WO2001014425A1 - Systemes de diagnostic a fins multiples utilisant des puces a proteines - Google Patents
Systemes de diagnostic a fins multiples utilisant des puces a proteines Download PDFInfo
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- WO2001014425A1 WO2001014425A1 PCT/KR2000/000928 KR0000928W WO0114425A1 WO 2001014425 A1 WO2001014425 A1 WO 2001014425A1 KR 0000928 W KR0000928 W KR 0000928W WO 0114425 A1 WO0114425 A1 WO 0114425A1
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- proteins
- protein chip
- protein
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- probe
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
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- B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
- G01N33/56988—HIV or HTLV
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
- G01N33/5761—Hepatitis B
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
- G01N33/5767—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis non-A, non-B hepatitis
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- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
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- C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
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Definitions
- the present invention relates to a protein chip which is prepared by fixing a plurality of probe spots on a solid micro substrate, wherein the probes are proteins or polypeptides capable of binding to target proteins in test samples.
- the present invention further relates to a method for manufacturing the protein chip, a method for diagnosing diseases of subjects and an automated diagnostic and/or analytic system using the protein chip.
- the automated diagnostic and/or analytic system comprises one or more protein chips of the invention, on which a number of probe spots are integrated in defined arrangement, a microarrayer performing a reaction between the probe and target proteins in test samples (e.g., sera of subjects), a reader for acquiring the signal of the reaction, and a means for transforming the signals into computer-readable data and analyzing the data.
- the protein chip of the invention combined with the diagnostic system can be used in analyzing such materials as an antigen comprised in samples quantitatively and qualitatively enabling multipurpose diagnoses of various diseases even with a small amount of samples for a number of subjects at a time, with a high throughput. Further advantages of the protein chip of the invention include feasibility for automation and rapidity of the diagnostic processes, and possibility of constructing profiles of specific diseases.
- a biological chip or a biochip also referred to as a biological array is a new terminology introduced only recently.
- the biochip refers to a solid support substrate on which biological substances such as nucleic acids are fixed in a defined arrangement suitable for the purpose of use.
- biological substances such as nucleic acids
- DNA chip there is a DNA chip on which the fixed substance is DNA fragments.
- the "substrate” refers to microplates or microchips, with small size and thin thickness, made of various materials.
- the principle of the biochip is based on the interaction of probe proteins immobilized on a micro-substrate and target proteins.
- this interaction is complementary hybridization of oligonucleotides immobilized on the microchips and DNA present in test samples; and, in the case of a protein chip, protein-protein interaction such as antigen-antibody interaction or ligand-receptor binding.
- DNA sequencing technique has revealed nucleotide sequence of DNAs contained in a genome of many organisms from bacteria to a human and the completion of the Genome Project will give us more information about structures and functions of the genomes.
- DNA sequencing technique since it is so laborious and time-consuming to analyze the huge quantities of genetic information newly disclosed everyday through conventional techniques, more rapid and accurate techniques which enables to analyze many genes simultaneously are required.
- DNA chips have been developed for genetic researches by combining conventional techniques in molecular biology with mechanical automation and electronically controlling techniques.
- DNA chip refers to a chip on which many DNA fragments are immobilized in a highly integrated manner for a gene analysis.
- DNA chips are expected to replace conventional research techniques because of its rapidness and accuracy for accumulating and processing data, the convenience of operation and feasibility for automation. DNA chips will have a wide range of applications, such as in the functional analysis of genes, screening of cancer or disease-related genes (Yershov K. et al., Genetics 93:4913-4918, 1996; Heller R.N. et al, Proc. Natl. Acad. Sci. USA 94:2150-2155, 1997; Derisi J. et al, Nat. Genet. 14:457-460, 1996), gene therapies, quarantines of animals or plants, examinations of food safety, development of new medicines (Winters M.A. et al, Antimicrob. Agents Chemother.
- DNA chips are classified into 4 types below, according to their manufacturing method, especially the method for fixing oligonucleotides on their substrate:
- a DNA chip of pin microarray type wherein oligonucleotides are inlaid at a predetermined location by a pin-type microarrayer.
- a DNA chip of ink jet type wherein oligonucleotides in a cartridge are sprayed onto the chip by electronic forces.
- a DNA chip of photolithographic array type wherein oligonucleotide arrays are directly synthesized on the chip using photosensitive chemicals in a light-directed manner.
- a DNA chip of electronic array type wherein (-) charged DNA attaches to (+) charged materials coated on the chip.
- Methods for diagnosing diseases developed so far include the use of reagents for blood cell agglutination, radioimmunoassay (RIA), enzyme immunoassay (EIA), chemiluminescence immunoassay (CLIA), etc.
- RIA radioimmunoassay
- EIA enzyme immunoassay
- CLIA chemiluminescence immunoassay
- These conventional diagnostic methods comprise the steps of: immobilizing antigens, incubating the immobilized antigens with antibodies in the sera or other samples of subjects, and detecting the antigen-antibody interaction with secondary antibodies labeled with radioactive chemicals, enzymes or fluorescence dyes, etc.
- a protein chip is different from a DNA chip in that the substance immobilized on the substrate is protein rather than DNA; it requires multiple-step reactions (such as antigen-first antibody interaction, primary antibody-secondary antibody interaction and washing between the reaction steps); and, therefore, different method and condition for manufacturing a protein chip and different reaction conditions are needed as well as delicate controlling technique. More specifically, many factors have to be considered for manufacturing protein chips. Firstly, antigenic proteins or polypeptides to be fixed on the chips have diverse electrical properties according to their kinds and structures unlike DNA having (-) charge commonly. Secondly, the proteins or polypeptides are relatively large in size compared with a DNA probe having the size ranging from 15-25 base pairs to approximately 500 base pairs. Thirdly, the antigenic proteins or polypeptides immobilized on chips must retain their antigenic conformation necessary for binding to antibodies in test samples.
- the protein chip should contain various antigenic proteins on a substrate, and, accordingly, it is required that the antigenic proteins are immobilized at an optimum condition determined by considering the optimum immobilization condition for each antigenic protein.
- a high accuracy in the reaction locations is required for a protein chip which employs an immunoassay method requiring multi-step reactions.
- different control programs from those used for DNA chips are required to be developed for the multi-step reactions of a protein chip.
- Protein chips have a wide range of applicability, e.g., in the diagnoses of various kinds of metabolic diseases such as virus- or bacteria-infected diseases, search for antagonists to various kinds of receptors by using a receptor-ligand binding reaction, and researches on the enzyme-substrate interactions and screening for the inhibitors of an enzyme by employing the chip wherein the enzyme is immobilized thereon.
- metabolic diseases such as virus- or bacteria-infected diseases
- search for antagonists to various kinds of receptors by using a receptor-ligand binding reaction
- researches on the enzyme-substrate interactions and screening for the inhibitors of an enzyme by employing the chip wherein the enzyme is immobilized thereon in the aspect of automation, early construction of a system therefor is possible by using the previously developed DNA chip analyzers.
- a further object of the present invention is to provide an optimum method for preparing a protein chip, conditions for the method being inevitably different from that for a DNA chip since the probe materials immobilized on the two kinds of chips are different from each other.
- Additional object of the present invention is to provide biological and chemical materials that are necessary for constructing a protein chip useful for diagnosing various diseases.
- a still another object of the present invention is to provide a protein chip that can perform simultaneously the diagnoses of plural diseases in a subject, of one disease in plural subjects, and of plural diseases in plural subjects.
- a still further object of the present invention is to provide an automated system where mass-diagnosis are made by using said protein chip and a database is constructed from the data acquired in a short time from said mass-diagnoses for use in the diagnoses, preventions, and treatment of diseases in a human body.
- the present invention provides a protein chip for mass-diagnosis, which comprises a micro solid substrate on which a plurality of spots of probe proteins are fixed in a defined arrangement, wherein,
- the probe proteins are selected from the group consisting of antigens, receptors and enzymes;
- the probe proteins are fixed on the micro solid substrate via bonds between amino groups of the probe proteins and functional groups of chemicals coated on the substrate;
- the probe proteins are capable of binding to target proteins in a test sample
- the quantity of the probe proteins per spot is minute, e.g., several to dozens pg, thereby reducing the required amount of a sample to a minimum.
- the present invention further provides a method for manufacturing a protein chip containing a micro solid substrate on which a plurality of spots of probe proteins are fixed in a defined arrangement, which comprises the steps of: 1) arraying mixtures of a coating buffer and one or more kinds of probe proteins at predetermined locations on the micro solid substrate, with the quantity of the proteins per spot in a minute amount, e.g., several to dozens pg;
- the present invention also provides a method for analyzing target proteins present in test samples quantitatively or qualitatively, which comprises the steps of:
- the present invention also provides an automated system for diagnosing a plurality of diseases in plural subjects comprising:
- a first microarrayer capable of arraying one or more probe proteins in plural spots on the protein chip
- a second microarrayer controlled to perform sequentially allotting test samples exactly to the locations at which the probe proteins are fixed on the protein chip, washing the protein chip after reaction, and adding secondary antibodies to react with target proteins in the test samples
- a fluorescence microscope or a micro chip reader for detecting the reaction between the probe proteins and the target proteins.
- the present inventors have endeavored to construct a multipurpose automated diagnosing system by developing a protein chip capable of mass diagnosing various viral diseases, and, consequently, have discovered that a clear discrimination between positive and negative serum samples are exhibited when antibodies in the samples are monitored by employing a protein chip prepared via coating various antigens on a solid substrate.
- the present inventors have accomplished the present invention by preparing said protein chip by using an automated microarrayer system which has been employed for preparing a DNA chip and confirming the correct discrimination between positive and negative samples by the protein chip with a high speed fluorescence scanner.
- Fig. 1 shows the genome of HIV used in the present invention and the cloning regions of gag and env antigens therein;
- Fig. 2 represents the genome of HCV used in the present invention and the cloning regions of antigen therein;
- Fig. 3 exhibits schematic view of a process of an antibody-antigen reaction using the protein chip of the present invention
- Fig. 4 displays a result of an antibody-antigen reaction for hepatitis B virus(HBV) which is analyzed using the protein chip of the present invention(a: a positive serum spread on the surface of slide and b: a negative serum spread on the surface of slide);
- Fig. 5 illustrates a result of an antibody-antigen reactions for human immunodeficiency virus(HIV) which is analyzed using the protein chip of the present invention(a: a positive serum spread on the surface of slide and b: a negative serum spread on the surface of slide);
- Fig. 6 depicts a result of an antibody-antigen reaction for hepatitis C virus(HCV) which is analyzed using the protein chip of the present invention(a: a positive serum spread on the surface of slide and b: a negative serum spread on the surface of slide);
- Fig. 7 describes a schematic view of a FITC conjugated antibody which is spread using a microarrayer
- Fig. 8 is a photograph of fluorescence microscope of FITC conjugated antibody which is spread using microarrayer
- Fig. 9 shows a schematic view of the HCV antigen which is spread using a microarrayer
- Fig. 10 represents a result of the antibody-antigen reactions for hepatitis C virus(HCV) which is analyzed using the protein chip of the present invention(a: positive serums spread on the surface of slide and b: negative serums spread on the surface of slide(beginning with stock solution and solutions diluted by factors of 10 "1 and 10 "2 in a and b, respectively));
- Fig. 11 exhibits a result of the specificity for HCV which is analyzed using the protein chip of the present invention(a: positive serums spread on the surface of slide and b: a negative serum spread on the surface of slide);
- Fig. 12 displays a result of the specificity for HIV which is analyzed using the protein chip of the present invention(a: positive serums spread on the surface of slide and b: a negative serum spread on the surface of slide); and
- Fig. 13 compares the sensitivities of the antigen-antibody reactions for HCV and HIV which is analyzed using the protein chip of the present of invention with those of conventional ELISA reaction(a: the sensitivity of antibody-antigen reaction for HCV and b: the sensitivity of antigen- antibody reaction for HIV(from stock solution to the diluted solution by 2 folds of positive serum and negative serum).
- the protein chip of the present invention comprises a micro solid substrate on which a plurality of spots of probe proteins are fixed in a defined arrangement, wherein, 1) the probe proteins are selected from the group consisting of antigens, receptors and enzymes, 2) the probe proteins are fixed on the micro solid substrate via bonds between amino groups of the probe proteins and functional groups of chemicals coated on the substrate, 3) the probe proteins are capable of binding to target proteins in a test sample; and 4) the quantity of the probe proteins per spot is minute, e.g., several to dozens pg.
- the protein chip of the present invention is characterized by its capability of performing simultaneous analysis or diagnosis on a plurality of samples at a time.
- probe protein refers to the proteins that are regularly arranged and immobilized on a micro solid substrate and can bind to the target proteins to be detected.
- the probe protein may be selected from the group consisting of antigens, antibodies, receptors, enzymes etc.
- antigens as probe proteins, it is possible to detect the presence of antibodies in a sample, thereby making it possible to diagnose various diseases.
- receptor proteins are used as probe proteins, it is possible to examine ligands which bind to the receptor proteins and inhibitors of said receptor-ligand binding.
- the inventive protein chip can be applied for the study on the enzyme-substrate reactions, and to screen for inhibitors of enzymatic activity.
- the probe proteins include antigenic proteins originated from unicellular organisms inclusive of virus, bacteria and fungi, animals, and plants.
- inventive protein chip prepared by employing one or more of the antigenic proteins originated from hepatitis B virus (HBV), human immunodeficiency virus(HIV) or hepatitis C virus(HCV) it is possible to diagnose simultaneously various diseases such as hepatitis B, AIDS, hepatitis C, etc.
- target protein refers to a high molecular-weight compound that is included in samples to be tested and can bind the probe proteins immobilized on the protein chip.
- target proteins include antibodies for the disease to be diagnosed in case when antigenic proteins are used as probe proteins; antigens for the disease to be diagnosed in case when antibodies are used as probe proteins; various ligands or - 12 -
- ligand-candidates in case when the receptor proteins are used as probe proteins; and substrates or inhibitor candidates in case when enzymes are used as probe proteins, and, in addition, various kinds of polysaccharides, carbohydrates, and compounds can also be used as targets.
- targets are those substances whose binding to various probe proteins can be regulated by changing physicochemical conditions.
- the substrate of said protein chip refers to a solid plate on which a plurality of spots of immobilized probe proteins are arranged regularly, and it is preferred that surface of the substrate is coated.
- the solid substrates may be made of glass; modified silicone; or commonly used polymers or gels such as tetrafluoroethylene, polystyrene, and polypropylene.
- the surface of the substrate is coated for the immobilization of the probe proteins with a chemical selected from the group consisting of polymers, plastics, resins, carbohydrates, silica, silica derivatives, carbon, metals, inorganic glasses and membranes.
- the chemicals used for coating the substrate have functional groups for binding with amino groups on the probe proteins, said functional groups having preferably an alkyl group, e.g., aminoalkylsilane.
- the substrate serves as a support for immobilizing the probe proteins and provides a space wherein the binding reactions between immobilized probe proteins and the targets in the samples occur.
- the size of the substrate, and the location, size, and shape (for example, dot or line) of the immobilized probe proteins may be varied with the purpose of the analysis, devices including automatic liquid-handling apparatus and reader, etc.
- Probe proteins are arranged in a predetermined defined area on the micro solid substrate, and preferable but not limitative example of said predetermined defined area is a circular spot having a diameter ranging from 150 to lOOO ⁇ m.
- said solid substrate include a tetragonal plate having a size of about 2.5 cm x 7.5 cm or a circular disk having a diameter of about 2-10 cm, and about 100-10,000 spots can be arranged per protein chip. The spots are arranged in plural columns and rows or they are arranged around the circumference of the round disk.
- a multipurpose protein chip can be prepared by dividing the substrate into one or more sectors and immobilizing proteins of a kind in each sector, said proteins in a sector being different from those in other sectors.
- the probe protein-coated chip In order to accelerate the immobilization of the probe protein by strengthening the binding of the probe proteins and the substrate, it is preferable to immerse the probe protein-coated chip in ethanol solution having a concentration of more than 98%, preferably, 100%.
- the present invention also provides a method for preparing a multipurpose protein chip having the features as described above.
- the inventive method comprises the steps of: 1) arraying mixtures of a coating buffer and one or more kinds of probe proteins at predetermined locations on a micro solid substrate, with the quantity of the proteins per spot ranging from several to dozens pg; 2) immobilizing the probe proteins by incubating the substrate at room temperature; 3) fixing the probe proteins on the substrate by immersing the substrate in 100% ethanol; and 4) drying the substrate obtained in step 3).
- sodium phosphate buffer or sodium carbonate buffer may be used and, preferably, about lOmM sodium phosphate buffer or 50 mM sodium carbonate buffer may be used.
- the kind and concentration of a coating buffer may be selected appropriately considering the kind and property of the probe protein to be immobilized on the substrate. For instance, it is preferable to use sodium phosphate buffer for antigens of HBV; and sodium carbonate buffer, for the antigens of HIV or HCV.
- an automatic microarrayer or liquid-handling devices corresponding thereto in order to arrange and immobilize the probe proteins on the substrate since they allow rapid and massive production or processing of the protein chips.
- a microarrayer suitable for the manufacture of a protein chip has not yet been developed, and, accordingly, a microarryer for manufacturing a DNA chip, e.g., GMS 417 ArrayerTM (Genetic MicroSystems Industry) may be provisionally used in the automated manufacturing process of the present invention.
- the method for analyzing quantitatively or qualitatively a target protein in a sample using the inventive protein chip comprises: 1) reacting a sample with the inventive protein chip; 2) washing the resulting protein chip;
- An embodiment of the method for analyzing a target protein may employ a protein chip which contains two or more kinds of antigen proteins of different diseases, each antigen protein being fixed on a separate portion of the micro solid substrate, and a serum sample taken from a subject to detect the resulting fluorescence using a fluorescence microscope or microchip reader, by which it can be determined whether subject carries such antigens.
- Another embodiment of the method for analyzing a target protein may employ a protein chip which contains a kind of antigen proteins fixed on the micro solid substrate and two or more serum samples taken from plural subjects to detect the resulting fluorescence, thereby determining the subject's infection of the disease.
- the method for analyzing a target protein may employ a protein chip which contains two or more kinds of antigen proteins generating different diseases, each antigen protein being fixed in array on the micro solid substrate, and two or more serum samples taken from plural subjects, thereby determining the subject's infection of such diseases.
- a protein chip which contains two or more kinds of antigen proteins generating different diseases, each antigen protein being fixed in array on the micro solid substrate, and two or more serum samples taken from plural subjects, thereby determining the subject's infection of such diseases.
- Binding of a probe protein fixed on the protein chip to a target protein contained in the sample may be detected by using any the conventional assay methods, e.g., radioimmuno assay(RIA), enzymeimmuno assay(EIA) or chemiluminiscenceimmuno assay(CLIA), among which a florescence detecting system is preferred to process automatically a large number of protein chips in a short time.
- an automated reader e.g., fluorescence-detecting biochip reader, may be used in detecting the binding of a probe protein to a target protein, it would be possible to industrialize directly without development of additional fluorescence-detecting device.
- An embodiment of the present invention describes the method for detecting the antigen-antibody reaction on the protein chip using the fluorescence-conjugated anti-human immunoglobulin G, particularly by reading a large number of protein chips automatically using high-speed scanner GMS 418 Array ScannerTM(Genetic Microsystems industry) which has been used in analyzing DNA chip.
- Use of the automated liquid-handling device and biochip reader may allow to manufacture and read a large number of protein chips at a time.
- the protein chip thus manufactured is particularly suitable in making a database of disease profiles obtained from individuals or groups thereof.
- the present invention further provides an automated diagnosis system for acquiring plural subjects' profiles for plural diseases in a short time, which comprises: the inventive protein chip; a first microarrayer for arranging automatically one or more kinds of probe proteins in plural spots on the protein chip; a second microarrayer for adding accurately a sample to target protein-fixed location of the protein chip, the second miroarrayer being controlled to perform sequentially allotting a sample to the location, washing the resulting reaction mixture and adding secondary antibodies thereto; and a fluorescence microscope or microchip reader for detecting the binding of probe protein to target protein.
- the automated diagnosis system further comprises a computerized device for making database of disease profiles with the data acquired by using the fluorescence microscope or microchip reader.
- the method for quantitative or qualitative analysis of target proteins is useful in diagnosing various diseases, preferably infectious diseases.
- the inventive diagnosis system using the protein chip is conducted by reading even small amount of antigen(10 "6 to 10 "5 orders of amount) and antibodies and detecting the resulting fluorescence, and, therefore, it is very economic and has high sensitivity and specificity.
- ELISA method requires at least 100 ng of antigens per well whereas the inventive diagnosis system requires at least 1 pg of antigens per spot. Therefore, the inventive diagnosis system examines plural blood samples using even a small amount of antigens.
- microarray system comprising automated liquid- handling device and reader may reduce necessary steps, thereby preventing waste of time, labor and resource, and may provides a good diagnosis only by a simple fluorescence-detecting step, thereby being effective in early diagnosis for various infectious diseases and prevention thereof.
- Another embodiment of the present invention describes an immunological diagnosis using a protein chip which contains Hepatitis B virus(HBV), human immunodeficiency virus(HIV) or Hepatitis C virus(HCV) antigens with positive and negative serum samples against each antigen(Fig. 3).
- Antigen-antibody reaction was detected in all positive serum samples but not at all in negative serum samples(Figs. 4 to 6 and 10).
- Fluorescein isothiocyanate(FITC)-conjugated antibody could sufficiently be detected with the biochip reader GMS 418 ScannerTM(Fig. 8), which suggested that the manufacturing time for a suitable detector can be reduced.
- specificity and sensitivity of HCV or HIV antigen- antibody reaction were examined and the results show that specificity and sensitivity thereof were excellent(Figs. 11 to 13).
- GMS 417 ArrayerTM for DNA chip can examine 60 chips simultaneously and the microarrayer can place 4 spots per sec. and give results using the analyzer having a capacity of reading 30 lines per sec.
- the inventive automated diagnosis system of the present invention using the protein chip is more efficient in terms of time, labor and resources than ELISA or CLIA method. That is, it is possible to diagnose simultaneously tens of diseases using a subject's blood sample, a viral disease using hundreds of subjects' blood samples or tens of diseases using hundreds of subjects' blood samples. Consequently, the automated diagnosis system can construct disease profiles of individuals constituting a specific group into database, which can be used actively in managing disease and preservation of health at nation level.
- HB V antigens were purchased from OEM CONCEPTS(#H6- V 19,
- HIV and HCV antigens were prepared as follows.
- HIV genome (GenBank Accession Number U26942) was subjected to polymerase chain reaction(PCR) to amplify polynucleotides encoding capsid protein p24 of gag protein and extracellular domain gp41of envelope protein.
- PCR polymerase chain reaction
- Each PCR product was cloned to vector pCRII(Invitrogen, Netherland) and Bglll/BamHI fragment of the recombinant vector was inserted at Bglll/BamHI sites of vector pMAL-c2(New England Biolabs Inc ;NEB, USA) to obtain an expression vector.
- Primers and reaction condition used in the PCR were shown in Table 2. Table 2
- the expression vector thus obtained was introduced into the E. coli Topi OF' to overexpress the antigen proteins. Then, the antigen proteins were purified by manufacturer's instruction and the purified antigen proteins were used as probe proteins of protein chip(Fig. 1).
- the polynucleotide(SEQ ID NO: 1) encoding NS3 antigen and the polynucleotide(SEQ ID NO: 2) encoding NS3core fusion antigen of Korean HCV were selected.
- NS3 antigens were prepared by conducting PCR; cloning the PCR product into vector pCRII; inserting the Ndel/EcoRI fragment of the resulting recombinant vector at Ndel/EcoRI sites of T7-tagged pET-17b vector(Novagen, USA); transforming E. coli Topi OF' with the resulting expression vector to overexpress antigens; transforming the resulting transformant with BL21; purifying antigens according to manufacturer's instructions.
- the purified antigens were used as probe proteins of protein chip.
- NS3 fusion antigens were prepared by conducting PCRs to amplify polynucleotides encoding core and NS3 antigens, respectively; cloning the polynucleotides into vector pCRII, respectively; inserting fragments of the recombinant vector at Bglll/Xbal and Bglll/BamHI sites of expression vector IciA vector; transforming E. coli Topi OF' with the resulting recombinant vector to overexpress antigens; transforming the resulting transformant with BL21 ; purifying the antigens according to manufacturer's instructions.
- the purified antigens were used as probe proteins of protein chip(Fig. 2).
- the PCR primers for amplifying the polynucleotide encoding the NS3 antigen and NS3core antigen of HCV are shown in Table3. PCR was performed as like the amplification of HIV antigen gene.
- HBV-positive and 2 HBV- negative serum samples provided by Institute of molecular biology & genetics of Seoul National University and, HBV-positive and HBV- negative control serum samples contained in ELISA kit of Green Cross Co. were used.
- HIV antigen 10 HIV-positive serum samples provided by medical college of Seoul National University, National institute for Health, Central Medical Center and Yonsei University, and 5 HIV-negative serum samples provided by Institute of molecular biology & genetics of Seoul National University were used.
- HCV 12 HCV-positive and 8 HCV-negative serum samples provided by Green Cross Co. were used.
- the HIV-positive serum samples were diluted by 2-fold and then heat-inactivated at 50 °C for 2 hours before use.
- HBV antigen-antibody reaction was conducted as shown in Fig. 3.
- HBV antigens obtained in Preparation Example 1 were diluted to a final concentration of 100 ⁇ glml with coating buffer(10mM sodium phosphate, pH 7.6). Each 1 [A of diluted antigen solution was added on the surface of aminoalkylsilane-coated microscope slide glasses(#S4651, Sigma Diagnostics, USA). The resulting slide glasses were kept at room temperature, and then, the coated antigens thereon were fixed by immersing the antigen-coated protein chip in 100% ethanol to reinforce bonding of amino group of antigen to alkyl group on the slide glasses and drying the resulting slide glasses completely. (1-2) Antigen-antibody reaction
- the slide glasses was washed three times with PBST and dried. Each fluorescence of the slide glasses was detected by fluorescence microscope and photographed. The HBV antigen-antibody reaction(Fig. 4) was repeated four times. All of the positive serum samples and positive control solution showed intensive fluorescence while the negative serum samples and negative control solution did not show any fluorescence and the control dilution solution did not.
- Example 1 The procedure of Example 1 was repeated except that HIV antigen and anti-HIV serum samples obtained in Preparation Example 1 and 2, respectively, were used in place of HBV antigen and anti-HBV serum samples.
- HIV antigens obtained in Preparation Examples 1 and 2 were diluted to a final concentration of 100 ⁇ g/m- ⁇ with coating buffer(50mM sodium carbonate, pH 9.6). HIV antigen-antibody reaction(Fig. 5) was repeated three times similarly with the above HBV antigen-antibody reaction. As the result, all positive serum samples showed evident fluorescence, while the negative serum samples and control did not show any fluorescence. Fluorescence intensities of the positive serum samples were materially different to those of the negative serum samples, and the coincident results were shown in ELISA.
- HCV antigens obtained in Preparation Examples 1 and 2 were diluted to a final concentration of 100 ⁇ g/m& with coating buffer(50 mM sodium carbonate, pH 9.6) .
- the procedure of Example 1 was repeated except that HCV antigens and anti-HCV serum samples obtained in Preparation Examples 1 and 2 were used in place of HBV antigens and anti-HBV serum samples.
- GMS418 Array ScannerTM (Genetic Microsystems, GM200), fluorescence of fluorescence-conjugated anti-human IgG (#31529,Pierce, USA) bound to antigen on the slide glass was determined.
- Fig. 8 Result of analyzing the slide thus manufactured using the high- speed fluorescence scanner is shown in Fig. 8, which suggest that the scanner known to only recognize DNA-staining fluorescence dyes Cy3 and Cy5 can read FITC conjugated antibody sensitively.
- Example 5 Construction of HCV protein chip using microarrayer and antigen-antibody reaction
- a protein chip for HCV antigen was constructed using GMS 417 Arrayer TM f as shown in Fig. 9.
- HCV antigen having an initial concentration of 100 ⁇ gl ⁇ l was serially diluted to a dilution factor of 10 "5 .
- the diluted antigen solutions were added to a 96 well microplate and the microplate was set in a microarrayer.
- Each 10 p-C of the diluted antigen solutions were spotted for three times in an array mode using 4 pins and rings to construct a protein chip including 4 lanes having 18 spots per lane, wherein the interval between spots was 375 ⁇ m; the interval between lanes, 0.9 mm; a diameter of spot, 150 ⁇ m; and unit amount of antigen based on the initial concentration, 1 pg/m- ⁇ .
- the protein chip was reacted with antibodies according to the procedure shown in Fig. 3, provided that the antibodies spotted on a parafilm was adhered closely to a surface of the slide glass rather than reacted with each spot, considering the size of the spots and the intervals between them.
- Example 6 Specificity of antigen-antibody reaction for HCV protein chip constructed by using the microarrayer
- a protein chip for HCV antigen was constructed using GMS 417
- HCV antigen 300 ⁇ glml was added to a 96 well microplate and the microplate was set in a microarrayer.
- HCV antigen was spotted on a slide glass(2.5 ⁇ 7.5 cm 2 ) (or chip) in an array mode using 4 pins and rings to construct a protein chip including 4 sets having 400 spots per set(see Fig.
- Example 7 Specificity of antigen-antibody reaction for HIV protein chip constructed by using the microarrayer
- a protein chip for HIV antigen was constructed using GMS 417
- HCV antigen was spotted on a slide glass(2.5x7.5 cm 2 ) (or chip) to construct a protein chip including 4 sets having 400 spots per set(see Fig. 7). 10 p£ of antigen was added per spot and, accordingly, 3 pg of antigen was used per sample.
- the protein chip prepared above was reacted with 10 positive and
- the chip was read at 532 nm with a flying objective microscope and the data obtained therefrom were analyzed using ImageneTM(Genetic MicroSystems, version 2.0), a software for analyzing the expressed data (see Fig. 12).
- Example 8 Sensitivities of antigen-antibody reactions for HCV and HIV protein chips constructed by using the microarrayer
- a protein chip for HCV and HIV antigens was constructed using GMS 417 Arrayer TMf as shown in Fig. 7, and its sensitivity for the antibodies in blood samples was examined and compared with that of the conventional ELISA method
- Each 300 g/m.£ of HCV and HIV antigens was added to a 96 well microplate and the microplate was set in a microarrayer. Antigens were spotted on a slide glass(2.5x7.5 cm ) (or chip) to construct a protein chip including 4 sets having 400 spots per set(see Fig. 7). 10 p£ of antigen was added per spot and, accordingly, 3 pg of antigen was used per sample.
- the protein chip prepared above was reacted with positive and negative sera according to the procedure of Fig. 3. One positive serum and one negative serum were adopted for HCV and HIV antigens, respectively, and they were subjected to serial double dilutions. Due to some technical problems, one antibody was reacted per set and the antibodies spotted on a parafilm was adhered closely to a surface of the slide glass in order to apply the antibodies uniformly.
- the chip was read at 532 nm with a flying objective microscope, the data obtained therefrom were analyzed using ImageneTM(Genetic MicroSystems, version 2.0), a software for analyzing the expressed data (see Fig. 12), and intensity of the fluorescence was determined for each set.
- An anti-human IgG- HRP(horseradish peroxidase) conjugate was diluted to 1/10,000 dilution and 100 ⁇ l of the dilution was then added to the well and reacted in 37 ° C incubator for 1 hour. After the reaction, wells were washed five times with a washing solution and 100 ⁇ l of tetramethylbenzidine (TMB) as a substrate solution was added thereto. The mixture was reacted at room temperature for 30 minutes and the reaction was terminated with a stopping solution (IN sulfuric acid solution). When the reaction was completed, absorptions were measured at 450 nm with an ELISA reader.
- TMB tetramethylbenzidine
- the protein chip analysis for HIV exhibited a very high sensitivity with the result that the S/CO ratio of the positive serum was at least 5 even at 2-8, while that of the negative serum was below the cutoff even at 2-1 and thereafter.
- the protein chip analysis for HCV also exhibited a high sensitivity with the result that the positive serum showed positive reaction up to 2-7, while the negative serum was below the cutoff even at 2-1 and thereafter.
- the result of the ELISA analysis for HIV was different from that of the protein chip; the S/CO ratio of the positive serum was at least 5 even at 2-8, as in the protein chip analysis, while that of the negative serum was above the cutoff up to 2-5.
- the positive serum showed a gentle decrease depending on the dilution factor, while the S/CO ratio of the negative serum was above the cutoff up to 2-5. This result showed that there may be an error in determining the infection of a disease by using the ELISA method.
- Examples 1 to 5 demonstrated that the reactions between antigen and antibody are conducted with a sufficient effectiveness on the protein chip at room temperature. Further, as shown in Examples 6 to 8 above, it was proved that the antigen-antibody reaction using the protein chip of the present invention was superior to that in ELISA method in terms of specificity and sensitivity, considering the reaction time and temperature.
- the diagnostic system using the protein chip of the present invention requires the smaller amount of antigen and antibody than those of conventional ELISA or CLIA method and provides excellent results in terms of sensitivity and specificity. Further, the inventive process is very economical in aspects of examining procedure and cost due to the reduced reaction steps and time.
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-
2000
- 2000-08-19 AU AU67356/00A patent/AU6735600A/en not_active Abandoned
- 2000-08-19 WO PCT/KR2000/000928 patent/WO2001014425A1/fr active Application Filing
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AU6735600A (en) | 2001-03-19 |
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