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EP1250675A2 - Creation d'une base de donnees biochimiques et procedes d'utilisation - Google Patents

Creation d'une base de donnees biochimiques et procedes d'utilisation

Info

Publication number
EP1250675A2
EP1250675A2 EP00965004A EP00965004A EP1250675A2 EP 1250675 A2 EP1250675 A2 EP 1250675A2 EP 00965004 A EP00965004 A EP 00965004A EP 00965004 A EP00965004 A EP 00965004A EP 1250675 A2 EP1250675 A2 EP 1250675A2
Authority
EP
European Patent Office
Prior art keywords
subjects
biochemical data
microspheres
test
map
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.)
Withdrawn
Application number
EP00965004A
Other languages
German (de)
English (en)
Inventor
Mark B. Chandler
Van S. Chandler
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.)
Luminex Corp
Original Assignee
Luminex Corp
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 Luminex Corp filed Critical Luminex Corp
Publication of EP1250675A2 publication Critical patent/EP1250675A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H10/00—ICT specially adapted for the handling or processing of patient-related medical or healthcare data
    • G16H10/40—ICT specially adapted for the handling or processing of patient-related medical or healthcare data for data related to laboratory analysis, e.g. patient specimen analysis
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H50/00—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics
    • G16H50/20—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics for computer-aided diagnosis, e.g. based on medical expert systems
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H70/00—ICT specially adapted for the handling or processing of medical references
    • G16H70/60—ICT specially adapted for the handling or processing of medical references relating to pathologies

Definitions

  • This invention relates to the creation and use of a database comprising biochemical data for a wide range of applications, including diagnosis of disease states, the prognosis for recovery, determination of the onset (or potential therefor) of future disease states, assessment of health or medical condition and the like
  • U S Pat No 4,874,693 discloses a method for detecting placental dysfunction, which is diagnostic of chromosomal abnormalities through quantifying the hormone human chorionic gonadotropin or its subumts in bodily fluids
  • U S Pat No 5,622, 171 discloses a method for diagnosis of a number of breast diseases based on analysis of radiographic images using a computer and a neural network
  • U S Pat No 5,724,983 discloses a method of periodically computing a diagnosis of a patient based on one or more continuous monitored clinical features as detected by an electrocardiograph A change-in-condition measure is periodically calculated, and an alarm is sounded when a threshold value of the change-in-condition measure is exceeded U.S.
  • Pat No 5,937,387 discloses a system and method for using a wide variety of factors such as smoking, blood pressure, and dietary cholesterol for an individual patient to compute the physiological age of the patient This information may be used by the patient to monitor and improve wellness Part of our inability to make strong a correlation is our lack of understanding of the function of genes
  • genes can be thought of as the "vocabulary" of biology, while the proteins they express are the “instructions” to biology Thus, if one could interpret these instructions, then the onset of disease could be detected earlier, and pharmaceuticals could be developed to change the instructions.
  • statisticians would set up controlled, randomized experiments to assign probability distributions in an attempt to associate one or more abnormal protein levels with a disease state. These statisticians would typically find weak correlation, presumably because there are often many different chains of protein interactions, which cause the same disease.
  • the present invention seeks to solve this problem by describing mathematically multiple paths that lead to the same outcome or multiple outcomes off of the same path.
