CN101196526A - Mass spectrometry reagent kit and method for rapid tuberculosis diagnosis - Google Patents
Mass spectrometry reagent kit and method for rapid tuberculosis diagnosis Download PDFInfo
- Publication number
- CN101196526A CN101196526A CNA2006101610927A CN200610161092A CN101196526A CN 101196526 A CN101196526 A CN 101196526A CN A2006101610927 A CNA2006101610927 A CN A2006101610927A CN 200610161092 A CN200610161092 A CN 200610161092A CN 101196526 A CN101196526 A CN 101196526A
- Authority
- CN
- China
- Prior art keywords
- protein
- tuberculosis
- mass
- serum
- slurry
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention relates to a method for capturing the phthisic Proteome in biological samples by supporting the stroma with the bead and separating the bead from the samples by the magnetic separator without the centrifuged samples. Then conduct the protein fingerprinting by the analysis of mass spectrography. The reagent kit tests the sensibility and the specificity of the normal person with the tuberculosis by Double Blind Test, which are all 100percent, among which the deviant SAA A can be used as blood serum in vitro for testing the normal person, minor tuberculosis patient, recrudescent tuberculosis patient, severe tuberculosis patient and tuberculosis patient after effective treatment. The elevated deviant SAA A shows poor treatment effectiveness, severe tuberculosis and the recrudescent tuberculosis, thus being a protein fingerprinting group for early warning of the early detection on tuberculosis worsening and prediction on tuberculosis recrudescence. The method provided in the invention can be used as the reagent kit to distinguish the protein fingerprinting or mass spectrum mapping between the normal person and the tuberculosis patient. The method is correct, convenient and fast.
Description
Technical field
The present invention relates to the kit of protein analysis method in a kind of new tuberculosis biological sample.A kind of by removing to catch biological marker with the matrix of protein bound, and detect the tuberculosis biological marker with the mass spectrophotometry that quantitative control is arranged.The present invention relates to the protein detection field referred in this, is a kind of external Mass Spectrometer Method method of new Noninvasive.The present invention can be applied to the detection method or the kit of the tuberculosis biological marker combination in the body fluid that has broken away from human body.
Background technology
Along with the enforcement of the Human Genome Project with finish, scientists has proposed the notion of back genome (post-genome) plan, and the research main points are transferred on the functional genomics, and biological function mainly to embody material be protein.1994, the Wilkins of Australia Macquarie university and the notion that Williams at first proposes protein group (proteome), refer to " all protein that a kind of genome is expressed ", promptly comprise a kind of cell and even the expressed all protein of a kind of biology.Research for protein group is the core of functional genomics research, is called proteomics (proteomics).Proteomics is considered to most important part in the genome research of back.Compare with genome, the composition of protein group is more complicated, and function is more active, and application prospect is more extensive.Proteomics is carried out the research of protein attribute from the cell integral level, as expression, posttranslational modification and interaction etc., and obtains extensive complete understanding for lysis, cell physiological biochemical character and regulated and control network thus.So proteomic techniques is progressively becoming the important research means of biology, medical science and pharmaceutics etc.
No matter the normal function or the pathology characteristic that are cell all are somewhat dependent upon the expressed protein function of cell.Therefore, the difference of the protein of surveyor's expression in vivo can be used for diagnosis of vitro disease sample and examination, and finally is used for drug development and disease treatment.And to carry out the differentiation analysis of protein expression and function, requirement can reach the degree of differentiating the complex mixture of molecule in the cell.But many materials often exist with trace in the cell, and the method that is used for analyzing proteins at present has limitation at above-mentioned everyway, is difficult to carry out chemical constitution and protein sequence identification and analysis with these conventional meanses.Can overcome this technical disadvantages with the mass spectrum associating.
Blood is because easy collection property and to the easy record of the physiological situation of body becomes one of best source of research biomarker.Protein fingerprint spectrum (mass spectrum) technology (MALDI-TOF-MS, SELDI-TOF-MS) is the laboratory diagnosis new technology that grew up in recent years, it and have that operation is easier, multisample detects, detect fast, sensitivity and specificity advantages of higher, is the progress of laboratory diagnosis technological revolution.The protein fingerprint spectral technology is mainly used in the early diagnosis of multiple disease, particularly disease in the application of medical domain.The protein fingerprint spectral technology is an extraordinary diagnostic techniques of development prospect, has broad clinical application prospect.Use the protein fingerprint spectral technology to become research comparison protein group and the available method of discovery biomarker, especially very useful in the research of polypeptide and low molecular weight protein mass spectrophotometry.But we are carrying out finding the standardized kit of the clinical tuberculosis ms diagnosis of neither one when proteomic image is analyzed.
Tuberculosis is the main respiratory system communicable disease that Much's bacillus causes, its etiological diagnosis mainly relies on phlegm nacterial smear and microbe growth, but the former positive rate is low, and the latter need last for 4～8 weeks again, length consuming time.The genetic quick diagnosis of Chu Xianing in recent years: tuberculosis polymerase chain reaction (PCR), but specificity is relatively poor.Higher because of tuberculosis polymerase chain reaction expense again, laboratory condition requires high, is difficult for extensively carrying out.Thereby urgent need is sought method of early diagnosis fast and effectively.
Summary of the invention:
The objective of the invention is to set up a kind of method that in the tuberculosis biological sample, detects.This method provides new approach for early detection lungy, and for finding that further new tuberculosis biological marker provides the foundation.
The present invention is intended to utilize mass-spectrometric technique, finds the marker protein kit and the method for tuberculosis morbidity early diagnosis as far as possible early, as much as possible, realizes early diagnosis, early warning and the effectively treatment of disease.Set up quality-control product kit and method that a cover is fit to mass spectrometry applications.Quality-control product is control test result's precision and an accuracy, makes the mass spectrum result have good repeatability.Obtain the tuberculosis marker protein by research, experiment and analysis, draw its special protein fingerprint spectrum.Research find some and tuberculosis patient prognosis that the protein of substantial connection is arranged and checked order, evaluation, functional analysis etc.
The present invention relates to a kind of stromal surface, and detect multiple biological marker state simultaneously with the quantitative property analysis of spectrum by the biological marker combination.
Biological marker among the present invention utilizes a mass spectrometer to find.The exactness high in quality of this equipment is about+and/-0.1%.
