WO2002073180A1 - Systeme de microtitrations de proteines base sur des anticorps - Google Patents

Systeme de microtitrations de proteines base sur des anticorps Download PDF

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WO2002073180A1
WO2002073180A1 PCT/US2002/007341 US0207341W WO02073180A1 WO 2002073180 A1 WO2002073180 A1 WO 2002073180A1 US 0207341 W US0207341 W US 0207341W WO 02073180 A1 WO02073180 A1 WO 02073180A1
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protein
proteins
detection
antibody
microarray
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PCT/US2002/007341
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Ruo-Pan Huang
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Ruo-Pan Huang
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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
    • 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
    • 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
    • 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
    • G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
    • 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
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
    • 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
    • G01N33/6854—Immunoglobulins
    • G01N33/686—Anti-idiotype
    • 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
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • 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
    • G01N33/6878—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids in eptitope analysis

Definitions

  • This invention relates to the analysis of polypeptides and proteins. More specifically, the invention relates to a novel method for the detection and identification of proteins by use of an antibody-based protein array assay system, which has the advantages of specificity associated with enzyme-linked immunosorbent assays (ELISA), of sensitivity associated with enhanced chemiluminescence (ECL) and of high-throughput associated with microarrays without requiring sophisticated equipment.
  • ELISA enzyme-linked immunosorbent assays
  • ECL enhanced chemiluminescence
  • proteomics with its underlying goal of assembling a complete library of all proteins, has a vital need for tools which will allow researchers to detect and analyze large numbers of proteins simultaneously. Tools for high throughput analysis of proteins in a manner like that enabled by DNA microarrays for gene analysis are simply not available.
  • the invention described and disclosed herein a novel method of analyzing proteins and other biomolecules, provides a much more capable method to analyze and evaluate protein expression than current analytical and diagnostic methods and does so with less complexity and at lower cost.
  • This invention would, therefore, be of great use in basic research, in medical diagnosis and in the biotechnology industry.
  • the present invention provides a method for detecting a specified protein comprising: immobilizing capture proteins in an array on a membrane; passing a solution containing proteins over the array of capture proteins, thereby capturing certain proteins; adding a detection protein which binds to a specified protein, or portion thereof; and detecting bound detection protein, the detection of bound detection protein indicating the presence of the specified protein, or portion thereof.
  • the present invention provides a method for detecting a specified protein comprising: immobilizing capture proteins in an array on a membrane; passing a solution containing detectable proteins over the array of capture proteins, thereby capturing certain proteins; and detecting bound detectable proteins.
  • the present invention provides a method for characterizing proteins by determination of interactions between the protein to be characterized and other proteins with defined properties. For example, use of an antibody specific for a given epitope can be used to determine that a protein of interest comprises the given epitope if the antibody binds to the protein of interest.
  • an antigen or antibody of interest is immobilized to a specific position on a microarray by specific binding interactions to a membrane-bound capture protein wherein the character of the capture protein provides information about the character of the protein of interest.
  • the binding of an antigen or antibody of known character to an antigen or antibody of interest immobilized on a membrane microarray can be used to provide information about the membrane-bound antigen or antibody of interest.
  • FIG. 1 Schematic representation of ELISA and ECL format to detect specific antigens in protein arrays. Antigens consisting of purified IgGs are immobilized on the PDVF membranes. Membranes are blocked with BSA. Specific antigens are detected using corresponding specific antibodies coupled with ECL.
  • FIG. 1 Raw image data for detection of individual antigen. 100 ⁇ l of individual IgG (50 ⁇ g/ml), TBS (negative control), BSA (negative control) and HRP-conjugated anti- Bovine IgG (as positive control) were immobilized onto PDVF membrane as indicated in the figure. The stripes were then incubated with different HRP-conjugated anti-IgGs (5 ng/ml) or without anti-IgG. The signals were then visualized by ECL. Abbreviations: Bov, bovine; Ck, chicken; Gt, goat; GP, guinea pig; Hu, human; Ms, mouse; Rb, rabbit; Shp, sheep.
  • FIG. 3 Sensitivity of detection. Different IgGs, negative control (TBS and BSA) and positive control (HRP-conjugated anti-Bovine IgG) were spotted onto PDVF membrane. The stripes were then incubated with different concentrations of HRP- conjugated anti-Bovine IgG and subjected to ECL. Images were obtained by short exposure (10 seconds) and long exposure (5 minutes).
  • Figure 4 Detection of an entire 96-well array. 100 ⁇ l of different Igs (50 ⁇ g/ml) were immobilized onto PDVF membranes. Each row contains one individual antigen. Individual membrane was then incubated with the indicated HRP-conjugated antibodies (5 ng/ml).
  • FIG. 5 Schematic representation of Sandwich ELISA and ECL format to detect specific cytokines or growth factors in protein arrays.
  • Specific capture antibodies are immobilized on PDVF membranes.
  • Specific antigens are captured by the correspondingly immobilized antibodies.
  • Biotin-conjugated detection antibodies then specifically bind to specific antigens. All detection antibody-binding events are indirectly detected using streptavidin-HRP and ECL.
  • FIG. 6 Specific detection of MCP-land IL-2 by sandwich format ELISA.
  • the stripes immobilized with different concentrations of captured anti-MCP- 1 antibody or anti-IL-2 antibody and other control reagents were incubated with MCP-1(5 ⁇ g/ml), or IL-2 (5 ⁇ g/ml), or EGF (5 ⁇ g/ml), or mock.
  • biotinylated anti-MCP 0.5 ⁇ g/ml
  • biotinylated anti-IL-2 0.5 ⁇ g/ml
  • a biotinylated control antibody was then incubated with the stripes.
  • the unbound biotinylated antibodies were then washed out and the stripes were incubated with HRP-conjugated streptavidin.
  • FIG. 8 Simultaneous detection of multiple cytokines. Different capture antibodies or controls were loaded onto PDVF membranes. Membranes were then incubated with different combinations of cytokines and their corresponding biotin-conjugated antibodies.
  • FIG. 9 Detection of MCP-1 from conditioned medium.
  • the membranes immobilized with different capture antibodies against different cytokines were incubated with 10 fold diluted conditioned media from U251cx43 (a cx43 -transfected human glioblastoma cell line) and U251N23 (a control-transfected human glioblastoma cell line). After washing, the membranes were incubated with a combination of biotin- conjugated antibodies as indicated in the Figure. Signals were detected by ECL system.
  • FIG. 10 Selection of membranes for protein arrays. Different membranes were spotted with IgGs and other controls as indicated. The membranes were then incubated with HRP-conjugated anti-bovine IgG after being blocked with 5 % BSA. The signals were visualized with ECL.
  • FIG. 11 Specificity and sensitivity of detection of HRP-conjugated anti-species- specific IgGs.
  • A MSI magnagraph membranes immobilized with different species- specific IgGs were incubated with individual HRP-conjugated anti-IgGs against specific species or controls as indicated in the figure. HRP-conjugated anti-bovine IgG was spotted onto membranes enable orientation of blots.
  • B Different concentrations of HRP-conjugated anti-bovine IgG as indicated beside each microarray row were used to test the detection sensitivity.
  • FIG. 12 High-density protein arrays to detect HRP-conjugated antibody.
  • A Array layout. Membranes were spotted with different IgGs or controls as indicated in the array layout. There are 504 spots in one array.
  • B Membranes were incubated individually or collectively with HRP-conjugated antibodies as indicated in the figure.
  • FIG. 13 Detection of cytokines in array format with high specificity and sensitivity.
  • A Hybond ECL membranes immobilized with different capture antibodies against different cytokines were incubated subsequently with single cytokine or all six cytokines or controls as indicated in figure, with corresponding biotin-conjugated anti- cytokines or controls and with HRP-conjugated streptavidin. The signals were detected by ECL.
  • B High sensitivity of detection is exemplified by IL-2. Membranes spotted with cytokines were incubated with different concentrations of cytokines. The signals were then detected with biotin-conjugated anti-IL-2 and HRP-conjugated streptavidin.
