WO2004103155A2 - Diagnostic medical base sur une ionisation/desorption laser avec exaltation en surface - Google Patents

Diagnostic medical base sur une ionisation/desorption laser avec exaltation en surface Download PDF

Info

Publication number
WO2004103155A2
WO2004103155A2 PCT/US2004/015210 US2004015210W WO2004103155A2 WO 2004103155 A2 WO2004103155 A2 WO 2004103155A2 US 2004015210 W US2004015210 W US 2004015210W WO 2004103155 A2 WO2004103155 A2 WO 2004103155A2
Authority
WO
WIPO (PCT)
Prior art keywords
antibodies
disease
sample
probe
antigens
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2004/015210
Other languages
English (en)
Other versions
WO2004103155A3 (fr
Inventor
Rebecca E. Caffrey
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Aspira Womens Health Inc
Original Assignee
Ciphergen Biosystems Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ciphergen Biosystems Inc filed Critical Ciphergen Biosystems Inc
Publication of WO2004103155A2 publication Critical patent/WO2004103155A2/fr
Anticipated expiration legal-status Critical
Publication of WO2004103155A3 publication Critical patent/WO2004103155A3/fr
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • 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
    • G01N33/6851—Methods of protein analysis involving laser desorption ionisation mass spectrometry

Definitions

  • Gas phase ion spectrometer refers to an apparatus that detects gas phase ions.
  • Gas phase ion spectrometers include an ion source that supplies gas phase ions.
  • Gas phase ion spectrometers include, for example, mass spectrometers, ion mobility spectrometers, and total ion current measuring devices.
  • Gas phase ion spectrometry refers to the use of a gas phase ion spectrometer to detect gas phase ions.
  • Mass spectrometer refers to a gas phase ion spectrometer that measures a parameter that can be translated into mass-to-charge ratios of gas phase ions.
  • the phrase includes molecules used in MALDI, frequently referred to as “matrix”, and explicitly includes cinnamic acid derivatives, sinapinic acid (“SPA”), cyano-hydroxy-cinnamic acid (“CHCA”) and dihydroxybenzoic acid, ferulic acid, hydroxyacetophenone derivatives, as well as others. It also includes EAMs used in SELDI. SEND is further described in United States patent 5,719,060 and United States patent application 60/408,255, filed September 4, 2002 (Kitagawa, "Monomers And Polymers Having Energy Absorbing Moieties Of Use In Desorption/ionization Of Analytes").
  • the "complexity" of a sample adsorbed to an adsorption surface of an affinity capture probe means the number of different protein species that are adsorbed.
  • Biochip refers to a chip to which a chemical moiety is attached.
  • the surface of the biochip comprises a plurality of addressable locations, each of which location has the chemical moiety attached there.
  • These protein biochips comprise an aluminum substrate in the form of a strip. The surface of the strip is coated with silicon dioxide.
  • silicon oxide functions as a hydrophilic adsorbent to capture hydrophilic proteins.
  • H4, H50, SAX-2, Q-10, WCX-2, CM-10, IMAC-3, IMAC-30, PS-10 and PS-20 biochips further comprise a functionalized, cross-linked polymer in the form of a hydrogel physically attached to the surface of the biochip or covalently attached through a silane to the surface of the biochip.
  • the H4 biochip has isopropyl functionalities for hydrophobic binding.
  • the H50 biochip has nonylphenoxy- poly(ethylene glycol)methacrylate for hydrophobic binding.
  • the SAX-2 and Q-10 biochips have quaternary ammonium functionalities for anion exchange.
  • the WCX-2 and CM-10 biochips have carboxylate functionalities for cation exchange.
  • the PG-20 biochip is a PS-20 chip to which Protein G is attached.
  • the LSAX-30 (anion exchange), LWCX-30 (cation exchange) and IMAC-40 (metal chelate) biochips have functionalized latex beads on their surfaces. Such biochips are further described in: WO 00/66265 (Rich et al., "Probes for a Gas Phase Ion Spectrometer," November 9,
  • Optical methods include, for example, detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, birefringence or refractive index (e.g., surface plasmon resonance, ellipsometry, a resonant mirror method, a grating coupler waveguide method or interferometry).
  • Optical methods include microscopy (both confocal and non-confocal), imaging methods and non-imaging methods.
  • Immunoassays in various formats e.g., ELISA
  • Electrochemical methods include voltametry and amperometry methods.
  • Radio frequency methods include multipolar resonance spectroscopy.
  • Polynucleotide and “nucleic acid” equivalently refer to a naturally- occurring or synthetic polymer comprising nucleotide monomers (bases).
  • Polynucleotides include naturally-occurring nucleic acids, such as deoxyribonucleic acid (“DNA”) and ribonucleic acid (“RNA”), as well as nucleic acid analogs.
  • Nucleic acid analogs include those which include non-naturally occurring bases, and those in which nucleotide monomers are linked other than by the naturally-occurring phosphodiester bond.
  • Nucleotide analogs include, for example and without limitation, phosphorothioates, phosphorodithioates, phosphorotriesters, phosphoramidates, boranophosphates, methylphosphonates, chiral -methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs), and the like.
  • Ligand refers to any compound that can participate in specific binding with a designated receptor or antibody.
  • Antibody refers to a polypeptide substantially encoded by at least one immunoglobulin gene or fragments of at least one immunoglobulin gene, that can participate in specific binding with a ligand.
  • the term includes naturally-occurring forms, as well as fragments and derivatives. Fragments within the scope of the term as used herein include those produced by digestion with various peptidases, such as Fab, Fab' and F(ab)'2 fragments, those produced by chemical dissociation, by chemical cleavage, so long as the fragment remains capable of specific binding to a target molecule, such as an antigen indicative of a disease.
  • a specific binding interaction will discriminate over adventitious binding interactions in the reaction by at least two-fold, more typically more than 10- to 100-fold.
  • specific binding is sufficiently discriminatory when determinative of the presence of the analyte in a heterogeneous (inhomogeneous) sample.
  • the affinity or avidity of a specific binding reaction is least about 10 "7 M, with specific binding reactions of greater specificity typically having affinity or avidity of at least 10 "8 M to at least about 10 "9 M.
  • the term "attached,” as used herein, encompasses interactions including, but not limited to, covalent bonding, ionic bonding, chemisorption, physisorption, and combinations thereof.
  • the term “disease” refers to an impairment of the normal state of a living organism, such as an animal, or one of its parts that interrupts or modifies the performance of vital functions and is a response, e.g., to environmental factors (e.g., as allergens, malnutrition, industrial hazards, climate, etc.), to specific infectious agents (e.g., bacteria, viruses, etc.), to inherited defects (e.g., genetic anomalies, etc.), or to combinations of these factors.