Matrix is any material that can combine with biological marker selectivity or specificity.Illustrate WCX negative ion, C8/C18 hydrophobic effect matrix adsorbent, these methods in the separating bio chemistry and have the purposes (being that anion adsorbent is caught cationic protein) of diagnosis reverse side by the adsorbent that these methods produce.The material that the base flush away does not adsorb.Any suitable washing lotion all can be used.
Biological marker at first can be had can catch with biomarker binding matrix absorption surface, and non-adsorbate can be from wash-out on the matrix, and the biomarker that is adsorbed onto base is detected in mass spectrometer.The source takes place by ion in biological marker, as laser, is ionized, and the ion of generation is experienced the collector collection by an ion, and mass analyzer is analyzed those ions that passes through then.Afterwards, detecting device is a mass-to-charge ratio with the ion information translation that detects.Quantitatively property control and mass spectrum laser energy regulation and control: before each test,, will be used for the maximal value that quantitative standards peak 6634.0Da intensity transfers to 50% mass signal intensity in the standardization quality controlled serum with mass spectral standardization quality controlled serum.The detection of biological marker is significantly with relevant with the detection of signal intensity.Like this, the quantity of biological marker and quality can be detected.
Flight mass spectrum generates time of flight spectrum to the analysis of analysans.The independent pulse signal that sample of ionization energy attack produces is not represented in the final analysis of this time of flight spectrum, but the signal sum of a series of pulses.Reduce interference like this, and increased dynamic range.These flight time data are subjected to the influence of data processing software.Data processing mainly comprises conversion flight time and mass-to-charge ratio and produces mass spectrum in the software, reduces baseline and reduces the side-play amount of instrument and filtration high frequency noise and alleviate high frequency noise.
Can utilize the DAP of computing machine to analyze by the data that absorption and detection to biological marker produce.These data of this computer program analysis to be showing the quantity of detected biological marker, and the intensity of shows signal and determine the molecular weight of each detected biological marker.Data analysis can also comprise the signal intensity of a series of definite biological marker and correct data departing from predetermined statistical distribution state.For example, by the height of calculating with each peak value of some parameter correlation, but the peak that standard observes.This parameter may be the unessential interference that is produced by chemical constitutions such as instrument and similar energy absorption molecules, and this can be provided with zeroing.
Computing machine can convert calculation result data to various forms and show.Its standard spectrum can represent, but has only peak height and quality information to keep in bands of a spectrum in one form, produces a figure more clearly, and makes and have easier the manifesting of biomarker of molecular weight much at one.In another form, two or more spectrums relatively are convenient to highlight the unique biological mark and are higher or lower than the biomarker of calibration sample with those.
Analysis generally comprises the evaluation at peak the collection of illustrative plates of the signal that displaying obtains from analysans.The peak can be selected by view, and software is available, and it is detected peaks automatically.Generally speaking, this software is tested and appraised signal and has signal to noise ratio (S/N ratio) and be higher than one and select threshold value and mark in the such mode of quality at the peak at the barycenter place of peak-to-peak signal to operate.In an effective program, more many spectral lines appear in the mass spectrum some same in a certain selected scope peaks with identification.A version of this software is assembled all peaks that appear at each the bar spectrum in definite mass range, near all peaks quality of appointment (mass-to-charge ratio) quality (mass-to-charge ratio) intermediate value bunch.
The biological marker that uses in the invention is that matrix is caught.These biomarkers are further to measure the identity that its different molecular weight knows that they are specific by mass spectrum (massspectrometry).
Detection to biological marker need be put a sample on the adsorption site of matrix, then cleans.Electrospray ionization mass spectrometry (electrospray ionizsation mass spectrometry, ESI-MS) be to apply a high voltage in exit capillaceous, the high electric field that is produced makes the liquid mist that flows out from kapillary change into tiny charged drop, along with solvent evaporation, the electric charge intensity on drop surface increases gradually, last drop disintegration causes analyte to enter gas phase with the form of single electric charge or multiple-charged ion in a large number with the ion of one or more electric charges.The ultimate principle of ground substance assistant laser parsing/ionization time of flight mass spectrometry (matrix-assisted laser desorption/ionization time of fight mass spectrometry.MALDI-TOF-MS) is to add SINAPINIC acid etc. and allow its drying on adsorption site, be dispersed in analyte in the molecule and form crystal, when with the laser radiation crystal, because substrate molecule is through energy that radiation absorbed, cause energy to be accumulated and heat production rapidly, thereby make the host crystal distillation, cause together with analyte and enter gas phase.Then, with mass spectroscopy matrix is analyzed, and a legacy figure who has shown protein molecule will generate, this figure is on the basis of the quality-charge ratio of protein molecule, shows with the form of the peak figure that is separated from each other.
Yet if necessary, these biological markers also can pass through, such as, determine that amino acid sequence of polypeptide differentiates.In protein chemistry and proteomics research, in order to increase the confidence level of identification of proteins, it is normally very important to obtain one section protein peptide fragment internal sequence information.For the sequencing of protein and polypeptide, traditional method is to adopt the Edman biodegrading process, and the weak point of this method maximum is time-consuming oversize (residue need spend 30-40 minute).In recent years, along with the develop rapidly of mass-spectrometric technique, the especially development of cracking technology such as (PSD) behind multi-stage ms (MS/MS) and the source, using the mass spectrum order-checking has become a kind of popular method.
For example, a biological marker can be depicted with many enzymes, for example V8 proteinase (V8 protease) or trypsase, and the molecular weight of digestion fragment (digestion fragments) can be used to search sequence in database, and these sequences match with the molecular weight of the digestion segment that is generated by plurality of enzymes.Perhaps, if this biological marker is not the protein molecule in the given data storehouse, on the basis of the N of biological marker utmost point amino acid sequence (N-terminal Amino Acid Sequence), can use the degraded probe, then, these probes can be used to describe by genome that sample generated that has detected biological marker or cDNA storehouse.At last, protein biological marker available protein scalariform ranking method (protein laddersequencing) sorts.By after molecule being broken into fragment and the method that fragment can be in order removed a single amino acids molecule from the fragment end with enzymolysis or other being handled, can generate protein gradient (protein ladders).Then, with mass spectrum this gradient is analyzed.Stepped fragment (ladder fragments) can identify the amino acid that is removed from molecular end in qualitative difference.Therefore, the present invention can be used for the goldstandard that biological marker is identified.
