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WO2008043256A1 - Kit d'essai de détection de groupes de biomarqueurs à variantes ou modifiés avec des groupes d'anticorps et par spectrométrie de masse et procédé associé - Google Patents

Kit d'essai de détection de groupes de biomarqueurs à variantes ou modifiés avec des groupes d'anticorps et par spectrométrie de masse et procédé associé Download PDF

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Publication number
WO2008043256A1
WO2008043256A1 PCT/CN2007/002728 CN2007002728W WO2008043256A1 WO 2008043256 A1 WO2008043256 A1 WO 2008043256A1 CN 2007002728 W CN2007002728 W CN 2007002728W WO 2008043256 A1 WO2008043256 A1 WO 2008043256A1
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Prior art keywords
antibody
biomarker
biomarkers
mass spectrometry
matrix
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PCT/CN2007/002728
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English (en)
Chinese (zh)
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Yang Xu
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Yang Xu
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Publication of WO2008043256A1 publication Critical patent/WO2008043256A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/96—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood or serum control standard
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph

Definitions

  • the present invention relates to a novel method for protein analysis in biological samples, a biomarker captured by a matrix capable of binding to an antibody, and a biomarker detected by quantitatively controlled mass spectrometry.
  • the invention referred to herein relates to the field of disease detection and is a novel non-invasive method for in vitro detection. More specifically, the invention relates to biomarkers which are capable of distinguishing multiple diseases at once by antibody sets with higher specificity and sensitivity and quantitatively controlled mass spectrometry.
  • the present invention can be applied to a detection method or kit development of a combination of biomarkers in body fluids that have been detached from the human body. Background technique
  • proteome refers to "all proteins expressed by a genome", including all proteins expressed by a cell or even a living organism. .
  • Research on proteomics is at the heart of functional genomics research, called proteomics. Proteomics is considered to be the most important part of post-genomic research. Compared with the genome, the composition of the proteome is more complex, more active, and has a wider application prospect.
  • proteomics studies protein properties from the overall level of cells, such as expression levels, post-translational modifications, and interactions, and thus gains a broad understanding of disease processes, cellular physiological and biochemical characteristics, and regulatory networks. Therefore, proteomics technology is gradually becoming an important research tool in biology, medicine and pharmacy.
  • the present invention uses a combination of an antibody group and a mass spectrometer to simultaneously detect a plurality of (three or more) biomarkers.
  • blood bank screening requires the detection of known markers of common viral microbial diseases.
  • the preparation of specific binding labels and the ability to identify labeled reagents in complex mixtures requires a significant amount of time, which hinders the development of such in vitro diagnostic kit methods.
  • An antibody such as an ELISA (enzyme-l inked immunosorbent assay) kit can be used to detect a disease marker. Using antibodies and three-color immunofluorescence, up to three disease markers can be detected, but three or more disease markers cannot be detected simultaneously.
  • the combination of an antibody group and a mass spectrometer can solve the problem of simultaneously identifying three or more viruses or microbial antigen markers.
  • anti-HBV (HBsAg) antibody anti-HCV antibody
  • anti-HIV p24 antigen Rosham SG et al. Performance of a quantitative human immunodeficiency virus type 1 p24 antigen assay on various HIV-1 subtypes for the follow-up of human Immunodeficiency type 1 seropositive individuals. J Virol Methods 2003; 113: 29-34) and anti-treponemal 17 kDa protein (George R et al.
  • the present invention can detect blood donors in the window phase, which is safer than the simple ELISA antibody kit (Lau DT, et al. A rapid immunochromatographic assay for hepatitis B virus screening. J Viral Hepat 2003; 10: 331-334).
  • the method of the present invention can simultaneously detect a plurality of mutated biomarkers.
  • the combination of an antibody and a mass spectrometer is a highly sensitive, highly accurate detection method that can examine and distinguish between different components and different molecular weights (between 1 or 2 amino acids) from a mixed system of different components. Biomarker (protein). In the future, it may become a new model for many disease detection in the clinic, as The usual method of bed inspection. For example, to distinguish between serum fibrin peptide A and variant fibrin peptide A in gastric cancer, it is impossible to determine which peptide fragment is related to the pathology by using the prior methods. Now, the antibody and the mass spectrometer can be combined, and the components and their components can be used.