  • Figure 14 Detection of cytokines in high-density protein array format.
  • A Different cytokines or controls were spotted on Hybond ECL membranes in high-density array format as indicated in figure.
  • B Detection of cytokines with high-density array format was demonstrated by incubation of the array membranes with individual or collective cytokines.
  • FIG. 1 Specific and sensitive detection of antibodies in array format.
  • A MSI magnagraph membranes loaded with different IgGs as indicated were incubated with single donkey anti-species-specific IgGs or all eight antibodies. Membranes were then incubated with HRP-conjugated anti-donkey IgG.
  • B Different concentrations of donkey anti-mouse IgG were used to test the detection limit in array format.
  • FIG. 16 High-density protein arrays to simultaneously detect multiple antibodies. Different IgGs or controls were loaded onto Hybond ECL membranes in an array format indicated in Figure 16 A. Simultaneous detection of multiple antibodies was demonstrated as indicated in Figure 16B.
  • Figure 17. (A) Detection of cytokines in array format with high specificity. Hybond ECL membranes immobilized with different captured antibodies against different cytokines were incubated subsequently with individual cytokine or control as indicated in figure, with corresponding biotin-conjugated anti-cytokine or control and with HRP- conjugated streptavidin. The signals were visualized by ECL.
  • B Detection of cytokines in array format with high sensitivity. Cytokine array membranes were incubated with different concentrations of MCP-1 and IL-2. The intensities of signals were detected by densitometry and plotted against the concentrations of MCP-1 or IL- 2.
  • FIG. 18 Simultaneous detection of multiple cytokines in an array format.
  • the combinations of multiple cytokines as indicated in Figure 18 were incubated with cytokine array membranes.
  • the membranes were then incubated with combination of multiple biotin-conjugated anti-cytokine antibodies. Signals were then visualized by ECL kit.
  • FIG. 19 Detection of multiple cytokine expression from conditioned media.
  • the cytokine array membranes were incubated with 50 fold diluted conditioned media from human glioblastoma cells U251 treated with or without TNF ⁇ . The membranes were then incubated with a cocktail of biotin-labeled antibodies against all twenty- four cytokines and signals were detected by ECL.
  • Figure 20 Detection of cytokine expression from patient's sera. 10 fold-diluted patient's sera were incubated with cytokine array membranes and bound species were detected using ECL.
  • FIG. 21 Conditioned medium arrays.
  • A Conditioned medium array design.
  • B 0.5 ⁇ l of conditioned media from different sources were spotted onto the anti-MCP- 1- coated membrane. The membrane was then incubated with biotin-conjugated anti- MCP-1 and HRP-conjugated streptavidin. The signals were then detected by ECL.
  • C The intensities of signals derived from standard MCP-1 were determined by densitometry and plotted against concentrations of MCP-1.
  • FIG 22 The human cytokine array systems to study the molecular mechanism of tumor suppression.
  • A). A template of human cytokine arrays.
  • B. Human cytokine arrays from Cx43 -transfected cells and control-transfected cells.
  • C). The immunoprecipitated complex separated by SDS PAGE and the levels of MCP-1 protein were detected by Western Blot using antibody against MCP-1.
  • FIG 23 IL-8 was specifically up-regulated in human breast cancer cells harboring mutant type p53.
  • Human cytokine arrays were used to profile cytokine expressions in a variety of human breast cancer cells. Conditioned media containing equal amounts of protein were subjected to protein arrays as described in Materials and Methods. B). The intensities of resulting signals were determined by densitometry. The relative expressions of MCP-1 were shown. C). Concentrations of IL-8 from conditioned media were determined by ELISA and the relative expression levels were shown. D). Cell proliferation rates were performed in the presence of antibody against IL-8 by using 3 H thymidine incorporation assays as described in experimental procedures. Antibody against IL-8 specifically inhibited cell proliferation in cells expressing high levels of IL-8 (BT-20), but not in the cells expressing low amount of IL-8 (MCF-10A).
  • FIG. 24 Supplementation of vitamin E led to down-regulation of MCP-1 expression.
  • FIG. 25 Human cytokine array system adapted to protein chips platform.
  • A). A map for the location of captured antibodies spotted onto protein chips.
  • B). Protein chips printed with 43 cytokine captured antibodies were incubated with samples from different sources: conditioned medium (human breast cancer cells BT549), serum, cell lysate (human MDA-MB-231), tissue lysate (human endometriosis tissue), urine (a normal individual), urine (a normal individual plus purified recombinant cytokines as indicated in the Fig.) and a control (PBS).
  • conditioned medium human breast cancer cells BT549
  • serum cell lysate
  • tissue lysate human endometriosis tissue
  • urine a normal individual
  • urine a normal individual plus purified recombinant cytokines as indicated in the Fig.
  • PBS a control
  • FIG. 26 Schematic representation of biotin-labeled and antibody-based protein arrays. Samples containing proteins are labeled with Biotin. The proteins of interest are captured by antibodies arrayed on protein chips. The signals are detected by Cy3- or cy5-streptavidin and laser scanner.
  • Figure 27 A) Location of captured antibody spot in human cytokine chips; B) Specific detection of individual cytokine labeled by biotin in human cytokine chips. Recombinant cytokine was labeled with biotin. The human cytokine chips were incubated with individual biotin-conjugated cytokines or with a control as indicated in the figure. The detectable signals were generated by incubation with cy3-streptavidin and the images were visualized by laser scanner. C). Simultaneous detection of multiple cytokines in human cytokine chips with high specificity. Human cytokine chips were incubated with different combinations of multiple cytokines labeled with biotin as shown in Fig. 2C. The chips were then incubated with cy3-conjugated streptavidin. Signals were visualized by laser scanner.
  • FIG. 28 Detection of cytokines in array format with high sensitivity.
  • Figure 29 Detection of multiple cytokine expression from real biological samples using biotin-labeled and antibody-based array system.
  • the human cytokine chips were incubated with 50 ⁇ l of biotin-labeled conditioned media from human breast cancer cells (MDA-MB-231), patient's plasma and BT549 cell lysate. The chips were then incubated with cy3 -streptavidin. The signals were detected as described before.
  • FIG. 30 Detection of cytokine expression from one pair of samples using the two color protein array system.
  • A a fixed amount of biotin labeled TNF ⁇ was incubated with cy3 -streptavidin and an increasing amount of biotin labeled TNF ⁇ incubated with cy 5 -streptavidin in the 1.5 ml centrifuge tubes. Both samples were then mixed together and added to the same human cytokine chip.
  • B a fixed amount of biotin labeled TNF ⁇ was incubated with cy5-streptavidin and an increasing amount of biotin labeled TNF ⁇ incubated with cy5 -streptavidin. Both samples were mixed and coincubated with the human cytokine chip. Signals generated by cy3 and cy5 flourescence were scanned using laser scanner with appropriate channels. The intensities of signals were plotted against concentrations of TNF ⁇ .
  • analyte or “analyte molecule” refers to a molecule, typically a macromolecule, such as a polynucleotide or polypeptide, whose presence, amount, and/or identity are to be determined.
  • the analyte is one member of a ligand/anti-ligand pair.
  • Antibody means a polyclonal or monoclonal antibody. Further, the term “antibody” means intact immunoglobulin molecules, chimeric immunoglobulin molecules, or Fab or F(ab') 2 fragments. Such antibodies and antibody fragments can be produced by techniques well known in the art which include those described in Harlow and Lane (Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989)) and Kohler et al. (Nature 256: 495-97 (1975)) and U.S. Patents 5,545,806, 5,569,825 and 5,625,126, incorporated herein by reference.
  • antibodies as defined herein, also include single chain antibodies (ScFv), comprising linked V H and V L domains and which retain the conformation and specific binding activity of the native idiotype of the antibody.
  • Single chain antibodies are well known in the art and can be produced by standard methods, (see, e.g., Alvarez et al., Hum. Gene Ther. 8: 229-242 (1997)).
