasts method of the present invention apparently higher than normal health, and testing cost is few, and cost is low, and the result is accurate, can diagnose in advance, has good medical effect.
Embodiment:
Further specify by the following examples the present invention, but not as limitation of the present invention.
1. prokaryotic expression, Purification and Characterization recombined human alpha-synapse nucleoprotein
WESTERN BLOT result shows, after recombined human alpha-synapse nucleoprotein behind the purifying of 50～1000ng and mouse anti human monoclonal antibody (3D5) combination, shown the positive band that differs in size, the size of molecular weight of albumen is approximately about 18kD, consistent with people's alpha-synapse nucleoprotein molecular weight, prove that recombinant expressed albumen is with people's alpha-synapse nucleoprotein (Fig. 3).
2.: the anti-human alpha-synapse nucleoprotein polyclonal antibody of rabbit is produced
After adopting recombinant expressed people's alpha-synapse nucleoprotein immunizing rabbit, the WESTERNBLOT result that the antiserum of results carries out shows, antiserum can obviously show recombinant expressed people's alpha-synapse nucleoprotein at 1: 30000 dilutability, shows that this rabbit anti-serum and antigen have good affinity; And the result of rat cerebral even slurry shows, the dilutability at 1: 10000, this antibody have only shown a band that coincide with the alpha-synapse nucleoprotein molecular size range, show that this antiserum has preferably specificity.
The preparation of kit:
Kit consists of: 96 hole elisa plates (Corning Incorporated), envelope antigen (recombined human alpha-synapse nucleoprotein), standard antibody (the anti-human alpha-synapse nucleoprotein polyclonal antibody of rabbit), antigenic dilution (0.05M carbonic acid buffer, pH 9.6), antibody and serum dilution (10%BSA/PBS), wash plate liquid (0.1% (v/v) Tween-20/PBS), confining liquid (10%BSA/PBS), mark two and resist (AP-goat anti-rabbit iggs; AP-mountain goat anti-human igg), chromogenic substrate (p-Nitrophenyl Phosphate Liquid Substrate System, Sigma)
Relevant dilution is washed plate liquid, confining liquid, and mark two is anti-, and composition and the compound method of chromogenic substrate belong to known technology.
In the serum sample from the detection of body people alpha-synapse nucleoprotein antibody
Adopt relative quantification ELISA method, to detecting from body alpha-synapse nucleoprotein antibody in patient Parkinson of 116 routine clinical diagnosises and the 78 routine normal healthy controls serum.The result shows that Parkinson's alpha-synuclein auto-antibodies in human sera content is 30.3 ± 11.69 antibody units, is 64.72567 from body alpha-synapse nucleoprotein antibody mxm. in the serum among patient Parkinson; And this antibody content of normal health control serum is 15.7 ± 5.99 antibody units, and mxm. is 27.665.Parkinson's alpha-synuclein auto-antibodies in human sera content is apparently higher than normal health contrast (p＜0.01).Fig. 5. relative quantification ELISA method detects alpha-synuclein auto-antibodies in human sera content.
**p＜0.01。
Claims (3)
1. kit that quantitatively detects alpha-synuclein auto-antibodies in human sera is characterized in that described kit comprises: people's alpha-synapse nucleoprotein and anti-human alpha-synapse nucleoprotein antibody,
And following auxiliary reagent,
Antigenic dilution: the 0.05M carbonic acid buffer,
Antibody and serum dilution: 10%BSA/PBS,
Wash plate liquid: 0.1% (v/v) Tween-20/PBS,
Confining liquid: 10%BSA/PBS,
Mark two is anti-: the AP-goat anti-rabbit igg; AP-mountain goat anti-human igg,
Chromogenic substrate: p-Nitrophenyl Phosphate Liquid Substrate System96 hole elisa plate.
2. the kit of claim 1, being prepared as follows of people's alpha-synapse nucleoprotein wherein: the conventional BL21 competent cell that transforms of pGEX-h α-SYN recombinant plasmid, get 1 bacterium colony and implant 10ml2 * 37 ℃ of shaken overnight of YTA-amp fluid nutrient medium, be transferred to after the preculture in 1 liter of fresh 2 * YTA-amp nutrient culture media, adding the IPTG inducible protein behind 37 ℃ of shaken cultivation 3h expresses, gather in the crops bacterium after continuing to cultivate 4h, with ice-cold PBS suspension bacterium, centrifugal behind the ultrasonication bacterium, supernatant obtains GST-h α-SYN fusion through glutathione-Sepharose affinitive layer purification, decompose through fibrin ferment again, enzyme was cut the post purifying and was obtained the recombined human alpha-synapse nucleoprotein
Wherein, every liter of described 2 * YTA-amp fluid nutrient medium contains 16g peptone, 10g yeast extract, 5g NaCl and 0.1g ampicillin.