  • the molecular weight is clearly distinguished and a special component related to the disease is found, which is a revolution in molecular medicine.
  • the method of the invention can simultaneously detect a plurality of modified biomarkers.
  • the modified biomarker group refers to a modified protein which is methylated, acetylated, hydroxylated, phosphorylated, and the like.
  • the clinical detection of tumors has remained at the cellular level in the past, so the clinician has long been hoping for a true early diagnosis (such as solid tumors before the formation of masses, leukemia in bone marrow cell examination) It cannot be achieved without being diagnosed. Summary of the invention:
  • This invention relates to biomarkers, which can be used to simultaneously distinguish patients with one or more diseases with greater specificity and sensitivity. This approach provides a new approach to early detection of disease and provides a basis for further discovery of new variant or modified biomarkers.
  • the present invention relates to a plurality of biomarkers or a plurality of disease states for simultaneously detecting a disease by labeling a specific antibody to a surface of a substrate capable of binding to the antibody and performing quantitative mass spectrometry.
  • Biomarkers can be non-mutated, mutated, and modified.
  • a variant of a biomarker group refers to a variant protein that is increased or decreased by one or more amino acids.
  • the modified biomarker group means that the modified protein is methylated, acetylated, hydroxylated, phosphorylated, or the like.
  • the biomarkers in the present invention were discovered using a mass spectrometer.
  • the mass accuracy of the device is approximately +/- 0.1%.
  • a matrix is any substance that binds selectively or specifically to an antibody.
  • the Protein A and G substrates adsorb the function of the Fc segment of the antibody.
  • Protein A and G substrates with absorbent function bind to antibodies that bind to biomarkers in serum. After a sufficient period of time, the biomarkers can bind to the antibodies -Protein A and G. The substrate washes away unadsorbed material. Any suitable lotion can be used.
  • the biomarker can first be captured by an antibody-matrix adsorption surface that binds to the biomarker, the non-adsorbed material can be eluted from the substrate, and the biomarker adsorbed to the substrate is detected in the mass spectrometer.
  • the biomarker is ionized by an ion generating source, such as a laser, and the generated ions are felt by an ion.
  • the aggregator collects and then the mass analyzer analyzes the ions that pass through. The detector then converts the detected ion information into a mass to charge ratio.
  • Quantitative Control and Mass Spectrometry Laser Energy Control Prior to each test, standardized QC serum from mass spectrometry was used to quantify the standard peak for quantification of 4091. IDa or 6634. 0 Da intensity to 50% mass spectrometry signal intensity The maximum value.
  • the detection of biomarkers will obviously be related to the detection of signal intensity. In this way, the quantity and quality of biomarkers can be
  • the analysis of the analytes by flight mass spectrometry generates a time-of-flight spectrum.
  • the final analysis of the time-of-flight spectrum does not indicate that the ionization energy attacks a single pulse signal produced by one sample, but the sum of a series of pulsed signals. This reduces interference and increases the dynamic range.
  • This time of flight data is affected by the data processing software.
  • Data processing in software mainly includes converting time-of-flight to mass-to-charge ratio to produce mass spectrum, reducing baseline and reducing instrument offset, and filtering high frequency noise to reduce high frequency noise.
  • Data generated by adsorption and detection of biomarkers can be analyzed using computer data analysis procedures.
  • the computer program analyzes the data to display the number of detected biomarkers and displays the strength of the signal and determines the molecular weight of each biomarker being detected.
  • Data analysis can also include a series of determinations of the signal strength of the biomarker and the deviation of the corrected data from the predetermined statistical distribution.
  • the observed peaks can be normalized by calculating the height of each peak associated with certain parameters. This parameter may be an unimportant disturbance caused by the chemical composition of the instrument and similar energy absorbing molecules, which can be set to zero.
  • the computer can convert the calculation result data into various forms to express. Its standard spectrum can be expressed, but in one form only peak height and mass information can be retained in the band, producing a sharper picture and making biomarkers with nearly identical molecular weights more visible. In another format, two or more spectral comparisons facilitate the visualization of unique biomarkers and biomarkers that are above or below the calibration sample.