Specificity is meant the single-minded attribute of a certain material or certain disease, and it is a feature of representing certain material or certain disease.Can discern certain material or certain disease by some feature, thereby it and other materials or disease are made a distinction.Identification to proprietary feature often depends on special detection method, for example will understand certain disease and whether have specific antigen and will detect with relevant specific antibody.Since proteomics research had new development, specific detection and confining method had had very big breakthrough on this traditional sense.As a protein different fragments variation is the sign of dissimilar tumours.
The present invention utilizes the WCX anionic substrates and utilizes C8/C18 hydrophobic matrix magnetic bead for example normal person and tuberculosis serum (slurry) to be carried out the protein comparative analysis.Quantitatively property control and mass spectrum laser energy regulation and control: before each test,, will be used for the maximal value that quantitative standards peak 6634.0 Da intensity transfer to 50% mass signal intensity in the standardization quality controlled serum (slurry) with mass spectral standardization quality controlled serum (slurry).
A kind of concrete operations step with judging normal person and kit lungy and method:
Below be with an operation scheme provided by the invention and tuberculosis kit example.
1. sample preparation and standardization quality controlled serum (slurry) preparation
Biological sample is diluted in the dilution buffer, optionally the centrifugal clarification sample.
Mass spectral standardization quality controlled serum (slurry) preparation definition meets following standard: blood donor 10 people, and 5 male 5 woman, blood group is the O type; Age is 18～30 years old; The national Chinese.Biochemical indicator is normal, comprising: T-CHOL, triglyceride, fasting blood-glucose, hepatitis B surface antigen, liver power checking, kidney merit are checked; There is not hereditary patient and his family family history; No serious infectious diseases history.The women can not be conceived, and the male sex is the non-smoker.
2. sample on matrix and tuberculosis, standardization quality controlled serum (slurry) preparation, the sample
Extract the health volunteer's (men and women half and half) of O type blood new blood: (a) treat under 4 ℃ after the blood clotting at once centrifugal, 4 ℃ of centrifugal 5min separation of serum.(b) 4 ℃ of following horse backs are centrifugal, 4 ℃ of centrifugal 5min separated plasmas.Standardization Quality Control or tuberculosis serum (slurry) sample packing 100 μ l one tubule are in-80 ℃ of storages; Or get pooled serum (slurry) and be diluted in U9 damping fluid (9M Urea, 2%CHAPS, 50mM Tris-HCL, a pH9.0) tubule, 25 ℃ of storages at 1: 2.Promptly become tuberculosis, standardization quality controlled serum (slurry) kit.With mass spectral standardization quality controlled serum and sample point sample on the site in the matrix of holder is arranged.The holder magnetic beads.Matrix is used for mark, in conjunction with serum (slurry).The matrix that is tagged on the liquid chromatography pillar can go to analyze with the standard method of liquid chromatography mass combined instrument (LC-MS).The quantivative approach that mass spectral standardization Quality Control O serum (slurry) is used for the mass spectrometer kit.
3. washing
Wash with binding buffer liquid.Before the sample bone dry, first part of wash solution is added to this site.Wash solution stopped on the site 10 seconds at least.Thoroughly remove first part of wash solution, repeat above step with second part of cleansing solution.Following steps: thoroughly wash whole magnetic beads array point with 0.05%～1% trifluoroacetic acid, biological marker is eluted on the mass spectrum special-purpose metal sheet (3 * 3mm circular hole is arranged), the air dry sheet metal, add 0.5 μ L energy-absorbing molecule (with 50% acetonitrile, the saturated standard solution of 0.5% trifluoroacetic acid preparation).
The energy-absorbing molecule can be used Sinapinic acid or alpha-Cyano-4-hydroxycinnamic acid etc.
3. mass spectral quantitative control and test
With laser desorption/ionization time of-flight mass spectrometer, with nitrogen laser (337nm) and 80cm or 120cm tof tube analysis array, or with removing to analyze biological marker or the protein that is stranded in each site with liquid chromatography mass combined instrument (LC-MS) standard method behind the biological marker of electron spray ionisation wash-out.Carry out the overlapping displaying of data with the Computer Analysis data.
Quantitative property spectrum regulation and control: before each test,, will be used for the maximal value that quantitative standards peak 6634.0Da equal strength transfers to 50% signal intensity in the standardization quality controlled serum with mass spectral standardization quality controlled serum (slurry).Quantitatively property control and mass spectrum laser energy regulation and control: before each test,, will be used for the maximal value that quantitative standards peak 6634.0Da intensity transfers to 50% mass signal intensity in the standardization quality controlled serum (slurry) with mass spectral standardization quality controlled serum (slurry).With 2746 ± 1Da, 5909 ± 1Da, 6634 ± 1Da base peak is mass spectrometry (molecular weight) quality control standard.
The present invention has been divided into several big classes with protein, i.e. albumen such as WCX negative ion, C8/C18 hydrophobic effect.Like this, available mass spectrometer is directly analyzed and quantitatively.
By analyzing hundreds of kind haemocyanin fingerprint peaks, find that following protein fingerprint or mass-spectrogram can be used for distinguishing normal and serum lungy.Peak C V＞30% or p＜0.01 is for there being significant difference:
The significant difference of normal and tuberculosis protein fingerprint or mass-spectrogram (± 15Da)
2742.52 3933.74 5061.78 6086.21 6847.88 8341.08 11682.95 28832.85
2873.31 3957.01 5336.94 6107.49 6872.55 8555.57 13740.09 37001.86
2954.47 4070.17 5541.18 6125.63 6998.47 8603.79 14015.41 43986.14
3159.65 4091.76 5732.49 6186.17 7149.35 8682.71 14132.22 46489.01
3219.19 4156.47 5808.41 6297.79 7568.94 9415.01 15114.90
3322.63 4175.24 5813.37 6359.01 7657.06 9699.29 15312.81
3379.73 4282.36 5839.91 6429.38 7763.09 11079.4 23383.41
3396.85 4346.63 5881.15 6476.47 7926.96 11389.1 23956.23
3449.31 4648.05 5902.36 6627.35 8048.73 11439.2 24097.22
3886.39 4962.98 5945.43 6832.45 8137.19 11526.7 27993.79
With therefrom filtering out several characteristic protein 28 73.3 ± 15Da, 3379.7 ± 15Da, 3449.3 ± 15Da, 5808.4 ± 15Da, 7568.9 ± 15Da, 8048.7 ± 15Da, 11439.2 ± 15Da, 11526.7 ± 15Da, 11683.0 ± 15Da, 15114.9 ± 15Da, 15312.8 ± 15Da, double-blind 100 routine normal persons and 100 routine tuberculosis.The susceptibility 100% of kit, specificity 100% (embodiment 2).