  • environmental factors e.g., as allergens, malnutrition, industrial hazards, climate, etc.
  • specific infectious agents e.g., bacteria, viruses, etc.
  • inherited defects e.g., genetic anomalies, etc.
  • autoimmune disease refers to a disease produced by an abnormal immune response against self-antigens of a subject.
  • infectious disease refers to a disease resulting from a pathogen, or a portion thereof, that infects or is otherwise introduced into a subject.
  • prion disease refers to a disease caused by a prion.
  • the present invention relates to the qualitative and quantitative detection of antibodies in samples derived from subjects to aid in the diagnosis and prognosis of disease. More specifically, the multiplexed detection methods of the invention combine the specificity of, e.g., antigen-antibody interaction assays with the resolving power and sensitivity of surface enhanced laser desorption/ionization (SELDI) gas phase ion spectrometry, including SELDI-mass spectrometry. In addition to greater sensitivity, the methods of the present invention can be performed with significantly higher throughput than preexisting approaches, such as RIAs, EIAs, and other immunoassays.
  • SELDI surface enhanced laser desorption/ionization
  • the methods of the present invention include a method of aiding in a disease diagnosis.
  • the method includes (a) capturing one or more antibodies, if any, present in at least one sample derived from a subject on a probe with one or more antigens indicative of the disease, which antigens specifically bind the antibodies.
  • a plurality of different antigens, each indicative of the disease are utilized, e.g., to provide added diagnostic assurance or verification.
  • the method also includes (b) detecting the captured antibodies, if any, by at least one version of SELDI gas phase ion spectrometry to provide antibody capture data.
  • the method also includes (c) correlating the antibody capture data with a probable diagnosis of the disease or a negative diagnosis for the subject.
  • the invention also provides a method of aiding in a disease prognosis, which includes (a) profiling at least a first sample derived from a subject diagnosed with a disease and (b) profiling at least a second sample derived from the subject diagnosed with the disease.
  • the profiling of (a) and (b) includes detecting antibodies in the first and second samples using the method described above.
  • the second sample is typically derived from the subject at a time that is subsequent to the first sample being derived from the subject, e.g., during course of treatment to monitor the efficacy of the treatment.
  • the method also includes (c) comparing the relative amounts of the antibodies in the first and second samples detected by profiling to aid in the prognosis of the disease for the subject.
  • any disease can be diagnosed or prognosticated using essentially any antigen that is indicative of the particular disease under consideration with the methods described herein. Accordingly, no attempt is made in this disclosure to describe all of the possible diseases, and antigens indicative thereof, that are the subject of the present invention. Based upon the description provided herein, one skilled in the art will readily appreciate how the methods, probes, and kits of the invention can be adapted to the specific disease to be diagnosed or prognosticated. Nonetheless, certain diseases and indicative antigens are described or referred to for purposes of illustration, but not to limit the present invention. In particular, certain general disease classifications that can be analyzed according to the methods of the invention include, e.g., autoimmune diseases, infectious diseases, prion diseases, and the like.
  • the antigens used in the methods of the invention are generally selected from, e.g., organic molecules, inorganic molecules, allergens, biomolecules, nucleic acids, proteins, peptides, peptide nucleic acids, prions, haptens, hapten-carrier conjugates, carbohydrates, lipids, and the like.
  • Exemplary autoimmune diseases that can be diagnosed or prognosticated using the methods of the invention include, e.g., Systemic Lupus Erythematosis (SLE), Systemic Rheumatic Disease, rheumatoid arthritis, diabetes, Sj ⁇ gren's Syndrome (SS), Progressive Systemic Sclerosis (PSS), Subacute Erythematosis, congenital complete heart block, neonatal complete heart block, Neonatal Lupus Dermatitis, Polymyositis, thyroid autoimmune disorders, mixed connective tissue disease (MCTD), Multiple Sclerosis (MS), and the like.
  • SLE Systemic Lupus Erythematosis
  • SLE Systemic Rheumatic Disease
  • rheumatoid arthritis diabetes
  • Sj ⁇ gren's Syndrome SS
  • PSS Progressive Systemic Sclerosis
  • Subacute Erythematosis congenital complete heart block
  • neonatal complete heart block Neonatal Lupus Dermatitis
  • Polymyositis Polymyositis
  • ssDNA Fc portions of human antibodies (rheumatoid factor), whole histones, and histone subclasses (e.g., distinct molecular fractions) have been used for detecting or evaluating systemic rheumatic disease.
  • Antibodies to the Sm antigen are typically less commonly found in patients with other rheumatic diseases.
  • Antibodies to ribosomal nuclear proteins (nRNP) have also been found in patients with rheumatoid arthritis, SS, PSS, and MCTD. Twenty to thirty percent of the patients with antibodies to Scl-70 antigen have progressive Systemic Sclerosis. Antibodies to Scl-70 are rarely found in patients with other systemic rheumatic diseases.
  • Antibodies to Ro (SS-A) antigen are found in half of Systemic Lupus Erythematosis patients, most patients with SS or Subacute Lupus Erythematosis and nearly all mothers of infants with congenital complete heart lock or Neonatal Lupus Dermatitis.
  • Antibodies to the La (SS-B) antigen usually occur in twenty to thirty percent of SS patients and with five to ten percent of SLE patients.
  • Antibodies to Jo-1 antigen are usually found in patients with polymyositis.
  • Antibodies to Ribosomal P antigens are found to occur in five to ten percent of systemic Lupus Erythematosis patients and ninety percent of those patients typically demonstrate signs of lupus psychosis.
  • Antibodies to mitochondrial antigens are typically found in all primary biliary cirrhosis patients.
  • Antibodies to histone antigens HI, H2A, H2B, H3, H4 are found in ninety-five to one hundred percent of drug-induced Lupus Erythematosis, fifteen to twenty percent rheumatoid arthritis, and thirty percent of all patients with Systemic Lupus Erythematosis.
  • Antibodies to cytoplasmic components of neutrophil granulocytes are present in the serum of patients with acute Wegener's granulomatosis and microscopic polyarteritis. Myeloperoxidase and proteinase 3 are the two major antigens present.
