CN101201360A - Novel mass spectrum analysis reagent box and method for detecting heavy hepatitis B - Google Patents
Novel mass spectrum analysis reagent box and method for detecting heavy hepatitis B Download PDFInfo
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Abstract
The invention relates to a kit and a novel method of detecting and evaluating the patients with the severe Hepatitis B, and belongs to a non-invasive external detection method. The method utilizes the mass spectrometry to identify and detect a novel variant beta2-microglubolin. The variant beta2-microglubolin or the peak value of 11729 minus and plus 15Da can be used to detect and evaluate the severe Hepatitis B. The rising of the variant beta2-microglubolin or the peak value of 11729 minus and plus 15Da promotes the poor prognosis. The variant beta2-microglubolin level in the serum of the severe Hepatitis B is obviously higher than that of the acute Hepatitis B. The variant beta2-microglubolin level in the serum of people died from the severe Hepatitis B is obviously higher that of the survivor. The method provided by the invention can detect the seriously injured patient, the sensitivity is 100 percent and the specificity is 100 percent. By detecting the peak value of 11729 minus and plus 15Da or the variant beta2-microglubolin captured on the adsorption surface medium of immune body via the quantitative spectrum analysis controlled by the serum of the standardized quality control, the method can be applied in the development of the detection method or the kit for the biological symbol combination in the body fluid separated from the human body. The method has the advantages of accuracy, convenience and rapidness.
Description
Technical field
The present invention relates to the new method that a kind of Noninvasive detects, assesses heavy hepatitis B patient kit, it is characterized in that adopting the method for mass spectroscopy accurately having been differentiated, detected by the B2M of magnetic bead or antibody capture in the sample.Method provided by the invention can detect the patient of heavy hepatitis B, and its sensitivity is 100%, and specificity is 100%.The B2M level of serum variation is a reflection hepatic tissue degree of damage good index, and to understanding heavy type hepatitis state of an illness severe degree, judging prognosis is significant.
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 antibody and mass spectrum associating.
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 characteristics of electro-spray ionization are to produce high charge ion rather than fragmention, make mass-charge ratio (mass to charge ratio, m/z) be reduced to the scope that most analytical instrument of quality can detect, thereby expanded the analyst coverage of molecular weight greatly, the validity score protonatomic mass of ion also can be calculated according to mass-to-charge ratio and charge number.
Ground substance assistant laser parsing/ionization time of flight mass spectrometry (matrix-assisted laserdesorption/ionization time of fight mass spectrometry, MALDI-TOF-MS) ultimate principle is to be dispersed in analyte in the energy-absorbing molecule and to form crystal, when with the laser radiation crystal, because the energy-absorbing molecule is through energy that radiation absorbed, cause energy to be accumulated and heat production rapidly, thereby make the distillation of energy-absorbing molecular crystal, cause together with analyte and enter gas phase.The mass spectrogram that MALDI-TOF-MS produced mostly is single charge ion, thereby the quality of the ion in the mass spectrogram and polypeptide and protein has one-to-one relationship.MALDI-TOF-MS and protein chip isolation technics use in conjunction be surface-enhanced laser desorb/ionization time of flight mass spectrometry (surface enhanced laser desorption/ionizationtime of fight mass spectrometry, SELDI-TOF-MS).
Summary of the invention:
The objective of the invention is to set up a kind of kit and method that in biological sample, detects normal person and heavy hepatitis B biological marker in the blood that exsomatizes.This method provides new approach for the early detection of heavy hepatitis B, and for finding that further biological marker new variation or that modify provides the foundation.
The present invention relates to the new method that a kind of Noninvasive detects, assesses the heavy hepatitis B patient, it is characterized in that adopting the method for mass spectroscopy accurately having been differentiated, detected by the B2M of the variation of WCX/ hydrophobic matrix or antibody capture in the sample.The B2M level of heavy hepatitis B serum variation is significantly higher than acute hepatitis B; The B2M level of heavy hepatitis B death person serum variation is significantly higher than the survivor.The B2M level of serum variation is a reflection hepatic tissue degree of damage good index, and to understanding heavy type hepatitis state of an illness severe degree, judging prognosis is significant.