Utilize the experimental result of C8 and C18 hydrophobic matrix magnetic bead consistent with the experimental result of above-mentioned WCX anionic substrates magnetic bead.
With one group of 11439.2 ± 15Da, 11526.7 ± 15Da, 11683.0 ± 15Da biological marker (Fig. 3) with multi-stage ms (MS/MS), source after cracking (PSD) and protein scalariform ranking method (protein ladder sequencing) sort.By molecule is broken into fragment, can generate protein gradient (protein ladders).Then, with mass spectrum this gradient is analyzed.Identify 11439.2 ± 15Da, 11526.7 ± 15Da, the serum amyloid A protein of 11683.0 ± 15Da biological marker for making a variation.Its chemical constitution is (hold to C end from N be arranged as 104 amino acid):
The chemical constitution of 11683 ± 15Da biological marker:
The N end
RSFFSFLGEAFDGARDMWRAYSDMREANYIGSDKYFHARGNYDAAKRGPG
GVWAAEAISDARENIQRFFGHGAEDSLADQAANEWGRSGKDPNHFRPAGL
PEKY
The C end.
The chemical constitution of 11527 ± 15Da biological marker:
The N end
SFFSFLGEAFDGARDMWRAYSDMREANYIGSDKYFHARGNYDAAKRGPG
GVWAAEAISDARENIQRFFGHGAEDSLADQAANEWGRSGKDPNHFRPAGL
PEKY
The C end.
The chemical constitution of 11439 ± 15Da biological marker:
The N end
FFSFLGEAFDGARDMWRAYSDMREANYIGSDKYFHARGNYDAAKRGPG
GVWAAEAISDARENIQRFFGHGAEDSLADQAANEWGRSGKDPNHFRPAGL
PEKY
The C end.
With the preparation of the serum amyloid A protein antibody of variation and from culture supernatant, extract required antibody.This antibody can be used for preparing the required kit of detection.
Find that with antiserum amyloid A antibody and mass spectrometer use in conjunction (embodiment 5) molecular weight and the embodiment 3 of analyte are consistent, then 11439.2 ± 15Da, 11526.7 ± 15Da, 11683.0 ± 15Da chemical constitution can be speculated as the serum amyloid A protein of variation.Be the associating of antibody and mass spectrum, can save the ordering of the serum amyloid A protein of variation and identify.The serum amyloid A protein (Fig. 3) of discovery variation can be used to distinguish normal person, slight tuberculosis patient, recur the serum after tuberculosis, severe tuberculosis patient, tuberculosis patient are effectively treated when antibody and mass spectrometer use in conjunction.Detection kit such as conventional ELISA can't detect the serum amyloid A protein of variation.
The present invention can be used for the quantitative control of the external tuberculosis Mass Spectrometer Method method of cell in vitro and Noninvasive, is used for the tuberculosis detection method of mass spectrometry clinical as the tuberculosis kit of the body fluid that exsomatizes.Can detect a plurality of tuberculosis protein biological indication marks groups and mass-spectrogram.
The external detection method of kit among the present invention and method and other Noninvasives relatively has following characteristics:
(1) accurate
Mass spectrum is directly analyzed very strong accuracy, and general error rate has only 0.1Da.Because protein is made up of amino acid, and amino acid whose average quality is known, if known the total molecular weight of antigen or biological marker, the variation of antigen so (referring to that amino acid changes) just is easy to be pushed and measures.As, the serum amyloid A protein of variation, but not common serum amyloid A protein can have the purposes of accurate discriminating normal person and slight tuberculosis patient, recurrence tuberculosis patient, severe tuberculosis patient and tuberculosis patient curative effect.Detection kit such as conventional ELISA can't detect the serum amyloid A protein of variation.
(2) convenient
This method has been divided into several big classes with protein, i.e. negative ion, hydrophobic effect albumen.Like this, available mass spectrometer is directly analyzed.The sorbent used holder of the method for magnetic bead supported matrix is magnetic particle or magnetic bead.Separate magnetic bead and sample with magnetic separator, need not centrifugal sample.Like this, available mass spectrometer is directly analyzed.
(3) quick
When carrying out medical diagnosis on disease, need not protein is checked order with protein fingerprint method provided by the invention.The present invention has adopted computing machine " barcode standard ", and signal intensity shows with the darkness intensity level along linear axes.This intensity can be analyzed the contrast power automatically with the scanner record and with analysis software, thereby helps the diagnosis of clinical complexity, carries out medical analysis as available this " protein fingerprint " or barcode standard.
Present national tuberculosis patient number is about 4,500,000, the row second place of the world.Wherein the infectiousness pulmonary tuberculosis patient is about 2,000,000, and annual death toll is about 250,000, ranks first in infectious disease, and surpassing hepatitis becomes the most serious infectious disease of China's harm.Tuberculosis is popular aggravates again in China, and four high one low situations do not change, and this disease might become incurable disease once more.Though the reason that causes China's tuberculosis to aggravate again is a lot, but, key issue is the incompatible needs of present employed tuberculosis examination, diagnostic techniques, they or positive rate are low, or length consuming time or expense are higher, or laboratory condition requires high reason, so that the tuberculosis patient can't in time find and treat.
This method is differentiated with WCX matrix, serum amyloid A protein antibody absorption surface matrix and mass spectroscopy and is detected the serum amyloid A protein of finding a kind of new form variation.The serum amyloid A protein of variation can be used for detecting normal person, slight tuberculosis patient, recurs the serum after tuberculosis, severe tuberculosis patient, tuberculosis patient are effectively treated.The serum amyloid A protein rising person of variation points out weak curative effect, severe tuberculosis and tuberculosis recurrence.So being an early detection tuberculosis, it worsens, predicts the early warning protein fingerprint group that the tuberculosis recurrence takes place.Its lasting negative expression can be pointed out tubercular's stable disease, and it is turned out cloudy through treatment from the positive and also can reach this purpose.Therefore, with it as a kind of clinical detection platform, with the prediction tuberculosis development, and the prompting corresponding treatment clinical meaning.By WCX matrix and by the serum amyloid A protein of the variation of catching on the antibody absorption surface matrix, detect with the quantitative property analysis of spectrum under the control of standardization quality controlled serum, can be applied to the detection method or the kit developing of the biological marker combination in the body fluid that has broken away from human body.A kit provided by the invention and method are used for Mass Spectrometer Method and quantitatively control.Thereby the tuberculosis vitro detection that helps clinical complexity.This method is accurate, convenient and fast.