  • the differences in protein expression between individuals may be rooted in each individual's unique genetic makeup or exposure to environmental factors
  • the present invention provides a method of creating a database containing biochemical data from at least about 1 ,000 subjects, preferably tens of thousands of subjects
  • the information compiled in the database of the present invention comprises biochemical data generated from one or more test samples obtained from the subjects and can be retrieved or correlated with identifiers of the subjects along with their medical histories
  • the method comprises (a) providing one or more test samples obtained from one or more subjects, (b) exposing a Multi-Anal yte Profile (MAP) Test Panel to at least a portion of the one or more test samples to provide one or more test mixtures, the MAP Test Panel comprising 20 or more subsets of microspheres, the microspheres of one subset being distinguishable from those of another subset and harboring at least one reagent designed to interact selectively, if not specifically, with, and to generate biochemical data concerning, a predetermined analyte, (c) optionally, adding one or more supplemental reagents to the one or more test mixtures to further the generation
  • a Multi-Analyte Profile (MAP) Test Panel which comprises 20 or more subsets of microspheres, the microspheres of one subset being distinguishable from those of another subset and harboring at least one reagent designed to interact selectively, if not specifically, with a predetermined analyte
  • the MAP Test Panel comprises 50 or more, 75 or more, 100 or more, 200 or more, or 300 or more subsets of microspheres
  • the microspheres of one subset are distinguishable from those of another subset by their characteristic fluorescence signatures Elsewhere in this specification, microspheres having this characteristic fluorescence signature might also be referred to as fluorescence addressable microspheres
  • the microspheres of the MAP Test Panel typically contain various concentrations of at least two or more fluorescent dyes, sometimes at least three or more fluorescent dyes and, preferably, at least four or more
  • the at least one reagent comprises any substance that can selective
  • the present invention also provides a kit for assaying 20 or more predetermined analytes in a single pass through a flow analyzer comprising a Multi-Analyte Profile (MAP) Test Panel comprising 20 or more subsets of microspheres, the microspheres of one subset being distinguishable from those of another subset and harboring at least one reagent designed to interact selectively, if not specifically, with a predetermined analyte
  • MAP Multi-Analyte Profile
  • MAP Multi-Analyte Profile
  • MAP Multi-Analyte Profile
  • the present invention provides a method of determining the efficacy or consequence(s) of experimental (or established) treatment, e g , drugs, radiation, surgery, gene or cell therapy, vaccine, diet and the like, by monitoring changes in biochemical data generated from a plurality of test samples obtained from a subject undergoing treatment over a given time interval
  • Yet another object of this invention is to diagnose a disease state or future disease state from the concentration profile of about 200-300, preferably more, biochemical analytes in a test sample Hence, probability and causal relationships between biochemical data and health effects are elucidated
  • Still another object of this invention is to provide methods of detecting side effects of drugs by determining the effect of drug administration on the concentration profile of 200-300 biochemical analytes in test samples obtained from subjects receiving drug and control subjects would have not received drug.
  • biochemical data obtained from the same subjects before and after drug administration can also be utilized
  • the present invention provides one or more electronic databases comprising biochemical data
  • a preferred electronic database can be described as comprising an electronically retrievable first set of information derived from a multiplexed analysis of a biological sample of an individual against a Multi-Analyte Profile (MAP) Test Panel comprising a plurality of predetermined analytes and at least an electronically retrievable second set of information which can be correlated with the first set and which is derived from the individual's medical history or medical condition
  • the first set of information may include quantitative information for each analyte of the MAP Test Panel, which is found in the biological sample
  • the second set of information may include the individual's phenotypic information and the individual's genetic information
  • the preferred database of the invention includes the first and second sets of information derived from 1 ,000 or more, 10,000 or more, 100,000 or more, 200,000 or more.