  • Exemplary infectious diseases that are optionally diagnosed or prognosticated using the methods of the present invention include those caused by bacterial pathogens, such as Systematic Lyme disease caused by an infection with Borrelia burgdorferi transmitted by the bite of an infected tick of the genus Modes, Leptospirosis caused by spirochetes of the genus Leptospira, and the like.
  • Antibodies to HMW (P83-100), Flagellin (P41), BmpA (P39), and OspC antigens are typically found in subjects with Systematic Lyme disease, whereas antibodies to serovar patoc 1 strain antigens are generally found in patients with Leptospirosis.
  • HTV Human Immunodeficiency Virus
  • SARS Severe Acute Respiratory Syndrome
  • HCV Severe Acute Respiratory Syndrome
  • Antigens indicative of certain of these viral pathogens include, e.g., HTLV, HCV, EBV, HTV, CMV HbsAg, Hbc, Hepatitis Surface, core, HJVI/I, HTL V ⁇ , Hepatitis C, and HJV- lp24 antigens.
  • HTV Human Immunodeficiency Virus
  • SARS Severe Acute Respiratory Syndrome
  • TSE neurodegenerative disorders that affect both humans and animals.
  • Prion diseases are referred to as sponiform encephalopathies due to the characteristic of forming holes or pores in cranial tissue.
  • Development of prion disease may be the result of mutations in the PrP gene.
  • Inherited prion diseases include Creutzfeldt- Jakob disease (CJD), fatal familial insomnia (FFI) and Gerstmann-Straussler-Scheinker syndrome or disease (GSS) in humans.
  • CJD Creutzfeldt- Jakob disease
  • FFI fatal familial insomnia
  • GSS Gerstmann-Straussler-Scheinker syndrome or disease
  • Prion diseases can also be contracted by an infectious mechanism.
  • This group of diseases includes iatrogenic CJD and a new variant of CJD, which may be the result of transmission of bovine spongiform encephalopathy (BSE, also referred to as “Mad Cow” disease) from cattle to humans.
  • BSE bovine spongiform encephalopathy
  • Prion diseases are described further in, e.g., U.S. Pat. No. 6,528,269, entitled “IMMUNOLOGICAL AGENTS SPECIFIC FOR PRION PROTEIN (PRP),” issued March 4, 2003 to Sy et al.
  • antigens used in the diagnostic and prognostic methods of the invention are obtained from essentially any source.
  • antigens are optionally chemically synthesized, obtained via recombinant DNA methods, purified from naturally-occurring sources, or the like.
  • samples comprising, e.g., antigens for analysis according to the methods described herein are optionally recovered and purified by any of a number of methods well known in the art, including electrophoresis, chromatography, precipitation, dialysis, filtration, centrifugation, crystallization and/or precipitation.
  • purification techniques such as ultra-centrifugation, ammonium sulfate or ethanol precipitation, acid extraction, ion exchange chromatography, high performance liquid chromatography, size exclusion chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography (e.g., as with C ⁇ -C 18 resins), affinity chromatography (e.g., as with immunoaffinity, immobilized metals, dyes, or other tagging systems), hydroxylapatite chromatography, and/or lectin chromatography are optionally used.
  • purification techniques such as ultra-centrifugation, ammonium sulfate or ethanol precipitation, acid extraction, ion exchange chromatography, high performance liquid chromatography, size exclusion chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography (e.g., as with C ⁇ -C 18 resins), affinity chromatography (e.g., as with immunoaffinity, immobilized metals, dyes, or other tagging systems),
  • samples are derived from mammalian subjects, particularly human subjects.
  • the samples typically include biological fluids such as blood, serum, plasma, saliva, urine, prostatic fluid, seminal fluid, seminal plasma, lymph, lung/bronchial washes, mucus, feces, nipple secretions, sputum, tears, or the like.
  • Samples also optionally include extracts from biological sources, such as organ extracts, etc.
  • biological samples such as these are optionally collected according to any known technique, such as venipuncture, biopsy, or the like.
  • the specific exemplary sample sources listed herein are offered to illustrate but not to limit the present invention. Additional sources of samples are known in the art and are readily obtainable.
  • the methods include fractionating biomolecules in an initial sample by one or a combination of fractionation techniques described above or otherwise known in the art to be useful for separating biomolecules to collect a sample fraction that may include the antibodies prior to mass profiling. Fractionation is typically utilized to decrease the complexity of analytes in the sample to assist detection and characterization of antibodies and/or inflammation markers, such as cytokines, leukotrienes, and the like.
  • fractionation protocols can provide additional information regarding physical and chemical characteristics of biomolecular components in a sample. For example, if a sample is fractionated using an anion-exchange spin column, and if an antibody is eluted at a certain pH, this elution characteristic provides information regarding binding properties of the antibody.
  • a sample can be fractionated to remove proteins or other molecules in the sample that are present in a high quantity and or which would otherwise interfere with the detection of a particular antibody, if present (e.g., antibodies that are expressed only at low levels in the subject, such as at the outset of a particular infection).
  • proteins in the samples of the invention are optionally fragmented or digested. This approach is particularly useful when components (e.g., antibodies, etc.) of a sample are to be identified. Fragmentation is optionally effected using any technique that produces peptide fragments from proteins in a sample. Many of these techniques are generally known in the art. For example, proteins are optionally fragmented enzymatically, chemically, or physically. In certain embodiments of the invention, antibodies, inflammation markers, and/or peptide fragments resulting from fragmentation are optionally modified to improve resolution upon detection.
  • the fragmentation of biomolecular components of a sample can be performed "on chip" in a SELDI environment by placing an aliquot of the sample on an adsorbent spot and adding, e.g., the proteolytic agent to the material on the spot. Additional details relating to the identification of biomolecules via fragmentation are described in, e.g., International Publication No. WO 02/074927 entitled "High accuracy protein identification” by Pham. [0079]
  • the antigens of the invention are optionally bound to or otherwise immobilized on probes (e.g., biochips, etc.) either before or after antibodies are captured with those affinity reagents.
  • the analysis of antibodies present in a given biological sample according to the present invention includes attaching or immobilizing selected antigens indicative of the disease to be diagnosed and/or prognosticated on the surface of a probe, e.g., a reactive surface
  • Probes suitable for use in the invention are described further herein, e.g., in the definitions provided above.
  • Antigens are optionally covalently or non-covalently attached to the probe.