Figure of description
Fig. 1 normal person and tuberculosis protein fingerprint spectrum and mass spectrum polypeptide spectrum
A characteristic peak of Fig. 2 normal person and tuberculosis protein fingerprint spectrum and mass-spectrogram
One stack features peak of Fig. 3 normal person and tuberculosis protein fingerprint spectrum and mass-spectrogram
Embodiment
The present invention will be described further in conjunction with specific embodiments, and these examples only are used for illustration purpose, and are not used in the restriction scope of the invention.
Embodiment 1 normal and differentiation lungy and the preparation of mass spectral kit
(1) experimental technique
One, material
The sample source: mass spectral standardization quality controlled serum (slurry) preparation definition meets following standard, blood donor men and women half and half, and blood group is 0 type; Age is 18～30 years old; Nationality, the Chinese.Biochemical indicator is normal, comprising: T-CHOL, triglyceride, fasting blood-glucose, hepatitis B surface antigen, liver power checking, kidney merit are checked; There is not hereditary patient and his family family history; No serious infectious diseases history.The women does not have pregnancy, and the male sex does not have smoking history person.Extract the health volunteer's (men and women half and half) of 10 0 type blood new blood, treat under 4 ℃ after the blood clotting at once centrifugal, 10,000rpm, 4 ℃ of centrifugal 5min; Sample is stored in-80 ℃.
Get pooled serum (slurry) 10 μ l, with 20 μ l U9 damping fluids (9M Urea, 2%CHAPS, 50mM Tris-HCL, pH9.0) dilution, then, with above sample ice bath vibration 30min.Sample after the above-mentioned sex change of 30 μ l is added the corresponding binding buffer liquid of 360 μ l, stand-by.The double of carrying out all sample standard deviations detects to reduce experimental error.
Brief operation step: with above-mentioned 200 μ l binding buffer liquid (50mM NaAC, pH 4.0～4.5) add to and install in the WCX negative ion magnetic bead tubule, put oscillator (MSl Minishaker) 400-600rpm, concussion 5min separates magnetic bead and sample with magnetic separator.Repeat twice of aforesaid operations.Thoroughly wash whole WCX negative ion magnetic bead array point with 0.05%～1% trifluoroacetic acid, biological marker is eluted on the mass spectrum special-purpose metal sheet (3 * 3mm circular hole is arranged), the air dry sheet metal adds 0.5 μ L energy-absorbing molecule (with 50% acetonitrile, the saturated standard solution of 0.5% trifluoroacetic acid preparation).Read with mass spectrophotometry then.Quantitatively property control and mass spectrum laser energy regulation and control: before each test,, will be used for the maximal value that quantitative standards peak 6634.0Da intensity transfers to 50% mass signal intensity in the standardization quality controlled serum (slurry) with mass spectral standardization quality controlled serum (slurry).
Data aggregation: before each experimental data is collected, rectify an instrument, make protein molecular weight error＜0.1% with All-in-one albumen.
Data analysis: use statistical method, the form of analyzing the gained data is with calculating machine-readable barcode standard of getting or peak, protein fingerprint is shown as the darkness intensity level signal along linear axes, and this intensity can be analyzed the contrast power automatically with the scanner record and with analysis software.All collection of illustrative plates have all carried out standardization, and are unified to they own whole population of ions (summation of peak area).To be used for the maximal value that quantitative standards peak 6634.0Da intensity transfers to 50% signal intensity in the standardization quality controlled serum, and proofread and correct, then carried out again in " minimizing baseline ", definition protein peak (s/n＞5) with the peak of 6634.0Da.Analyzed the protein peak between all 700～50000Da, and scrutiny each corresponding peak, calculate average, standard error (SD) and the coefficient of variation (CV%) at peak.By analyzing hundreds of kind haemocyanin fingerprint peaks, find that following protein fingerprint can be used for distinguishing normal and tuberculosis.Peak C V＞30% or p＜0.01 is for there being significant difference (Fig. 1,2, signal intensity shows with the darkness intensity level along linear axes):
The significant difference of normal and tuberculosis protein fingerprint or mass-spectrogram (± 15Da)
2742.52 3933.74 5061.78 6086.21 6847.88 8341.08 11682.95 28832.85
2873.31 3957.01 5336.94 6107.49 6872.55 8555.57 13740.09 37001.86
2954.47 4070.17 5541.18 6125.63 6998.47 8603.79 14015.41 43986.14
3159.65 4091.76 5732.49 6186.17 7149.35 8682.71 14132.22 46489.01
3219.19 4156.47 5808.41 6297.79 7568.94 9415.01 15114.90
3322.63 4175.24 5813.37 6359.01 7657.06 9699.29 15312.81
3379.73 4282.36 5839.91 6429.38 7763.09 11079.4 23383.41
3396.85 4346.63 5881.15 6476.47 7926.96 11389.1 23956.23
3449.31 4648.05 5902.36 6627.35 8048.73 11439.2 24097.22
3886.39 4962.98 5945.43 6832.45 8137.19 11526.7 27993.79
The double-blind of embodiment 2 kits
With from embodiment 1, filtering out several characteristic protein peak 2873.3 ± 15Da, 3379.7 ± 15Da, 3449.3 ± 15Da, 5808.4 ± 15Da, 7568.9 ± 15Da, 8048.7 ± 15Da, 11439.2 ± 15Da, 11526.7 ± 15Da, 11683.0 ± 15Da, 15114.9 ± 15Da, 15312.8 ± 15Da, the normal and 100 routine tuberculosis of double-blind 100 examples:
| Double-blind | Tuberculosis (100) | Normally (100) |
| Tuberculosis | 100 | 0 |
| Normally | 0 | 100 |
Susceptibility 100% specificity 100%
Utilize the experimental result of C8 and C18 hydrophobic matrix magnetic bead consistent with the experimental result of above-mentioned WCX anionic substrates magnetic bead.