The B2M biological marker of the variation among the present invention utilizes a mass spectrometer to find.The exactness high in quality of this equipment is about+and/-0.1%.
The B2M biomarker at first can be had the antibody or the hydrophobic absorption surface matrix of WCX/ that can combine with biomarker catches, and non-adsorbate can be from wash-out on the matrix, and the biomarker that is adsorbed onto base is detected in flight mass spectrometer.The source takes place by ion in biomarker, 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 4091.1Da or 6634.0Da intensity transfer to 50% mass signal intensity in the standardization quality controlled serum with mass spectral standardization quality controlled serum.The detection of biomarker is significantly with relevant with the detection of signal intensity.Like this, the quantity of biomarker 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 biomarker produce.These data of this computer program analysis to be showing the quantity of detected label, and the intensity of shows signal and determine the molecular weight of each detected biomarker.Data analysis can also comprise the signal intensity of a series of definite biomarker 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 label 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.
Detection to biological marker need be put a sample on the adsorption site of matrix, then cleans.On adsorption site, add SINAPINIC acid etc. and allow its drying.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.
By Mann-Whitney U check analysis hundreds of kind haemocyanin fingerprint peaks, find that following protein fingerprint or mass-spectrogram can be used to distinguish the serum of normal and hepatitis patient.Peak C V＞30% or p＜0.01 is for there being significant difference:
The significant difference of normal and hepatitis patient protein fingerprint or mass-spectrogram (± 15Da)
2045.10 4132.67 5193.66 8558.99
2815.90 4155.88 5297.89 8685.47
3262.12 4279.06 5474.44 8764.03
3387.19 4294.94 5630.33 8900.28
3939.42 4343.05 5838.52 9409.52
3954.05 4471.67 6080.09 11654.9
3971.58 4593.82 6235.71 11729.9
4070.81 4960.67 6874.87 13730.4
4089.02 4989.61 7620.60 13947.7
4112.88 5061.49 7963.47 16113.6
With therefrom filtering out a characteristic protein 11729.9Da, double-blind normal person and heavy hepatitis B, find that following a kind of molecular weight is 11729 ± 15Da protein fingerprint (Fig. 1) serum that can be used to distinguish normal human serum, heavy hepatitis B patient, the urgent patient of heavy hepatitis B, acute hepatitis B patient's serum:
| Shaker test | The normal person | Heavy hepatitis B | The urgent patient of heavy hepatitis B | |
| 11729±15Da | (-) | (++) | (+++) | (+) |
| Add up to (example) | 50 | 25 | 10 | 10 |
The discovery molecular weight is that the variation of 11729 ± 15Da Da protein fingerprint is significant for discriminating heavy hepatitis B patient.By the discriminating at this peak, The above results shows that the sensitivity of this method is 100% (45/45), specificity 100% (50/50).Utilize the experimental result of C8 and C18 hydrophobic matrix consistent with the experimental result of above-mentioned WCX anionic substrates.
Because the biomarker in this invention identifies by quality and antibody, thereby they can detect by mass spectroscopy and directly know the identity that they are specific.This method than antibody be the basis ELISA and immunofluorescence technique more accurate.Technology such as present ELISA mainly rely on indirect chemistry or radiometric determination, thereby can't directly identify the variation of antigen, and can measure the molecular weight of antigenic variation fragment with immunity-mass-spectrometric technique.
Find that with anti-B2M antibody matrix and mass spectrometer use in conjunction analyte is a molecular weight and above-mentioned consistent, but not the molecular weight (13705.9Da) of B2M molecule in the blood in the known cDNA database.Then 11729 ± 15Da chemical constitution can be speculated as the B2M of variation.Find that the B2M of following variation can be used to distinguish the urgent patient of normal human serum, heavy hepatitis B patient's serum, heavy hepatitis B, acute hepatitis B patient's serum:
| Shaker test | The normal person | Heavy hepatitis B | The urgent patient of heavy hepatitis B | Acute hepatitis B |
| The B2M of variation | (-) | (++) | (+++) | (+) |
| Add up to (example) | 50 | 25 | 10 | 10 |
The B2M level of heavy hepatitis B serum variation is significantly higher than acute hepatitis B; The B2M level of heavy hepatitis B death person serum variation is significantly higher than the survivor.
