CN101661032A - Biomarker of Parkinson disease - Google Patents

Abstract

The invention relates to a biomarker of Parkinson disease, in particular to a method used for detecting pathogeny of the Parkinson disease and identifying the biomarker of the Parkinson disease (PD).The invention also provides a kit used for diagnosing or monitoring PD. A proteinic isoform or a monomer or polymer form thereof can be used for early diagnosing vulnerable subjects who are possible to suffer from PD. For example, the increase of the level of monomer transthyretin (TTR) in serum or the reduction of the level of dipolymer TTR can be used as a tool for diagnosing and monitoring PD.

Description

Parkinsonian biomarker

Technical field

The present invention relates in general to the field of molecular biology, Neurobiology, pharmacology and diagnosis, prognosis or preventing/treating Parkinson's (PD), especially relates to Parkinsonian biomarker.

Background technology

Parkinson's are a kind of carrying out property nerve retrograde affections, are characterized in having occlusion body (Lewy corpusculum) in the loss of dopaminergic neuron in the black substance and the survived neuronal.Parkinsonian typical clinical feature comprises that carrying out property is trembled, tetanic and bradykinesia.Yet at its commitment, Parkinson's are to be difficult to diagnosis, and this is owing to lack clinical symptoms and lack reliable biomarker.In addition, Parkinson's may be very similar with other diseases, this comprising but be not limited to following several disease, as essential tremor and multiple system atrophy, this makes possible diagnosis more complicated.Can based on only in brain susceptible disease become neurology that cell in the zone significantly just occurs after the forfeiture and show and make the PD diagnosis.

Summary of the invention

The present invention relates generally to the analytical test of blood serum sample, also relate to the explanation of carrying out for Parkinsonian diagnosis, prognosis or preventing/treating in certain aspects biomarker.In one aspect of the invention, this paper provides Parkinsonian diagnosis or monitoring method, it comprises: the level of (a) measuring at least a biomarker in experimenter's sample, wherein said biomarker be selected from haptoglobin, transferrin, apolipoprotein A-1 precursor, alpha1-antitrypsin precursor and transthyretin (transthyretin, TTR); (b) level and the reference expression level with biomarker in the sample compares, and wherein the difference of the level of biomarker and reference expression level has been indicated the existence of PD among the experimenter or developing stage that should disease in the sample.

In one embodiment, reference levels are meant the level of not suffering from least a biomarker in the Parkinsonian control population.In one embodiment, reference levels are meant the level of at least a biomarker among more earlier certain time experimenter.In one embodiment, reference levels are meant before the scheme of receiving treatment the level of at least a biomarker among the experimenter.

In one embodiment, described sample is selected from blood plasma, serum, whole blood and cerebrospinal fluid (CSF).

In one embodiment, determination step comprises the amount of at least a biomarker polypeptide in the measuring samples.In one embodiment, determination step comprises the amount of the posttranslational modification polypeptide of at least a biomarker in the measuring samples.

In one embodiment, comprise the monomer or the polymer of at least a biomarker in the measuring samples in the determination step, perhaps monomer and polymer are all measured.In one embodiment, having a kind of biomarker at least is transthyretin.In one embodiment, the relative quantity that the determination step of at least a biomarker level is comprised monomer in the measuring samples or dimeric forms transthyretin.In one embodiment, the level of monomeric form transthyretin is compared with reference levels and is increased, thereby the indication experimenter suffers from Parkinson's, or indicates this sick stage.In one embodiment, dimeric forms transthyretin level is compared with reference levels and is decreased, thereby the indication experimenter suffers from Parkinson's, or indicates this sick stage.In one embodiment, described determination step is undertaken by immunoassays.In one embodiment, described immunoassays are ELISA or western trace.In one embodiment, described measuring process utilizes two dimensional gel electrophore-sis and mass spectrum to carry out.

In one embodiment, selecting the step of therapeutic scheme to be based on the experimenter suffers from this situation of Parkinson's or carries out based on this sick stage.In one embodiment, the step of measuring at least a biomarker is carried out after the experimenter being used this therapeutic scheme.

In another aspect of the present invention, this paper provides screening to can be used for treating the method for Parkinsonian compound, comprise: (a) measure the expression of at least a biomarker in the experimenter's who uses test compounds sample, wherein said biomarker is selected from haptoglobin, transferrin, apolipoprotein A-1 precursor, alpha1-antitrypsin precursor and transthyretin; (b) expression and the reference expression level with described biomarker compares; (c) determine the effectiveness of test compounds, the difference that the expression of wherein said biomarker is compared with the reference expression level indicates this test compounds to can be used for treating Parkinson's.In one embodiment, reference levels are to accept before the test compounds expression of at least a biomarker among this experimenter.

In another aspect of the present invention, this paper provides the Parkinsonian kit that is used to diagnose or monitor the experimenter, it comprises: (a) be used for measuring one or more detectable of at least a biomarker of experimenter's sample, wherein said biomarker is selected from haptoglobin, transferrin, apolipoprotein A-1 precursor, alpha1-antitrypsin precursor and transthyretin; (b) at least a from the control sample of not suffering from the Parkinson's control population; And (c) operation instruction of described one or more detectable.

In one embodiment, one or more detectable that the invention provides at least a biomarker are used to prepare the purposes of Parkinsonian diagnosis or monitoring reagent box, and wherein said at least a biomarker is selected from haptoglobin, transferrin, apolipoprotein A-1 precursor, alpha1-antitrypsin precursor and transthyretin.

In one embodiment, wherein said kit is used to measure the level from least a biomarker described in experimenter's the sample, and compare with reference levels, wherein the difference of the level of at least a biomarker described in the sample and reference levels indicates this experimenter to suffer from Parkinson's, perhaps indicates the Parkinsonian stage.

In one embodiment, wherein said reference levels are levels of not suffering from least a biomarker described in Parkinsonian experimenter's control population.

In one embodiment, wherein said reference levels are levels of at least a biomarker described in this experimenter in early time.

In one embodiment, wherein said reference levels are levels of this experimenter described at least a biomarker before the scheme of receiving treatment.

In one embodiment, wherein said sample is selected from blood plasma, serum, whole blood and cerebrospinal fluid.

In one embodiment, wherein said mensuration comprises the amount of the polypeptide of at least a biomarker described in the measuring samples.

In one embodiment, wherein said mensuration comprises the amount of the posttranslational modification polypeptide of at least a biomarker described in the measuring samples.

In one embodiment, wherein said mensuration comprises the amount of the described at least a biomarker of monomer in the measuring samples or polymer form, perhaps these two kinds of forms is all measured.

In one embodiment, wherein said at least a biomarker is a transthyretin.

In one embodiment, the level of at least a biomarker of wherein said mensuration comprises the relative quantity of transthyretin monomer in the measuring samples or dimeric forms.

In one embodiment, wherein the level of monomeric form transthyretin is compared with reference levels and is improved, and indicates this experimenter to suffer from Parkinson's, perhaps indicates the Parkinsonian stage.

In one embodiment, wherein the level of dimeric forms transthyretin is compared with reference levels and is reduced, and indicates this experimenter to suffer from Parkinson's, perhaps indicates the Parkinsonian stage.

In one embodiment, wherein said mensuration is undertaken by immunoassays.

In one embodiment, wherein said immunoassays provide ELISA or western to carry out.

In one embodiment, wherein said determination step is realized by dielectrophoresis and mass spectrophotometry.

Above general introduction only is used for explanation, and and be not intended to carry out any type of restriction.Except above-mentioned illustrative aspect, embodiment and feature, other more aspect, embodiment and feature can be with reference to the accompanying drawings and will be clearly after the detailed description hereinafter.

The accompanying drawing summary

The two-way gel images of this exemplary of protein spots of differential expression in the serum of Figure 1A and 1B demonstration Parkinsonian and control population.

Fig. 2 shows that the two-way gel polypeptide sample to downcutting carries out picture in this illustrated examples scheme of tandem mass spectrum (MS), and the mass-to-charge ratio of described polypeptide sample (m/z) is 697.93.

Fig. 3 A shows the picture that carries out this exemplary of TTR western trace with the serum of Parkinsonian and contrast.