Experiment conclusion
Kit with this method can be differentiated normal person and tuberculosis, susceptibility 100%, specificity 100%.
The ordering at embodiment 3 tuberculosis protein fingerprint spectrums one stack features peak is identified
With one group of 11439.2 ± 15Da, 11526.7 ± 15Da, 11683.0 ± 15Da biological marker (Fig. 3) with multi-stage ms (MS/MS), source after cracking (PSD) and protein scalariform ranking method (protein ladder sequencing) method such as sort.By molecule is broken into fragment, can generate protein gradient (protein ladders).Then, with mass spectrum this gradient is analyzed.Identify 11439.2 ± 15Da, 11526.7 ± 15Da, the serum amyloid A protein of 11683.0 ± 15Da biological marker for making a variation.Its chemical constitution is (hold to C end from N be arranged as 104 amino acid):
The chemical constitution of 11683 ± 15Da biological marker:
The N end
RSFFSFLGEAFDGARDMWRAYSDMREANYIGSDKYFHARGNYDAAKRGPG
GVWAAEAISDARENIQRFFGHGAEDSLADQAANEWGRSGKDPNHFRPAGL
PEKY
The C end.
The chemical constitution of 11527 ± 15Da biological marker:
The N end
SFFSFLGEAFDGARDMWRAYSDMREANYIGSDKYFHARGNYDAAKRGPG
GVWAAEAISDARENIQRFFGHGAEDSLADQAANEWGRSGKDPNHFRPAGL
PEKY
The C end.
The chemical constitution of 11439 ± 15Da biological marker:
The N end
FFSFLGEAFDGARDMWRAYSDMREANYIGSDKYFHARGNYDAAKRGPG
GVWAAEAISDARENIQRFFGHGAEDSLADQAANEWGRSGKDPNHFRPAGL
PEKY
The C end.
The preparation of the serum amyloid A protein antibody of embodiment 4 variations
Synthetic and the sequence of serum amyloid A protein:
The N end
RSFFSFLGEAFDGARDMWRAYSDMREANYIGSDKYFHARGNYDAAKRGPG
GVWAAEAISDARENIQRFFGHGAEDSLADQAANEWGRSGKDPNHFRPAGL
PEKY
The C end.
With the serum amyloid A protein immune mouse of synthetic variation, treat that immune response occurs after, from peripheral blood, separate the B cell.Select and isolate the single lymphocyte that to secrete required antibody with haemolysis plaque analytic approach.Individual cells is expanded to 1 * 10
7More than individual, extract mRNA with Quick mRNA Purification Kit.MRNA with extraction is a template, synthetic cDNA chain.With this cDNA is template, adds murine antibody variable region of heavy chain (V
H) universal primer, variable region of light chain (V
L) universal primer, carry out polymerase chain reaction, obtain the V of amplification
HGenetic fragment and V
LGenetic fragment.Amplified production is identified and separated at the 15g/L agarose gel electrophoresis.Reclaim the V of amplification with glass milk
HGenetic fragment and V
LGenetic fragment.With etc. the Limker Primer Mix that attempts of mole mixes, carry out polymerase chain reaction, connection V
HAnd V
LAfter the amplified production separation and purification, obtain specific single-chain antibody (ScFV).This ScFV can be used for preparation and detects required DNA sheet.These amplified production two ends are added restriction enzyme site, after purifying is quantitative, be connected to P
UC19On the carrier.To connect product and transform ehec infection TOP10.Cut evaluation through blue hickie screening and enzyme, filter out recombinant plasmid.Form monoclonal antibody at 96 orifice plates.Filter out the monoclonal antibody strain of tiring the highest with the ELISA method, a large amount of preparations, and from culture supernatant, extract required antibody.This antibody can be used for preparation and detects required kit.
Serum amyloid A protein molecular antibody and mass spectrum are identified in embodiment 5 blood
To have with Carbodiimide method (Carbodiimide Method) that matrix combines (Gunn DL, et al.Preparation ofsensitive and stable erythrocytes by the carbodi imide method for the detectionof primary and secondary IgM and IgG antibody.J Immunol Methods.1972 on the magnetic beads of hydroxy-acid group mark with the amino group of antiserum amyloid A antibody; 1 (4): 381-389.).
With the sample point sample on the site of an antiserum amyloid A antibody matrix.
Wash with binding buffer liquid.Before the sample bone dry, first part of wash solution is added to this site.Wash solution stopped on the site 10 seconds at least.Thoroughly remove first part of wash solution, repeat above step with second part of cleansing solution.Thoroughly wash whole array point with 1% trifluoroacetic acid, biological marker is eluted on the mass spectrum special-purpose metal sheet (3 * 3mm circular hole is arranged), the air dry sheet metal adds 0.5 μ L Sinapinic acid energy-absorbing molecule (with 50% acetonitrile, the standard solution of the preparation of 0.5% trifluoroacetic acid).
Use mass spectrometer, analyze biological marker or the protein that array goes to analyze each site with nitrogen laser (337nm) and 80cm or 120cm tof tube.Carry out the overlapping displaying of data with the Computer Analysis data.
Experimental result
Find that with antiserum amyloid A antibody matrix and mass spectrometer use in conjunction the molecular weight and the embodiment 3 of analyte are consistent, then 11439.2 ± 15Da, 11526.7 ± 15Da, 11683.0 ± 15Da chemical constitution can be speculated as the serum amyloid A protein of variation.The serum amyloid A protein of finding following variation can be used to distinguish normal person, slight tuberculosis patient, recur the serum after tuberculosis, severe tuberculosis patient, tuberculosis patient are effectively treated:
| Shaker test | The normal person | Slight tuberculosis, recurrence tuberculosis | Severe tuberculosis | After tuberculosis is effectively treated |
| The serum amyloid A protein of variation | (-) | (+) | (++) | (-) |
| Add up to (example) | 50 | 50 | 50 | 50 |
50 routine normal persons' serum: feminine gender
The serum of the slight tuberculosis of 50 examples, recurrence tuberculosis patient: the positive
The serum of 50 routine severe tuberculosis patients: strong positive
Serum after 50 routine tuberculosis patients are effectively treated: feminine gender
With embodiment 5 " serum amyloid A protein molecular antibody and mass spectrum are identified in the blood ", i.e. antibody and mass spectrum associating, can save the ordering of the serum amyloid A protein of variation and identify.