Yet if necessary, these biological markers also can pass through, such as, determine that amino acid sequence of polypeptide differentiates.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.
With 11729 ± 15Da biological marker 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.Look into the blood B2M molecule in the known cDNA database molecular weight (molecular weight is: 13705.9Da, pI:6.1).Identify the B2M of 11729 ± 15Da biological marker for variation.Its chemical constitution is (holding the amino acid of arranging to the C end from N):
The chemical constitution of 11729 ± 15Da biological marker:
The N end
IQRTPKIQVYSRHPAENGKSNFLNCYVSGF
51 HPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYAC
101 RVNHVTLSQPKIVKWDRDM
The C end.
The B2M of variation is prepared antibody.
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.According to the complex process of gene to protein expression, a kind of product-protein of specific gene must have relevant polycomponent protein expression.Form an aggregative model figure (protein fingerprint spectrum) by detection to these different components, this collection of illustrative plates (as certain tumour) is compared with other collection of illustrative plates (as normal person or other diseases), and then discern this differential protein (as tumour antigen or its fragment), thereby the normal person is made a distinction with certain disease patient blood of trouble.
The concrete operations step:
Below be with an operation scheme example provided by the invention.
1. sample preparation and standardization quality controlled serum preparation
Biological sample is diluted in the dilution buffer, optionally the centrifugal clarification sample.Mass spectral standardization quality controlled serum preparation definition meets following standard: blood donor 10 people, and 5 male 5 woman, blood group is the O type; Age is 18-30 year; 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 the sample
[a. can be with the magnetic bead of WCX or hydrophobic matrix on the matrix of holder with mass spectral standardization quality controlled serum and sample point sample, anionic surface (WCX, Weak Cationic Exchanger ,-hydroxy-acid group) matrix magnetic bead (embodiment 1) and C8 and C18 hydrophobic surface magnetic bead; Or with the magnetic bead (embodiment 2) of anti-B2M antibody matrix on b. mark].Mass spectral standardization quality controlled serum can be used for mass spectral quantivative approach.The holder of matrix can be sheet metal, ceramic bead, magnetic beads, polymer or Capillary Electrophoresis pillar.
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.Below different step can be arranged:
(1) water thoroughly washs whole array point, and the biological marker of air dry matrix and delay adds 0.5 μ L energy-absorbing molecule (with 50% acetonitrile, the saturated standard solution of the preparation of 0.5% trifluoroacetic acid);
(2) or with the thorough whole array point of washing (when with ceramic bead, magnetic beads, when polymer is holder) of 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);
(3) or with electrospray ionization mass spectrometry (when liquid chromatography pillar or Capillary Electrophoresis pillar are holder).
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.Series connection quadrupole rod mass spectrum or linear ion hydrazine mass spectrum identify modification with variation biological marker and the order-checking of polypeptide de novo.Carry out the overlapping displaying of data with the Computer Analysis data.
Quantitative property spectrum regulation and control: before each test,, the standardization quality controlled serum is used for the maximal value that quantitative standards peak 4091.1Da or 6634.0Da equal strength transfer to 50% signal intensity in the WCX magnetic bead with mass spectral standardization quality controlled serum.
The external detection method of the present invention and other Noninvasives relatively has following characteristics:
(1) accurately reaches accurately
Characteristics that accurately detect multiple biological marker method with antibody and mass spectrum associating are to tell analyte from the complicated sample potpourri exactly.The accuracy rate that antibody combines with antigen surpasses 95%.This is the basic Chu of ELISA and immunofluorescence kit etc.
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.But ELISA and immunofluorescence kit can't be known the variation of antigen.So, unite the direct information that accurate detection of biological denotation approach can provide analyte (antigen or biological marker) chemistry or architectural feature with antibody and mass spectrum.For example:
The molecular weight of known fiber protein peptides A molecule is 1536Da, and chemical constitution is 16 amino acid (N end Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg C ends).The antibody of fibrinopeptide A and mass spectrometer use in conjunction find that molecular weight is the fibrinopeptide A of 1536Da.Then 100% can determine that analyte is a fibrinopeptide A, i.e. the most accurate discriminating (goldstandard).