The picture of Fig. 3 B displaying monomer and this exemplary of dimer TTR relative intensity.

Embodiment

This paper openly is used for detecting protein and the method whether experimenter exists PD or detect its stage to small part based on the sample test result.This paper also openly is used for monitoring protein and the method that is diagnosed as Parkinsonian experimenter's disease progression state based on the sample test result to small part.Specimen disclosed herein includes but not limited to as blood (or the fraction of blood, include but not limited to for example blood plasma or serum), lymph liquid, mucus, tear, saliva, capsule liquid, urine, ight soil, cerebrospinal fluid (CSF), ascites, whole blood and systemic biopsy samples.

The present invention generally provides detection, measurement and the comparison to for example (but being not limited only to) haptoglobin, transferrin, apolipoprotein A-1 precursor, alpha1-antitrypsin precursor protein, transthyretin or its any posttranslational modification variant in the sample.Correspondingly, many aspects of the present invention relate to collection, preparation, separation, evaluation, sign and the comparison to instantaneous biomarker (instant-biomarker) in the test sample.The invention still further relates to and detect and/or monitor the sample that contains haptoglobin, transferrin, apolipoprotein A-1 precursor, alpha1-antitrypsin precursor protein, TTR or its any posttranslational modification variant, above mark can separately or unite to be used for determining whether the experimenter suffers from Parkinson's, or determines its morbidity stage.

Another aspect comprise based on the existence of PD among the experimenter whether or its degree select therapeutic scheme.Correspondingly, the invention still further relates to and select and monitor the influence of medicament (for example medicine, compound) the biomarker expression, as described herein.Another aspect of the present invention relates to the diagnostic kit that is used to detect and monitor PD, describes in further detail in its part hereinafter.

Implement when of the present invention, used the routine techniques of molecular biology, protein biochemistry, cell biology, immunology, microbiology and DNA reorganization.These technology are well-known, and have obtained elaboration respectively in many documents, Current Protocols inMolecular Biology for example, and the I-III volume, Ausubel edits. (1997); Sambrook etc., Molecular Cloning:A Laboratory Manual, second edition. (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, NY, 1989); DNACloning:A Practical Approach, the first volume and second volume, Glover edits (1985); Oligonucleotide Synthesis, Gait edit (1984); Nucleic Acid Hybridization, Hames ﹠amp; Higgins edits. and (1985); Transcription and Translation, Hames﹠amp; Higgins edits. and (1984); Animal Cell Culture, Freshney edits. and (1986); Immobilized Cells and Enzymes (IRL Press, 1986); Perbal, A PracticalGuide to Molecular Cloning; Meth.Enzymol. book series, (Academic Press, Inc., 1984); Gene Transfer Vectors for Mammalian Cells, Miller ﹠amp; Calos edits. and (Cold Spring Harbor Laboratory, NY, 1987); And Meth.Enzymol., the 154th and 155 volumes, Wu ﹠amp; Grossman and Wu edit.The method that is used for detection and measurement polypeptide gene product expression levels (being the gene translation level) is well-known in the art, comprises and uses the polypeptide detection method, and it includes but not limited to for example antibody test and quantitative technique.(also can consult Strachan ﹠amp; Read, Human Molecular Genetics, second edition. (John Wiley and Sons, Inc., NY, 1999)).

Unit, prefix and symbol are all represented with the SI form that it is generally acknowledged.Except as otherwise noted, amino acid sequence is according to writing from left to right to the c-terminus direction from aminoterminal.Amino acid herein can be represented according to the trigram symbol or the one-letter symbol of generally acknowledging that IUPAC-IUBMB term NK is recommended.

Many terms have extensively been used in the following description.These usages of this paper are for convenience to understanding of the present invention.Following term is defined more fully by the reference instructions is whole.In this article, unless clearly indicate, when not having numeral-classifier compound to modify or when represent with " a kind of (individual) ", noun is interpreted as " one or more (individual) " accordingly.

This paper employed to the experimenter " use " thus medicament or medicine comprise introduces compound or be delivered to any approach that the experimenter brings into play its expectation function.Use and can be undertaken by any suitable way, described approach comprises in the per os, nose, stomach and intestine outer (in intravenous, intramuscular, the peritonaeum or subcutaneous), rectum or part.Use and comprise that the oneself uses or used by other people.Should also be understood that described multiple treatment or avoidance mode to medical condition is intended to expression " basically ", this comprises treatment fully or prevention, but also comprises non-treatment fully or the prevention that has obtained some biology or medical science correlated results.

Term " evaluation " and " assessment " are used interchangeably, and are meant any type of measurement, comprise determining that whether factor exists.Term " is determined ", " measurement ", " assessment " and " mensuration " can be exchanged use, and quantitatively and observational measurement include interior.Assessment can be relative or absolute." assessment ... have a situation " comprise and measure its amount, and determine whether it exists.

Term as used herein " biomarker " is meant polypeptide (it has specific apparent molecular weight) under background of the present invention, it is at the sample of taking from PD patient and take between the compared sample of contrast experimenter or control population and there are differences.

Term used herein " control population " is meant negative diagnosis of PD or the detection individuality less than PD, i.e. normal or health volunteer.

Term used herein " level difference " is meant the amount difference compared with the control of the biomarker that exists in the sample of taking from PD patient.In one embodiment, biomarker can be a polypeptide, and its amount in PD patient's sample is compared with control level and occurred raising or reducing.In another embodiment, biomarker can be at differential expression aspect the amount of biomarker or the variant form (being isoform, poly variant and/or posttranslational modification variant).

" validity " of term test compounds used herein is meant the amount that treats and/or prevents effect of enough realizing ideal, and comprises as preventing or alleviate the amount of the symptom of being controlled disease (being PD).The compound amount that the experimenter is used will depend on the order of severity and the type and the individual feature of disease, and this includes, but are not limited to as general health situation, age, sex, body weight with to the tolerance of medicine.This also will depend on degree, seriousness and the stage of disease.Those skilled in the art can determine proper dosage according to these factors and other factors.

Term used herein " immunoassays " is meant and uses high sensitivity and specific immune response detects or the quantitative test chemical of the predetermined substance (as biomarker) in the biological sample that described biological sample includes but not limited to as blood or humoral sample.Its high sensitivity is owing to use antibody and purifying antigen as reagent.The antibody that is used for immunoassays herein is to respond to antigenic stimulus and the protein (immunoglobulin (Ig)) that produces by bone-marrow-derived lymphocyte (immunocyte).Immunoassay is measured the formation of antibody-antigenic complex, and by Indicator Reaction it is detected.Realize high sensitivity by the indicator system (as enzyme labeling) of using amplifying signal.

Term used herein " monomer " is meant can aggregate into peptide, polypeptide or the protein that polymeric single component form exists.The monomer that exists with single component form contains all fundamental characteristics of this peptide, polypeptide or protein integral body, unless those characteristics only occur after assembling.

Term used herein " polymer " (comprising term " dimer ") is meant the peptide, polypeptide or the protein that exist with the form more than single component.Dimer is meant by be cascaded two monomers of performance function of covalent bond, ionic link, hydrogen bond or Van der Waals force.

Term used herein " polypeptide ", " peptide " and " protein " are used interchangeably, and refer to the polymer of amino acid residue.This term is applicable to such amino acid polymer, and promptly wherein one or more amino acid residues are artificial chemical analogs of corresponding natural amino acid, and this term is applicable to naturally occurring amino acid polymer too.

Term used herein " colony " can be the group of any at least two individualities.Colony can include but are not limited to control population, patient colony, reference group, population groups, colony of family, clinical colony and with sex colony etc.

Term used herein " polypeptide of posttranslational modification, peptide and protein " also comprises modification, such as but not limited to glycosylation, fat adhere to, sulphation, carboxylated, hydroxylation, ADP ribosylation and other complex polysaccharide of interpolation.

Term used herein " reference levels " is meant the amount or the concentration of the biomarker that is used for the comparison purpose.In one embodiment, reference levels can be the average levels of at least a biomarker in health volunteer's control group sample.In another embodiment, reference levels can be the levels of at least a biomarker among (promptly at this measure before) same experimenter in early time.In another embodiment, reference levels can be the levels of at least one biomarker among this experimenter before the scheme of receiving treatment.