  • the analysis generally involves the identification of peaks in the map of the signal obtained from the analyte. Peaks can be selected from the view, software is available, and it automatically detects peaks. In general, the software operates by identifying that the signal has a signal to noise ratio above a selected threshold and marking the quality of the peak at the centroid of the peak signal. In an efficient procedure, compare many lines to identify the same peaks that appear in a selected range of the mass spectrum. One version of the software aggregates all peaks of each spectrum that occur within a defined mass range, assigning a mass (mass-to-charge ratio) cluster to all peaks near the median of the mass (mass-to-charge ratio).
  • biomarkers used in the invention are captured by antibodies. These biomarkers are further passed through mass spectrometry (mass spectrometry) Determine their different identities to determine their specific identity.
  • Electrospray ionization mass spectrometry is to apply a high voltage at the outlet of the capillary.
  • the high electric field generated causes the liquid flowing out of the capillary to be atomized into tiny charged droplets.
  • the charge intensity of the droplet surface gradually increases, and finally the droplet collapses into a large number of ions with one or more charges, causing the analyte to enter the gas phase in the form of singly or multiply charged ions.
  • the basic principle of (matrix-assisted laser desorption/ ionization time of fight mass spectrometry, MALDI-TOF-MS) is to add SINAPINIC acid and the like to the adsorption point and let it dry, disperse the analyte in the molecule and form crystals.
  • the laser illuminates the crystal the energy absorbed by the matrix molecules through the radiation causes energy to accumulate and rapidly heats up, thereby sublimating the matrix crystals, causing them to enter the gas phase along with the analyte.
  • the matrix is analyzed by mass spectrometry, and a remnant map showing the protein molecules is generated. This graph is based on the mass-to-charge ratio of the protein molecules and is displayed as peak maps of each other. from.
  • biomarkers in the present invention are identified by mass spectrometry and antibody matrices, they can be directly detected by mass spectrometry to know their specific identity. This method is more accurate than antibody-based ELISA and immunofluorescence.
  • Example 1 Application of the differential expression of serum fibrin peptide A in gastric cancer. Check the chemical structure of the normal fibrin peptide A molecule in the known genomic or cDNA library database (16 amino acids from the N-terminus to the C-terminus with a molecular weight of 1536 Da):
  • the fibrin peptide A antibody was used in combination with a mass spectrometer to find fibrin peptide A having a molecular weight of 1465 + 1 Da.
  • the chemical structure and molecular weight can be arranged from the N-terminus to the C-terminus to 15 amino acids:
  • Antibody-based ELISA and immunofluorescence methods cannot distinguish between serum fibrin peptide A and variant fibrin peptide A in gastric cancer.
  • these biomarkers can also be identified by, for example, determining the amino acid sequence of the polypeptide.
  • determining the amino acid sequence of the polypeptide In protein chemistry and proteomics research, in order to increase protein identification The credibility of obtaining a piece of internal sequence information of a protein peptide fragment is usually very important.
  • the traditional method is to use the Edman degradation method, and the biggest disadvantage of this method is that it takes too long (a residue takes 30-40 minutes).
  • mass spectrometry technology especially the development of multi-stage mass spectrometry (MS/MS) and post-source cleavage (PSD) technologies, the application of mass spectrometry has become a popular method.
  • a biomarker can be characterized by a number of enzymes, such as V8 protease or trypsin, and the molecular weight of the digestion fragments can be used to search for sequences in the database, which are generated by multiple enzymes. The molecular weight of the digested fragments is consistent.
  • a degradation probe can be used based on the N-terminal Amino Acid Sequence of the biomarker, and then these probes are used Describe the genomic or cDNA library generated by the sample from which the biomarker was detected.
  • protein biomarkers can be sorted using protein ladder sequencing.
  • Protein ladders can be generated by breaking the molecules into pieces and treating them with enzymatic or other methods that remove a single amino acid molecule from the ends of the fragments in sequence. This gradient was then analyzed by mass spectrometry. The difference in mass between the ladder fragments identifies the amino acids that are removed from the end of the molecule. Therefore, the present invention can be applied to the gold standard for biomarker identification.
  • Specificity refers to the specific property of a substance or disease, which is a characteristic of a substance or a disease.
  • a certain substance or disease can be identified by certain characteristics to distinguish it from other substances or diseases.