Antibody and mass spectrometer use in conjunction with fibrinopeptide A find that analyte is that molecular weight is the fibrinopeptide A of 1465 ± 1Da variation.Then chemical constitution can (be held the end to C from N) and is speculated as 15 amino acid: N end Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg C ends.
(2) convenient
This method has been divided into protein in the protein of negative ion, hydrophobic, antibody effect.Like this, available mass spectrometer is directly analyzed.After being identified, protein can remain forevermore with digital form (bar code band).Can save the area in sample storehouse like this.
(3) quick
Unite accurate detection of biological denotation approach with known antibodies provided by the invention and mass spectrum and carry out disease blood serum when detecting, need not protein check order promptly as can be known " biological marker variation or that modify ".Unlike the immunofluorescence technique kit, can't know " biological marker variation or that modify ".The biological marker of modifying refers to methylate, the high expressed or low expression of acetylation, hydroxylation, phosphorylation modification etc. change.When carrying out the disease blood serum diagnosis, need not protein is checked order with protein fingerprint method provided by the invention.Technology such as present ELISA mainly rely on indirect chemistry or radiometric determination, thereby can't directly identify the variation of antigen, and can measure the molecular weight of antigenic variation fragment with immunity-mass-spectrometric technique.
Description of drawings
11729 ± 15Da protein fingerprint peak value among Fig. 1 normal person, heavy hepatitis B patient, the acute hepatitis B patients serum
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.
The mass spectrometry reagent kit and the method for 1 one kinds of novel detection heavy hepatitis Bs of embodiment
(1) experimental technique
One, material
1. sample is originated: the serum of A.50 routine normal controls group; B.25 the patient's of routine heavy hepatitis B serum; C.10 the urgent patient's of routine heavy hepatitis B (in preceding two skies of death) serum; E.10 routine acute hepatitis B patient's serum.
2. quality control: A. people's standardization quality controlled serum B. mass spectrum laser energy regulation and control: before each test, with above-mentioned standardization quality controlled serum.
Two, method
1. the collection of sample: draw serum after the whole blood collection, place-80 ℃ of preservations; Take out blood serum sample in-80 ℃ of refrigerators, put on the ice chest and melt; With 10,000 rev/mins, 4 ℃ centrifugal 2 minutes; Get supernatant.
2. the preparation of sample: each adsorbent matrix supports that object point needs serum 2.5 μ l.Get serum 5 μ l, with 10 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 15 μ l is added the corresponding binding buffer liquid of 185 μ l, stand-by.
3. sample detection: go up sample, above-mentioned sample 100 μ l are added in the PCR pipe (Beijing Sai Erdi company) that installs WCX (Weak Cationic Exchanger, anionic surface, hydroxy-acid group) magnetic bead, put and hatch 30 minutes on the magnetic processor, remove liquid.(50mM NaAC pH4.0-4.5) to the PCR pipe that installs the WCX magnetic bead, puts and hatches 2 minutes on the magnetic processor, removes liquid, repeats twice of aforesaid operations to add 100 μ l binding buffer liquid.Add 10 μ l Elution Buffer2 minute, the wash-out sample is to supernatant.Getting 5 μ l supernatants moves in another PCR pipe, add the abundant mixing of 5 μ l SPA saturated solutions, get 1 μ l mixed solution application of sample to 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 SINAPINIC acid (with 50% acetonitrile, the standard solution of the preparation of 0.5% trifluoroacetic acid).The double of carrying out all sample standard deviations detects to reduce experimental error.
4. above-mentioned sample is added in the mass spectrum, will generate flight time mass spectrum.The outside peptide molecule quality standard of using is come the correction mass accuracy.