Term used herein " sample " can include but not limited to the tissue or the body fluid of health, include but not limited to as blood (or the fraction of blood, as blood plasma or serum), lymph liquid, mucus, tear, saliva, gall-bladder liquid, urine, seminal fluid, ight soil, CSF, ascites or whole blood, and comprise from systemic biopsy samples.Sample can be from any experimenter, as suffers from PD or experimenter/patient and the contrast experimenter who suffers from the PD risk arranged.

Term used herein " screening " is meant determines whether test compounds has the ability or the feature of preventing or delaying (alleviating) purpose pathological state as herein described (being PD).The sensitivity of diagnostic method can be different.The sensitivity of diagnostic assay is meant the number percent that detection compound is produced the diseased individuals of sound response.

Term used herein " experimenter " is meant mammal, including but not limited to as human, also can be animal, as domestic animal (as dog, cat etc.), domestic animal (as ox, sheep, pig, horse etc.) and animal used as test (as monkey, rat, mouse, rabbit, cavy or the like).Term " patient " is meant to suffer from " experimenter " that PD or suspection suffer from PD.

Term used herein " treatment " is meant therapeutic treatment and prevention or safeguard procedures, its objective is prevention or delays (alleviating) purpose pathological state or disease.If after the test compounds according to the inventive method amount of receiving treatment, the experimenter shows observing of one or more PD S or Ss and/or detectablely weakens or disappear, or the improvement of Hoehn-Yahr and/or UPDRS classification appears, or the improvement of one or more Quality Of Well Being Indexs appears, can think that then patient's PD illness has successfully obtained treatment.

Term used herein " variant " is meant polypeptide, peptide and/or protein or its fragment of interchangeabling form (as monomer, dimer or polymer) or any posttranslational modification form." variant " includes but not limited to polypeptide, peptide and/or protein or its fragment difference on secondary, three grades or quaternary structure.

I. diagnose and/or monitor the method for PD

A. Measure the biomarker of PD

On the one hand, this paper provides from sample (including but not limited to as blood, urine, cerebrospinal fluid and brain tissue) and separates the PD biomarker.CSF can be used for detecting the change of brain chemistry aspect, and described change can influence protein expression, function or stability, makes CSF directly contact born of the same parents' external space of brain thus.Lumbar puncture is to obtain one of several existing methods of CSF sample.But lumbar puncture is a kind of invasive method, and this makes the cerebrospinal fluid sample that obtains the experimenter become difficult.Without wishing to be held to theory, 500 milliliters the CSF of having an appointment every day is absorbed into blood, and this prompting blood measuring can be used as the method that substitutes that CSF measures.Therefore, the blood sample that obtains by venipuncture or other conventional method of knowing can provide the source of cerebrospinal fluid, is used for the mensuration of the corresponding biomarker of PD phenotype.

In one embodiment, can in people's tissue sample, screen biomarker as herein described.These samples can comprise the albumen of tissue sample, intact cell, cell lysate or separation, comprising but be not limited to the core (core) as needle puncture biopsy, sample, lymph node tissue or the serum of surgical excision.

In one embodiment, the preparation of sample described herein, separation and/or extraction can be undertaken by the conventional method of knowing.In one embodiment, can be applied to or applied in any combination in separating and/or the technology of purifying biological mark comprises: chemical extraction (as phenol or chloroform extraction), dialysis, precipitate (as ammonium sulfate precipitation), electrophoresis and chromatographic technique.Chemical separation technology does not generally provide the specific isolation to single protein, but can be used for removing nonprotein material or contaminated materials a large amount of in the sample.Electrophoretic separation comprises that the sample that will extract places the hole of gel, and described gel can be the polyacrylamide gel or the Ago-Gel of sex change or non-sex change.Gel is applied direct current or Pulse Electric, and the multiple one-tenth branch in the sample separates according to the combination of its molecular weight size, configuration, electric charge and/or its physical characteristics.After can distinguishing on glue, the part that comprises separated protein can be taken off from glue.Method for purifying proteins is known in the art on polyacrylamide gel and the Ago-Gel, and commercialization.

In one aspect, this paper provides the immunoassays that are used to detect the polypeptide biomarker.The polypeptide biomarker can be by mark antibody or the antibody that carries out mark thereafter detect.Can use various ways to determine whether to comprise in the sample protein in conjunction with given antibody.The method of immunity that can be used for the detection of biological mark includes but not limited to, as Dot blot, western trace, protein-chip, immunoprecipitation (IP), competitiveness and noncompetitive protein bound measure, enzyme linked immunosorbent assay (ELISA) (ELISA) and other generally use and broadly described method, wherein much commercializations in scientific literature and patent documentation.Those skilled in the art can make known protein matter/antibody testing method be applicable to whether to contain biomarker in the working sample at an easy rate, and the relative concentration or the variant form of this specific expression of polypeptides product in the working sample.Protein from the experimenter can utilize technology well known to those skilled in the art to separate.Method of separating protein includes but not limited to as method (the Harlow ﹠amp described at " Antibodies:A LaboratoryManual "; Lane, Antibodies:A LaboratoryManual.Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988).

Antibody can be used to detect the biomarker of expression in several different methods, include but not limited to as western trace or ELISA.In these purposes, can be on solid support with antibody or proteopexy.But holder or carrier comprise the holder of any conjugated antigen or antibody.Holder of knowing or carrier include but not limited to cellulose, polyacrylamide, graniton and the magnetic iron ore as glass, polystyrene, polypropylene, tygon, glucosan, nylon, diastase, natural and modification.

In one embodiment, the western trace is measured and can be used for the detection of biological mark.This paper employed " western trace " is intended to contain all changes type of basic protein determination.The western engram technology is very ripe in the art, generally comprises by by PAGE protein being separated according to molecular weight under sex change or non-sex change condition, detects thereafter.Immunofluorescence and enzyme immunoassay (EIA) (EIA) are all very ripe in the art.Yet, also can use other reporter molecules, include but not limited to as radioactive isotope, chemiluminescence or bioluminescent molecules.To those skilled in the art, how adjusting experimental technique, to adapt to required purposes be conspicuous.

In another embodiment, IP measures and can be used for the detection of biological mark.This paper employed " immunoprecipitation " or " IP " are intended to contain all changes type that basic protein detection is measured.The IP technology is very ripe in the art, generally comprises and utilizes specificity or non-specific antibody that protein is precipitated from sample.Then, the albumen that precipitates can utilize SDS-PAGE to separate, and detects again.Yet, also can use other reporter molecules, include but not limited to as radioactive isotope, chemiluminescence or bioluminescent molecules.To those skilled in the art, how adjusting experimental technique, to adapt to required purposes be conspicuous.

In another embodiment, exist the ELISA of multiple different versions and/or sandwich ELISA can be used in the middle of method of the present invention and the mensuration.This paper employed " ELISA " is intended to contain all changes type of basic immunosorbent assay.This paper employed " sandwich assay " is intended to contain all changes type of basic two site immunoadsorption combination technologies.Immunofluorescence and EIA technology are all very perfect in the art.Yet, also can use other reporter molecules, include but not limited to as radioactive isotope, chemiluminescence or bioluminescent molecules.To those skilled in the art, how adjusting experimental technique, to adapt to required purposes be conspicuous.

On the other hand, this paper provides the two dimensional gel electrophore-sis technology that is used to detect polypeptide and monomer whose and polymer form.Two dimensional gel electrophore-sis is known in the art, is usually included on first dimension isoelectric focusing and carries out the SDS-PAGE electrophoresis subsequently on second dimension.To those skilled in the art, how adjusting experimental technique, to adapt to required purposes be conspicuous.Consult as Hames etc., Gel Electrophoresis of Proteins:A Practical Approach (IRL Press, NY, 1990); Shevchenko etc., Proc.Natl.Acad.Sci.USA, the 93rd volume, 14440-14445 page or leaf, (1996); Sagliocco etc., Yeast, the 12nd volume, 1519-1533 page or leaf, (1996); And Lander, Science, the 274th volume, 536-539 page or leaf (1996).