  • Non-covalent attachment methods include electrostatic attachment (e.g., using poly-lysine, aminosilane, etc.), molecular recognition (e.g., streptavidin-biotin, etc.), hydrophobic attachment, and the like.
  • covalent attachment methods include those using moieties, such as aldehyde, epoxide, thiol, carbodiimide, or other groups.
  • the probe further includes affinity reagents that specifically bind inflammation markers, such as cytokines, leukotrienes, and/or the like.
  • the methods further include capturing and detecting the inflammation markers, if any, present in the sample to provide inflammation marker capture data. This data is also correlated with, e.g., the probable diagnosis or prognosis of the disease or the negative diagnosis for the subject.
  • the immobilized antigen is typically contacted with the biological sample to be analyzed to effect capture of the antibodies and/or inflammation markers, if any, present in the sample prior to analyte detection.
  • the antigens and/or other affinity reagents are contacted with the biological sample to effect capture of the antibodies and/or inflammation markers, if any, present in the sample prior to immobilizing the antigens and/or affinity reagents on the probe.
  • Arrays or other chips are optionally directly analyzed by, e.g., SELDI TOF-MS.
  • this process generally includes loading energy adsorbing molecules (EAM) on the probe surface, followed by a drying operation, and analyzing the analyte mixture by laser desorption/ionization mass spectrometry.
  • EAM energy adsorbing molecules
  • antigens and/or other affinity reagents disposed on the surfaces of other solid supports, e.g., chromatography beads are used to capture antibodies in samples.
  • the captured antibodies are typically desorbed and collected from the beads by an acidic or other treatment.
  • the collected antibodies fraction is then typically analyzed using various detection methods, including mass spectrometry (e.g., SELDI, MALDI, electrospray, etc.). Analyte detection is described further below.
  • mass spectrometry e.g., SELDI, MALDI, electrospray, etc.
  • Analyte detection is described further below.
  • samples and these affinity reagents are contacted for a period of between about 30 seconds and about 12 hours, and preferably, between about 30 seconds and about 15 minutes.
  • samples are generally contacted with affinity reagents under ambient temperature and pressure conditions.
  • modified temperature typically between about 0°C and about 100°C and more typically 4°C through 37°C
  • pressure conditions can be desirable, which conditions are determinable by those skilled in the art.
  • a sample volume of about 1 ⁇ l to 500 ⁇ l is contacted with, e.g., an antigen or other affinity reagent in a particular capture step.
  • the sample volume typically contains from a few attomoles to 100 picomoles of biomolecules (e.g., antibodies, inflammation markers, etc.).
  • affinity reagents are also typically provided in volumes of about 1 ⁇ l to 500 ⁇ l.
  • affinity reagents are optionally directly immobilized on a probe surface or via a linker or capture molecule, such as receptors, linker antibodies, Protein A, Protein G, a mercaptoheterocyclic ligand, or the like.
  • linker or capture molecule such as receptors, linker antibodies, Protein A, Protein G, a mercaptoheterocyclic ligand, or the like.
  • samples are analyzed without being fractionated prior to examination by, e.g., SELDI.
  • a sample is optionally analyzed directly from a subject to assess the presence of antibodies and/or inflammation markers.
  • Samples are also optionally analyzed, according to the methods of the present invention, after fractionation of the samples (e.g., before or after being captured by antigens or other affinity reagents, etc.). Fractionation of a sample aliquot typically increases the total information content about biomolecules present in the particular sample.
  • fractionation may result in the detection of trace amounts of antibodies that would otherwise be undetectable, or not accurately detected, in an unfractionated sample by eliminating signals attributable to more abundant biomolecules that would otherwise suppress the signals of less abundant components.
  • biomolecules remaining in the sample after fractionation are typically detected with improved, e.g., mass accuracy as a result of an increased signa noise ratio.
  • the use of information about sample components from fractionated samples as well as unfractionated samples generally leads to a higher confidence level that a given antibody or inflammation marker has been accurately detected.
  • the fractionation steps that generate sample fractions can be performed by, e.g., any of the purification/fractionation methods described above.
  • biomolecules in the sample are optionally separated into fractions using, e.g., centrifugation, dialysis, HPLC, SEC or the like.
  • fractionated samples are then analyzed by the methods of detection described herein.
  • fractionating and analyzing the sample is performed by SELDI/retentate chromatography.
  • Retentate chromatography involves directly contacting a sample with adsorbents (e.g., antigens or other affinity reagents) bound to a surface of a probe in which the adsorbents capture one or more antibodies and/or inflammation marker.
  • adsorbents e.g., antigens or other affinity reagents
  • This embodiment also includes removing non-captured material from the probe, e.g., by one or more washes prior to gas phase ion spectrometric analysis.
  • the sample is indirectly contacted with a probe surface after being contacted with, e.g., an affinity reagent bound to a chromatographic resin, which affinity reagent captures one or more components of the sample.
  • affinity reagent bound to a chromatographic resin
  • non-captured materials are optionally removed (e.g., by one or more washes) before or after the adsorbent is contacted with the probe surface. Additional details relating to retentate chromatography are provided in, e.g., U.S. Patent Application 20020177242 (Hutchens).
  • Washing to remove non-captured materials can be accomplished by, e.g., bathing, soaking, dipping, rinsing, spraying, or washing the surface of the probe or other solid support (e.g., a chromatographic resin, etc.) following exposure to the sample with an eluant.
  • a microfluidics process is preferably used when an eluant is introduced to small spots (e.g., surface features) of affinity reagents on the probe.
  • the eluant can be at a temperature of between 0°C and 100°C, preferably between 4°C and 37°C.
  • Any suitable eluant e.g., organic or aqueous
  • each of the one or more washes optionally includes an identical or a different elution condition relative to a preceding wash.
  • FIG. 2 schematically illustrates another surface enhanced laser deso ⁇ tion/ionization assay of a sample. As depicted, sample 200 is applied to biochip 202 which includes antigen 204 bound to surface feature 206.
  • sample 200 Components of sample 200 that are not bound to antigen 204 are washed away (e.g., eluted or the like) from biochip 202 prior to mass analysis, as described above.