  • the database also includes a relationship amenable to mathematical or computational manipulation comprising (1) one or more rules de ⁇ ved at least in part from a database comprising a first set of information derived from a multiplexed analysis of a biological sample of an individual against a Multi-Anahte Profile (MAP) Test Panel comprising a plurality of predetermined analytes and at least a second set of information which can be correlated with the first set and which is derived from the individual's medical history or medical condition, and (n) one or more variables dependent at least on input comprising information derived from a multiplexed analysis of a biological sample of the patient against a panel comprising a pluralit
  • MAP Multi-Anahte Profile
  • a database is compiled comprising biochemical data, including the concentrations of biochemicals in blood samples taken from a large number of persons selected to be representative of the population, the blood samples taken annually over a period of at least 5 years
  • biochemical data including the concentrations of biochemicals in blood samples taken from a large number of persons selected to be representative of the population, the blood samples taken annually over a period of at least 5 years
  • a medical history is also determined for each person
  • concentrations of biochemicals and changes in concentrations of biochemicals are correlated with the medical histories and changes in medical histories of the persons involved
  • an algorithm is derived for correlating the concentrations or changes in concentrations of biochemicals in the blood sample with the presence of a disease state or future disease state in the person whose blood is being tested
  • This technology allows the simultaneous determination of the concentrations of multiple biochemicals in a single sample of blood or other biological fluids
  • this technology will be referred to as the "Luminex” technology
  • the profile of concentrations of biochemicals derived is referred to as a Multi-Analyte Profile (MAP)
  • MAP Multi-Analyte Profile
  • database will be used interchangeably with “electronic database”
  • Other terms, which can be equivalently used for “database,” include “automated information retrieval system,” “computer readable database,” or “database accessible by a computer”
  • database does not refer to conventional medical records as, for example, kept in a doctor's office, hospital, or health maintenance organization even if in electronically searchable form
  • the database created by this effort is the largest and most comprehensive repository of information about the complex biochemical processes underlying health and disease. It is expected that the present invention will enable the detection of cancer years earlier than is now possible with conventional technologies Heart disease and diabetes are predicted in time to allow pre-symptomatic intervention Ultimately, the fundamental defect and the complete characterization of every disease is identified by this invention
  • test samples comprise biological fluids, mixtures, or preparations thereof More preferably, the one or more test samples comprise blood samples, mixtures, or preparations thereof
  • preferred reagents bound to the microspheres comprises a small molecule, natural product, synthetic polymer, peptide, polypeptide, polysaccha ⁇ de, hpid, nucleic acid, or combinations thereof
  • the predetermined analyte comprises a drug, hormone, antigen, antibody, protein, enzyme, DNA, RNA, or combinations thereof
  • supplemental reagents may comprise a substrate, antibody, affinity reagent, label, or combinations thereof
  • supplemental reagents may comprise a substrate, antibody, affinity reagent, label, or combinations thereof
  • biochemical data is broadly meant to capture a wide range of information of potential interest to medical investigators, but this term includes at least the presence, absence, or quantity of predetermined analyte present in the one or more test samples
  • the biochemical data preferably includes data concerning 20 or more predetermined analytes. More preferably, 100 or more predetermined analytes, and, most preferably, 300 or more predetermined analytes
  • some or all of the subjects in a particular pool of subjects hav e been diagnosed with a disease or other pathological condition have been diagnosed with a neoplastic, neurodegenerative, skeletal, muscular, connective tissue, skin, organ, metabolic, addictive, psychiatric disease, or combinations thereof
  • test samples may be obtained from one or more subjects at least every month, quarter, biannually, or annually
  • one or more test samples are obtained from one or more subjects annually over a period of at least three, five, seven, or nine years
  • the examinations or questioning of the one or more subjects are conducted or performed, or their medical histories determined or obtained, annually over the same period
  • a relationship, if any, is determined between the one or more changes in the biochemical data and the one or more changes in the medical conditions or histories of the one or more subjects In so doing, one finds that one or more changes in the biochemical data correlate with one or more changes in the medical conditions or histories of the one or more subjects
  • Additional medical information is derived from approximately monthly surveys
  • information concerning the person's phenotype such as height, weight, sex, race, hair and eye color is recorded
  • the blood sample is analyzed for genetic information which contributes to the diagnosis of disease state and prospective disease state of this invention
  • the database of the invention continues to grow, becoming an all- encompassing and increasingly powerful diagnostic platform. Some original participants have significantly different profiles in year two, allowing the biochemical manifestations of ongoing or incipient disease, or even a lifestyle change, to be recognized.