On the other hand, this paper provides and has utilized mass spectrum (MS) to come polypeptide is identified.Can determine the identity and the expression of target polypeptide by mass-spectrometric technique.Analytic approach based on MS can be used for analyzing the target polypeptide of separation and the target polypeptide in the sample.The mass spectrum form that is used to analyze the target polypeptide comprises ionization (ionization, I) technology, it includes but not limited to as substance assistant laser desorpted (MALDI), continues or pulse electro-spray ionization (ESI) and correlation technique, include but not limited to as ionspray or thermal spray, the collision of bulk cluster (massive cluster impact, MCI).These ion guns can be complementary with test format, comprise linearity or nonlinear reflection flight time (TOF), single or multiple quadrupole rod, single or multiple sectorial magnetic field, Fourier transform ion cyclotron resonance (Fourier transform ion cyclotron resonance, FTICR), ion trap with and the combination, include but not limited to as ion trap/TOF.With regard to ionization, can use many matrix/wavelength combinations (as MALDI) or solvent combination (as ESI).One of ordinary skill in the art will readily recognize that these technology and liquid chromatography technology (LC) are made up the sensitivity that can strengthen the MS method.To those skilled in the art, how adjusting experimental technique, to adapt to required purposes be conspicuous.

With regard to MS analyzes, the target polypeptide can be dissolved in the suitable solvent or reagent system.The selection of solvent or reagent system (for example organic or inorganic solvent) will be depended on the characteristic of target polypeptide and the type of used MS, and carry out based on technology well known in the art.MALDI can consult as Vorm etc., Anal.Chem. the 61st volume, 3281 pages, (1994); ESI can consult as Valaskovic etc., Anal.Chem. the 67th volume: 3802 pages (1995).Select such solvent: it can make the energy that the target polypeptide introduces because of vaporescence and the risk that is degraded is as far as possible little.Can realize the reduction of target polypeptide degraded risk by for example the sample embedding being advanced matrix.Suitable matrix can be organic compound, and it includes but not limited to sugar, as pentose or hexose, or includes but not limited to polysaccharide, as cellulose.Such compound is degraded into carbon dioxide and water by the thermal decomposition effect, thereby can not form the residue that can produce chemical reaction.Matrix can be mineral compound also, includes but not limited to as ammonium nitrate, and it can thoroughly be decomposed and not stay any residue.Especially, the compound member of phenylpropanol family (including but not limited to as alpha-cyano-4-hydroxycinnamic acid) also can be used as matrix described herein.The use of these solvents and other solvent is well known by persons skilled in the art.Electron spray MS is described in Fenn etc., J.Phys.Chem. the 88th volume: 4451-4459 page or leaf, (1984)), some summary documents have been summed up its present application.Consult Smith etc., Anal.Chem. the 62nd volume: 882-89 page or leaf (1990), and Ardrey etc., Spectroscopy the 4th volume: 10-18 page or leaf, (1992).

The target polypeptide quality of measuring by MS can compare with the quality of corresponding known peptide.For example, when the target polypeptide was misfolded proteins, its corresponding known peptide can be corresponding non-misfolded proteins, such as wild-type protein or reference protein.Utilize ESI accurately to measure and fly mole molecular weight of (femtomole) rank sample size, this is that they all can be used for carrying out the calculating of molecular weight owing to there are a plurality of quasi-molecular ions.Ah's mole (attomole) protein below horizontal by as ESI MS (Valaskovic etc., Science the 273rd volume: 1199-1202 page or leaf, (1996)) and MALDI MS (Li etc., J.Am.Chem.Soc. the 118th volume: 1662-1663 page or leaf, (1996)) technology be detected.

The level of target protein can be measured by mass spectrometry method in the sample, include but not limited to technology well known in the art, as substance assistant laser desorpted/ionization-flight time mass spectrum (MALDI-TOF-MS) and surface-enhanced laser desorb/ionization-flight time mass spectrum (SELDI-TOF-MS).The method of carrying out MALDI is well known to those skilled in the art.Consult as Juhasz etc., Analysis, Anal.Chem. the 68th volume: 941-946 page or leaf (1996).Many technology that are used to improve solubleness also are known.MALDI-TOF-MS is described in Hillenkamp etc., Biological Mass Spectrometry, and Burlingame and McCloskey edit. (Elsevier Science Publ., Amsterdam, 1990) 49-60 page or leaf.

Target protein also can detect by series connection MS method in the sample, and it includes but not limited to technology known in the art, as liquid chromatography electro-spray ionization tandem mass spectrum (LC-ESI-MS/MS).Consult as Cagney and Emili Nat.Biotech. the 20th volume: 163-170 page or leaf (2002), and Gu etc., J.Am.Soc.Mass Spectrom. the 14th volume: 1-7 page or leaf, (2003).The method of using and improving LC-ESI-MS/MS is well known to those skilled in the art.

B. The level that compares the PD biomarker

In yet another aspect, this paper provides biomarker level and reference levels in experimenter's sample is compared.Biomarker is biochemical characteristics or the performance that can be used for measuring disease progression, recovery or result of treatment among the experimenter.When comparing with reference levels, the expression of biomarker described herein can be associated with experimenter's morbid state.Therefore, biomarker can be used to refer to experimenter's disease or health status.In another embodiment, the similarity of biomarker level and reference levels can be indicated does not have this morbid state among the experimenter.In another embodiment, the difference that biomarker is compared with reference levels can be indicated and be had this disease among the experimenter, or indicates the stage of this disease.In another embodiment, can compare, with the progress of monitoring of diseases or disappear at name a person for a particular job biomarker and reference levels of different time.

In yet another aspect, this paper provides and relatively has been selected from following biomarker: haptoglobin, transferrin, apolipoprotein A-1 precursor, alpha1-antitrypsin precursor and/or transthyretin.Haptoglobin is the plasma proteins of 45KDa, the free hemoglobin that it discharges in conjunction with red blood cell.See NCBI nonredundancy Protein Data Bank accession number 4826762.In one embodiment, PD patient's haptoglobin level raises compared with the control.

In another embodiment, transferrin is the iron transfer albumen relevant with Parkinsonian pathology, and its theoretical molecular is 77KDa.See NCBI nonredundancy Protein Data Bank accession number 4557871.Iron level in the postmortem PD patient black substance is higher than control level.Consult as Sofic etc., J.Neurochem. the 56th volume: 978-982 page or leaf (1991).Therefore, the level of transferrin is higher than reference levels in PD patient.

The apolipoprotein A-1 precursor is the precursor protein of the main apolipoprotein of high-density lipoprotein (HDL) (HDL), its abundance in blood plasma higher relatively (1.0-1.5mg/ml).Consult as Brewer etc., Biochem.Biophys.Res.Commun. the 80th volume: 623-630 page or leaf (1978).The theoretical molecular of apolipoprotein A-1 precursor is 31KDa.See NCBI nonredundancy Protein Data Bank accession number 4557321.

In one embodiment, the apolipoprotein A-1 precursor is lower than reference levels in the Parkinsonian.The alpha1-antitrypsin precursor is the precursor protein of alpha1 Anti-trypsin, and the latter is the abundantest protease inhibitors of content in the blood, and is the member of SERPIN family protein enzyme inhibitor.Consult as Forsyth etc., Genomics. the 81st volume, the 3rd phase: 336-345 page or leaf (2003).The theoretical molecular of alpha1-antitrypsin precursor is 47KDa.See NCBI nonredundancy Protein Data Bank accession number 1703025.In one embodiment, the alpha1-antitrypsin precursor is lower than reference levels in PD patient.