  • the antibodies of the present invention can be of any isotype IgG, IgA, IgD, IgE and IgM.
  • Antigen includes substances that upon administration to a vertebrate are capable of eliciting an immune response, thereby stimulating the production and release of antibodies that bind specifically to the antigen.
  • Antigen includes molecules and/or moieties that are bound specifically by an antibody to form an antigen/antibody complex.
  • antigens may be, but are not limited to being, peptides, polypeptides, proteins, nucleic acids, DNA, RNA, saccharides, combinations thereof, fractions thereof, or mimetics thereof.
  • an antigen/antibody complex can form as well as assays for the detection of the formation of an antigen/antibody complex and quantitation of the detected protein are standard in the art.
  • assays can include, but are not limited to, Western blotting, immunoprecipitation, immunofluorescence, immunocytochemistry, immunohistochemistry, fluorescence activated cell sorting (FACS), fluorescence in situ hybridization (FISH), immunomagnetic assays, ELISA, ELISPOT (Coligan, J.E., et al., eds. 1995. Current Protocols in Immunology. Wiley, New York.), agglutination assays, flocculation assays, cell panning, etc., as are well known to the person of skill in the art.
  • Bind means the well understood antigen/antibody binding as well as other nonrandom association between an antigen and an antibody.
  • Specifically bind describes an antibody or other ligand that does not cross react substantially with any antigen other than the antigen, or antigens, specified. For instance, “specific binding” of an antibody to a class of antigens having an epitope in common is contemplated.
  • Capture protein is an immobilized protein which binds, is bound by, or forms a complex with, one or more analytes of interest in a sample to be tested.
  • Detection protein is a protein comprising a detectable label which binds, is bound by, or forms a complex with, one or more analytes of interest in a sample to be tested or is a protein which binds, is bound by, or forms a complex with, one or more analytes of interest which can be bound by further species that comprise a detectable label.
  • detectable labels include, but are not limited to, nucleic acid labels, chemically reactive labels, fluorescence labels, enzymic labels and radioactive labels.
  • Discrimination antibodies means antibodies which bind to a specific subset of proteins, wherein binding indicates the presence of some characteristic of any protein bound by the discrimination antibody.
  • the characteristic may be the presence of a specific structure, a specific epitope, a specific sequence or an ability to be bound by a specific antibody, e.g., the discrimination antibody.
  • Fusion protein is a recombinant protein comprising amino acid sequence derived from more than one protein.
  • Membrane means the well understood material of commerce and widespread use in the field of biotechnology, as well as other flexible, non-rigid sheets of polymeric or elastomeric materials. Examples include, but are not limited to, nylon, nitrocellulose, or equivalent materials known to those of skill in the art.
  • “Microarray,” as used herein, is an ordered arrangement of array elements (e.g., capture proteins) capable of binding other species. The elements are arranged so that there are preferably at least one or more different array elements, more preferably at least 10 array elements, and most preferably at least 100 array elements, and even more preferably 10,000, on a 1 cm 2 substrate surface.
  • Mimetic includes a chemical compound, or an organic molecule, or any other mimetic, the structure of which is based on or derived from a binding region of an antibody or antigen. For example, one can model predicted chemical structures to mimic the structure of a binding region, such as a binding loop of a peptide. Such modeling can be performed using standard methods. In particular, the crystal structure of peptides and a protein can be determined by X-ray crystallography according to methods well known in the art. Peptides can also be conjugated to longer sequences to facilitate crystalization, when necessary.
  • the conformation information derived from the crystal structure can be used to search small molecule databases, which are available in the art, to identify peptide mimetics which would be expected to have the same binding function as the protein (Zhao et al., Nat. Struct. Biol. 2: 1131-1137 (1995)).
  • the mimetics identified by this method can be further characterized as having the same binding function as the originally identified molecule of interest according to the binding assays described herein.
  • mimetics can also be selected from combinatorial chemical libraries in much the same way that peptides are.
  • Ostresh et al. Proc. Natl. Acad. Sci. USA 91 : 11138-11142 (1994); Oorner et al, Bioorg. Med. Chem. 4: 709-715 (1996); Eichler et al., Med. Res. Rev. 15: 481-96 (1995); Blondelle et al., Biochem. J. 313: 141-147 (1996); Perez-Paya et al., J. Biol. Chem. 271 : 4120-6 (1996)).
  • Solid support means the well-understood solid material to which various components of the invention are physically attached, thereby immobilizing the components of the present invention.
  • solid support means a non-liquid substance.
  • a solid support can be, but is not limited to, a membrane, sheet, gel, glass, plastic or metal.
  • Immobilized components of the invention may be associated with a solid support by covalent bonds and/or via non-covalent attractive forces such as hydrogen bond interactions, hydrophobic attractive forces and ionic forces, for example.
  • the invention provides arrays useful for the detection, characterization and/or quantitation of antigens or antibodies.
  • the invention further provides methods for the production of arrays of the invention.
  • the invention further provides methods for the use of the arrays of the invention.
  • the present invention relates to a method of detecting a specified protein. More specifically, a method of detecting a protein that comprises the immobilization of capture proteins onto a membrane to form a microarray.
  • a capture protein of the invention is able to bind to the specified protein, also referred to herein as the protein of interest, thereby "capturing" the protein of interest.
  • the method can further comprise the use of a detection protein.
  • a detection protein is able to bind to the immobilized specified protein.
  • the method further comprises the detection of bound detection protein, thereby indicating the presence of the specified protein.
  • Detection may be accomplished by use of a detectable label attached directly to the detection protein, or detection may be accomplished by use of a detectable label which may, through manipulation known to those of skill in the art, be associated with the detection protein. Examples of the latter include, but are not limited to, the use of a biotinylated detection protein and the a detectable label conjugated to streptavidin or avidin.
  • any sample or preparation containing the "protein of interest” can be detectably labeled.
  • a sample containing a protein of interest can be biotinylated and then contacted with the microarray, thereby allowing it to be bound by capture proteins contained in the array.
  • the presence of the protein of interest at a particular location, such as that corresponding to a particular capture agent, can be determined by addition of streptavidin- or avidin- that is conjugated to a detectable label.
  • Other types of detectable labeling of proteins of interest can also be employed, including, but not limited to, Cy3 and Cy5 labels, gold particles, and radiolabels.
  • the capture protein is an antigen and the protein detected is an antibody.
  • the protein detected is a specific antibody.
  • the detection protein can be an antibody.
  • the detection protein is an anti-antibody and still further, it may be an idiotype-specific anti-antibody.
  • the capture protein is an antibody and the protein detected is an antibody.
  • the protein detected is a specific antibody.
  • the detection protein is an antibody.
  • the detection protein is an anti-antibody and still further it may be an idiotype-specific anti-antibody.
  • the capture protein is an antibody and the protein detected is an antigen.
  • the protein detected is a specific antigen.
  • the detection protein is an antibody.
  • the detection protein is an epitope-specific antibody.
  • the capture protein is a protein which is able to bind a protein of interest wherein the capture protein and the protein of interest are not antigen-antibody cognates.
  • the first protein may be immobilized to a membrane so as to act as the capture protein
  • the second protein may be immobilized by the interaction between the first and second proteins and the presence of the second protein in a sample may be determined by use of a detection protein.
  • the capture protein and protein of interest may be such that they normally do not form complexes or interact with one another, but that the conditions or proteins may be altered so as to effect complex formation and thereby effect capture of the protein of interest. It is contemplated that one protein, either the capture protein or the protein of interest can interact with different proteins under different conditions or may interact with many different proteins under identical conditions.
  • the capture proteins are specific for specified proteins and therefore, specifically bind specific proteins.
  • Specific proteins may mean a single species of protein, or a number of proteins which share common characteristics, such as, but not limited to, common structural motifs, common sequence or an ability to interact with other species in an identical or very similar manner.
  • the capture proteins are specific for specified epitopes.