This method is differentiated with WCX matrix, serum amyloid A protein antibody absorption surface matrix and mass spectroscopy and is detected the serum amyloid A protein of finding a kind of new form variation.The serum amyloid A protein of variation can be used for detecting normal person, slight tuberculosis patient, recurs the serum after tuberculosis, severe tuberculosis patient, tuberculosis patient are effectively treated.The serum amyloid A protein rising person of variation points out weak curative effect, severe tuberculosis and tuberculosis recurrence.So being an early detection tuberculosis, it worsens, predicts the early warning protein fingerprint group that the tuberculosis recurrence takes place.Its lasting negative expression can be pointed out tubercular's stable disease, and it is turned out cloudy through treatment from the positive and also can reach this purpose.Therefore, with it as a kind of clinical detection platform, with the prediction tuberculosis development, and the prompting corresponding treatment clinical meaning.By WCX matrix and by the serum amyloid A protein of the variation of catching on the antibody absorption surface matrix, detect with the quantitative property analysis of spectrum under the control of standardization quality controlled serum, can be applied to the detection method or the kit developing of the biological marker combination in the body fluid that has broken away from human body.This method is accurate, convenient and fast.
All documents of mentioning in the present invention are incorporated by reference in this application all, is just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (8)
1. the method with magnetic bead supported matrix is caught the kit and the method for tuberculosis protein group in the biological sample, it is characterized in that adopting the protein fingerprint method that protein group in the tuberculosis or mass-spectrogram are differentiated detection.Detect with the quantitative property analysis of spectrum under standardization quality controlled serum (slurry) control.This method realizes by following steps:
(1) sample preparation and mass spectrum standardization quality controlled serum (slurry) preparation;
(2) sample on matrix and serum (slurry) binding reagents box preparation, the sample;
(3) washing;
(4) mass spectral quantitative control and Mass Spectrometer Method;
Wherein said step (1) is diluted in biological sample in the dilution buffer.With O type blood, the men and women equates, is mixed with mass spectral standardization quality controlled serum (slurry); Described step (2) is with mass spectral standardization quality controlled serum (slurry) and sample point sample on the site in the matrix of holder is arranged.The holder magnetic beads.Matrix is with WCX anionic substrates and C8/C18 hydrophobic matrix and serum (slurry) selective binding; Described step (3) is washed with binding buffer liquid.Before the sample bone dry, first part of wash solution is added to this site.Wash solution stopped on the site 10 seconds at least.Thoroughly remove first part of wash solution, repeat above step with second part of cleansing solution.Thoroughly wash whole array point with trifluoroacetic acid, biological marker is eluted on mass spectrum special-purpose metal sheet or the site.Described step (4) adds 0.5 μ L energy-absorbing molecule (with 50% acetonitrile, the saturated standard solution of the preparation of 0.5% trifluoroacetic acid) and goes to analyze delay with the biological marker in each site or with going to analyze with mass spectrometer behind the biological marker of electron spray ionisation wash-out with mass spectrometer.Blood is carried out the protein comparative analysis, with mass-to-charge ratio data in the computing machine analyzing blood.This kit is according to several characteristic protein peak 2873.3 ± 15 Da, 3379.7 ± 15 Da, 3449.3 ± 15 Da, 5808.4 ± 15 Da, 7568.9 ± 15 Da, 8048.7 ± 15 Da, 11439.2 ± 15 Da, 11526.7 ± 15 Da, 11683.0 ± 15 Da, 15114.9 ± 15 Da, 15312.8 ± 15 Da, double-blind normal person and susceptibility lungy 100%, specificity 100%.Quantitatively property control and mass spectrum laser energy regulation and control: before each test,, will be used for the maximal value that quantitative standards peak 6634 ± 1 Da intensity transfer to 50% mass signal intensity in the standardization quality controlled serum (slurry) with mass spectral standardization quality controlled serum (slurry).With 2746 ± 1 Da, 5909 ± 1 Da, 6634 ± 1 Da base peaks is mass spectrometry (molecular weight) quality control standard.
2. the described protein analysis method of claim 1 comprises negative ion, hydrophobic effect adsorbent in the used matrix of the method for magnetic bead supported matrix.
3. the sorbent used holder of the method for magnetic bead supported matrix is magnetic particle or magnetic bead in the claim 2.
Described one group of claim 1 (bunch) molecular weight is that the variation of protein fingerprint of 11439.2 ± 15 Da, 11526.7 ± 15 Da, 11683.0 ± 15 Da variation is for the purposes of differentiating normal person, slight tuberculosis patient, recurrence tuberculosis patient, severe tuberculosis patient, tuberculosis patient curative effect reagent box.
5. described 11683.0 ± 15 Da protein fingerprints of claim 4 are serum amyloid A protein, 11526.7 the chemical constitution of ± 15Da protein fingerprint is the serum amyloid A protein (its aminoterminal has been removed arginine) of variation, the chemical constitution of 11439.2 ± 15 Da protein fingerprints is the serum amyloid A protein (its aminoterminal has been removed arginine and serine) of variation.
6. the kit of the serum amyloid A protein of the described detection variation of claim 5 is characterized in that it comprises: a container and the antibody (group) that is loaded on the serum amyloid A protein of the described detection variation of claim 4 in the container.
7. kit as claimed in claim 6, its feature in, described antibody (group) is monoclonal antibody (group) or polyclonal antibody (group).