TTR is synthetic in the choroid plexus of liver and brain.Consult as Schreiber etc., Comp.Biochem.Phys. the 105th volume, the 2nd phase: 317-325 page or leaf (1993).It participates in the serum transhipment of thyroxine and blood plasma RBP ELISA.Consult as Zheng etc., Toxicol.Sciences. the 61st volume: 107-114 page or leaf (2001).In CSF, TTR is main TBP.Consult as Hagen etc., J.Clin.Endocrinol.Metab. the 37th volume: 415-422 page or leaf (1973).TTR is the homotetramer albumen of molecular weight 55KDa, and wherein each monomer all comprises by hydrogen bond and forms dimeric two βZhe Die sheets.See document Soprano etc., J.Biol.Chem. the 260th volume: 11793-11798 page or leaf (1985).The TTR dimer is preponderated in many biological samples, and is not destroyed under the sex change condition.Consult as Sanchez etc., Electrophoresis the 16th volume: 1131-1151 page or leaf (1995).As previously mentioned, TTR can exist with monomer, dimer in specimen or the two all has.The estimated molecular weight of monomer TTR is about 14KDa, and the TTR estimated molecular weight of dimeric forms is about 28-36KDa.In one embodiment, compare with reference levels, can have the decline of dimer TTR amount or the raising of monomer TTR amount in PD patient's the sample, perhaps two kinds of situations occur simultaneously.In another embodiment, compare with reference levels, serum T TR dimer/monomer ratio also may be lower in PD patient.Therefore, PD diagnoses and the early stage biomarker of morbidity than can be used as to TTR dimer/monomer.

Mass discrepancy between the protein shown in this article can detect by multiple Measurement for Biochemistry, includes but not limited to as rapidly and efficiently reverse phase liquid chromatography (HPLC) method, mass spectroscopy, hydrophobic chromatography, DEAE cellulose, Bio-Gel P-60 and DEAE-Sephadex ^TMContinuous chromatography on the post and/or gel electrophoresis (including but not limited to as SDS-PAGE or two dimensional gel electrophore-sis) utilize commercialization antibody to detect thereafter as required.Be used for by the antibody that any immunoassays described herein detect transthyretin be can buy (Santa CruzBiotechnology, Santa Cruz, CA).See catalog number (Cat.No.) sc-13098 (called after prealbumin (FL-147), anti-rabbit igg).Experimenter's sample and reference levels will be measured any independent biomarker or any possibility that may make up is taken into account thereby the techniques described herein also will compare.

II. the method for selecting therapeutic scheme and being used to screen useful compound

Method as herein described can be used for prognosis, diagnosis, classification, treatment and selection and/or the filler test compound of PD.In some embodiments, these methods can be used to detect the progress of PD, select effective particular treatment, and/or screening has the compound of treatment or preventive effect to the treatment of PD and prevention.Therefore, method described herein can be used to determine whether PD exists after adopting particular treatment and/or compound.Described method also can be used for monitoring the reproduction of PD treatment and biomarker or PD symptom, or is used for determining in advance whether multiple treatment and/or compound be effective.For this reason, embodiments more of the present invention provide and have been used for determining whether the experimenter has the PD onset risk, select experimenter, the detection clinical effectiveness of clinical trial and divide experimenter's prognosis or predict and measure.

In one embodiment, mensuration can be used for prognosis or prediction purpose, thereby determines whether the experimenter has the Parkinsonian risk of generation, and randomly before experimenter's morbidity it is implemented prophylactic treatment subsequently.In addition, prognosis as herein described is measured can be used for determining whether the experimenter can be used as the possible candidate of clinical testing, can determine wherein whether the Parkinson's treatment of being used is effective.For example, these methods can be used for determining whether the experimenter can effectively treat the experimenter with the compound that influences haptoglobin, transferrin, apolipoprotein A-1 precursor and alpha1-antitrypsin precursor protein and/or TTR and/or its any variant.Therefore, the invention provides and be used for determining whether the experimenter can use the method for effectively treating at the compound of PD, wherein obtain specimen and biomarker as herein described is compared (for example, wherein the rising of experimenter's biomarker expression or reduction are to use described compound to prevent and/or treat Parkinsonian diagnosis basis) with reference levels.Therefore, the experimenter can classify according to diagnostic assay result as herein described before clinical testing.To this, the compound that one embodiment of the invention provide can be used for effectively treating Parkinsonian screening, and wherein experimenter classification can obtain medical treatment/reappraising behind the clinical test results.

In order to derive, need obtain the clinical response data of the population of subjects (being clinical colony) of receiving treatment to the clinical response of treatment and the correlativity between the particular organisms marker levels.Can obtain these clinical datas by retrospective analysis to clinical test results.In one embodiment, can and implement one or more new clinical testings by design and obtain described clinical data.Clinical population data analysis can be used for defining canonical reference colony, and then can be used for to participate in clinical testing and to select therapeutic scheme is that purpose is classified the experimenter.In one embodiment, the experimenter in the clinical colony is carried out classification at Parkinsonian existence.Classification to potential experimenter includes but are not limited to: Hoehn ﹠amp for example; Yahr stage method or the analysis of UDPRS score also determine that subsequently clinical colonial organism marker expression level raises or reduction with respect to control population.In one embodiment, the similarity according to one or more observed in the measurement level of the one or more biomarkers of experimenter and canonical reference colony biomarker levels can or be assigned as specific group or class with its division.

In another embodiment, the therapeutic interest scheme is put on each experimenter in the experimental population, by the reaction (i.e. Zhi Liao effectiveness) of one or more each experimenter of predetermined canonical measure to treatment.In many cases, the expection trial flock is known from experience and to be shown a series of response, the number (for example high, medium and low) that the respondent that the researchist will select to be made up of multiple response divides into groups.In addition, the expression of biomarker (as TTR monomer or the dimer that reduces or improve) is carried out quantitatively, this can be in enforcement before or after the treatment.Next these results are analyzed to determine whether that observed any clinical response (being curative effect) change is that statistics is significant between the group.Available statistical analysis technique is at L.D.Fisher ﹠amp; G.vanBelle has description among the Biostatistics:A Methodology for theHealth Sciences (Wiley-Interscience, NY, 1993).

Those skilled in the art can set up mathematical model with clinical response or the Parkinson's classification of prediction as biomarker expression function in the above-mentioned analysis.The evaluation of correlativity can be used as the basis of design diagnostic method between clinical response and the biomarker expression, described diagnostic method is in order to determine whether the experimenter has response to treatment, perhaps may need more treatment thus, promptly more heavy dose of compound, medicine or treatment with the reduced levels response.Diagnostic method can be taked one of various ways, for example ELISA or carry out mass spectrophotometry thereafter based on the test of antibody, serology test, the test of western trace, IP test or two dimensional gel electrophore-sis.Only requirement is to have correlativity between the result of diagnostic test and its PD behind.In one embodiment, this diagnosis utilizes the western trace to determine whether to exist among the experimenter rising or the dimeric reduction of serum T TR of aforesaid serum T TR monomer.In another embodiment, this diagnosis utilizes the western trace to determine that the experimenter compares the level difference that whether has above-mentioned haptoglobin, transferrin, apolipoprotein A-1 precursor and/or alpha1-antitrypsin precursor protein and/or its any posttranslational modification variant with reference levels.

In one embodiment, mensuration described herein can be used for determining whether and can treat or prevent PD to experimenter's administered compound or other treatment scheme that described compound includes but not limited to as activator, antagonist, plan peptide, polypeptide, peptide, nucleic acid, micromolecule or other drug candidate.

In another embodiment, goal treatment or prophylactic treatment can be selected from following: albumen, polypeptide, peptide, nucleic acid, virus, virus-like particle amino acid, amino acid analogue, modified amino acid, modified amino acid analogue, steroids, proteoglycan, lipid and carbohydrates, or its combination (for example: the therapy that comprises albumen and DNA component, or therapy combination, wherein one or more compositions are converted into activity form with other composition, for example catalysis).

In another embodiment, therapeutic or prophylactic treatment also can comprise nucleic acid, it includes but not limited to: for example, oligonucleotides or modified oligonucleotide, antisense oligonucleotides or modified antisense oligonucleotides, aptamers, cDNA, genomic DNA, artificial or natural dyeing body (for example, yeast artificial chromosome) or its part, RNA (comprising siRNA, shRNA, mRNA, tRNA, rRNA or ribozyme); Peptide nucleic acid (peptide nuleic acid, PNA); Virus or virus-like particle; Nucleotide or ribonucleotide or its synthetic analogues, they can be to modify or unmodified.Described treatment can also be amino acid or its analog, and it can be the hormone or non-peptide class (as the cholesterol) hormone of modification or unmodified; Proteoglycans; Lipid or carbohydrates.