  • the identification of proprietary features often relies on specific detection methods, such as the detection of the presence or absence of specific antigens in a disease. Since the new development of proteinomics research, this traditional method of specific detection and definition has made a great breakthrough.
  • Different fragment variations such as a protein are markers of different types of tumors. According to the complex process of gene-to-protein expression, the product-protein of a specific gene must have the expression of related multi-component proteins.
  • this map (such as a tumor) is compared with other maps (such as normal people or other diseases) to identify this specific protein. (such as tumor antigens or fragments thereof) to distinguish normal people from patients with certain diseases.
  • kits and methods for detecting variants or modifying biomarker populations by antibody sets and mass spectrometry The following is an example of an operating protocol and kit provided by the present invention.
  • the biological sample is diluted in a dilution buffer and the sample is clarified by centrifugation as needed.
  • the standardized quality control serum preparation definition of mass spectrometry meets the following criteria: 10 donors, 5 males and 5 females, blood type 0; age 18-30 years; ethnic Han.
  • the biochemical indicators were normal, including: total cholesterol, triglycerides, fasting blood glucose, hepatitis B surface antigen, liver function test, renal function test; family history without genetic disease; no major infectious disease history. Women cannot be pregnant, and men are not smokers.
  • the normalized quality control serum and samples of the mass spectra were spotted at a site in the matrix with the support.
  • Samples come from blood, body fluids, secretions, cell lysates, tissue lysates and organ lysates.
  • the support may be a metal piece, a glass piece, a ceramic piece, a ceramic bead, a magnetic bead, a polymer, a liquid chromatography column or a Sepharose beads.
  • the matrix is used to label and bind a variety of antibodies.
  • the antibody is a monoclonal antibody or a polyclonal antibody.
  • the antibody group is increased indefinitely to achieve unlimited detection of multiple or multiple biomarkers or antigen markers (only molecular weight differences in biomarkers are within the mass spectrometric error rate).
  • the synthetic mutated or modified biomarker is immunized to the mouse, and after the immune response occurs, the B cells are isolated from the peripheral blood.
  • the monoclonal antibody strain with the highest titer was screened by ELISA, prepared in large quantities, and the desired antibody was extracted from the culture supernatant.
  • This antibody can be used to prepare kits for detecting variants or modifying biomarkers.
  • the method of preparing a kit by combining a matrix with a plurality of antibodies can be carried out by any method capable of binding to an antibody and any substance capable of selectively or specifically binding to the antibody, for example: using proteins A and G
  • the substrate on the labeled Sepharose beads binds to the Fc portion of the antibody; the substrate on the magnetic beads with the carboxylate-group label is bound to the amino-groups of the antibody by the Carbodiimide method (Carbodiimide Method) Gunn DL, et al. Preparation of sensitive and stable erythrocytes by the carbodi imide method for the detection of primary and secondary IgM and IgG antibody. J Immunol Methods. 1972 ; 1 (4) : 381-389. ); labeled with streptavidin The matrix binds to the biotin-labeled antibody; the MEP HyperCel-labeled ceramic beads bind to the antibody
  • the matrix labeled with antibodies to the liquid chromatography column can be analyzed by standard methods of liquid chromatography mass spectrometry (LC-MS). Standardized quality control serum for mass spectrometry can be used in a quantitative method for mass spectrometry kits. 3. Washing
  • wash with binding buffer The first wash solution was added to the site before the sample was completely dried. The wash solution was left at the site for at least 10 seconds. Thoroughly remove the first wash solution and repeat the above steps with the second wash. There are different steps below:
  • the energy absorbing molecule may be Sinapinic acid or alpha- Cyano - 4_hydroxycinnamic acid.
  • a laser desorption/ionization time-of-flight mass spectrometer analyzing the array with a nitrogen laser (337 nm) and an 80 cm or 120 cm flight tube, or electrospray ionization of the eluted biomarker followed by a liquid chromatography mass spectrometer (LC-MS) Standard method to analyze biomarkers or proteins that are retained at each point.
  • Tandem quadrupole mass spectrometry or linear ion trap mass spectrometry was used to identify modified and variant biomarkers and sequencing of the peptide de novo. Analyze data using a computer for data overlay display.