  • the detection proteins are horseradish-peroxidase labeled proteins. In a further embodiment, the detection proteins are horseradish-peroxidase labeled antibodies. In a further embodiment, the detection protein is detected by chemiluminescence. In a further preferred embodiment, the detection protein is detected by enhanced chemiluminescence.
  • the capture proteins bind cytokines.
  • the detection proteins bind cytokines.
  • Anti-cytokine antibodies are preferred examples of both capture and detection proteins. Examples of suitable anti-cytokine antibodies include, but are not limited to, anti-human G-CSF, anti-human IL-10, anti -human GM-CSF, anti-human IL-13, anti-human GRO ⁇ anti-human IL-15, anti -human IFN- ⁇ , anti-human MCP-1 anti-human IL-l ⁇ , anti-human MCP-2, anti-human IL-2, biotinylated anti-human MCP-3, anti-human IL-3, biotinylated anti-human MIG, biotinylated anti-human IL-5, biotinylated anti-human/mouse/pig TGF ⁇ l, anti-human IL-6, polyclonal rabbit anti -human RANTES, anti-human IL-7, biotinylated anti-human TNF
  • the capture proteins bind growth factor related proteins, angiogenesis or anti-angiogenesis related proteins, particularly secreted angiogenesis factors.
  • the detection proteins bind growth factor related proteins, angiogenesis or anti-angiogenesis related proteins, particularly secreted angiogenesis factors.
  • the capture proteins and detection proteins are selected from species which bind to infection-associated antibodies or antigens. These antibodies or antigens may be proteins or antigens from the pathogenic species which infects the infected subject, or may be protein, antigens or antibodies elicited in response to infection of a subject.
  • the capture proteins bind more than one specified protein.
  • An example of such a capture protein includes, but is not limited to, an antibody which binds structurally related proteins or an antibody which binds a common, or closely related, amino acid sequence.
  • the detection proteins bind more than one specified protein.
  • An example of such a detection protein includes, but is not limited to, an antibody which binds structurally related proteins or an antibody which binds a common, or closely related, amino acid sequence.
  • the specified protein is a fusion protein comprising an engineered epitope and further amino acid sequence.
  • the capture protein binds the engineered epitope.
  • the detection protein binds the engineered epitope.
  • the present invention relates to a method for detecting protein. More specifically, the method of detecting protein comprises immobilizing proteins in a microarray on membrane, contacting the microarray with a solution containing discrimination antibodies capable of binding to selected idiotypes to form discrimination complexes, contacting the microarray with a solution containing detection protein capable of binding discrimination complexes and detecting bound detection protein.
  • the detection proteins are antibodies. In a further embodiment, the detection proteins are horseradish-peroxidase labeled antibodies. In a further embodiment, the detection proteins are detected by chemiluminescence and still further, they may be detected by enhanced chemiluminescence. In a further embodiment, the detection protein is an anti-IgA, anti- IgD, anti-IgE, anti-IgG or anti-IgM antibody and the detection protein can bind to more than a single species of discrimination antibody.
  • arrays comprising antibodies against proteins of interest can be used to capture proteins and the phosphorylation or modification of the proteins may be determined by detection of phosphorylation by use of detection antibodies specific for phosphorylated amino acids.
  • suitable anti- phosphorylation antibodies include those raised against phosphotyrosine, phosphoserine or phosphothreonine.
  • proteins which specifically recognize carbohydrate moieties such as concavalin A and wheat germ agglutinin may be used to detect carbohydrate moieties of glycoproteins.
  • Antibodies specific for ubiquitin may be used to detect ubiquitination.
  • the present invention relates to a method of forming a microarray of proteins on a membrane for use in the practice of other aspects of the invention.
  • the method of applying a protein onto defined positions on a membrane, whereby repeated application of different proteins allows the formation of an ordered arrangement of protein array elements.
  • the array elements consist of capture proteins capable of binding other species.
  • Each array element may be formed by a single species of capture protein or multiple species of capture proteins. If an array element is formed from multiple species of capture proteins, they may be selected so as to bind a single species of analyte or they may be selected so as to bind multiple species of analytes.
  • An array element formed from multiple species of capture proteins, each selected so as to bind a single species of analyte may be formed from a group of capture proteins which differ from one another in characteristics other than their binding specificity.
  • a multitude of capture proteins which differ in their binding capacity for the analyte at different pH values can be combined to provide an array element which operates effectively over a broader range of pH values.
  • Another example of an array element using multiple capture proteins to capture a single species would be an element comprising multiple antibodies which recognize different epitopes of the same analyte species.
  • Different embodiments of this aspect of the invention which provide array elements comprising capture proteins which bind a single protein of interest over a wide range of environmental conditions are contemplated and are provided herein to the practitioner of skill in the art without undue experimentation.
  • a capture protein that binds a specific epitope such as an antibody which specifically binds a particular structural motif that is common to a number of proteins, is contemplated herein.
  • An example of such a capture protein would be the antibody against the carboxy terminus of Mekl disclosed by Giroux et al. (Current Biology 9: 369-372 (1999)), the antibodies disclosed by Wang et al. (Mol. Cell Biol. 20: 4505- 4512 (2000)) or any other antibody identified by one of skill in the art to bind to multiple species. Many such antibodies are known to those of skill in the art and the use of such antibodies is contemplated in the use of the current invention.
  • detection proteins that bind a specific epitope such as an antibody which specifically binds a particular structural motif that is common to a number of proteins, is also contemplated herein.
  • a multispecific detection protein may be used to identify the presence of a specific motif, amino acid sequence or other characteristic in a bound analyte or bound protein of interest.
  • such a multispecific detection protein may be used to characterize immobilized proteins.
  • the membrane is made from polyvinylidene fluoride, nitrocellulose, nylon and/or other suitable materials.
  • membranes contemplated herein include, but are not limited to, PVDF, Biotrans (ICN), Zeta-probe (Bio-Rad), Colony/Plaque Screen (NEN), Hybond-N + (Amersham), Magnacharge (MSI), Magnagraph (MSI) and Hybond ECL (Amersham).
  • the array of the present invention can be provided on other solid support materials as are known to those of skill in the art, including, but not limited to, polyacrylamide layers on glass or other solids.
  • the proteins are antibodies, antigens or are unknown proteins from biological samples.
  • each discrete region of the array contains proteins from a selected group of cells, viruses or other protein-containing species and still further, the selected species may be clonal in nature or may be obtained from a tissue sample.
  • the proteins are cytokines.
  • the present invention relates to the microarray formed by the method of forming a microarray of proteins on a solid support.
  • solid supports include, but are not limited to, membranes, plastics, gels, sols, glass, ceramics and metal.
  • microarrays and suitable supports see Shalon et al. (Genome Research 6: 639-645 (1996), LeGendre (BioTechniques 9: 788-805 (1990)), U.S. Patent Serial No. 6,197,599 and U.S. Patent Serial No. 6,140,045, each of which is incorporated herein by reference.
  • the microarray is formed on a membrane made from polymeric, elastomeric or other suitable membrane material.
  • membranes which are desirable to optimize the invention include the ability to bind large amounts of protein, the ability to bind protein with minimal denaturation and the inability to bind proteins that are not proteins of interest when used as described herein.
  • membranes displays minimal "wicking" when protein solution containing proteins to be used as capture proteins are applied to the membrane.
  • a membrane with minimal wicking allows small aliquots of protein containing solution applied to the membrane to result in small, defined spots of immobilized protein.
  • membranes with less suitable wicking characteristics results in larger, more diffuse spots of immobilized protein when applied to the membrane.
  • Methods of generating the arrays and microarrays of the invention include, but are not limited to, any of the methods commonly used for application of protein solution to a membrane, as well as other methods used to apply a liquid solution to a flexible, solid support (for example, inkjet or bubble jet printing).
  • the use of a pipetman, as described in the examples is one method for applying a protein solution to a membrane to generate an array.