In one embodiment, can also use micromolecule, it comprises inorganic and organic substance.In another embodiment, described micromolecule is a pharmaceutically active agents.Available pharmaceutically active agents kind includes but are not limited to microbiotic, anti-inflammatory drug, angiogenesis or vasoactive agent, growth factor and chemotherapeutics.

In another embodiment, interested therapeutic or prophylactic treatment can be selected from PD medicine levodopa, COMT inhibitor, Entacapone, Tolcapone, dopamine agonist, bromocriptine, Pramipexole, Ropinirole, apomorphine, amantadine, anticholinergic agents, benztropine mesylate, third ring pyridine, Biperiden and/or the selegiline.

In another embodiment, in fact interested therapeutic or preventative therapy can be operations, its program can be selected from pallidotomy, thalamotomy, thalamus stimulation, neural graft and/or deep brain stimulation (deep brain stimulation, DBS).

In one embodiment, the classification of the expression of one or more biomarkers and PD has correlativity.Can monitor the development of PD progressively by the patient being classified based on the Hoehn that is divided into five different phases and Yahr stage method.Each stage has all been described symptom and the clinical manifestation of PD.These stages are made up of following Pyatyi: (I) only symptom occurs at a side body; (II) symptom all occurs in the body both sides, wherein do not have disequilibrium; (III) with light disequilibrium to the moderate disease association, wherein the patient still can live on one's own life; (IV) handicap appears in the patient, but the patient still can independently walk or stand; (V) unless get help, the patient can only wheelchair or bed.Consult Hoehn and Yahr etc., Parkinsonism:onset, progression andmortality.Neurology. the 17th volume: 427-442 page or leaf (1967).In another embodiment, can be according to unified Parkinson's marking scales (Unified Parkinson ' s Disease RatingScale, UPDRS) to the monitoring of carrying out property of PD, this realizes by based on the clinical manifestation that is divided into four separate phases the patient being classified.Each stage is used 5 fens standards estimating the PD clinical manifestation.Stage is by forming with following relevant symptom: (I) spirit, behavior and mood, wherein Mental retardation, the disturbance of thought, enterprising spirit/dynamic role and depression are estimated; (II) daily routines, wherein to speech, hydrostomia, swallow, write, incision of food, wear the clothes, clean, stand up on the bed, fall down, stiff, walk, tremble and do not feel like oneself and estimate; (III) action is checked, wherein to supination before speech, facial expression, static tremor, action tremor, stiff, finger kneading, hands movement, the hand-screw, leg dirigibility, from chair stand up, the stability and the body action of posture, gait, posture slowly estimate; (IV) Zhi Liao complication, wherein estimate following: dyskinetic duration, dyskinetic anergy, dyskinesia pain, morning dystonia; Predictable " pass " state (off), uncertain " pass " state (off), unexpected " pass " state (off), the duration of " pass " state (off); And apositia, feel sick, vomiting, sleep disordered and symptomatic positional obstacle (Symptomatic orthostasis).Consult as Rascol etc., Treatment interventions for Parkinson ' s disease:an evidencebased assessment.Lancet. the 359th volume: 1589-1598 page or leaf (2002).Therefore, can there be correlativity in the expression of biomarker disclosed herein with Hoehn and Yahr and/or UPDRS stage division, and selection, monitoring and/or the screening of prognosis, diagnosis, methods of treatment that is used for PD is to the treatment compound.

In one embodiment, first time point determining from experimenter's specimen in the expression of biomarker (as monomer or dimer TTR), and it is compared with the specimen that derives from experimenter level of biomarker when the time point thereafter.The specimen that derives from the experimenter can be compared in the level of the biomarker at second time point place with the specimen that derives from the experimenter in the raising or the reduction of first time point place biomarker expression, and wherein biomarker indicates the experimenter who needs the Parkinson's treatment in the difference of first time point and second time point place expression.Perhaps, biomarker indicates the experimenter that treatment responds to Parkinson's in the difference of first time point and second time point place expression.The level of biomarker can be further and Hoehn ﹠amp among the experimenter; Yahr and/or UPDRS classification are associated.May need further test to make positive diagnosis.

In another embodiment, the present invention also comprises the method for monitoring Parkinsonian progress, it is the therapeutic scheme and/or the purpose of screening useful compound by choice, wherein measure in the specimen derive from the experimenter biomarker at the expression (for example rising or the reduction of monomer or dimer TTR) of first time point, and with its with the specimen that derives from the experimenter in biomarker compare in the level at a back time point place.Derive from biomarker in experimenter's the specimen in the raising of first time point place expression or reduce and can compare in the level at second time point place with biomarker in the specimen that derives from the experimenter, wherein biomarker indicates effective Parkinson's treatment or compound in the raising (for example raising of the reduction of monomer TTR and/or dimer TTR) of the expression at first time point and second time point place.Those skilled in the art understand easily, use all biomarkers as herein described (being haptoglobin, transferrin, apolipoprotein A-1 precursor, alpha1-antitrypsin precursor protein, TTR or its variant, the perhaps variant of its any posttranslational modification) all can use to be used to the method for selecting therapeutic scheme and/or screening useful compound.Have several different methods can be used to above-mentioned Parkinsonian diagnostic or prognostic evaluation, comprise TTR and all biomarkers as herein described, and whether the experimenter has the discriminating/treatment of easy trouble physique to these diseases.

In one embodiment, the standard control level of the variant of haptoglobin, transferrin, apolipoprotein A-1 precursor, alpha1-antitrypsin precursor protein, TTR or its variant or its any posttranslational modification can be determined by detecting in different control groups.Then reference levels are compared with the measurement level of given experimenter's as herein described biomarker.According to the similarity that the measurement level is compared with given group of control level, the experimenter can be divided or be assigned as specific group.

Skilled person in the art will appreciate that making this mensuration has in a way uncertainty.Therefore, the standard deviation of control group expression can be used to make probabilistic to be judged, the method for this paper all is suitable in the judging based on the genotype of probability or phenotype group of wide region.Therefore, for example, but be not limited only to, in one embodiment, if the measurement level of biomarker falls in 2.5 times of standard deviations of any control group mean value, then the experimenter can be assigned in this group.In another embodiment, if the measurement level of biomarker falls in 2.0 times of standard deviations of any control group mean value, then the experimenter can be assigned in this group.In another embodiment, if the measurement level of biomarker falls in 1.5 times of standard deviations of any control group mean value, then the experimenter can be assigned in this group.In another embodiment, if the measurement level of biomarker falls in 1.0 times of standard deviations of any control group mean value, then the experimenter can be assigned in this group.Therefore, this process allows to make a determination under multiple degree of probability, particular subject is dispensed in the group that be in, and this distribution then can be determined to classification of risks the position that individuality should be in.

Be used to diagnose the kit of PD

On the other hand, this paper be provided for that PD to the experimenter diagnoses, the kit of prognosis or monitoring.Such detection kit also can be used for the experimenter is classified, to select in clinical testing.Especially, the present invention includes and be used for the kit whether test sample exists the corresponding polypeptide of biomarker of the present invention.For example, this detection kit can comprise the compound or the reagent of mark, and the means that are used for the amount of working sample polypeptide, described compound or reagent can test sample in the corresponding polypeptide of biomarker of the present invention.

In one embodiment, method of the present invention can utilize the diagnostic kit of pre-packing to carry out, and comprises the necessary reagent of implementing any the inventive method in this kit.For example, this kit can comprise at least a specific antibody at biomarker described herein, described specific antibody can be advantageously used in as under the clinical setting, and in order to examination and diagnosis patient, and examination and evaluation show the experimenter who PD is had neurological susceptibility.This kit also can comprise enzyme and the damping fluid that can be used in the inventive method, and the electrophoresis mark, and it includes but not limited to as molecular weight marker.This kit can comprise the instructions about reagent uses and the result explains.