  • Quantitative mass spectrometry Before each test, the intensity of the standard peak 4091. IDa or 6634. 0 Da used for quantification in the standardized quality control serum was adjusted to the maximum value of 50% signal intensity using the standardized quality control serum of the mass spectrometer.
  • kits and methods of the present invention have the following characteristics compared to other non-invasive in vitro assay methods -
  • a feature of a method for accurately detecting multiple biomarkers using a combination of multiple antibodies and mass spectrometry is the ability to The analyte is accurately resolved in a complex sample mixture. The accuracy of antibody binding to antigen exceeds
  • Mass spectrometry has a high degree of accuracy, and the general error rate is only 0.1 Da. Since proteins are composed of amino acids, and the average mass of amino acids is known, if the total molecular weight of the antigen or biomarker is known, the variation of the antigen (referred to as amino acid changes) can be easily inferred. However, ELISA and immunofluorescence kits are not known for antigenic variation. Therefore, the combination of antibodies and mass spectrometry to accurately detect biomarkers provides direct information on the chemical or structural characteristics of the analyte (antigen or biomarker).
  • the fibrin peptide A molecule is known to have a molecular weight of 1536 Da and a chemical structure of 16 amino acids (N-terminal Ala-Asp- Ser_Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu_Gly-Gly-Gly-Val- Arg C end).
  • the fibrin peptide A antibody was combined with a mass spectrometer to find fibrin peptide A having a molecular weight of 1536 Da. Then 100% determines that the analyte is fibrin peptide A, the most accurate identification (gold standard).
  • the antibody with fibrin peptide A was used in combination with a mass spectrometer to find that the analyte was a fiber egg with a molecular weight of 1465 soil 1 Da variant.
  • the chemical structure can be (from the N-terminus to the C-terminus) presumed to be 15 amino acids: N-terminal
  • Multiple or more antibody markers and one or more variant or modified biomarkers can be detected simultaneously using three or more antibody sets on a substrate in combination with mass spectrometry.
  • Three-color immunofluorescence can simultaneously analyze three biomarkers, but cannot achieve analysis of more than three biomarkers or combine antibodies and mass spectrometry to accurately and efficiently determine a variant or modified analyte (antigen or Biomarker).
  • the matrix can be any material that binds selectively or specifically to the antibody.
  • variant or modified biomarkers can be known without sequencing the protein. Unlike immunofluorescence kits, one kit can simultaneously label up to three antibodies, not Know "variant or modified biomarkers.” Modified biomarkers refer to high or low expression changes in methylation, acetylation, hydroxylation, phosphorylation, and the like.
  • the direct error analysis of the mass spectrometer is very accurate, and the general error rate is only 0.1 Da. For example, 4302 Da positively charged proteins were found in the serum of liver cancer, while in normal people there were only 4287 Da positively charged proteins.
  • the molecular weight difference between the two proteins is 15 Da, which is methyl (-CH3).
  • the deproteinase was used to verify that the 4302 Da protein was methylated 4287 Da protein, ie, under the treatment of demethylase, the 4302 Da protein was converted to 4287 Da protein.
  • the modified protein can be verified by demethylase, deacetylase, dehydroxylation enzyme, dephosphorylation enzyme.
  • the application of the methylation kit to tumor cell detection can be used to distinguish between normal human samples and cancer patient population samples.
  • a plurality of antibody kit methods provided by the present invention are used for simultaneous detection by mass spectrometry, and are not limited to three antibodies. This can help clinically complex detection, such as the use of this method for disease detection kits, forensic analysis, population gene mutation analysis, tuberculosis resistant strain changes in the population and so on. detailed description
  • Specimen source A. Serum of 50 normal controls; B. 50 patients with gastric cancer without lymph node invasion; C. 50 patients with gastric cancer with lymph node invaders.
  • Quality Control A. Human standardized quality control serum
  • Sample collection After the whole blood is collected, the serum is taken and stored at 80 °C. - The serum sample is taken out in the refrigerator at 80 °C, and melted on the ice box; centrifuged at 10,000 rpm, 4 °C. Minute; take the supernatant.