  • energy absorbing molecules 210 (not shown in Figure 1) are added to biochip 202 to absorb energy from ionization source 212 (i.e., a laser) to aid deso ⁇ tion of antibodies 208 from the surface of biochip 202.
  • Mass spectrum 214 is produced by mass spectral analysis of desorbed/ionized antibodies 208.
  • any suitable gas phase ion spectrometer is used as long as it allows biomolecular components on the substrate to be resolved and detected.
  • Tandem mass spectrometers can usefully be selected from the group that includes orthogonal quadrupole time-of-flight (Qq-TOF), ion trap (IT), ion trap time-of-flight (IT-TOF), time-of-flight time-of-flight (TOF-TOF), and ion cyclotron resonance (ICR) varieties.
  • Tandem mass spectrometers and associated instrumentation which can be adapted to perform the methods described herein are described further in, e.g., Patent Application Publication No. US 2002/0182649 by Weinberger et al., which published December 5, 2002. Additional details relating to various mass spectrometry techniques and instrumentation are included in, e.g., Skoog et al., Principles of Instrumental Analysis, 5 th Ed., Harcourt Brace & Co.
  • SELDI- based analysis is typically coupled with a quantitative surface scanning technique, such as a method that includes the detection refractive index or diffraction (e.g., surface plasmon resonance, ellipsometry, resonant mirror methods, grating-coupled waveguide methods, interferometry, multi-polar resonance spectroscopy, etc.).
  • a quantitative surface scanning technique such as a method that includes the detection refractive index or diffraction (e.g., surface plasmon resonance, ellipsometry, resonant mirror methods, grating-coupled waveguide methods, interferometry, multi-polar resonance spectroscopy, etc.).
  • an ion mobility spectrometer or total ion current measuring device is optionally used to detect biomolecular components.
  • the detectors utilized in practicing the invention typically further comprise interfaced digital computers, e.g., to control device operation (e.g., ion generation in a gas phase ion spectrometer, etc.) and to participate in data acquisition and analysis.
  • Analysis software can be local to the computer or can be remote, but communicably accessible to the computer.
  • the computer can have a connection to the internet permitting use of analytical packages such as Protein Prospector, PROWL, or the Mascot Search Engine, which are available on the world wide web.
  • the analysis software can also be remotely resident on a LAN or WAN server.
  • Exemplary systems that include digital computers are described further below.
  • mass spectrometry e.g., on the surface of a probe, such as a ProteinChip ® Array
  • the results would show a pattern of different peaks of given molecular masses.
  • Detected masses typically correspond to single antibodies or inflammation markers (except, e.g., for multiple charged-species, subunits or fragments of the same protein, etc.).
  • the size of the signal compared to a standard curve is generally proportional to the amount of the particular antibody or inflammation marker.
  • Pattern analysis software containing data related to these biomolecules typically informs on the identity of detected antibody or inflammation marker and provides quantitative information regarding these contaminating proteins.
  • Data generated by deso ⁇ tion and detection of biomolecules, such as antibodies or inflammation markers is optionally analyzed using any suitable method, e.g., to identify and/or quantify detected components and to correlate that data with a diagnosis or prognosis of the disease under consideration.
  • antibody detection and quantification data is typically compared to positive controls (e.g., a
  • SELDI analysis of a sample comprising a known quantity of an identified antibody) and/or negative controls e.g., a SELDI analysis of a sample that lacks antibodies and/or inflammation markers related to the disease under consideration.
  • data is analyzed with the use of a logic device, such as a programmable digital computer that is included, e.g., as part of a system.
  • the computer generally includes a computer readable medium that stores logic instructions of the system software. Certain logic instructions are typically devoted to memory that includes the location of each feature on a probe, the identity of the antigen(s) or other adsorbent(s) at that feature, the elution conditions used to wash the adsorbent(s), or the like.
  • the computer' also typically includes logic instructions that receives as input, data on the strength of the signal at various molecular masses received from a particular addressable location or surface feature on the probe, and instructions for entering data into a database.
  • This data generally indicates the number and masses of components detected, including the strength of the signal generated by each component.
  • data generation in mass spectrometry typically begins with the detection of ions by an ion detector.
  • a typical laser deso ⁇ tion mass spectrometer can employ a nitrogen laser at 337.1 n .
  • a useful pulse width is about 4 nanoseconds.
  • power output of about 1-25 ⁇ j is used.
  • Ciphergen's ProteinChip ® system employs an analog-to-digital converter (ADC) to accomplish this.
  • ADC analog-to-digital converter
  • the ADC integrates detector output at regularly spaced time intervals into time-dependent bins. The time intervals typically are one to four nanoseconds long.
  • the time- of-flight spectrum ultimately analyzed typically does not represent the signal from a single pulse of ionizing energy against a sample, but rather the sum of signals from a number of pulses. This reduces noise and increases dynamic range. This time-of-flight data is then subject to data processing.
  • TOF-to-M Z transformation involves the application of an algorithm that transforms times-of-flight into mass-to-charge ratio (M/Z).
  • M/Z mass-to-charge ratio
  • the signals are converted from the time domain to the mass domain. That is, each time-of-flight is converted into mass-to-charge ratio, or M/Z.
  • Calibration can be done internally or externally. In internal calibration, the sample analyzed contains one or more analytes of known M/Z. Signal peaks at times-of-flight representing these massed analytes are assigned the known M/Z.
  • parameters are calculated for a mathematical function that converts times-of-flight to M/Z.
  • a function that converts times-of-flight to M/Z such as one created by prior internal calibration, is applied to a time-of-flight spectrum without the use of internal calibrants.
  • Baseline subtraction improves data quantification by eliminating artificial, reproducible instrument offsets that perturb the spectrum. It involves calculating a spectrum baseline using an algorithm that inco ⁇ orates parameters such as peak width, and then subtracting the baseline from the mass spectrum.
  • High frequency noise signals are eliminated by the application of a smoothing function.
  • a typical smoothing function applies a moving average function to each time-dependent bin.