  • MAP Multi-Analvte Profiling
  • a test of an individual's blood includes at least about 200-300 analyte MAP, and comparative analysis of patient results with the growing database.
  • Profiling becomes an essential part of the routine annual check-up, offering all the common screening tests plus substantially more diagnostic information obtained by testing for hundreds of additional analytes and checking the results against the database.
  • the MAP of 200-300 analytes that initiates the study is growing into the thousands as the role of more blood biochemicals is defined. It is also important to note that the database is only "seeded" by the original 200,000 participants. As the MAP is expanded, each of the millions of annual tests becomes part of the database. The database even suggests effective therapeutic regimens based on a patient's MAP and the availability of advanced technology in a given country. For example, a diagnosis in the U.S. that would suggests organ transplantation may provide other options for a patient in Venezuela. Genetic information derived from blood or biological fluids is optionally included in the database as supplemental information, which aids in deriving the correlation between changes in biological fluids and disease states and the development of disease states.
  • the medical and scientific value derived from the study resides in the integrative database. Access to the database is strictly controlled in order to prevent corruption or alteration of the data. Worldwide interaction with the database occurs over the Internet. Results of a patient's profile are sent over the "net" to a secure central server where they are evaluated against the database. A diagnostic report including suggestions for possible therapeutic modalities is then returned via the Internet to the lab where the MAP was performed.
  • Every patient test that is evaluated by the database also expands the database. It quickly changes from a database built upon hundreds of thousands of patient profiles into one sifting information from hundreds of millions of patient profiles from around the world.
  • Luminex technology is extended to animal studies, developing MAPs for laboratory mice (used in biomedical research) and for veterinary applications.
  • the present invention relates to a novel combination of large scale protein measurements and causal mathematics and statistics, which results in a series of mathematical models of human and animal biology. These models are created by measuring 20 to 10,000 proteins in blood, developing a profile of these proteins as they compare to observations of medical history, and, using causal methods, deriving a directed graph of protein interactions representative of normal and abnormal biological conditions.
  • causal methods as used in the present invention define a mathematical language for expressing that measurement "A” causes disease “X.” In doing so, equations are possible that describe a chain of protein interactions that eventually lead to disease.
  • an important aspect of the invention relates to a method of predicting a disease in a subject comprising providing measurements of gene products in a sample obtained from the subject, applying causal mathematics and statistics, and determining causal interactions of gene products to predict the disease.
  • the gene products comprise proteins.
  • the method can further comprise a comparison of at least one gene product to a control sample. Determining the causal interaction can involve deriving a graph.
  • the method can utilize multiple measurements conducted at various times. Alternatively, a plurality of measurements can be made at one or a plurality of times. In a preferred embodiment, 20 or more measurements are made.
  • the invention includes the derivation of an algorithm for predicting a disease in a subject comprising causal mathematics and statistics for evaluating information on protein levels in the subject and an output predictive of disease. Moreover, the mathematical relationship derived correlates the protein levels to disease. The mathematical relationship permits comparisons of the protein levels of test subjects with those of control subjects.
  • the invention comprises a system for predicting a disease in a subject comprising a microprocessor, and an algorithm using causal mathematics and statistics for evaluating information on protein levels in the subject to provide an output, wherein the output is predictive of the disease.
  • the system can further comprise a database of medical profiles for comparison with the subject.
  • the invention comprises a method for developing a mathematical model predictive of disease comprising the steps of iterative application of an algorithm to a set of standard data to provide an output; and comparison of the output to a disease profile.
  • Yet a further embodiment of the invention is directed to a method for treating a disease comprising diagnosing a disease by the steps of providing measurements of gene products in a sample obtained from the subject, applying causal mathematics and statistics, and determining causal interactions of gene products to predict the disease; and applying a pharmacologic treatment specifically tailored to the disease.