  • Further examples for the preparation of such protein arrays on membranes, and the use of such protein arrays to detect the presence of specified proteins or analytes is described in U.S. Patent Serial No. 6,197,599, incorporated herein by reference.
  • a vacuum manifold transfers a plurality, e.g., 96, aqueous samples of proteins from 3 millimeter diameter wells to a porous membrane.
  • a common variant of this procedure is a "slot-blot" method in which the wells have highly-elongated oval shapes.
  • a more efficient technique employed for making ordered arrays of proteins uses an array of pins dipped into the wells, e.g., the 96 wells of a microtiter plate, for transferring an array of samples to a substrate, such as a porous membrane.
  • One array includes pins that are designed to spot a membrane in a staggered fashion, for creating an array of 9216 spots in a 22 x 22 cm area (Lehrach, et al., Hybridization
  • Another method for generating a microarray contemplated involves dispensing a known volume of a reagent at each selected array position, by tapping a capillary dispenser on the support under conditions effective to draw a defined volume of liquid onto the support, wherein this process is repeated using selected reagents at each selected array position to create a complete array.
  • the method may be practiced in forming a plurality of such arrays, where the solution-depositing step is applied to a selected position on each of a plurality of solid supports at each repeat cycle. Further description of such a method may be found in U.S. Patent Serial No. 5,807,522, incorporated herein by reference.
  • devices normally utilized for printing on paper can be utilized to generate the microarrays of the invention.
  • the desired reagent can be loaded into the printhead of a desktop jet printer and printed onto suitable membranes.
  • Silzel et al. (Clinical Chemistry 44: 2036-2043 (1998)), incorporated herein by reference, describes the use of a similar method to generate a multianalyte array on a solid support.
  • the microarray generated on the membrane has a density of at least 5 spots/cm 2 , preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000 or 9000 spots/cm 2 and most preferably at least 10,000 spots/cm 2 .
  • the spots on the membrane may each represent a different species of protein or that the multiple spots on the membrane may represent the same species of protein. In a further embodiment, the spots each represent an array element of differing identity or characteristics.
  • the present invention also relates to a method of forming a microarray of proteins using a first and second binding partner wherein each discrete region in the microarray has a selected composition, character or identity. More specifically, the method relates to the formation of a microarray wherein a first binding partner, which can bind a second binding partner, is immobilized on a membrane and a second binding partner, or second binding partners, is applied to the membrane under conditions wherein the first and second binding partners form a stable complex, thereby immobilizing the second binding partner.
  • the first binding partner can be selected so as to maximize the binding of the second binding partner, thereby increasing the effective density of second binding partner on the membrane.
  • first binding partners may be chosen so as to effectively block the non-specific binding of sample of the membrane, thereby increasing the sensitivity of each array element by reducing the level of background signal.
  • the combined effects of the improvements in capture protein density and in reduced background allow the effective use of microarrays of significantly higher density.
  • the first binding partner is a protein.
  • the first binding partner may be applied to selected portions of the microarray or may be used to coat the entire membrane.
  • the first binding partner is an idiotype-specific antibody and still further, the second binding partner may be an antibody containing the idiotype.
  • the first binding partner specifically orients the second binding partner so as to maximize its effectiveness as a capture protein, for example, the first binding partner may be a monoclonal antibody specific for an epitope of capture antibodies that positions the capture antibodies onto the membrane in an optimal spatial orientation for binding a protein of interest.
  • binding partners described herein are not limited to being proteins or antibodies.
  • the first binding partner can be biotin or a biotinylated protein and the second binding partner is avidin, streptavidin or comprises portions thereof or the first binding partner can be avidin, streptavidin or comprise portions thereof and the second binding partner is biotin or a biotinylated protein.
  • the second binding partner is a fusion protein comprising a first amino acid sequence whose presence allows binding to a first binding partner and a second amino acid sequence whose presence allows binding to at least one other protein such that the second binding protein can be a capture protein.
  • the present invention relates to the microarray formed by the use of any embodiment of the method whereby a first and second binding partner is used to structure an microarray, orient elements of the microarray for greater efficiency or effectiveness or wherein the use of the first and second binding partners increases the density of the microarray and the sensitivity of the microarray-based assay.
  • the present invention relates to a method of characterizing proteins which relies on binding interactions between proteins.
  • the method comprises immobilizing proteins onto a solid support to form a microarray of immobilized proteins, contacting the immobilized proteins of the microarray with detection proteins of known characteristics and detecting binding of detection proteins to the immobilized proteins, thereby determining characteristics of the immobilized proteins.
  • the solid support can be a membrane and the detection proteins can be antibodies or antigens.
  • the proteins immobilized on the membranes are unknown proteins and the detection proteins are used to characterize and/or identify the unknown proteins. However, it is not necessary that the proteins bound to the membrane be unknown proteins for this aspect of the invention to provide a benefit.
  • known proteins may be immobilized on the membranes and the binding of known detection proteins may be used to characterize properties of the known proteins immobilized in the microarray.
  • known proteins could be immobilized in a microarray and probed with antibodies specific for certain modifications, such as glycosylation, phosphorylation and ubiquitination, or probed with antibodies specific for certain structural motifs, whereby binding of the selected detection protein indicates the presence of the modification or structural motif in the known, but not entirely characterized, immobilized proteins of the microarray.
  • the microarrays are microarrays of immobilized nucleic acids.
  • immobilized nucleic acids act as capture nucleic acids, in a manner analogous to capture proteins, to immobilize species of interest, which are then detected using detection proteins.
  • a nucleic acid array is contacted with transcription factors. Transcription factors, and the sequences to which they bind, are detected by detecting the presence of protein at the position of the specific sequences, and by identifying the transcription factors.
  • Immobilized transcription factors can be identified either through the sequence to which they bind, wherein the sequence must be known or determined, by use of detection antibodies, specific for the transcription factor to be identified, or by other methods known to those of skill in the art.
  • the present invention relates to a method of detecting a specified protein. More specifically, a method of detecting a specified protein that comprises immobilizing capture proteins in a microarray on a membrane, labeling the proteins contained in a solution that contains the specified protein, passing the solution containing the specified labeled protein over the microarray of capture proteins and detecting bound specified protein. As described earlier, capture proteins are able to bind to the specified protein.
  • the immobilized capture proteins can be immobilized to known positions and can be of known binding characteristics
  • the presence of the protein of interest, the specified protein can be determined by detection of protein at a site on the microarray that corresponds to a capture protein that binds the protein of interest.
  • the capture protein can be a capture protein that captures a specific protein.
  • the labeling used to label the proteins in the solution, including the specified protein can be biotinylation. If the labeling used is biotinylation, use of detectably labeled avidin or streptavidin can be used to generate a detectable signal.
  • Example 1 Simultaneous detection of multiple proteins with an Array-based Enzyme-Linked Immunosorbant Assay (ELISA) and Enhanced Chemiluminescence (ECL).
  • ELISA Enzyme-Linked Immunosorbant Assay
  • ECL Enhanced Chemiluminescence
  • PDVF polyvinylidine difloride
  • the membranes were then placed in a plastic box and incubated with ECL substrate according to the manufacturer's instructions (Amersham, Aylesbury, UK). Finally the membranes were placed on a sheet of Kodak X-OMAT film (Eastman Kodak, Rochester, NY, USA) for development by autoradiography.
  • cytokines For detection of cytokines, a Sandwich ELISA approach was adapted. Each pair of antibodies was purchased from BD PharMingen (San Diego, CA, USA). All cytokines, except growth-regulated oncogene ⁇ (GRO ⁇ ), were obtained from Peptotech (Rochy Hill, NJ, USA). GRO ⁇ was the product of BD PharMingen. The capture antibodies were immobilized onto PDVF membranes (Immobilon, Millipore, Bedford, MA). . The membranes were then incubated with cytokines, either individually or collectively, with other controls or with conditioned media.
  • GRO ⁇ growth-regulated oncogene ⁇
  • the membranes were incubated with corresponding detection antibodies. Extensive washing was then carried out again. The membranes were then incubated with 20,000 fold diluted HRP-conjugated streptavidin (BD PharMingen, San Diego, CA, USA) and the pattern of detection antibody binding was determined by development of the blot according to the manufacturer's instructions for ECL Western blotting detection reagents, incorporated herein by reference (Amersham, Aylesbury, UK).
  • One antibody is immobilized onto PDVF membrane and functions as a capture protein.
  • a second antibody is labeled with biotin and functions as a detection protein.
  • the binding of the detection antibody, thereby detecting the presence of a bound antigen, is detected by use of an ECL system ( Figure 5).
  • Monocyte chemotactic protein- 1 (MCP-1) and Interleukin-2 (IL-2) were used in the model system.
  • Anti-MCP- 1 antibody or anti-IL-2 antibody (capture) or other controls were then immobilized onto PDVF membranes as previously described for immobilization of other proteins.
  • the membranes were then incubated with MCP-1 or IL-2 or different negative controls respectively.
  • the membranes were then incubated with biotin-conjugated anti-MCP-1 or biotin-conjugated anti-IL-2 or other negative controls.
  • MCP-1 and IL-2 are specifically detected.
  • the sensitivity of detection of MCP-1 was such that concentrations as low as 500 pg/ml of MCP-1 were detected ( Figure 7).
  • the specificity of detection was such that no signal was detected in the control spots where ⁇ -CXCR-1, a receptor for CXC class of chemokines, was immobilized.
  • cytokines can be detected simultaneously using this sandwich assay.
  • Membranes with different capture antibodies immobilized on them were incubated with different combinations of cytokines and their corresponding detection antibodies, in this case, biotin-conjugated antibodies.
  • the specific signals were detected as expected as shown in Figure 8.
  • a microspot approach was developed.
  • capture proteins either antibodies or antigens
  • the membrane is then exposed to a sample containing a protein, or proteins, of interest.
  • the protein of interest is bound by its cognate, either an antibody or antigen, spotted onto membrane, is thereby immobilized, and its presence is determined by the binding of a detection protein, specifically an HRP-labeled antibody.
  • Detection of the HRP-labeled antibody's presence by enhanced chemiluminescence indicates the presence of the bound antigen of interest, thereby indicating its presence in the sample.
  • the sensitivity of the array was demonstrated by incubation of a membrane with different antigens and controls spotted on it with different concentrations of HRP- conjugated anti-bovine IgG. As shown in Figure 1 IB, concentrations of HRP- conjugated antibody (anti-bovine IgG) as low as 5 pg/ml of can be detected. Similarly high sensitivity was also seen in other HRP-conjugated antibodies.
  • Array assay of different species of HRP-conjugated IgGs Different IgGs (0.25 ⁇ l of 100 ⁇ g/ml) were loaded onto membranes as described above. Membranes were blocked with 5% BSA (Bovine Serum Albumin) /TBS (0.01 M Tris HCI pH7.6/0.15 M NaCI) for 1 hour at room temperature and incubated individually or collectively with HRP-conjugated antibodies for 2 hours at room temperature. Arrays were then washed three times with TBS/0.1% Tween 20 and then twice with TBS. The presence of HRP- conjugated antibodies, and therefore, the presence of the specified IgGs, was determined by enhanced chemiluminescence as described previously.
  • BSA Bovine Serum Albumin
  • TBS Tris HCI pH7.6/0.15 M NaCI
  • Array assay of multiple cytokines Pairs of antibodies that recognize different epitopes of the same antigen were used to capture and detect a certain antigen.
  • 0.25 ⁇ l of an individual capture protein, an antibody, at a concentration of 200 ⁇ g/ml was spotted onto membranes as described above. After blocking with 5% BS A/TBS, membranes were incubated with one or more cytokines prepared in 5% BSA/TBS for 2 hours at room temperature. Unbound cytokines were removed from the membrane by washing with TBS/Tween 20 and TBS. The presence of bound cytokines was detected by binding of biotin-conjugated anti-cytokine antibodies to the bound cytokine.
  • membranes were incubated with rabbit anti-Donkey IgG, which was pre- absorbed with agarose-immobilized guinea pig IgG, goat IgG, human IgG and sheep IgG, to remove cross-reaction components. Imaging of bound donkey anti-IgGs was carried out with enhanced chemiluminescence.
  • Array assay of multiple cytokines The protein array system described above was extended to assay human cytokines. The principle of this assay is based upon the sandwich ELISA and utilizes ECL for detecting antigen binding. Pairs of antibodies, which recognize two different epitopes of same antigen, are used. One antibody is spotted onto membrane and served as a capture protein. The second corresponding antibody is labeled with biotin and served as a detection protein. The binding of the detection protein was detected by use of ECL as described previously.
  • Hybond ECL membrane showed the highest sensitivity and lowest background. Consequently, Hybond ECL membrane was used in the assay and analysis of cytokines. Six cytokines were assayed using this array format. The specificity of this assay was first demonstrated with individual cytokines. As shown in Figure 13, the assay exhibited great specificity for each of the cytokines tested, consistent with ELISA data. All six cytokines were specifically recognized, and bound, by their corresponding capture antibodies and were specifically detected by their cognate detection antibodies. No cross-reaction was observed among the six cytokines or the controls, including EGF, BSA and buffer only.
  • FIG 14. Another example of this approach being used in a high density array format is shown in Figure 14.
  • anti-cytokine antibodies and controls were spotted on the membranes to act as capture proteins (Figure 14A). These membranes were then incubated with a control, INF ⁇ , IL-2 and INF ⁇ , IL-6 and TNF ⁇ , IL-2, IL-6, IL-8 and MCP-1, or IL-2, IL-6, IL-8, INF ⁇ , TNF ⁇ and MCP-1.
  • Figure 14B Specific binding and detection of the bound antigens can be seen in Figure 14B.
  • Array assay of multiple antibodies The protein array system also can be used to detect antibodies.
  • different known antigens are spotted onto membranes.
  • the membranes are then incubated with samples containing antibodies to be detected. After extensive washing to remove unbound antibodies, the membranes are incubated with HRP-conjugated antibody(ies) against species-specific IgGs, thereby detecting the presence of bound antibodies.
  • HRP-conjugated antibody(ies) against species-specific IgGs, thereby detecting the presence of bound antibodies.
  • the detectable signals from the HRP-conjugated antibodies are then analyzed by use of the ECL methodology as described above.
  • IgGs are the antigens of the antigen/antibody complex and act as the capture proteins.
  • membranes were incubated with individual donkey anti-IgGs against given species.
  • anti-donkey IgG the detection protein in this case, to detect the presence of bound donkey anti-IgGs. against given species, the proteins of interest. Extensive washing was then repeated and the pattern of antibody binding was determined using ECL.
  • FIG. 16 Another example of this approach being used in a high density array format is shown in Figure 16.
  • IgGs from various species and controls were spotted on the membranes to act as capture proteins (Figure 16 A). These membranes were then incubated with a control or various combinations of donkey antibodies against the selected IgGs (anti-chicken IgG, anti-chicken IgG and anti-guinea pig IgG, anti-mouse IgG and anti-rabbit IgG, anti-human IgG and anti-rabbit IgG or anti-chicken IgG, anti- goat IgG, anti-guinea pig IgG, anti-human IgG, anti-mouse, IgG, anti-rat IgG, anti- rabbit IgG and anti-sheep IgG). Specific binding and detection of the bound antibodies of interest can be seen in Figure 16B.
  • This example describes a highly sensitive ELISA-based protein array system in which multiple cytokines can be simultaneously detected from the experimental model system, from tissue culture media and from sera from patients. After identifying a candidate protein for analysis, this system allows hundreds of biological samples to be analyzed quantitatively using a single array membrane.
  • Antibodies were purchased from BD PharMingen (San Diego, CA). All of cytokines except GRO ⁇ and granulocyte colony stimulating factor (G-SCF) were obtained from Peptotech (Rochy Hill, NJ). GRO ⁇ and G-SCF were the products of BD PharMingen. HPR-conjugated streptavidin was also purchased from BD PharMingen.
  • Array assay of purified cytokines Membranes with arrays of immobilized capture antibodies were blocked with 5% BSA (Bovine serum albumin)/TBS (0.01 M Tris HCI pH7.6/0.15 M NaCI) for 1 hour. Membranes were then incubated with a single cytokine or various combinations of different cytokines (0.25 ⁇ g/ml) prepared in 5% BSA/TBS for 2 hours at room temperature.
  • BSA Bovine serum albumin
  • TBS Tris HCI pH7.6/0.15 M NaCI
  • Protein array assay for detection of cytokines from conditioned media and sera Ten ml of 50 fold diluted conditioned media and one ml of 10 fold-diluted sera were incubated with membrane arrays for detecting cytokines.
  • human glioblastoma cells U251 Human glioblastoma cells U251 (Huang et al., Cancer Res. 58: 5089-5096 (1998); Huang et al., Cancer Res. 55: 5054-5062 (1995)) were plated in 35 mm tissue culture dishes at a density of 4xl0 5 cells per dish. Cells were cultured with Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS) for 24 hours.
  • DMEM Dulbecco's modified Eagle's medium
  • the complete culture medium was then replaced with serum-free DMEM.
  • Cells were then stimulated for 48 hours in the presence or absence of 50 ng/ml of recombinant human TNF ⁇ .
  • the supernatants were then collected, centrifuged at 1,000 g, aliquoted and stored at -80 °C until testing.
  • Patient's sera were obtained from Department of Gynecology and Obstetrics, Emory University School of Medicine.
  • Hybond ECL membranes were soaked with anti- MCP- 1 capture antibody at a concentration of 20 ⁇ g/ml for 4 hours at 4°C. Membranes were then air-dried. 0.5 ⁇ l of conditioned media from different sources were spotted onto the membranes. After blocking with 5%>BSA/TBS, membranes were incubated with biotin-conjugated anti-MCP- 1 antibody. After washing, membranes were incubated with HRP-conjugated streptavidin. The signals were visualized by ECL.
  • ELISA ELISA.
  • Conventional ELISA was performed as per manufacturer's instructions. Essentially 96 well ELISA plates were coated overnight at 4 °C using 50 ⁇ l of 8 ⁇ g/ml capture antibodies. 1% BSA/PBS was used as a blocking buffer. 100 ⁇ l of conditioned media and 2 fold diluted patients' sera and different concentrations of standard cytokines were added to each well in duplicate. Unbound materials were removed by washing with PBS/1 % Tween. 100 ⁇ l of 1 ⁇ g/ml biotinylated anti-cytokine detection antibody were added to each well. The plates were incubated for 1 hour at room temperature.
  • Densitometry The intensities of signals were scanned and quantitated by densitometry in accordance with the manufacturer's instructions (Bio-Rad, Hercules, CA).
  • GRO antibody recognizes all three members of GRO: GRO ⁇ , GRO ⁇ and GRO ⁇ .
  • EGF epidermal growth factor
  • MCP-1 myel growth factor
  • MCP-1 myel growth factor
  • MCP-1 myel growth factor-1
  • membranes with different immobilized antibodies were incubated with different combinations of cytokines and corresponding antibodies. Described in Figure 18, these membrane array assays resulted in the expected patterns of binding and demonstrate the ability to detect specific analytes.
  • cytokine expression from conditioned media and patient's sera.
  • a challenging question facing the antibody-based microarray was whether it could be used to directly detect protein expression, i.e., the presence of a particular protein product, in conditioned media, patient's specimen, crude cell lysate or crude tissue lysate.
  • conditioned media collected from different sources were tested for the presence of cytokines.
  • cytokines are differentially expressed in human glioblastoma U251 cells treated with or without tumor necrosis factor ⁇ (TNF ⁇ ).
  • TNF ⁇ tumor necrosis factor ⁇
  • Conditioned medium microarrays for high throughput molecular profiling are provided.
  • membranes homogenously with an antibody specific for a corresponding antigen.
  • membranes were coated with capture anti-MCP antibody. Different samples were then spotted onto the membranes. The expression levels of MCP-1 were then detected by biotin-conjugated anti-MCP- 1 antibody coupled with ECL system. We found that coating of the membrane with a specific antibody increased detection sensitivity at least 100 fold. Then we tested the potential of this array system. 324 loci in a single membrane pre-coated with anti-MCP- 1 capture antibody were spotted with conditioned media from different sources ( Figure 21 A). Membrane was then incubated with biotin conjugated anti-MCP antibody.
  • Example 4 Profiling of human cytokines in a variety of biological processes by protein arrays.
  • This example demonstrates the manufacture and use of a higher density array system to simultaneously detect 35 cytokines.
  • monocyte chemoattatic protein- 1 may be implicated in tumor suppression by gap junction protein, connexin 43; interleukin 8 levels are associated with p53 status in breast cancer cells and the levels of monocyte chemoattactic protein- 1 were significantly decreased in patient in response to vitamin E supplementation.
  • our system can be easily adapted to chip platform. Multiple cytokine expression from different sources can be detected using protein chips.
  • the human cytokine array system can also be used to identify possible targets of chemoprevention.
  • cytokine levels from 11 patient's serum before and after vitamin E supplementation.
  • vitamin E supplementation increased vitamin E levels in patient's sera (Santanam et al., "Vitamin E supplementation decreases autoantibodies to oxidized lipid-protein complexes," J. Medicinal Food 1 :247-251 (1998)).
  • Representative results were shown in Fig. 24 A.
  • supplementation of vitamin E decreased a number of cytokine expression.
  • the relative levels of MCP- 1 were then determined by densitometry as shown in Fig.24 B and the difference was statistically analyzed (Fig. 24 C).
  • MCP-1 levels were significantly down-regulated after supplementation with vitamin E, suggesting that MCP- 1 may be a molecular target of antioxidant supplementation. This observation represents the first report of a potential molecular target of vitamin E in vivo. Further studies may reveal valuable information about molecular mechanisms of vitamin E supplementation in the chemoprevention and identify novel targets.
  • the system can be used to detect multiple human cytokines from a variety of biological sources such as, but not limited to, conditioned medium, serum, cell lysate, tissue lysate and urine.
  • Our system can be both a membrane-based system and a chip-based system.
  • the membrane format allows designing arrays in a simple, cheap and flexible way. Among the particular advantages to the membrane-based system are that no sophisticated equipment is required in the entire process. Consequently, it is adaptable for use by nearly all of the research community.
  • the protein chip format also has its own particular advantages. Among these are its ability to be used in a high-throughput approach using methods and compositions that are in widespread use in the field. Variation from one assay to the next is quite acceptable for almost all purposes and is usually within the range of approximately 10%>. This sensitivity is high enough to detect the change of most biological processes.
  • array membranes were essentially as described elsewhere in this application and in the references incorporated herein by reference.
  • a computed generated-template was used to guide to spot solution onto membranes.
  • 0.20 ⁇ l of capture antibodies 200 ⁇ g/ml were manually loaded onto membranes by a 2 ⁇ l pipetman in duplicate.
  • HRP-conjugated antibody was spotted onto membranes as positive control and identification of orientation of arrays.
  • Membranes that included immobilized capture antibodies were blocked with 5% BSA (Bovine serum albumin)/TBS (0.01 M Tris HCI pH7.6/0.15 M NaCI) for 1 hour. Membranes were then incubated with 1 ml of a single or a combination of different cytokines (100 ng/ml) or 1 ml of conditioned media or 1 ml of 10 fold diluted patient's sera prepared in 5% BSA TBS for 2 hours at room temperature. After an extensive wash with TBS/0.1% Tween 20 (3 times, 5 min each) and TBS (2 times, 5 min each) to remove unbounded cytokines, membranes were incubated individually or collectively with biotin-conjugated anti-cytokine antibodies.