(2) experimental result
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 Mann-Whitney U check analysis hundreds of kind haemocyanin fingerprint peaks, find that following protein fingerprint or mass-spectrogram can be used to distinguish the serum of normal and hepatitis patient.Peak C V＞30% or p＜0.01 is for there being significant difference:
The significant difference of normal and hepatitis patient protein fingerprint or mass-spectrogram (± 15Da)
2045.10 4132.67 5193.66 8558.99
2815.90 4155.88 5297.89 8685.47
3262.12 4279.06 5474.44 8764.03
3387.19 4294.94 5630.33 8900.28
3939.42 4343.05 5838.52 9409.52
3954.05 4471.67 6080.09 11654.9
3971.58 4593.82 6235.71 11729.9
4070.81 4960.67 6874.87 13730.4
4089.02 4989.61 7620.60 13947.7
4112.88 5061.49 7963.47 16113.6
With therefrom filtering out a characteristic protein 11729.9Da, double-blind normal person and heavy hepatitis B, find that following a kind of molecular weight is 11729 ± 15Da protein fingerprint (Fig. 1) serum that can be used to distinguish normal human serum, heavy hepatitis B patient, the urgent patient of heavy hepatitis B, acute hepatitis B patient's serum:
| Shaker test | The normal person | Heavy hepatitis B | The urgent patient of heavy hepatitis B | |
| 11729±15Da | (-) | (++) | (+++) | (+) |
| Add up to (example) | 50 | 25 | 10 | 10 |
The discovery molecular weight is that the variation of 11729 ± 15Da Da protein fingerprint is significant for discriminating heavy hepatitis B patient.By the discriminating at this peak, The above results shows that the sensitivity of this method is 100% (45/45), specificity 100% (50/50).
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.
B2M Molecular Identification in embodiment 2 blood
To have with Carbodiimide method (Carbodiimide Method) that matrix combines (Gunn DL, et al.Preparation of sensitiveand stable erythrocytes by the carbodiimide method for the detection of primaryand secondary IgM and IgG antibody.J Immunol Methods.1972 on the magnetic beads of hydroxy-acid group mark with the amino group of anti-B2M antibody; 1 (4): 381-389.).
With the sample point sample on the site of an anti-B2M 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 anti-B2M antibody matrix and mass spectrometer use in conjunction analyte is that molecular weight and embodiment 1 are consistent, but not the molecular weight (13705.9Da) of B2M molecule in the blood in the known cDNA database.Then 11729 ± 15Da chemical constitution can be speculated as the B2M of variation.Technology such as present ELISA mainly rely on indirect chemistry or radiometric determination, thereby can't directly identify the variation of antigen, and can measure the molecular weight of antigenic variation fragment with immunity-mass-spectrometric technique.Find that the B2M of following variation can be used to distinguish the urgent patient of normal human serum, heavy hepatitis B patient's serum, heavy hepatitis B, acute hepatitis B patient's serum:
| Shaker test | The normal person | Heavy hepatitis B | The urgent patient of heavy hepatitis B | Acute hepatitis B |
| The B2M of variation | (-) | (++) | (+++) | (+) |
| Add up to (example) | 50 | 25 | 10 | 10 |
The B2M level of heavy hepatitis B serum variation is significantly higher than acute hepatitis B; The B2M level of heavy hepatitis B death person serum variation is significantly higher than the survivor.
The ordering of embodiment 3 heavy hepatitis B protein fingerprint spectrums one characteristic peak is identified
With 11729 ± 15Da biological marker 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.Look into the blood B2M molecule in the known cDNA database molecular weight (molecular weight is: 13705.9Da, pI:6.1).Identify the B2M of 11729 ± 15Da biological marker for variation.Its chemical constitution is (holding the amino acid of arranging to the C end from N):
The chemical constitution of 11729 ± 15Da biological marker:
The N end
IQRTPKIQVYSRHPAENGKSNFLNCYVSGF
51 HPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYAC
101 RVNHVTLSQPKIVKWDRDM
The C end.
The preparation of the B2M antibody of embodiment 4 variations
Synthetic and the sequence of the B2M of variation:
The N end
IQRTPKIQVYSRHPAENGKSNFLNCYVSGF
51 HPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYAC
101 RVNHVTLSQPKIVKWDRDM
The C end.
With the B2M 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.