  • the moving average filter is a variable width digital filter in which the bandwidth of the filter varies as a function of, e.g., peak bandwidth, generally becoming broader with increased time-of-flight. See, e.g., WO 00/70648, November 23, 2000 (Gavin et al., "Variable Width Digital Filter for Time-of-flight Mass Spectrometry").
  • a computer can transform the resulting spectrum into various formats for displaying.
  • spectrum view or retentate map a standard spectral view can be displayed, wherein the view depicts the quantity of analyte reaching the detector at each particular molecular weight.
  • peak map a standard spectral view
  • peak map only the peak height and mass information are retained from the spectrum view, yielding a cleaner image and enabling analytes with nearly identical molecular weights to be more easily seen.
  • gel view each mass from the peak view can be converted into a grayscale image based on the height of each peak, resulting in an appearance similar to bands on electrophoretic gels.
  • this software functions by identifying signals having a signal-to-noise ratio above a selected threshold and labeling the mass of the peak at the centroid of the peak signal.
  • many spectra are compared to identify identical peaks present in some selected percentage of the mass spectra.
  • One version of this software clusters all peaks appearing in the various spectra within a defined mass range, and assigns a mass (M/Z) to all the peaks that are near the mid-point of the mass (M/Z) cluster.
  • Classification models can be formed using any suitable statistical classification (or "learning") method that attempts to segregate bodies of data into classes based on objective parameters present in the data.
  • Classification methods may be either supervised or unsupervised. Examples of supervised and unsupervised classification processes are described in Jain, "Statistical Pattern Recognition: A Review", IEEE Transactions on Pattern Analysis and Machine Intelligence, Vol. 22, No. 1, January 2000, which is herein inco ⁇ orated by reference in its entirety.
  • supervised classification training data containing examples of known categories are presented to a learning mechanism, which learns one more sets of relationships that define each of the known classes. New data may then be applied to the learning mechanism, which then classifies the new data using the learned relationships.
  • supervised classification processes include linear regression processes (e.g., multiple linear regression (MLR), partial least squares (PLS) regression and principal components regression (PCR)), binary decision trees (e.g., recursive partitioning processes such as CART - classification and regression trees), artificial neural networks such as backpropagation networks, discriminant analyses (e.g., Bayesian classifier or Fischer analysis), logistic classifiers, and support vector classifiers (support vector machines).
  • linear regression processes e.g., multiple linear regression (MLR), partial least squares (PLS) regression and principal components regression (PCR)
  • binary decision trees e.g., recursive partitioning processes such as CART - classification and regression trees
  • artificial neural networks such as backpropagation networks
  • discriminant analyses e.g., Bayesian classifier or Fischer analysis
  • logistic classifiers e.g., Bayesian classifier or Fischer analysis
  • support vector classifiers support vector machines
  • a preferred supervised classification method is a recursive partitioning process.
  • Recursive partitioning processes use recursive partitioning trees to classify spectra derived from unknown samples. Further details about recursive partitioning processes are in U.S. Provisional Patent Application Nos. 60/249,835, filed on November 16, 2000, and 60/254,746, filed on December 11, 2000, and U.S. Non- Provisional Patent Application Nos. 09/999,081, filed November 15, 2001, and 10/084,587, filed on February 25, 2002. All of these U.S. Provisional and Non Provisional Patent Applications are herein inco ⁇ orated by reference in their entirety for all pu ⁇ oses.
  • the classification models that are created can be formed using unsupervised learning methods.
  • Unsupervised classification attempts to learn classifications based on similarities in the training data set, without pre- classifying the spectra from which the training data set was derived.
  • Unsupervised learning methods include cluster analyses. A cluster analysis attempts to divide the data into "clusters" or groups that ideally should have members that are very similar to each other, and very dissimilar to members of other clusters. Similarity is then measured using some distance metric, which measures the distance between data items, and clusters together data items that are closer to each other.
  • Clustering techniques include the MacQueen's K-means algorithm and the Kohonen's Self-Organizing Map algorithm.
  • the classification models can be formed on and used on any suitable digital computer. Suitable digital computers include micro, mini, or large computers using any standard or specialized operating system such as a Unix, WindowsTM or LinuxTM based operating system. The digital computer that is used may be physically separate from the mass spectrometer that is used to create the spectra of interest, or it may be coupled to the mass spectrometer. [0116]
  • the training data set and the classification models according to embodiments of the invention can be embodied by computer code that is executed or used by a digital computer.
  • the computer code can be stored on any suitable computer readable media including optical or magnetic disks, sticks, tapes, etc., and can be written in any suitable computer programming language including C, C++, visual basic, etc.
  • Software included in the systems utilized in performing the methods of the invention typically has logic instructions, e.g., capable of quantifying detected antibodies and/or inflammation markers, capable of determining closeness-of-fit between one or more detected biomolecule masses in sets of mass data and database entries to aid in the diagnosis or prognosis of the particular disease under consideration.
  • logic instructions e.g., capable of quantifying detected antibodies and/or inflammation markers, capable of determining closeness-of-fit between one or more detected biomolecule masses in sets of mass data and database entries to aid in the diagnosis or prognosis of the particular disease under consideration.
  • Various software packages are currently available for querying databases, improving the speed of mass spectrometric protein identification processes, and otherwise integrating mass spectrometry into bioinformatics.
  • Mascot is a search engine that uses mass spectrometry data to identify proteins from primary sequence databases. See, e.g., Perkins et al.
  • ProFound which performs rapid database searching combined with Bayesian statistics for protein identification. Profound is described further in, e.g., Zhang and Chait (2000) "ProFound-An expert system for protein identification using mass spectrometric peptide mapping information,” Anal. Chem.