  • the Multi-Analyte Profile (MAP) Test Panel of the present invention comprises a collection of subsets of microspheres, the microspheres of each subset differing from those of another subset by at least one classification parameter (e.g., size, fluorescent color, non- fluorescent color, refraction index, magnetic property, density, etc.). Furthermore, the microspheres of each subset carry at least one distinct type of reagent.
  • classification parameter e.g., size, fluorescent color, non- fluorescent color, refraction index, magnetic property, density, etc.
  • antibody as used herein includes within its scope any of the various classes or sub-classes of immunoglobulins, e.g., IgG, IgA, IgM, or IgE derived from any of the animals conventionally or unconventionally used as a source of sera, such as sheep, rabbits, goats, or mice, to name a few. Antibody also encompasses monoclonal antibodies whether produced by cell fusion with immortalized cells or by recombinant techniques in eukaryotic or prokaryotic cells.
  • Antibody also includes intact molecules or "fragments" of antibodies, monoclonal or polyclonal, the fragments being those which contain the binding region of the antibody, i.e., fragments devoid of the Fc portion (e.g., Fv, Fab, Fab'. F(ab') 2 or fragments obtained by reductive cleavage of the disulfide bonds connecting the heavy chain components in the intact antibody, so long as they retain antigen binding capabilities).
  • fragments devoid of the Fc portion e.g., Fv, Fab, Fab'. F(ab') 2 or fragments obtained by reductive cleavage of the disulfide bonds connecting the heavy chain components in the intact antibody, so long as they retain antigen binding capabilities.
  • antigen is understood to include both naturally antigenic species (for example, drugs, proteins, bacteria, bacterial fragments, cells, cell fragments, carbohydrates, nucleic acids, lipids, and viruses, to name a few) and haptens, which may be rendered antigenic under suitable conditions and recognized by antibodies or antibody fragments
  • analyte is meant to be construed broadly and includes “antigens,” “antibodies,” “enzymes,” “nucleic acids, “and the like, but is not solely limited to “antigens”.
  • analytes are conceived, including, for example, environmental contaminant analytes, agricultural products, industrial chemicals, water treatment polymers, pharmaceutical drugs, drugs of abuse, and biological analytes, such as antigenic determinants of proteins, polysaccharides, glycoproteins, hpoproteins, nucleic acids, hormones, and parts of organisms, such as viruses, bacteria, fungi, parasites, plants and microbes.
  • reagent refers to the reaction partner or binding partner of an analyte.
  • the molecular interactions between reagent and analyte are generally selective, preferably specific.
  • Preferred analyte:reagent couples, however, include, but are not limited to, antigemspecific lmmunoglobuhn; hormone:hormone receptor; nucleic acid strand omplementary polynucleotide strand; avidimbiotin; protein A: ⁇ mmunoglobulin; protein G:IgG immunoglobulins; enzyme:substrate; lectin: specific carbohydrate; drug:protem; small molecule:prote ⁇ n, and the like.
  • Synthetic or recombinant peptides, polypeptides and proteins can also be prepared from the sequence information from any of a number of publicly accessible protein databases, including those available on the Internet.
  • databases include PubMed, SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq (http://www.ncbi.nlm.nih.gov/PubMed).
  • Bioquest - (monoclonal, polyclonal), BioSource International - (monoclonal, polyclonal); Bio- Synthesis - (monoclonal, polyclonal, peptides), Biotrend - (monoclonal, polyclonal, peptides); Biovendor - (monoclonal, polyclonal), Bioworld - (monoclonal, polyclonal, peptides); Babraham Technix - (monoclonal, polyclonal), Capralogics - (polyclonal), Cell Essentials - (monoclonal, polyclonal, peptides) - bioreactor production and antibody purification; Charles River Laboratories - (polyclonal), Charles River Laboratories SPAFAS - (polyclonal) - custom manufacturing of antibodies (antiserum or IgY), Cosmix - phage-display based services including custom mouse Fab antibodies, Covalab
  • neoplastic human host derived from a neoplastic human host are cloned by fusion with immortalized human cell lines to provide hybridomas secreting monoclonal antibodies (MAbs) specific for a cell surface antigen of a neoplastic cell.
  • MAbs monoclonal antibodies specific for antigens of solid tumor cells, such as breast cancer cells or leukemic cells which are not found on normal cells of the same tissue type, are provided for use in diagnostics and therapy.
  • the peptide-adjuvant mixture is emulsified in Freund's complete adjuvant and injected subcutaneously into one rabbit. Two weeks later the peptide/enhancer mixture is emulsified in Freund's incomplete adjuvant and is injected again subcutaneously. Three days after this injection, five ml of blood is drawn through an ear vein and the resultant sera is tested for antibody titers. Approximately two weeks after the second injection, each rabbit is boosted with only the peptide/enhancer mixture and bled four days later. Subsequent injections, containing only the peptide enhancer mixture, and bleeds are performed once a month.
  • a five ml blood sample is drawn and the serum tested for antibody titer.
  • a two log dilution of the neat sera i.e., 1:100 dilution to 1 : 10,000 dilution
  • the neat sera can not be used for further assay development due to rather high background color generation. Consequently, antisera is purified as described hereinafter.
  • Each polyclonal antiserum is purified by column chromatography using a mixed ion exchange resin (J. T. Baker, Inc., Phillipsburg, NJ).
  • the resin fractionates the serum into two major fractions: one fraction containing serum contaminants such as albumin and transferrin and the other fraction containing a highly enriched immunoglobulin fraction.
  • the resin-bound antibody is eluted from the column using a linear gradient of 0 to 0.75 M NaCl in 25 mM MES (2-N-Morphohnoethanesulfonic acid) (pH 5.6 without NaCl, pH 7 0 at 0.75 M NaCl). Five ml fractions are collected and analyzed for protein content (absorption at 280 nm).
  • rabbit polyclonal antibodies are prepared by immunizing a rabbit with polyacryiamide gel material containing affinity-purified protein of interest.
  • the IgG fraction is isolated from the obtained antiserum and absorbed by passage through columns with immobilized human protein.
  • mice of the BALB/c strain are immunized by giving three lntrape ⁇ toneal injections with
  • Myeloma cells in logarithmic phase of growth are resuspended in serum-free medium and readied for fusion with BALB/c spleen lymphocytes.
  • the spleen and lymph node lymphocytes and myeloma cells are mixed in a ratio of 1: 1.25 and 1:2, respectively.
  • Cells were fused by dropwise addition of 50% (wt/vol) polyethylene glycol 4000 (PEG) at 37 °C at about 5 ml to 10 8 and 1 ml to 4.5x 10 7 for the spleen and lymph node lymphocytes, respectively.
  • the fusion is stopped by gentle addition of serum-free medium After cent ⁇ fugation, the supernatant is removed and the cells are washed once in serum containing medium.
  • hypoxanthine-aminopte ⁇ n-thymidine (HAT)-conta ⁇ n ⁇ ng medium The fused cells at an amount of approximately 7x l0 5 cells/well (spleen fusion) and 5x l0 5 cells/well (lymph node fusion) are distributed in 50 microhter ahquots to wells of flat-bottomed microtiter plates containing 150 microhter of selection medium.
  • the cells are incubated at 37 °C in 5% CO 2 in a humidified incubator
  • the selection medium is renewed after a week or when needed.
  • the wells are inspected for hybridoma growth.
  • ELISA-positive wells are transferred into cups of 24-well plates and then to small 25 cm culture flasks. ELISA-positive hybrid cells are frozen in liquid nitrogen as early as possible. Hybridomas from ELISA positive wells are cloned by limited dilution.
  • Antibodies are then purified as follows: The Protein G Sepharose 4 FF column is opened by removing the top cap first. This will avoid air bubbles being drawn into the gel. The 20% ethanol storage solution is poured off and the Protein G Sepharose 4 FF column is equilibrated by filling it to the top with Binding Buffer (-30 ml) whereafter the column is allowed to drain. The column will stop flowing automatically as the meniscus reaches the top frit, preventing the column from drying out. The culture supernatants are centrifuged, filtered and 50-150 ml of the prepared sample is applied and allowed to absorb into the gel.
  • Binding Buffer -30 ml
  • Unbound proteins are washed away by filling the columns to the top with Binding Buffer and the buffer is allowed to pass through the column, eluting unbound materials.
  • the bound IgG is eluted by filling the column with Elution Buffer on the column.
  • One ml fractions of eluted antibodies are collected in minisorb tubes containing neutralizing buffer, and the purity of the elution fractions is checked on a 8-25% gradient gel employing Phast gel System (Pharmacia) followed by silver staining. Isotyping of obtained monoclonal antibodies is achieved by Mouse Typer Sub-Isotyping kit (Bio- Rad).
  • Twenty or more reagents are coupled to uniformly sized microspheres according to the literature.
  • Each of the twenty or more reagents is coupled to a specific subset of microspheres, which are dyed with two types of fluorescent materials, such that each subset exhibits a characteristic fluorescence signature.
  • the characteristic fluorescence signature allows a flow analyzer to distinguish the members of one subset from those of another.
  • Twenty or more unique subsets of microspheres are prepared, each according to methods similar to those described, e.g., in PCT Application Number US98/21562.
  • Kits are also prepared comprising the MAP Test Panel and associated buffers, vials and supplemental reagents.
  • test samples typically, blood samples
  • the test samples are exposed to the MAP Test Panel and the results (i.e., biochemical data generated) are recorded using a flow analyzer.
  • Biochemical data from thousands of volunteers are compiled in a database, which can be cross-checked with the identities and accompanying medical conditions or histories of the individuals from which the biochemical entries originated.
  • Test samples are periodically (e.g., biannually) withdrawn from the volunteers over a period of five years. Each time the health, condition and medical records of each volunteer are updated. Careful examination of the information presented in the database, even after a short period of 18 months, reveals relationships between features of the biochemical data and the relative health or medical condition of the subjects. Indeed, the development of pathological conditions is foretold by the biochemical data generated in advance of a formal diagnosis or of the appearance of clinical disease. Similarly, a separate group of subjects are followed over the course of drug administration or experimental therapy, to obtain direct information about the effects of same on protein expression levels and their consequences on the health, recovery, or the occurrence of unwanted complications.

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  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Medical Informatics (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Primary Health Care (AREA)
  • Biomedical Technology (AREA)
  • Data Mining & Analysis (AREA)
  • Databases & Information Systems (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention est basée sur l'observation que toutes les maladies peuvent être diagnostiquées par analyse d'au moins 200/300 substances biochimiques présentes dans le sang du patient. De plus, on peut également prédire l'évolution d'une maladie à partir du profil de ces substances biochimiques dans le sang. L'invention met en application des procédés automatisés rapides afin de déterminer les concentrations de ces substances biochimiques dans le sang et compare ces concentrations à une base de données contenant des informations obtenues à partir d'une étude prolongée sur de nombreuses années et effectuée sur environ 200 000 personnes. En se basant sur les corrélations découvertes entre les concentrations biochimiques dans le sang et l'existence d'états pathologiques, l'invention permet de diagnostiquer un état pathologique actuel chez le patient et a la capacité de prédire l'apparition de maladies futures chez ce patient.
EP00965004A 1999-09-15 2000-09-15 Creation d'une base de donnees biochimiques et procedes d'utilisation Withdrawn EP1250675A2 (fr)

Applications Claiming Priority (3)

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US15394199P 1999-09-15 1999-09-15
US153941P 1999-09-15
PCT/US2000/025183 WO2001020533A2 (fr) 1999-09-15 2000-09-15 Creation d'une base de donnees biochimiques et procedes d'utilisation

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