  • Serum 3 ⁇ l is required for each adsorbent support point, and the serum is diluted with 2 volumes of buffer (for example: 3 ⁇ l of serum diluted with 6 ⁇ l buffer). Mix the sample thoroughly. 9 ⁇ l of the above sample was added to the corresponding binding buffer of ⁇ so that the total dilution of the serum reached about 40 times. Will be The prepared serum sample 40 ⁇ 1 was loaded onto the adsorbent.
  • Sample detection Load, add 40 ⁇ 1 treated sample to the adsorbent support (antibody with labeled fibrin peptide ⁇ ), set the oscillator, 400-600 rpm, shake for 1 hour at 4 °C . The sample was decanted and 200 ⁇ l of binding buffer was added to each well. The oscillator was placed at room temperature for 400-600 rpm, shaken for 5 minutes, the liquid in the well was removed, and the binding buffer 200 ⁇ 1 was added again, and the operation was repeated once. 200 ⁇ l of HPLC water was added to each well and immediately removed. After removing the adsorbent support, after the sample was dried, the sample was added with 0.5 ⁇ M of SINAPINIC acid (5 mg/mL 50% acetonitrile; 0.5% trifluoroacetic acid), and allowed to dry naturally.
  • SINAPINIC acid 5 mg/mL 50% acetonitrile; 0.5% trifluoroacetic acid
  • Table 1 distinguish normal people, early gastric adenocarcinoma without lymph node invasion and gastric adenocarcinoma patients with lymph node violation
  • the fibrin peptide A antibody was used in combination with a mass spectrometer to find fibrin peptide A having a molecular weight of 1465 ⁇ 1 Da.
  • the chemical structure and molecular weight can be arranged from the N-terminus to the C-terminus to 15 amino acids:
  • the 1465 + 1 Da biomarkers were sorted by multi-stage mass spectrometry (MS/MS), post-source lysis (PSD), and protein ladder sequencing. Protein ladders can be generated by breaking the molecules into fragments. This gradient is then analyzed by mass spectrometry.
  • the 1465 + 1 Da biomarker was identified as Fibrinopeptide A with alanine truncation at N-terminal. Its chemical structure is (15 amino acids arranged from the N-terminus to the C-terminus):
  • Example 1 Application of the variation of serum fibrin peptide A in gastric cancer, that is, the combination of antibody and mass spectrometry, can eliminate the sorting and identification of the fibrin peptide A which is mutated.
  • Example 3 Preparation of Mutated or Modified Biomarker Antibodies
  • Transthyretin fragment (2380. 1 Da, LGISPFHEHAEVVFTANDSGPR), Bradykinin fragment (904. 5 Da, RPPGFSPF), Methyl- Arg- Bradykinin (1075. 2 Da, Methyl- RPPGFSPFR), Dehydro-Ala-FPA (1518. 7 Da), FPA fragment (1465. 7 Da, DSGEGDFLAEGGGVR), Fibrinogen alpha fragment (3190. 4 Da, SSSYSKQFTSSTSYNRGDSTFESKSYKM), Apo E fragment (2409. 1 Da, AATVGSLAGQPLQERAQAWGERL), C3f fragment ( 1865. 0 Da, SSKITHRIHWESASLL).
  • the synthetic mutated or modified biomarker is used to immunize the mouse, and after the immune response occurs, the B cells are isolated from the peripheral blood.
  • a single lymphocyte capable of secreting the desired antibody is selected and isolated by hemolytic plaque assay.
  • a single cell was expanded to 1 ⁇ 10 7 or more, and mRNA was extracted using a Quick mRNA Purification Kit.
  • the cDNA strand was synthesized using the extracted mRNA as a template.
  • a murine antibody heavy chain variable region ( VH ) universal primer and a light chain variable region (V) universal primer were added to carry out polymerase chain reaction to obtain an amplified VH gene fragment and gene fragment.
  • the amplified product was identified and isolated by electrophoresis on a 15 g/L agarose gel.
  • the amplified VH gene fragment and V gene fragment were recovered using glass milk. Mix with the Timol Primer Mix and perform polymerase chain reaction to connect V H and VL. After isolation and purification of the amplified product, a specific single chain antibody (ScFV) was obtained. This ScFV can be used to prepare a DNA piece for detection.
  • the amplified product was ligated with restriction enzyme sites, purified and quantified, and ligated to the P ue19 vector.