In another embodiment, the invention provides kit, it includes at least a biomarker that is packaged in one or more tubules in contrast.This kit also can contain other component, includes but not limited to as being packaged in the hybridization buffer (antibody of biomarker is therein as probe) in the autonomous container.With regard to regard to the kit of antibody, kit can comprise as (1) first antibody, as is bonded to the first antibody on the solid support, and it combines with the corresponding polypeptide of mark of the present invention; (2) different second antibody, it can combine with described polypeptide or described first antibody, and is conjugated with detectable label.This kit also can comprise as buffering agent, antiseptic or protein stabilizing agent.This kit can also comprise the required component of the described detectable label of detection, as enzyme or substrate.This kit also can comprise control sample or a series of control sample, can measure and compare with specimen it.Each component in the kit can be loaded on independently in the container, and all containers can be in instructions be included in individual packaging, and described instructions is used to explain the result who uses this kit to experimentize.

Embodiment

The following examples have been carried out further instruction to the present invention, and these embodiment should not be interpreted as any type of restriction.

Material and method

Foreword and sample colony.The purpose of embodiment described herein is a proteomic map of identifying associated biomolecule mark of the present invention in order to describe.Blood serum sample changes to identify the protein group that promotes PD or can be used as the PD diagnosis marker from 12 the special fully property sent out PD patients.All PD patients belong to Hoehn and Yahr I level or II level, and the mean age is 58.3 ± 7.9 years old, and the range of age is 43-68 year (table 1).Before collecting sample, PD patient does not accept any treatment of PD in advance.Control sample is from the healthy individual of 12 sexes and age-matched, and its mean age is 58.5 ± 8.2 years old, and the range of age is 44-68 year.Control population and PD patient have all accepted the synthetic medicine evaluation, and it comprises, and medical history is looked back, neurological status is reported and lab investigation.Laboratory result shows that total serum protein, blood urea nitrogen (BUN), kreatinin, lipid and blood sugar concentration are all within NL.All blood serum sample is all collected in BeiJing, China's Xuanwu Hospital of Capital University of Medical Science neurology department.

The preparation blood serum sample.Obtain the blood sample of PD patient and control population by venipuncture.In incubated at room after 60 minutes, sample in 4 ℃ with 3,000 * g centrifugal 10 minutes.Collect supernatant subsequently and be kept at-80 ℃.Supernatant and chloroform are in 1: 1 ratio (volume/volume), to extract and to remove the lipid pollutant.The fraction that does not contain lipid is applied to rProtein A Sepharose, and (NJ) post is collected and is handled twice again for GEHealthcare, Piscataway.The effluent (flow though) that to remove immunoglobulin (Ig) (Ig) is applied to Cibachron blue, and (MO) post is collected also and is handled twice again to remove albumin for Sigma, St.Louis.Protein subsequently under-20 ℃ with the sample of 1: 4 ratio (volume/volume) and acetone precipitation 12 hours, being deposited under the room temperature of obtaining is air-dry.It is resuspended with the following damping fluid of 450 microlitres to comprise 1 milligram of protein example: 7M urea, 2M thiocarbamide, 20mM Tris-HCl (pH7.5), 0.5%CHAPS, 0.5% (volume/volume) IPG solution (pH3-10), 6%2,2 '-dithiothreitol (DTT) and trace bromophenol blue.(Pierce, Rockford IL) measure serum proteins concentration to utilize BCA protein determination kit.

Statistical analysis.Utilize the Mann-Whitney check that the value of two dimensional gel electrophore-sis gained protein spots is analyzed.Same, use the Mann-Whitney check that has standard deviation to compare the relative band intensity of western trace.All statistics are measured the SPSS software of all using version 13.0, and (Chicago IL) analyzes for SPSS, Inc..For all assays, if the p value less than 0.05, is then thought significantly.

Embodiment 1-utilizes two dimensional gel electrophore-sis isolated protein and identification of protein variant/isoform

Utilize two dimensional gel electrophore-sis serum analysis protein example.Separation on first direction is undertaken by 1 milligram of protein example is added in 24 centimetres of IPG adhesive tape, wherein uses the non-linear pH gradient of 3-7.(NJ), under 20 ℃, by being forced into 130,000Vh came protein isolate quality sample: 40V 10 hours under the following conditions for GE Healthcare, Piscataway to utilize IPGphor equipment; 100V 20 hours; 200V 2 hours; 500V 2 hours; 1,000V 2 hours; 8,000V gradient 2 hours; 8,000 volts 12.5 hours.Subsequently the IPG adhesive tape was hatched 15 minutes in level pad (50mM Tris-HCl, 6M urea, 30% (volume/volume) glycerine, 2% (weight/volume) SDS, trace bromophenol blue, 1% (weight/volume) dithiothreitol (DTT)).The IPG adhesive tape was hatched 15 minutes at the level pad that contains 2.5% (weight/volume) iodoacetamide (IAA) again.After the isoelectric focusing, (Piscataway NJ) further makes sample separation for EttanDALT II system, GE Healthcare with 5W/ glue to re-use 12%SDS-PAGE.Utilize Coomassie brilliant blue G-250 (CBB G250) with gel-colored 12 hours subsequently, with ImageScanner II technology (GE Healthcare, Piscataway, NJ) imaging.The volume of protein spots is carried out standardization at total some volume of glue, use thereafter ImageMaster 2D Elite software (GE Healthcare, Piscataway, NJ) quantitative.Shown in Figure 1A, about 430 protein spots have been detected.Figure 1A also shows, when PD patient's proteinogram and contrast were compared, the abundance difference that 12 protein spots are arranged was greater than 1.2 times.Further characterize the protein spots of these 12 differential expressions by MALDI-TOF-MS and ESI-LC-MS/MS.

Protein spots is downcut from two-way gel, then in (Nutley NJ) digests for 12.5ng/mL, Roche with trypsase in ammonium bicarbonate buffers (50mMol/L) under 37 ℃.According to manufacturer's instructions, use ZipTip C18 (Millipore, Billerica, MA) pipettor tip purified peptide.Subsequently, peptide is applied to on the saturated alpha-cyano-4-hydroxycinnamic acid matrix of 5% trifluoroacetic acid and 50% acetonitrile.(Applied Biosystems, Foster City CA) obtain the peptide spectrum to utilize Voyager DEPRO MALDI-TOF mass spectrometer.(Matrix Science, Boston MA) retrieve the data that obtain in NCBI nonredundancy Protein Data Bank to utilize the Mascot searching algorithm.Retrieval limits and comprises: for single isotopic mass, the permission of quality testing is 100ppm; Maximum trypsase mistake is cut to once; Halfcystine is by the modification that methylates of carbamyl amine.Identify in order to carry out tandem mass spectrum, the peptide extract that vacuum is drained is resuspended in 20 microlitres, 0.1% formic acid and 2% acetonitrile, be applied to 180 μ m * 10cm BioBasic C18 post (Thermo Finnigan, Waltham, MA) on, and with the 10-30% acetonitrile linear gradient elution that contains 0.1% formic acid 30 minutes.The tandem mass spectrum of precursor peptide (precursor peptide) uses+2 ,+3 and+4 electriferous state (covering the mass-to-charge ratio of 400-1800) carries out.(MA) record is composed, and utilizes the SEQUEST algorithm to retrieve in NCBI nonredundancy Protein Data Bank for Thermo Finnigan, Waltham to utilize LCQ DECAXP PLUS.For one times/two times/three times charged peptides, SEQUEST cross correlation score (Xcorr) threshold value is respectively 1.9/2.2/3.75.In addition, the threshold value of Delta correlation (Δ Cn) is 0.1.Protein is identified by MALDI-TOF-MS, and is confirmed by LC-ESI-MS/MS.The result proves that 10 in the protein spots of 12 cutting-outs by the polypeptide fragment of being made up of haptoglobin, transferrin, apolipoprotein A-1 precursor, alpha1-antitrypsin precursor or TTR.The NCBI accession number of institute's identification of protein, sequence table, theoretical isoelectric point and molecular weight are listed in table 2

As shown in Fig. 1 .A, haptoglobin is with a 1-5 point representative, and wherein the 1-4 point demonstrates significantly improving of abundance.But No. 5 remarkable decline has appearred in point compared with the control in PD patient.In addition, transferrin (6 and No. 7 points) also significantly raises in PD patient.Yet remarkable decline has all appearred in apolipoprotein A-1 precursor (No. 8 points), alpha1-antitrypsin precursor (No. 9 points) and TTR (No. 10 points) in PD patient.Importantly, shown in Fig. 1 .B, TTR compares in PD patient according to having descended 2 times, and its relative abundance is respectively 0.011 ± 0.004 and 0.023 ± 0.007 (p＜0.05).Fig. 2 shows the tandem mass spectrum of TTR, wherein SEQUEST and b-and y-series ion coupling.Comprise as shown in Figure 2 ,+mass-to-charge ratio of the precursor peptide of 2 state of charge is determined as 697.93.The amino acid sequence of its associated precursors peptide is accredited as AADDTWEPFASGK (SEQ ID NO:1), corresponding to the 56-68 position residue of TTR.Therefore, to multiple proteins described herein with and the evaluation of relative abundance difference can be used for diagnosing in the method for PD.

The checking of embodiment 2-Western trace

In order to verify among the embodiment 1 result who obtains, the serum of PD patient and contrast is further carried out the Western engram analysis.12.5%SDS-PAGE separation of serum protein example (50 microgram), and transfer to pvdf membrane (Bio-Rad, Hercules, CA) on.Film is sealed, and with the TTR polyclonal antibody (1: 5000, Santa Cruz Biotechnology, Santa Cruz is CA) 4 ℃ of following overnight incubation.(CA) with 1: 10,000 ratio adds for Santa CruzBiotechnology, Santa Cruz will to resist rabbit igg-HRP second antibody.Calculate the relative intensity of band with QuantityOne software (Bio-Rad, Hercules, CA, version 4.6.3).As shown in Figure 3A, in the serum proteins (50 milligrams) of PD patient's (1-3 swimming lane) or contrast (4-6 swimming lane), detect the situation that exists of TTR.As positive control, in the swimming lane that the people TTR adding of 50ng purifying is independent (+).Molecular weight marker marks with corresponding KDa quality.Shown in Fig. 3 B, detected two bands that molecular weight is 36KDa and 14KDa, they correspond respectively to the estimated molecular weight of dimer and monomeric form TTR.

Shown in Fig. 3 B, detect among the PD patients serum and significantly be lower than control group (4.5 ± 1.7, p＜0.05) through standardized dimer band intensity (3.2 ± 1.5).Correspondingly, the intensity (10.5 ± 2.7) of the monomer band among the PD patients serum is significantly higher than contrast (8.9 ± 2.2, p＜0.05).Consistent with the result among the embodiment 1, the western engram analysis shows, has occurred the dimer TTR level that reduces among the PD patients serum, and/or the monomer TTR level (value of p shown in the asterisk is less than 0.05) that improves.It is therefore, described herein that multi-form detection can be used for diagnosing in the method for PD to TTR.

Equivalent

The present invention is not limited only to the specific embodiments described in the application.It is obvious to the skilled person that and to carry out many modifications and change, and do not depart from its design and scope.In conjunction with description above, the method and apparatus of the functional equivalent in the scope of the invention except that this paper is cited will be clearly to those skilled in the art.These modifications and change are intended to fall in the scope of claims with the full breadth of the described equivalent of claims.The present invention only is subject to the full breadth of equivalent described in appending claims and these claims.Should be appreciated that the present invention is not subject to concrete method, reagent, compound, composition or living things system, yes can change for they.It is also understood that term used herein only is used to describe specific embodiment, and be not intended to restriction.

In addition, when formal description feature of organizing with Ma Kushi of the present invention or aspect, those skilled in the art can understand that any individual member of this Ma Kushi group or the description of member's subgroup also contained in this paper.

Just as what it will be appreciated by those skilled in the art that, for any and whole purpose, particularly with regard to written description is provided, all scopes disclosed herein also comprise any and all possible among a small circle and combination among a small circle.Any scope of listing can be thought same scope is divided into equal two parts at least, three parts, four parts, five parts, ten parts etc. and is described at an easy rate.As non-limiting instance, each scope that this paper discussed can easily be divided into first three/one, middle 1/3rd and back three/first-class.Those skilled in the art also will appreciate that, word include but not limited to " as many as ", " at least ", " more than ", " being less than " etc. comprise the numerical value of being mentioned, and refer to be divided into as mentioned above scope among a small circle.At last, skilled person in the art will appreciate that scope comprises each individual member.Therefore, for example, the group that contains 1-3 cell is meant the group that contains 1,2 or 3 cell.Similarly, the group that contains 1-5 cell is meant the group that contains 1,2,3,4 or 5 cell, and the rest may be inferred.

Although herein disclosed is many aspects and embodiment, other aspects and embodiment also are conspicuous to those skilled in the art.Many aspects disclosed herein and embodiment only are used for illustration purpose and unrestricted, and real scope and design are described by following claim.

Claims (19)

1. one or more detectable of at least a biomarker are used to prepare the purposes of Parkinsonian diagnosis or monitoring reagent box, and wherein said at least a biomarker is selected from haptoglobin, transferrin, apolipoprotein A-1 precursor, alpha1-antitrypsin precursor and transthyretin.

2. the purposes of claim 1, wherein said kit is used to measure the level from least a biomarker described in experimenter's the sample, and compare with reference levels, wherein the difference of the level of at least a biomarker described in the sample and reference levels indicates this experimenter to suffer from Parkinson's, perhaps indicates the Parkinsonian stage.

3. the purposes of claim 2, wherein said reference levels are levels of not suffering from least a biomarker described in Parkinsonian experimenter's control population.

4. the purposes of claim 2, wherein said reference levels are levels of at least a biomarker described in this experimenter in early time.

5. the purposes of claim 2, wherein said reference levels are levels of this experimenter described at least a biomarker before the scheme of receiving treatment.

6. each purposes among the claim 2-5, wherein said sample is selected from blood plasma, serum, whole blood and cerebrospinal fluid.

7. each purposes among the claim 2-6, wherein said mensuration comprises the amount of the polypeptide of at least a biomarker described in the measuring samples.

8. each purposes among the claim 2-6, wherein said mensuration comprises the amount of the posttranslational modification polypeptide of at least a biomarker described in the measuring samples.

9. each purposes among the claim 2-6, wherein said mensuration comprise the amount of the described at least a biomarker of monomer in the measuring samples or polymer form, perhaps these two kinds of forms are all measured.

10. each purposes among the claim 1-9, wherein said at least a biomarker is a transthyretin.

11. the purposes of claim 10, the level of at least a biomarker of wherein said mensuration comprise the relative quantity of transthyretin monomer in the measuring samples or dimeric forms.

12. the purposes of claim 11, wherein the level of monomeric form transthyretin is compared with reference levels and is improved, and indicates this experimenter to suffer from Parkinson's, perhaps indicates the Parkinsonian stage.

13. the purposes of claim 11, wherein the level of dimeric forms transthyretin is compared with reference levels and is reduced, and indicates this experimenter to suffer from Parkinson's, perhaps indicates the Parkinsonian stage.

14. each purposes among the claim 2-13, wherein said mensuration is undertaken by immunoassays.

15. the purposes of claim 14, wherein said immunoassays are ELISA or western trace.

16. each purposes among the claim 1-13, wherein said determination step is realized by dielectrophoresis and mass spectrophotometry.

17. screening can be used for treating the method for Parkinsonian compound, it comprises

(a) measure the expression of at least a biomarker in the experimenter's used test compounds the sample, wherein said at least a biomarker is selected from haptoglobin, transferrin, apolipoprotein A-1 precursor, alpha1-antitrypsin precursor and transthyretin;

(b) expression and the reference expression level with described at least a biomarker compares;

(c) determine the effectiveness of this test compounds, the difference of the expression of wherein said at least a biomarker and reference expression level indicates this test compounds to can be used for treating Parkinson's.

18. the method for claim 17, wherein said reference expression level are the expressions of this experimenter described at least a biomarker before accepting this test compounds.

19. be used to diagnose or monitor experimenter's Parkinsonian kit, it comprises:

(a) be used for measuring one or more detectable of level of this experimenter's at least a biomarker of sample, wherein said at least a biomarker is selected from haptoglobin, transferrin, apolipoprotein A-1 precursor, alpha1-antitrypsin precursor and transthyretin;

(b) from a at least control sample of not suffering from Parkinsonian experimenter's control population; With

(c) be used to explain the explanation of using the result that described one or more detectable measure.

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