8. kit as claimed in claim 1, its feature in, described biological sample is serum or blood plasma.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2006101610927A CN101196526A (en) | 2006-12-06 | 2006-12-06 | Mass spectrometry reagent kit and method for rapid tuberculosis diagnosis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2006101610927A CN101196526A (en) | 2006-12-06 | 2006-12-06 | Mass spectrometry reagent kit and method for rapid tuberculosis diagnosis |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101196526A true CN101196526A (en) | 2008-06-11 |
Family
ID=39547063
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2006101610927A Pending CN101196526A (en) | 2006-12-06 | 2006-12-06 | Mass spectrometry reagent kit and method for rapid tuberculosis diagnosis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101196526A (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102150043A (en) * | 2008-06-25 | 2011-08-10 | 贝勒研究院 | Blood transcriptional signature of mycobacterium tuberculosis infection |
| CN101424661B (en) * | 2008-07-23 | 2012-05-30 | 中国人民解放军总医院第二附属医院 | Serodiagnosis model establishing method for active tuberculosis disease |
| CN102661884A (en) * | 2012-05-03 | 2012-09-12 | 浙江大学 | Sample containing tuberculosis serum characterized protein and preparation method thereof |
| CN105181837A (en) * | 2015-08-28 | 2015-12-23 | 程永娟 | Mycobacterium tuberculosis detection method |
| CN105209907A (en) * | 2013-06-05 | 2015-12-30 | Dh科技发展私人贸易有限公司 | SWATHTM data-independent acquisition technology for the detection of host cell protein contaminants in biotherapeutics protein products |
| CN114858904A (en) * | 2021-02-04 | 2022-08-05 | 北京毅新博创生物科技有限公司 | Mass spectrometry model comprising characteristic polypeptides for diagnosing neocoronary pneumonia |
| CN114858903A (en) * | 2021-02-04 | 2022-08-05 | 北京毅新博创生物科技有限公司 | Characteristic polypeptide composition for diagnosing neocoronary pneumonia |
| CN114858905A (en) * | 2021-02-04 | 2022-08-05 | 北京毅新博创生物科技有限公司 | Application of characteristic polypeptide composition and mass spectrum model in preparation of new coronary pneumonia detection product |
| CN114858906A (en) * | 2021-02-04 | 2022-08-05 | 北京毅新博创生物科技有限公司 | Kit for diagnosing neocoronary pneumonia |
-
2006
- 2006-12-06 CN CNA2006101610927A patent/CN101196526A/en active Pending
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102150043A (en) * | 2008-06-25 | 2011-08-10 | 贝勒研究院 | Blood transcriptional signature of mycobacterium tuberculosis infection |
| CN101424661B (en) * | 2008-07-23 | 2012-05-30 | 中国人民解放军总医院第二附属医院 | Serodiagnosis model establishing method for active tuberculosis disease |
| CN102661884A (en) * | 2012-05-03 | 2012-09-12 | 浙江大学 | Sample containing tuberculosis serum characterized protein and preparation method thereof |
| CN102661884B (en) * | 2012-05-03 | 2015-04-15 | 浙江大学 | Sample containing tuberculosis serum characterized protein and preparation method thereof |
| CN105209907A (en) * | 2013-06-05 | 2015-12-30 | Dh科技发展私人贸易有限公司 | SWATHTM data-independent acquisition technology for the detection of host cell protein contaminants in biotherapeutics protein products |
| CN105209907B (en) * | 2013-06-05 | 2019-08-16 | Dh科技发展私人贸易有限公司 | The SWATH of detection for the host cell proteins matter pollutant in biological therapy protein productTMData independence acquisition technique |
| CN105181837A (en) * | 2015-08-28 | 2015-12-23 | 程永娟 | Mycobacterium tuberculosis detection method |
| CN114858904A (en) * | 2021-02-04 | 2022-08-05 | 北京毅新博创生物科技有限公司 | Mass spectrometry model comprising characteristic polypeptides for diagnosing neocoronary pneumonia |
| CN114858903A (en) * | 2021-02-04 | 2022-08-05 | 北京毅新博创生物科技有限公司 | Characteristic polypeptide composition for diagnosing neocoronary pneumonia |
| CN114858905A (en) * | 2021-02-04 | 2022-08-05 | 北京毅新博创生物科技有限公司 | Application of characteristic polypeptide composition and mass spectrum model in preparation of new coronary pneumonia detection product |
| CN114858906A (en) * | 2021-02-04 | 2022-08-05 | 北京毅新博创生物科技有限公司 | Kit for diagnosing neocoronary pneumonia |
| WO2022166485A1 (en) * | 2021-02-04 | 2022-08-11 | 北京毅新博创生物科技有限公司 | Kit for diagnosing covid-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110057955B (en) | Method for screening specific serum marker of hepatitis B | |
| CN101196526A (en) | Mass spectrometry reagent kit and method for rapid tuberculosis diagnosis | |
| US7180056B2 (en) | Mass spectrometry and mass spectrometry system | |
| EP2005172A2 (en) | Apolipoprotein fingerprinting technique | |
| Umar et al. | NanoLC‐FT‐ICR MS improves proteome coverage attainable for∼ 3000 laser‐microdissected breast carcinoma cells | |
| CN101611313A (en) | Mass spectrometry biomarker assay | |
| JP2003533672A (en) | Methods for untargeted complex sample analysis | |
| JP2004347604A (en) | System for analyzing compound mixture of biological fluid, and other fluid for identifying information on biological condition | |
| Stevenson et al. | Evolution of seed allergen quantification–from antibodies to mass spectrometry | |
| CN101158666B (en) | Antibody group and mass spectrometric detection variation or modifying biological indication marks group kit and method | |
| US20160293394A1 (en) | MALDI-TOF MS Method And Apparatus For Assaying An Analyte In A Bodily Fluid From A Subject | |
| CN108426970A (en) | The method that enzymolysis stability by investigating candidate peptide fragment filters out the feature peptide fragment of milk powder allergen protein | |
| CN101169417A (en) | Reagent kit and method for judging normal person and liver cancer using magnetic bead supported matrix | |
| CN109791158A (en) | The method that more attributes for complex sample monitor | |
| CN107064390A (en) | The detection method of anaphylactogen in a kind of chocolate | |
| CN101246176B (en) | Mass spectrum kit for detecting squamous-cell carcinoma antigen feminine cervical carcinoma serum protein and preparation method thereof | |
| CN101153872B (en) | Novel reagent kit for detecting and estimating critical patients and method thereof | |
| CN101191795A (en) | Reagent kit and method for detecting digestive system tumor biological mark group by immunomic mass spectrometry | |
| Merkley et al. | A proteomics tutorial | |
| CN108020669B (en) | Application of urinary osteopontin and polypeptide fragment thereof in lung adenocarcinoma | |
| CN101201355A (en) | Immunity group mass spectrometric detection individuation knubble biological flag as well as curative effect reagent box and method | |
| CN101354379A (en) | Reagent for detecting multiple myeloma characteristic protein by mass spectrum | |
| CN101271088A (en) | Mass spectrum reagent kit and method for detecting and prognosis judging CEA negative gastric cancer | |
| CN101201360A (en) | Novel mass spectrum analysis reagent box and method for detecting heavy hepatitis B | |
| CN103454428A (en) | Novel immunomic mass spectrometry kit for detecting individualized insulin and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20080611 |