  • the invention also provides for the storage and retrieval of a collection of data in a computer data storage apparatus, which can include magnetic disks, optical disks, magneto-optical disks, DRAM, SRAM, SGRAM, SDRAM, RDRAM, DDR RAM, magnetic bubble memory devices, and other data storage devices, including CPU registers and on-CPU data storage arrays.
  • a computer data storage apparatus can include magnetic disks, optical disks, magneto-optical disks, DRAM, SRAM, SGRAM, SDRAM, RDRAM, DDR RAM, magnetic bubble memory devices, and other data storage devices, including CPU registers and on-CPU data storage arrays.
  • the target data records are stored as a bit pattern in an array of magnetic domains on a magnetizable medium or as an array of charge states or transistor gate states, such as an array of cells in a DRAM device (e.g., each cell comprised of a transistor and a charge storage area, which may be on the transistor).
  • the invention also preferably provides a magnetic disk, such as an IBM- compatible (DOS, Windows, Windows95/98/2000, Windows NT, OS/2) or other format (e.g., Linux, SunOS, Solaris, AIX, SCO Unix, VMS, MV, Macintosh, etc.) floppy diskette or hard (fixed, Winchester) disk drive, comprising a bit pattern encoding data from an assay of the invention in a file format suitable for retrieval and processing in a computerized sequence analysis, comparison, or relative quantitation method.
  • a magnetic disk such as an IBM- compatible (DOS, Windows, Windows95/98/2000, Windows NT, OS/2) or other format (e.g., Linux, SunOS, Solaris, AIX, SCO Unix, VMS, MV, Macintosh, etc.) floppy diskette or hard (fixed, Winchester) disk drive, comprising a bit pattern encoding data from an assay of the invention in a file format suitable for retrieval and processing
  • the invention also provides a network, comprising a plurality of computing devices linked via a data link, such as an Ethernet cable (coax or lOBaseT), telephone line, ISDN line, wireless network, optical fiber, or other suitable signal transmission medium, whereby at least one network device (e.g., computer, disk array, etc.) comprises a pattern of magnetic domains (e.g., magnetic disk) and/or charge domains (e.g., an array of DRAM cells) composing a bit pattern encoding data acquired from an assay of the invention.
  • a network device e.g., computer, disk array, etc.
  • a pattern of magnetic domains e.g., magnetic disk
  • charge domains e.g., an array of DRAM cells
  • the invention also provides a method for transmitting assay data that includes generating an electronic signal on an electronic communications device, such as a modem, ISDN terminal adapter, DSL, cable modem, ATM switch, or the like in which the signal includes (in native or encrypted format) a bit pattern encoding data from an assay or a database comprising a plurality of assay results obtained by the method of the invention.
  • an electronic communications device such as a modem, ISDN terminal adapter, DSL, cable modem, ATM switch, or the like in which the signal includes (in native or encrypted format) a bit pattern encoding data from an assay or a database comprising a plurality of assay results obtained by the method of the invention.
  • the invention provides a computer system for comparing a query target to a database containing an array of data structures, such as an assay result obtained by the method of the invention, and ranking database targets based on the degree of identity and gap weight to the target data.
  • a central processor is preferably initialized to load and execute the computer program for alignment and/or comparison of the assay results.
  • Data for a query target is entered into the central processor via an I/O device.
  • Execution of the computer program results in the central processor retrieving the assay data from the data file, which comprises a binary description of an assay result.
  • the target data or record and the computer program can be transferred to secondary memory, which is typically random access memory (e.g., DRAM, SRAM, SGRAM, or SDRAM).
  • Targets are ranked according to the degree of correspondence between a selected assay characteristic (e.g., binding to a selected binding functionality) and the same characteristic of the query target and results are output via an I/O device.
  • a central processor can be a conventional computer (e.g., Intel Pentium, PowerPC, Alpha, PA-8000, SPARC, MIPS 4400, MIPS 10000, VAX, etc.);
  • a program can be a commercial or public domain molecular biology software package (e.g., UWGCG Sequence Analysis Software, Darwin);
  • a data file can be an optical or magnetic disk, a data server, a memory device (e.g., DRAM, SRAM, SGRAM, SDRAM, EPROM, bubble memory, flash memory, etc.);
  • an I/O device can be a terminal comprising a video display and a keyboard, a modem, an ISDN terminal adapter, an Ethernet port, a punched card reader, a magnetic strip reader, or other suitable I/O device.
  • FIG. 3 schematically illustrates an exemplary surface enhanced laser deso ⁇ tion/ionization time-of-flight mass spectrometry system 300.
  • photon energy produced by laser source 302 impacts biochip 304 at surface feature 306, which includes one or more selected antigens with captured antibodies.
  • the photon energy causes captured antibodies at surface feature 306 to desorb and ionize.
  • the desorbed ions are then accelerated through flight tube/mass analyzer 308. Ions are separated according to mass/charge ratios, which as depicted is simply the mass of the ionic species, because each ion is singly charged. As further illustrated, smaller ions travel faster than larger ions, thereby resolving the species according to mass.
  • the present invention also provides probes and kits for aiding in the diagnosis and/or prognosis of a disease (e.g., an autoimmune disease, an infectious disease, a prion disease, etc.).
  • a probe according to the present invention includes a biochip derivatized with one or more antigens indicative of the disease, which antigens specifically bind antibodies present in a sample.
  • the probe also includes one or more affinity reagents that specifically bind inflammation markers, such as cytokines, leukotrienes, and the like. Additional aspects of the probes of the invention are described above, including in the provided definitions.
  • Kits generally include one or more probes comprising biochips derivatized with one or more capture molecules that specifically bind at least one antigen indicative of a disease.
  • the kits of the invention optionally also include affinity reagents, such as antigens indicative of one or more diseases, e.g., either bound to the capture molecules on the probe or packaged separately. Suitable antigens and other affinity reagents are described in greater detail above or are otherwise known in the art.
  • Kits may further include a pre-fractionation spin column (e.g., K-30 size exclusion column) that can be used to prepare samples as described herein.
  • a pre-fractionation spin column e.g., K-30 size exclusion column