  • BSA Bovine serum albumin
  • TBS Tris HCI pH7.6/0.15 M NaCI
  • ELISA Conventional ELISA was performed according to the manufacturer's instructions. Essentially 96 well ELISA plates were coated overnight at 4 °C using 50 ⁇ l of 8 ⁇ g/ml capture antibodies. 1%> BSA/PBS were used as a blocking buffer. 100 ⁇ l of conditioned media and different concentrations of standard cytokines were added to each well in duplicate. Unbound materials were washed out with PBS/1% Tween. 100 ⁇ l of 1 ⁇ g/ml biotinylated anti-cytokine detection antibody were added to each well. The plates were incubated for 1 hour at room temperature. After washing, 100 ⁇ l of streptavidin-HRP conjugated antibodies were added to well and incubation was continued for 30 minutes at room temperatures.
  • 300 pL of capture antibodies (500 ⁇ g/ml) were printed onto Hydrogel chips (Packard Bioscience, Meriden, CT) using the Biochip Arrayer (Packard Bioscience). After blocking, the chips were incubated with 50 ⁇ l of different samples at room temperature for 2 hr. The chips were then washed to remove unbound components. Biotin-labeled detection antibody cocktail was added (50 ⁇ l/chip) and incubated at room temperature for 1 hr. After the washing, cy3 labeled streptavidin was added and the chips were incubated at room temperature for 1 hr. The excess amount of cy3 streptavidin was removed and the resulting signals were scanned by laser scanner (Affymetrix, Santa Clara, CA). A series of diluted Cy3 streptavidin, cy5 streptavidin and Biotin IgG (BlgG) were included as a positive control. BSA was used as negative control.
  • MacBeath G, Schreiber SL Printing proteins as microarrays for high- throughput function determination [see comments]. Science 2000, 289 :1760- 1763.
  • Ge H UPA, a universal protein array system for quantitative detection of protein-protein, protein-DNA, protein-RNA and protein-ligand interactions. Nucleic Acids Res 2000, 28:e3-
  • Huang RP Detection of multiple proteins in an antibody-based protein microarray system. J Immunol Methods 2001, 255:1-13.
  • Huang RP Huang R, Fan Y, Lin Y: Simultaneous detection of multiple cytokines from conditioned media and patient's sera by an antibody-based protein array system. Anal Biochem 2001, 294:55-62. 19. Huang RP: Simultaneous detection of multiple proteins with an array-based enzyme-linked immunosorbent assay (ELISA) and enhanced chemiluminescence (ECL). Clin Chem Lab Med 2001, 39:209-214.
  • ELISA enzyme-linked immunosorbent assay
  • ECL enhanced chemiluminescence
  • Example 5 Profiling of protein expression by biotin-labeled and antibody-based protein arrays.
  • proteins from a variety of biological sources are labeled with biotin.
  • the biotin-labeled proteins are then incubated with arrays having antibodies as capture proteins.
  • the particular support used here is a chip, but the method is equally applicable to the use of membrane-based arrays.
  • targeted proteins were captured by the array antibodies spotted on protein chips.
  • the presence of targeted proteins was detected using cy3- or cy5-conjugated streptavidin and the signals were imaged by laser scanner.
  • Most proteins at concentration of pg/ml can be detected with a detection limit at low to mid fg levels.
  • the useful dynamic detection range of this system is at least about 4 to 5 orders of magnitude.
  • the system also can be easily adapted to two- color binding assay, allowing measurement of levels of proteins in a test sample with respect to a reference sample at the same chip.
  • this technology we applied this technology to profile human cytokines, chemokines, growth factors, angiogenic factors and proteases in a variety of human breast cancer cell lines and identify the key molecular targets from monocytes upon activation by lipopolysaccharide (LPS).
  • LPS lipopolysaccharide
  • the excess amount of streptavidin was removed and the signals were scanned by laser scanner (Packard, Meriden, CT).
  • a series of diluted Cy3 streptavidin, cy5 streptavidin and Biotin IgG (BlgG) were included as positive control.
  • BSA was used as negative control.
  • a second approach is to label proteins to be detected with fluorescent dye, gold particles or biotin, so that the use of a separate detection protein is not required.
  • the second approach is demonstrated in this example.
  • the proteins to be detected/identified were labeled with biotin.
  • the biotinylated proteins were then incubated with array membranes or chips having specific, known sets or subsets of capture proteins. After extensive washes to remove any unbound proteins, the array membranes or chips were incubated with streptavidin conjugated with cy3 or cy5 or HRP as shown in Fig. 26.
  • the detection sensitivity of individual cytokines, in this example is different and is, as one would expect, dependent on the binding affinity between antigen and antibody. Nevertheless, a linear increase in spot intensity was observed with increasing amounts of cytokine in several cytokines we tested.
  • Antibody microarrays can also be used to measure the levels of protein in a test sample with respect to a reference sample, e.g., cancer versus normal cells, stimulated cells versus control, treated versus untreated cells and so on.
  • a reference sample e.g., cancer versus normal cells, stimulated cells versus control, treated versus untreated cells and so on.

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Abstract

Cette invention concerne des microtitrations de protéines et des techniques basées sur des microtitrations de protéines permettant de déterminer la présence et les quantités de protéines d'intérêt. Ces microtitrations et leurs méthodes d'utilisation peuvent servir à détecter simultanément de multiples antigènes ou anticorps par des analyses à fort débit reposant sur l'affinité différentielle de molécules entre elles. Pour l'obtention des microtitrations, on peut immobiliser des protéines de capture dans une matrice sur une membrane. Des analysats d'intérêt peuvent être liés par les protéines de capture et être détectés soit par la position dans laquelle ils sont immobilisés, soit par l'identité des protéines ou agents de détection qui se lient avec lesdits analysats. Les interactions susceptibles d'être décelées au moyen de la présente invention peuvent également être utilisées pour caractériser des protéines d'identité ou de caractère inconnu.
PCT/US2002/007341 2001-03-08 2002-03-08 Systeme de microtitrations de proteines base sur des anticorps WO2002073180A1 (fr)

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WO2007056411A2 (fr) * 2005-11-08 2007-05-18 Genentech, Inc. Procede de production d'anticorps pan-specifiques
CN102353767A (zh) * 2011-07-07 2012-02-15 贺福元 一种全成分群同时测算方法
CN104569418A (zh) * 2013-10-12 2015-04-29 广州瑞博奥生物科技有限公司 一种用于生物标记物筛选的抗体芯片试剂盒
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CN116735883A (zh) * 2023-08-14 2023-09-12 天津理工大学 一种便携式检测乳腺癌标志物的比色传感芯片制备方法

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007056411A2 (fr) * 2005-11-08 2007-05-18 Genentech, Inc. Procede de production d'anticorps pan-specifiques
WO2007056411A3 (fr) * 2005-11-08 2007-08-30 Genentech Inc Procede de production d'anticorps pan-specifiques
CN102353767A (zh) * 2011-07-07 2012-02-15 贺福元 一种全成分群同时测算方法
CN104569418A (zh) * 2013-10-12 2015-04-29 广州瑞博奥生物科技有限公司 一种用于生物标记物筛选的抗体芯片试剂盒
CN104569418B (zh) * 2013-10-12 2016-06-29 广州瑞博奥生物科技有限公司 一种用于生物标记物筛选的抗体芯片试剂盒
CN114935655A (zh) * 2022-06-16 2022-08-23 安徽农业大学 一种猪il-17胶乳增强免疫比浊法检测试剂盒及其制备使用方法
CN114935655B (zh) * 2022-06-16 2024-02-20 安徽农业大学 一种猪il-17胶乳增强免疫比浊法检测试剂盒及其制备使用方法
CN116735883A (zh) * 2023-08-14 2023-09-12 天津理工大学 一种便携式检测乳腺癌标志物的比色传感芯片制备方法
CN116735883B (zh) * 2023-08-14 2023-10-20 天津理工大学 一种便携式检测乳腺癌标志物的比色传感芯片制备方法

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