3. the kit of claim 1, wherein being prepared as follows of anti-human alpha-synapse nucleoprotein antibody: 4 of rabbit, blood sampling before the immunity, preparation serum is for subsequent use; The Fu Shi Freund's complete adjuvant that the warp of 2ml alpha-synapse nucleoprotein solution and equivalent fully shakes is fully emulsified; Antigenic solution hypodermic injection after every animals received 0.8ml emulsification, initial immunity are after 14 days, and blood sampling preparation antiserum detects immune effect; Initial immunity is after 4 weeks, the animals received reinforced immunological, and after 2 weeks, blood sampling preparation antiserum; Take ammonium sulfate salting-out process separation and purification antibody.
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| JP6263473B2 (en) * | 2011-11-02 | 2018-01-31 | バイオジェン インターナショナル ニューロサイエンス ゲーエムベーハー | Use of anti-α-synuclein antibodies to diagnose high levels of α-synuclein in the brain |
| US9534044B2 (en) * | 2013-02-28 | 2017-01-03 | United Arab Emirates University | Alpha-synuclein antibodies and uses thereof |
| CN103983778B (en) * | 2014-05-08 | 2016-04-13 | 首都医科大学宣武医院 | A kind of method detecting subject's body fluid external promotion disease related protein polymerizing power |
| CN104215779B (en) * | 2014-09-18 | 2016-01-20 | 首都医科大学宣武医院 | A kind ofly detect the method for haemoglobin in conjunction with alpha-synapse nucleoprotein |
| CN104711280B (en) * | 2014-12-10 | 2018-04-13 | 中国医学科学院医学生物学研究所 | The method that expression and purifying lack exon 3 and 5 α synapse nucleoprotein alternative splicing body proteins at the same time |
| CN106568969B (en) * | 2016-10-19 | 2017-09-26 | 首都医科大学 | A kind of ELISA detection method of 129 phosphorylation alpha synuclein aggregation bodies of serine |
| EP3583124A1 (en) | 2017-02-17 | 2019-12-25 | Bristol-Myers Squibb Company | Antibodies to alpha-synuclein and uses thereof |
| CN111537738A (en) * | 2020-05-18 | 2020-08-14 | 南通大学附属医院 | Kit for detecting early Parkinson's disease |
| CN112710850A (en) * | 2020-12-29 | 2021-04-27 | 苏州百志生物科技有限公司 | Kit for detecting content of alpha-synuclein in human body fluid |
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| WO2003069332A2 (en) * | 2002-02-14 | 2003-08-21 | The University Of Lancaster | Detection and/or monitoring of synuclein-related diseases |
| CN101052417A (en) * | 2004-08-09 | 2007-10-10 | 艾兰制药公司 | Prevention and treatment of synucleinopathic and amyloidogenic disease |
| CN101308144A (en) * | 2007-05-16 | 2008-11-19 | 首都医科大学宣武医院 | Method for detecting subject humoral disease related protein polymerizing power |
| CN101460161A (en) * | 2006-03-29 | 2009-06-17 | 弗尔德里克斯制药股份有限公司 | Inhibition of alpha-synuclein toxicity |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2003069332A2 (en) * | 2002-02-14 | 2003-08-21 | The University Of Lancaster | Detection and/or monitoring of synuclein-related diseases |
| CN101052417A (en) * | 2004-08-09 | 2007-10-10 | 艾兰制药公司 | Prevention and treatment of synucleinopathic and amyloidogenic disease |
| CN101460161A (en) * | 2006-03-29 | 2009-06-17 | 弗尔德里克斯制药股份有限公司 | Inhibition of alpha-synuclein toxicity |
| CN101308144A (en) * | 2007-05-16 | 2008-11-19 | 首都医科大学宣武医院 | Method for detecting subject humoral disease related protein polymerizing power |
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