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Hematology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Cell Biology (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Optics & Photonics (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)

Abstract

La présente invention se rapporte à des procédés permettant de faciliter un diagnostic ou un pronostic médical. Lesdits procédés consistent à capturer des anticorps dans des échantillons prélevés sur des sujets, et ce sur des sondes et au moyen d'antigènes qui sont indicateurs de maladies particulières. Ces procédés consistent également à détecter les anticorps capturés au moyen d'au moins une version d'une spectrométrie ionique en phase gazeuse à désorption/ionisation laser avec exaltation en surface. L'invention se rapporte en outre à des sondes et à des trousses permettant la mise en oeuvre des procédés décrits ci-dessus.
PCT/US2004/015210 2003-05-16 2004-05-14 Diagnostic medical base sur une ionisation/desorption laser avec exaltation en surface Ceased WO2004103155A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US47122903P 2003-05-16 2003-05-16
US60/471,229 2003-05-16

Publications (2)

Publication Number Publication Date
WO2004103155A2 true WO2004103155A2 (fr) 2004-12-02
WO2004103155A3 WO2004103155A3 (fr) 2007-12-06

Family

ID=33476813

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2004/015210 Ceased WO2004103155A2 (fr) 2003-05-16 2004-05-14 Diagnostic medical base sur une ionisation/desorption laser avec exaltation en surface

Country Status (1)

Country Link
WO (1) WO2004103155A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021234396A1 (fr) * 2020-05-22 2021-11-25 The Binding Site Group Limited Procédé d'identification ou de caractérisation d'une réponse immune chez un sujet

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0700521B1 (fr) * 1993-05-28 2003-06-04 Baylor College Of Medicine Procedes et spectrometre de masse pour la desorption et l'ionisation d'analytes

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021234396A1 (fr) * 2020-05-22 2021-11-25 The Binding Site Group Limited Procédé d'identification ou de caractérisation d'une réponse immune chez un sujet
CN115698723A (zh) * 2020-05-22 2023-02-03 结合点集团有限公司 鉴定或表征受试者中的免疫应答的方法
JP2023526556A (ja) * 2020-05-22 2023-06-21 ザ バインディング サイト グループ リミティド 対象における免疫応答を同定又は特徴付ける方法
JP7769639B2 (ja) 2020-05-22 2025-11-13 ザ バインディング サイト グループ リミティド 対象における免疫応答を同定又は特徴付ける方法

Also Published As

Publication number Publication date
WO2004103155A3 (fr) 2007-12-06

Similar Documents

Publication Publication Date Title
JP4587954B2 (ja) タンパク質相互作用相違マッピング
US20060084059A1 (en) Serum biomarkers in hepatocellular carcinoma
US7605003B2 (en) Use of biomarkers for detecting ovarian cancer
EP2005172A2 (fr) Procédé de caractérisation biochimique de l'apolipoprotéine
EP2088426B1 (fr) Spectrométrie de masse d'un échantillon biologique par immunoprécipitation
EP1573044A2 (fr) Biomarqueurs seriques du cancer du poumon
US20050064516A1 (en) Biological markers for diagnosing multiple sclerosis
WO2017150681A1 (fr) Procédé de diagnostic de sclérose latérale amyotrophique utilisant un peptide signal comme index
JP2003532055A (ja) 前立腺癌マーカー
WO2004111654A2 (fr) Biomarqueurs de serum dans une maladie cardiaque ischemique
EP1977250A1 (fr) Méthode et marqueurs pour le diagnostic de maladies rénales
WO2017150680A1 (fr) Procédé de diagnostic de la maladie d'alzheimer en utilisant un peptide signal comme index
US20030119063A1 (en) High accuracy protein identification
US20060286602A1 (en) Method and markers for the diagnosis of renal diseases
US20060205010A1 (en) Methods of host cell protein analysis
WO2004103155A2 (fr) Diagnostic medical base sur une ionisation/desorption laser avec exaltation en surface
US20040033613A1 (en) Saliva-based protein profiling
JP2024120022A (ja) 全身型若年性特発性関節炎および川崎病を診断および識別するための組成物および方法
US20040096820A1 (en) Comparative proteomics of progressor and nonprogressor populations
CN112147344B (zh) 动脉粥样硬化性脑梗死的代谢标志物及其在诊疗中的应用
WO2002074927A2 (fr) Identification haute precision de proteines
US20060177870A1 (en) Immunoassays
WO2005071421A1 (fr) Detection specifique de troponine et de formes modifiees de troponine
WO2004030521A2 (fr) Utilisation de biomarqueurs pour detecter un rejet aigu de transplantation renale
RU2381505C1 (ru) Способ диагностики сифилиса с использованием метода прямого протеомного профилирования сыворотки крови

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase