CN101191795A - Reagent kit and method for detecting digestive system tumor biological mark group by immunomic mass spectrometry - Google Patents
Reagent kit and method for detecting digestive system tumor biological mark group by immunomic mass spectrometry Download PDFInfo
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Abstract
The invention relates to a biomarker captured on a surface matrix adsorbed with an antibody group, which is detected by a quantitative mass spectrum analysis under the control of standard quality control serum. The definition of immunomic mass spectrometry analysis, namely IMS, is a method which combines a group of various antibodies with a spectrometry to accurately identify a variant or modified biomarker group. A plurality of biomarkers can be captured on one antibody group matrix at the same time, and a spectrometry accurate analysis is processed to the captured variant or modified biomarker. The invention can detect a plurality of biomarker groups at the same time. The method of the invention can be used for detecting a biomarker combination in fluid which is separated from a human body. The biomarker combination is applicable to the detection method of a kit which identifies in vitro fluid of a normal person and patients with different types of tumours of the digestive system. The method is accurate, convenient and rapid.
Description
Technical field
The present invention relates to protein analysis method in a kind of new biological sample, a kind of by removing to catch biological marker with the matrix of antibodies, and come the detection of biological sign with the mass spectrophotometry that quantitative control is arranged.The present invention relates to the disease detection field referred in this, is a kind of external detection method of new Noninvasive.Say that more properly this invention relates to biological marker (biomarkers), and these antigens or biological marker can be distinguished with the mass spectrum of the antibody group of higher specificity and sensitivity and quantitatively control with multiple disease is disposable.The present invention can be applied to the detection method or the kit developing of the digestive system tumor biological sign 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 antibody and mass spectrum associating.
The present invention can detect the method for the biological marker of multiple (more than three kinds) simultaneously with antibody group and mass spectrum associating.As, blood bank's examination requires to detect specifically the known mark of common virus microbial diseases.But preparation specificity incorporation of markings and the reagent that can identify mark in the potpourri of complexity need the plenty of time, and this has hindered the development of this type of external diagnosis reagent case method.At present, also there is not a kind of method disease marker more than four kinds can be detected simultaneously.ELISA (enzyme-linked immunosorbent assay) kit etc. utilizes antibody can be used to detect a kind of disease marker.Utilize antibody and three look immunofluorescences, can accomplish at most to detect three kinds of disease markers, but disease marker just can't detect simultaneously more than three kinds.Utilize antibody group and mass spectrometer use in conjunction, promptly can solve virus or the microbial antigen mark differentiated simultaneously more than three kinds.Say for example, to resist HBV (HBsAg) antibody, HCV antigen/antibody combination, anti-HIV p24antigen (Ribas SG et al.Performance of a quantitative human immunodeficiency virus type1 p24 antigen assay on various HIV-1 subtypes for the follow-up of humanimmunodeficiency type 1seropositive individuals.J Virol Methods 2003; 113:29-34) and anti-syphilis antibody (anti-treponemal 17kDa protein) (George R et al.An analysis ofthe value of some antigen-antibody interactions used as diagnostic indicators ina treponemal Western blot (TWB) test for syphilis.J Clin Lab Immunol 1998; On 50:27-44) etc. the antibody combined holder (magnetic pearl, chip etc.) that is tagged to Protein A or G.Because the molecular weight of every kind of special body antibody capture biological antigens is different, so when Protein A/G-antibody and mass spectrometer use in conjunction, mass spectrometer has just separated these four kinds of antigens easily simultaneously.Reasoning thus, if select antibody more than four kinds simultaneously, and the antigen molecular of this antibody institute combination more than four kinds is different, then the present invention can distinguish different types of disease more than four kinds simultaneously.The present invention can the detection window phase the blood donor, this is than simple ELISA antibody kit safer (Lau DT, et al.A rapid immunochromatographic assay for hepatitis B virus screening.J ViralHepat 2003; 10:331-334).
In addition, method of the present invention can detect the biological marker of multiple variation simultaneously.Antibody and mass spectrometer use in conjunction are the detection methods of a kind of high sensitivity, high accuracy, and it can check and distinguish the biological marker (protein) of different component, different molecular weight (difference is between 1 or 2 amino acid) from the mixed system of a heterogeneity.In the future, it may become the new model of numerous disease detection clinically, as the conventional method of clinical examination.For example, the fibrinopeptide A of serum fibrinopeptide A and variation in the difference cancer of the stomach, use previous methods and can not determine which kind of peptide class fragment is relevant with morbidity, now can antibody and mass spectrometer use in conjunction, with this component wherein and their clear the making a distinction of molecular weight, and find and the relevant particular components of this disease morbidity, this is the revolution of molecular medicine.Method of the present invention can detect multiple modified biological sign simultaneously.The modifying protein that the biomarker group who modifies refers to for methylate, acetylation, hydroxylation, phosphorylation modification etc.In the generation evolution of human tumor, past is detected clinical tumor and rests on the cellular level always, therefore the early diagnosis truly looked forward to for a long time of clinician (do not forming as yet before the enclosed mass as solid tumor, leukaemia is the bone marrow cell inspection can not be made a definite diagnosis before) is impossible realization.
Summary of the invention:
The objective of the invention is to set up a kind of method that in biological sample, detects normal person and certain or multiple disease biological marker in the blood that exsomatizes.This invention relates to biological marker (biomarkers), and these biological markers can be used to higher specificity and sensitivity certain or multiple disease patient be distinguished simultaneously.This method provides new approach for the early detection of disease, and for finding that further biological marker new variation or that modify provides the foundation.(Immunomic mass spectrometry analysis IMS) is defined as one group of multiple (class) antibody and mass spectrum and unites and accurately differentiate method variation or modifying biological indication marks group the immune group Mass Spectrometer Method.
The present invention relates to a kind of by mark specific antibody group to can with the stromal surface of antibodies, and detect the multiple biological marker or the various disease states of certain disease simultaneously with the quantitative property analysis of spectrum.Biological indication marks group can be that do not make a variation, variation, modification.Variation biological indication marks group be meant that variant protein matter is for increasing or reduce one or more amino acid.The biomarker group who modifies be meant modifying protein for methylate, acetylation, hydroxylation, phosphorylation modification etc.
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 antibody selectivity or specificity.Illustrate the function of Protein A and G matrix absorption antibody Fc section.Protein A and G base and antibodies with absorbing agent function, biological marker in the antibodies serum.Through one section time enough biological marker can be combined with antibody-Protein A and G.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 the antibody-matrix absorption surface 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 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 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 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 caught by antibody.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.Ground substance assistant laser parsing/ionization time of flight mass spectrometry (matrix-assisted laser desorption/ionization time of fight mass spectrometry, MALDI-TOF-MS) ultimate principle 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.
Because the biological marker in this invention identifies by mass spectrum and antibody matrix, 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.
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 (V8protease) 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.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 mass spectrogram) 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 normal person and certain disease of trouble patient are made a distinction.
The concrete operations step of the kit of detecting digestive system tumor biological mark group by immunomic mass spectrometry and method:
Below be with an operation scheme provided by the invention and kit example.
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 0 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 matrix and multiple antibodies preparation, the sample
With mass spectral standardization quality controlled serum and sample point sample on the site in the matrix of holder is arranged.Sample comes in blood, body fluid, secretion, cytolysate, histolysate and organ dissolved matter.Holder available metal sheet, glass sheet, potsherd, ceramic bead, magnetic beads, polymer, liquid chromatography pillar or Sepharose beads etc.Matrix is used for mark, in conjunction with multiple antibody.Antibody is monoclonal antibody or polyclonal antibody.Increase the antibody group reaches and detects a plurality of or multiple biomarker or the antigenic mark molecular weight difference of biological marker (need only in the Mass Spectrometer Method error rate) endlessly endlessly.With biological marker immune mouse synthetic variation or that modify, treat that immune response occurs after, from peripheral blood, separate the B cell.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 preparing the required kit that detects variation or modified biological sign.Matrix and multiple antibodies prepare kit method can with any can with method and any material that can combine of antibodies with antibody selectivity or specificity, for example: combine with antibody Fc section with matrix on the Sepharose beads of albumin A and G (Protein A and G) mark; To have with Carbodiimide method (Carbodiimide Method) that matrix combines (Gunn DL, et al.Preparation of sensitive and stable erythrocytes by the carbodiimide methodfor the detection of primary and secondary IgM and IgG antibody.J Immunol Methods.1972 on the magnetic beads of carboxylate-groups mark with the aminoterminal (amino-groups) of antibody; 1 (4): 381-389.); With the matrix of streptavidin mark and the antibodies of biotin mark; Ceramic bead and antibodies (through high capacity and high selectivityinteraction and the cooperative influence of a thioether group) with the MEPHyperCel mark; Or the like method.Multiple antibody labeling to the matrix 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 controlled serum can be 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.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 0.05%～1% trifluoroacetic acid thoroughly wash whole array point (when being holder) with ceramic bead, magnetic beads, polymer or Sepharose beads, 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.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,, will be used for the maximal value that quantitative standards peak 4091.1Da or 6634.0Da equal strength transfer to 50% signal intensity in the standardization quality controlled serum with mass spectral standardization quality controlled serum.
The present invention can be used for the clinical disease external detection method of cell in vitro and Noninvasive, is used for the detection method of clinical disease as the kit of the body fluid that exsomatizes.
Use statistical method, by serum analysis protein fingerprint peak, find that 1465 ± 15Da, 8938 ± 15Da, 28078 ± 15Da, 8707 ± 15Da biological marker can be used to distinguish the normal person, the variation of digestive system tumor biological marker group is expressed, the sensitivity of this method is: cancer of the stomach 95% (198/208 example), liver cancer 81% (183/226), colorectal cancer patient 87% (158/182).And the AFP of cancer of the stomach, liver cancer, colorectal cancer and CEA sensitivity are in the digestive system tumor: 46% to 60%.
With 1465 ± 15Da, 8938 ± 15Da, 28078 ± 15Da, 8707 ± 15Da biological marker 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 digestive system tumor biological and be masked as the biological indication marks group that variation is expressed:
(A) cancer of the stomach 1465+15Da:FPA-degAla (its aminoterminal of fibrinopeptide A has been removed alanine) sequence FPA fragment (1465.7Da, DSGEGDFLAEGGGVR).
(B) liver cancer 8938+15Da:C3a-degArg (its C end of complement C 3 has been removed arginine) sequence N end
Ser-Val-Gln-Leu-Thr-Glu-Lys-Arg-Met-Asp-Lys-Val-Gly-Lys-Tyr-Pro-Lys-Glu-Leu-Arg-Lys-Cys-Cys-Glu-Asp-Gly-Met-Arg-Glu-Asn-Pro-Met-Arg-Phe-Ser-Cys-Gln-Arg-Arg-Thr-Arg-Phe-Ile-Ser-Leu-Gly-Glu-Ala-Cys-Lys-Lys-Val-Phe-Leu-Asp-Cys-Cys-Asn-Tyr-Ile-Thr-Glu-Leu-Arg-Arg-Gln-His-Ala-Arg-Ala-Ser-His-Leu-Gly-Leu-AlaC end.
(C) colorectal cancer 28078+15Da:ApoA-I (apolipoprotein A-1) sequence:
1 DEPPQSPWDRVKDLATVYVDVLKDSGRDYVSQFEGSALGKQLNLKLLDNW
51 DSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPY
101 LDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRD
151 RARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHL
201 STLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ
The ApoA-I Molecular Mass:28078.652Da of variation, pI:5.3
(D) colorectal cancer 8707+15Da:ApoA-II (apolipoprotein A-1 I) sequence:
1 QAKEPCVESLVSQYFQTVTDYGKDLMEKVKSPELQAEAKSYFEKSKEQLT
51 PLIKKAGTELVNFLSYFVELGTQPATQ
The ApoA-II Molecular Mass:8707.921Da of variation, pI:5.1
Antibody and mass spectrum associating can be saved the ordering of the biological indication marks group of variation and be identified.With the biomarker FPA-degAla of variation, C3a-degArg, ApoA-I, ApoA-II is synthetic, preparation antibody.
The external detection method of kit among the present invention and method and other Noninvasives relatively has following characteristics:
(1) accurately reaches accurately
Characteristics that accurately detect multiple biological marker method with multiple 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.Be the associating of antibody and mass spectrum, can save the ordering of the fibrinopeptide A of variation and identify (embodiment 1 and embodiment 2).
(2) be convenient to joint-detection
Be marked on the matrix and the mass spectrum associating with antibody group more than three kinds, can detect the biological marker and the biological marker one or more variations or that modify of multiple (more than three kinds) simultaneously.Produce a kind of available mass spectrometer like this and directly analyzed the method (embodiment 1) of the biological marker and the biological marker one or more variations or that modify of multiple (more than three kinds).Three look immunofluorescence techniques can be analyzed three kinds of biological markers simultaneously, but can't reach the analysis of the biological marker more than three kinds or unite to come accurately, determine efficiently analyte (antigen or biological marker) a kind of variation or that modify as antibody and mass spectrum.Matrix can be with any material that can combine with antibody selectivity or specificity.
(3) quick
When accurately detecting multiple biological marker method and carry out multiple disease detection, need not protein check order promptly as can be known " biological marker variation or that modify " with the associating of multiple known antibodies group provided by the invention and mass spectrum.Unlike the immunofluorescence technique kit, a kit can can't be known " biological marker variation or that modify " with three kinds of antibody of tense marker at most.
A multiple antibody reagent cassette method provided by the invention is used for mass spectrum and detects simultaneously, can be not limited to three kinds of antibody.Thereby help the detection of clinical complexity, carry out the application that the detecting digestive system tumor biological mark group by immunomic mass spectrometry variation is expressed as available this method.
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 application that the variation of embodiment 1 detecting digestive system tumor biological mark group by immunomic mass spectrometry is expressed
(1) experimental technique
One, material
1. sample is originated: the serum of A.920 routine normal controls group; B.208 the serum of routine Patients with Gastric Cancer; C.226 the serum of routine hepatocarcinoma patient; D.182 routine colorectal cancer 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.
3. matrix contains the equivalent antibody of mark: anti-FPA (fibrinopeptide A), anti-C3a (complement C 3), anti-ApoA-I (apolipoprotein A-1), anti-ApoA-II (apolipoprotein A-1 I).
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 supports that object point needs serum 1 μ l, serum is diluted with damping fluid, with the abundant mixing of sample.
3. sample detection: go up sample, in adsorbent holder (matrix contains the equivalent antibody of mark: antifibrin peptide A, anticomplement 3a, anti-apolipoprotein A-1, anti-apolipoprotein A-1 I), add the sample of handling well, put oscillator, 400-600 rev/min, shook 30～60 minutes.The binding buffer liquid X2 that adds 200 μ l.Add 200 μ l HPLC water X1.After taking out the adsorbent holder, after waiting to do, sample adds SINAPINIC acid (5mg/mL 50% acetonitrile of 0.5 μ l; 0.5% trifluoroacetic acid), the drying of giving free rein to.
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.
Experimental result
Use statistical method,, find that following biological marker can be used to distinguish the normal person, (table one) expressed in the variation of digestive system tumor biological marker group by serum analysis protein fingerprint peak.
Table one, differentiation normal person, the variation of digestive system tumor biological marker group are expressed
| Shaker test | Patients with Gastric Cancer | Hepatocarcinoma patient | Colorectal cancer patient |
| 1466Da(+) | 8938Da(+) | 28078Da(+)、8707Da(+) | |
| Add up to (example) | 198 | 183 | 158 |
The serum of 198 routine Patients with Gastric Cancer: the positive
The serum of 183 routine hepatocarcinoma patients: the positive
158 routine colorectal cancer patients' serum: the positive
The discovery molecular weight is that the normal person is distinguished in the variation of 1465 ± 15Da, 8938 ± 15Da, 28078 ± 15Da, 8707 ± 15Da biological marker, the variation of digestive system tumor biological marker group is expressed significant.By the discriminating at this peak, the serum of the serum of 198/208 routine Patients with Gastric Cancer, 183/226 routine hepatocarcinoma patient, 158/182 routine colorectal cancer patient's seropositivity in this experiment.This result shows that the sensitivity of this method is: cancer of the stomach 95% (198/208 example), routine liver cancer 81% (183/226), routine colorectal cancer patient 87% (158/182).
The AFP of digestive system tumor cancer of the stomach, liver cancer, colorectal cancer and CEA sensitivity are: 46% to 60%.
The ordering of embodiment 2 digestive system tumor biological marker groups is identified
With 1465 ± 15Da, 8938 ± 15Da, 28078 ± 15Da, 8707 ± 15Da biological marker 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 the biological indication marks group that biological marker is expressed for variation:
Experimental result
(A) 1465 ± 15Da:FPA-degAla (its aminoterminal of fibrinopeptide A has been removed alanine) sequence FPA fragment (1465.7Da, DSGEGDFLAEGGGVR)
(B) 8938 ± 15Da:C3a-degArg (its C end of complement C 3 has been removed arginine) sequence N end
Ser-Val-Gln-Leu-Thr-Glu-Lys-Arg-Met-Asp-Lys-Val-Gly-Lys-Tyr-Pro-Lys-Glu-Leu-Arg-Lys-Cys-Cys-Glu-Asp-Gly-Met-Arg-Glu-Asn-Pro-Met-Arg-Phe-Ser-Cys-Gln-Arg-Arg-Thr-Arg-Phe-lle-Ser-Leu-Gly-Glu-Ala-Cys-Lys-Lys-Val-Phe-Leu-Asp-Cys-Cys-Asn-Tyr-Ile-Thr-Glu-Leu-Arg-Arg-Gln-His-Ala-Arg-Ala-Ser-His-Leu-Gly-Leu-AlaC end.
(C) 28078+15Da:ApoA-I (apolipoprotein A-1) sequence:
1 DEPPQS PWDRVKDLATVYVDVLKDSGRDYVSQFEGSALGKQLNLKLLDNW
51 DSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPY
101 LDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRD
151 RARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHL
201 STLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ
The ApoA-I Molecular Mass:28078.652Da of variation, pI:5.3
(D) 8707 ± 15Da:ApoA-II (apolipoprotein A-1 I) sequence:
1 QAKEPCVESLVSQYFQTVTDYGKDLMEKVKSPELQAEAKSYFEKSKEQLT
51 PLIKKAGTELVNFLSYFVELGTQPATQ
The ApoA-II Molecular Mass:8707.921Da of variation, pI:5.1
With embodiment 1, i.e. antibody and mass spectrum associating can be saved the ordering of the biological indication marks group of variation and be identified.
The preparation of embodiment biomarker antibody 3 variations or that modify
Synthesizing of biomarker variation or that modify: synthetic FPA-degAla, C3a-degArg, the ApoA-I of variation, the ApoA-II of variation.With the biomarker 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.
The present invention relates to a kind ofly by by the biological marker of catching on the antibody group absorption surface matrix, detect with the quantitative property analysis of spectrum under the control of standardization quality controlled serum.On an antibody group matrix, catch a plurality of biological markers simultaneously, and biological marker variation or that modify of catching is carried out the mass spectrum Accurate Analysis.Can detect a plurality of biological indication marks groups simultaneously.Method of the present invention can be used for detecting the biological marker combination in the body fluid that has broken away from human body.The combination of these biological markers can be used for differentiating simultaneously the exsomatize detection method of kit of body fluid of normal person and multiple digestive system tumor patient.
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 (10)
1. one kind is gone to catch the analytical approach of biological marker in the biological sample with the matrix that contains the antibody group, it is characterized in that the method that adopts mass spectroscopy that the biological marker of having been caught by the antibody group 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) sample on matrix and multiple antibodies kit preparation, the sample;
(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) is with mass spectral standardization quality controlled serum and sample point sample on the site in the matrix of holder is arranged.Holder available metal sheet, glass sheet, potsherd, ceramic bead, magnetic beads, polymer, liquid chromatography pillar or Sepharose beads etc.Matrix is any material that can combine with antibody selectivity or specificity; 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.Water is the whole array point of washing thoroughly, the biological marker of air dry matrix and delay; Or, biological marker is eluted on mass spectrum special-purpose metal sheet or the site with the thorough washing of trifluoroacetic acid whole array point (when being holder) with ceramic bead, magnetic beads, polymer, liquid chromatography pillar or Sepharose beads.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.Use the Computer Analysis data.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. the described matrix of claim 1 is used to catch multiple antibody, and antibody is to be used to catch known biological marker.The analytical approach of described biological marker can detect biological indication marks group a plurality of, variation or that modify.
3. the analytical approach of the described biological marker of claim 1, the biological indication marks group of being caught derives from the blood that breaks away from human body.
In the methods analyst biological sample with claim 1 special biological marker to detect the method for normal and digestive system tumor disease biological marker.
5. with the method for claim 2, can detect the method for the biological marker of the biological marker of digestive system tumor disease more than three kinds and one or more amino acid variations simultaneously.
6. biological marker described in the claim 5 is: cancer of the stomach 1465 ± 15Da (its aminoterminal of fibrinopeptide A has been removed alanine), liver cancer 8938 ± 15Da (its C end of complement C 3 has been removed arginine), colorectal cancer 28078 ± 15Da (apolipoprotein A-1 of variation) and 8707 ± 15Da (the apolipoprotein A-1 I of variation).
7. the holder of claim 1 mesostroma can be sheet metal, glass sheet, potsherd, ceramic bead, magnetic beads, polymer, liquid chromatography pillar or Sepharose beads.
8. the purposes of the described biological marker of claim 6 is characterized in that, is used to prepare the kit that detects variation or modify the digestive system tumor biological sign.
9. the kit of described detection variation of claim 8 or modified biological sign is characterized in that it comprises: a container and be loaded on the described detection variation of claim 8 in the container or the antibody more than three kinds and the antibody group of modified biological sign.
10. kit as claimed in claim 9, its feature in, described antibody (group) is monoclonal antibody (group) or polyclonal antibody (group).
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102308215A (en) * | 2009-02-10 | 2012-01-04 | 株式会社日立高新技术 | Immunoanalytical method and system using mass spectrometry technology |
| CN101451975B (en) * | 2008-12-29 | 2012-01-25 | 浙江大学 | Method for detecting cancer of stomach prognosis and staging blood serum protein |
| CN102507941A (en) * | 2011-10-25 | 2012-06-20 | 北京正旦国际科技有限责任公司 | Hepatocirrhosis immuno-mass spectrometric detection reagent kit |
| CN102507936A (en) * | 2011-11-09 | 2012-06-20 | 北京正旦国际科技有限责任公司 | Multi-antibody immunomic mass spectrum kit for liver cancer marker |
| CN106872712A (en) * | 2016-12-26 | 2017-06-20 | 许洋 | In real time, the immunomic mass spectrometry kit and preparation method of trace, modified mark |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101451975B (en) * | 2008-12-29 | 2012-01-25 | 浙江大学 | Method for detecting cancer of stomach prognosis and staging blood serum protein |
| CN102308215A (en) * | 2009-02-10 | 2012-01-04 | 株式会社日立高新技术 | Immunoanalytical method and system using mass spectrometry technology |
| CN102308215B (en) * | 2009-02-10 | 2014-06-25 | 株式会社日立高新技术 | Immunoanalytical method and system using mass spectrometry technology |
| DE112010000814B4 (en) | 2009-02-10 | 2021-09-09 | Hitachi High-Tech Corporation | Immunoanalytical method and system using mass spectrometer technology |
| CN102507941A (en) * | 2011-10-25 | 2012-06-20 | 北京正旦国际科技有限责任公司 | Hepatocirrhosis immuno-mass spectrometric detection reagent kit |
| CN102507936A (en) * | 2011-11-09 | 2012-06-20 | 北京正旦国际科技有限责任公司 | Multi-antibody immunomic mass spectrum kit for liver cancer marker |
| CN102507936B (en) * | 2011-11-09 | 2013-10-23 | 北京正旦国际科技有限责任公司 | Multi-antibody immunomic mass spectrum kit for liver cancer marker |
| CN106872712A (en) * | 2016-12-26 | 2017-06-20 | 许洋 | In real time, the immunomic mass spectrometry kit and preparation method of trace, modified mark |
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Application publication date: 20080604 |