Conclusion
To compare from sample with statistical significance heavy hepatitis B and control group (normal specimens), the B2M biological marker that 11729 ± 15Da organizes or anti-B2M antibody absorption surface is caught of being caught with the WCX magnetic bead is consistent.New form variation B2M that anti-B2M antibody absorption surface is caught or the kit of organizing with 11729 ± 15Da that the WCX magnetic bead is caught can detect, assess the heavy hepatitis B patient.The B2M level of heavy hepatitis B serum variation is significantly higher than acute hepatitis B; The B2M level of heavy hepatitis B death person serum variation is significantly higher than the survivor.The method of the invention provides can detect heavy hepatitis B, and its sensitivity is 100%, and specificity is 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.
This method is differentiated with WCX matrix, B2M antibody absorption surface matrix and mass spectroscopy and is detected the B2M of finding a kind of new form variation.The B2M of variation can be used for the detection and the assessment of heavy hepatitis B.The B2M rising person of variation points out poor prognosis.By WCX matrix and by the B2M 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 (7)
1. the present invention relates to a kind of detection, assessment heavy hepatitis B patient's kit and new method, the method that it is characterized in that adopting mass spectroscopy that the B2M biological marker of the variation of having been caught by matrix in the sample is accurately differentiated, detected.This method realizes by following steps:
(1) sample preparation and mass spectrum standardization quality controlled serum preparation;
(2) on the sample sample to matrix;
(3) washing;
(4) mass spectral quantitative control and mass spectrometric measurement;
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; Described step (2) with mass spectral standardization quality controlled serum and sample point sample on matrix.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 magnetic bead with 0.5～2% trifluoroacetic acid, biological marker is eluted on the sheet metal of mass spectrum special use, the air dry sheet metal adds 0.5 μ L with 50% acetonitrile, the energy-absorbing molecular criteria solution of 0.5% trifluoroacetic acid preparation.The energy-absorbing molecule can be used Sinapinic acid or alpha-Cyano-4-hydroxycinnamic acid etc.; Described step (4) is removed the B2M of analytical variance with mass spectrometer.With the Computer Analysis data is 11729 ± 15Da peak value.Quantitative property spectrum regulation and control: before each test,, will be used for the maximal value that quantitative standards peak 4091.1Da or 6634.0Da intensity transfer to 50% signal intensity in the standardization quality controlled serum with mass spectral standardization quality controlled serum.
2. described B2M kit of claim 1 and analytical approach, the B2M of the variation of being caught derives from whole blood, serum and the blood plasma that breaks away from human body.
3. the described matrix of claim 1 comprises anionic surface matrix, hydrophobic surface matrix, anti-B2M antibody matrix.The holder of matrix comprises ceramic bead, magnetic beads, polymer or Capillary Electrophoresis pillar.
4. the described molecular weight of claim 1 is that the variation of B2M of 11729 ± 15Da or variation is for the purposes of differentiating normal person, heavy hepatitis B, acute hepatitis B and prognosis kit.
5. the described 11729 ± 15Da albumen of claim 4 is the B2M of variation, holds the amino acid of arranging to the C end to be from N: IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSF SKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM.
6. the kit of the described detection variation of claim 5 B2M is characterized in that it comprises: a container and the antibody (group) that is loaded on the B2M 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).
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113933373A (en) * | 2021-12-16 | 2022-01-14 | 成都健数科技有限公司 | Method and system for determining organic matter structure by using mass spectrum data |
| CN114460190A (en) * | 2022-01-19 | 2022-05-10 | 西安乐析医疗科技有限公司 | Method for measuring content of beta 2-microglobulin |
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2006
- 2006-12-15 CN CNA2006101681161A patent/CN101201360A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113933373A (en) * | 2021-12-16 | 2022-01-14 | 成都健数科技有限公司 | Method and system for determining organic matter structure by using mass spectrum data |
| CN113933373B (en) * | 2021-12-16 | 2022-02-22 | 成都健数科技有限公司 | Method and system for determining organic matter structure by using mass spectrum data |
| CN114460190A (en) * | 2022-01-19 | 2022-05-10 | 西安乐析医疗科技有限公司 | Method for measuring content of beta 2-microglobulin |
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