  • the ligation product was transformed into E. coli T0P10.
  • the recombinant plasmid was screened by blue-white spot screening and restriction enzyme digestion.
  • Monoclonal antibodies were formed in 96-well plates.
  • the monoclonal antibody strain with the highest titer was screened by ELISA, prepared in large quantities, and the desired antibody was extracted from the culture supernatant. This antibody can be used to prepare a kit for testing.
  • Example 4 Accurate detection of multiple, variant or modified biomarkers using a combination of multiple antibodies and mass spectrometry
  • Specimen source Standardized quality control serum for mass spectrometry. ⁇ group (plus Transthyretin
  • Bradykinin Trans group (Transthyretiru Transthyretin fragment), group C (Bradykinin ⁇ Bradykinin fragment), group D (FPA, Dehydro-Ala-FPA, FPA fragment, Bradykinin, Methyl- Arg- Bradykinin), E Group (Fibrinogen alpha> Fibrinogen alpha fragment C3f, C3f fragment, Bradykinin fragment), group F (Apo E, Apo E fragment, C3f, C3f fragments FPA fragments Bradykinin, Fibrinogen alpha).
  • Reagents Transthyretin (2451.1 Da, ALGISPFHEHAEVVFTANDSGPR), Transthyretin fragment (2380.1 Da, LGISPFHEHAEVVFTANDSGPR), Bradykinin (1060.6 Da, RPPGFSPFR), Bradykinin fragment (904.5 Da, RPPGFSPF), Methyl- Arg- Bradykinin (1075.2 Da, Methyl-RPPGFSPFR ) , FPA (1536. 7 Da, ADSGEGDFLAEGGGVR) , Dehydro - Ala- FPA (1518. 7 Da) , FPA fragment (1465. 7 Da, DSGEGDFLAEGGGVR) , Fibrinogen alpha (3261.
  • Transthyretin antibodies Bradykinin antibodies, FPA antibodies, Fibrinogen alpha antibodies, Apo E antibodies, C3f antibodies are all derived from synthesis, preparation or donation.
  • the matrix is combined with a variety of antibodies to prepare a kit, sample loading
  • the sample was spotted on a site in the matrix with the support.
  • the support uses magnetic beads. Take 50 ⁇ l of magnetic beads labeled with carboxy late-groups in a 500 ⁇ tube. Using the Carbodiimide method, the magnetic beads on the substrate with the carboxylate-groups label and the amino terminus of various antibodies (Transthyretin antibody, B dykinin antibody, FPA antibody, Fibrinogen alpha antibody, Apo E antibody, C3f antibody) (ami no-groups) (Gunn DL, et al. Preparation of sensitive and stable erythrocytes by the carbodiimide method for the detection of primary and secondary IgM and IgG antibody. J Immunol Methods. 1972; 1 : 381-389.).
  • the multi-antibody magnetic bead kit is available for mass spectrometry. Standardized quality control serum from mass spectrometry was used for quantification of mass spectrometry.
  • the first wash solution was added to the site before the sample was completely dried.
  • the wash solution was left at the site for at least 10 seconds. Thoroughly remove the first wash solution and repeat the above steps with the second wash.
  • the entire array was thoroughly washed with 1% trifluoroacetic acid, and the biomarker was eluted onto a mass spectrometer (with 3 x 3 ⁇ round holes), and the metal piece was naturally dried, and 0.5 LSinapinic acid energy absorbing molecule (50% acetonitrile) was added. , 0. 5% trifluoroacetic acid prepared saturated standard solution).
  • Quantitative mass spectrometry Prior to each test, the standard peak 4091. IDa or 6634. 0 Da intensity used for quantification in standardized quality control serum was adjusted to the maximum of 50% signal intensity using standardized quality control serum from mass spectrometry.
  • Fibrinogen alpha Fibrinogen alpha 3190. 4 Da ( + )
  • the present invention utilizes a plurality of antibodies and mass spectrometry to perform protein contrast analysis on the A to F groups, and the results show that the sensitivity of the group detection is 100% and the specificity is 100%.
  • the present invention combines an antibody group and a mass spectrometer to simultaneously detect multiple, mutated (groups A to F) or modified (group D) biomarkers.
  • the present invention relates to a method for detecting a biomarker captured on a surface substrate by an antibody group and performing quantitative mass spectrometry under the control of standardized quality control serum. Simultaneous capture of multiple biomarkers on an antibody set matrix and accurate mass spectrometry analysis of captured variant or modified biomarkers. Multiple biomarker populations can be detected simultaneously.
  • the method of the present invention can be used to detect biomarker combinations in body fluids that have been detached from the body. These biomarker combinations can be used in a kit for simultaneously identifying a normal human body and a plurality of patient body fluids. All documents mentioned in the present application are incorporated by reference in the present application as if each document is individually incorporated by reference. In addition, it should be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

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Abstract

L'invention concerne un kit d'essai pour détecter les groupes de biomarqueurs à variantes ou modifiés avec des groupes d'anticorps et par spectrométrie de masse, et son procédé. Les groupes d'anticorps sont absorbés sur les biomarqueurs de capture de substrat, qui sont détectés par spectrométrie de masse dans le contrôle de sérum de contrôle de qualité standardisée. Un groupe d'anticorps peut capturer simultanément de multiples biomarqueurs. Les biomarqueurs à variantes ou modifiés capturés sont analysés par spectrométrie de masse destinée à détecter simultanément de multiples groupes de biomarqueurs.
PCT/CN2007/002728 2006-10-08 2007-09-17 Kit d'essai de détection de groupes de biomarqueurs à variantes ou modifiés avec des groupes d'anticorps et par spectrométrie de masse et procédé associé WO2008043256A1 (fr)

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CN2006101406520A CN101158666B (zh) 2006-10-08 2006-10-08 一种用含有抗体组的基质去捕获生物样品中生物标志的分析方法

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106872712A (zh) * 2016-12-26 2017-06-20 许洋 实时、痕量、修饰性标志物的免疫质谱试剂盒及制备方法

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102308216B (zh) * 2009-02-09 2014-07-09 罗切格利卡特公司 免疫球蛋白糖基化模式分析
CN105254715B (zh) * 2015-10-16 2018-05-08 上海市肺科医院 结核病检测多肽及其应用
CN106872630B (zh) * 2017-03-29 2018-07-24 山东大学 与重度少弱精子症相关的生物标志物的筛选与应用
CN109293762A (zh) * 2018-10-17 2019-02-01 湖北民族学院 DRC3f抗原多肽，抗DRC3f的多克隆抗体及应用
US20230314427A1 (en) * 2019-06-14 2023-10-05 Arizona Board Of Regents On Behalf Of Arizona State University Detection of antigens
CN112745374B (zh) * 2019-10-31 2022-11-25 中国科学院生物物理研究所 一种血液样本质量评估方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6814870B2 (en) * 2001-02-20 2004-11-09 Advion Biosciences, Inc. Microchip electrospray device and column with affinity adsorbents and use of the same
CN1614423A (zh) * 2003-11-05 2005-05-11 许洋 用生物芯片及质谱仪联合检测多种病毒微生物标记的用途
CN1657941A (zh) * 2004-02-19 2005-08-24 许洋 用抗体吸附表面物质及质谱联合检测多种抗原标记的用途
CN1714145A (zh) * 2002-10-03 2005-12-28 诺曼·利·安德森 用质谱法高灵敏度定量肽

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1537170A (zh) * 2000-11-20 2004-10-13 ����ҽѧԺ 定量检测前列腺特异性膜抗原和其它前列腺标记的方法和装置

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6814870B2 (en) * 2001-02-20 2004-11-09 Advion Biosciences, Inc. Microchip electrospray device and column with affinity adsorbents and use of the same
CN1714145A (zh) * 2002-10-03 2005-12-28 诺曼·利·安德森 用质谱法高灵敏度定量肽
CN1614423A (zh) * 2003-11-05 2005-05-11 许洋 用生物芯片及质谱仪联合检测多种病毒微生物标记的用途
CN1657941A (zh) * 2004-02-19 2005-08-24 许洋 用抗体吸附表面物质及质谱联合检测多种抗原标记的用途

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106872712A (zh) * 2016-12-26 2017-06-20 许洋 实时、痕量、修饰性标志物的免疫质谱试剂盒及制备方法

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