WO2007130549A1 - Monoclonal antibody microarray - Google Patents

Abstract

The present invention relates generally to the field monoclonal antibody microarrays and their use including the detection and differential expression between abnormal cells and normal cells using monoclonal antibody microarrays. These microarrays and methods enable protein profiling of different sources of samples such as tumor tissue, drug-treated cells, human cells infected with different pathogens (parasites, virus) etc. The protein expression profile of clinical samples may allow us to identify disease markers and enable the development of new anti-disease drugs. Furthermore the correlation of protein expression profile with clinical symptoms may allow us to use the antibody microarray as a diagnostic and/or prognostic tool for different diseases. The invention further relates to the identification of proteins differentially expressed in adenocarcinoma as compared to borderline tumor.

Description

MONOCLONAL ANTIBODY MICROARRAYS REFERENCE TO RELATED APPLICATIONS This application claims priority to US Provisional Patent Application Serial

No. 60/797,301 filed on May 2, 2006, which is incorporated herein in its entirety.

GOVERNMENT SUPPORT

Research supporting this application was carried out by the United States of America as represented by the Secretary, Department of Health and Human Services.

FIELD OF THE INVENTION

The present invention relates to the use of novel monoclonal antibody arrays for the identification of proteins that are differentially expressed from abnormal cells, such as neoplastic or diseased cells versus normal cells.

BACKGROUND OF THE INVENTION

Proteins are the major components of cells. They determine the shape, structure, and function of the cell. Proteins are assembled by 20 different amino acids each with a distinct chemical property. This variety allows for enormous versatility in the chemical and biological properties of different proteins. Human cells have up to 100,000 genes for encoding different proteins. Despite the fact that new proteins are being discovered at an unprecedented rate, protein structure and function studies are lagging behind, mainly due to a lack of high throughput methods.

Antibodies and recombinant proteins are powerful tools for protein studies. Antibodies are a large family of glycoproteins that specifically bind antigens. A protein can be identified by using antibodies generated against the protein in immunochemical methods such as Western blot, immunoprecipitation, and enzyme linked immunosorbent assay (ELISA). Monoclonal and polyclonal antibodies against most known proteins have been generated and are widely used in both research and therapy. Recombinant proteins can be readily expressed in organisms like bacteria and yeast and this has made such proteins convenient and indispensable tools in protein structure and function studies. There is a growing demand for recombinant proteins, especially in large scale screening of drug targets and in clinical medicine. Today, numerous antibodies and recombinant proteins have been produced. One important issue is how to analyze proteins in large scale by using a large number of antibodies or recombinant proteins in a single experiment.

Protein analysis and binding studies can be facilitated by immobilizing one or more proteins on a support. For example, in Western blot analysis, proteins of interest are first separated by electrophoresis and then transferred onto a nitrocellulose or a polyvinylidene difluoride (PVDF) membrane. In phage display screening methods, several hundred thousand proteins expressed by phages are bound to membranes. In both Western blotting and phage display screening, proteins are bound (i.e., immobilized) non-covalently. The protein of interest is then selected by a unique property, e.g., interaction with an antibody. In some other applications such as immunoprecipitation and affinity purification, agents (e.g., antibodies, ligands) are bound to solid supports (e.g., agarose beads) through their primary amines, sulfhydryls or other reactive groups, or by non-covalent linkages. In general, proteins retain their abilities of interacting with other proteins or ligands after immobilization. Monitoring the expressions and properties of a large number of proteins is desired in many important applications. One such application is to reveal protein expression profiles. A cell can express a large number of different proteins. And the expression patterns (the number of proteins expressed and the expression levels) vary in different cell types. This difference is the primary reason that different cells have different functions. Since many diseases are caused by the change in protein expression pattern, comparing protein expression patterns between normal and disease conditions may reveal proteins whose changes are critical in causing the disease and thus identify appropriate therapeutic targets. Methods of detecting protein expression profiles will also have other important applications including tissue typing, forensic identification, and clinical diagnosis. Protein expression pattern can be examined with antibodies in an immunoassay, but usually in a small scale. Therefore, one major obstacle in profiling protein expression pattern is a lack of large scale protein screening methods. Microarrays have been developed for screening potential binding partners (e.g., proteins, small molecules) for binding to proteins on an array, in a high- throughput manner. Microarrays are typically comprised d of a large number of a library of target or capture reagents (peptides, proteins, tissues, affinity reagents) robotically arrayed or spotted in high density onto a solid support. Potential binding partners for screening are labeled (usually with fluorescence) and contacted with to a target or capture reagent immobilized on the array under conditions to allow for binding. Following a wash step, binding to the individual targets is measured and quantified. Each collection of binding partners is tested individually and results from individual arrays are compared.

Protein posttranslational modifications (e.g., phosphorylation, glycosylation, and ubiquitination) play critical roles in regulating protein activity. One of the modifications is phosphorylation at either serine, threonine or tyrosine residues. Protein phosphorylation is an important mechanism in signal transduction. Aberrant protein phosphorylation contributes to many human diseases. Among the methods of detecting protein phosphorylation, metabolic labeling of cells with radioisotopes and immuno-detection of phosphoproteins with antibodies are the most commonly used. However, these methods are only applicable to analyzing at best a few proteins each time. Antibodies specific for phosphorylated amino acids, such as PY20, can reveal that multiple proteins are phosphorylated, but fail to provide the identity of the phosphorylated proteins. A new method for simultaneously detecting and identifying multiple phosphorylated proteins is highly desirable for signal transduction studies and clinical diagnosis

Protein-protein interactions are critical for protein function. Currently, there are several methods to detect protein-protein interactions; however, many of the methods are either low throughput or require large quantities of reagents or both. The methods include co-immunoprecipitation (Harlow and Lane, 1988, Antibodies, A Laboratory Manual. Cold Spring Harbor Laboratory), yeast two-hybrid screening (Fields and Song, 1989, Nature, 340:245-246) and phage display library screening (Smith, 1985, Science 228:1315-1317) are the most commonly used. In co-immunoprecipitation, a protein of interest can be precipitated with its antibody that is immobilized on agarose beads. Any other protein(s) that co-immunoprecipitated with the protein of interest can be identified by either blotting with its antibody when it is known or purification and sequencing when it is a novel protein. However, this method cannot be applied to large-scale identification of protein-protein interactions. Yeast two-hybrid screening is a technique for detecting protein-protein interaction. Although a single yeast two-hybrid screening assay can detect many interacting proteins, it is time-consuming and prone to false positive results. Moreover, many protein- protein interactions only occur in the presence of additional cellular factors or after posttranslational modifications, which may not be present in yeast. Therefore, yeast two- hybrid screening fails to identify many important protein-protein interactions that only take place in mammalian cells. Phage display screening of protein-protein interaction suffers similar limitation. Therefore, there is a need for improved techniques which allow rapid and detailed analysis of multiple proteins for both basic research and clinical medicine. Such techniques will be extremely valuable in monitoring the overall patterns of protein expression, protein posttranslational modification, and protein-protein interaction in different cell types or in the same cell type under different physiological or pathological conditions Proteins arrays have been developed for screening for the presence or absence of binding partners that can be bound by proteins in the array; however, these methods are also limited. Typically, potential binding partners from a single source are applied and then detected. The number of binding partners that can be detected can be limited by cross- reactivity of both primary and secondary antibodies, and the ability to detect differentially tagged secondary antibodies. To test multiple collections of binding partners (e.g., cell extracts from different sources), multiple arrays must be run in parallel. Alternatively, arrays can frequently be stripped and reused; however, this increases the time required to perform assays and possibly decreases reproducibility and sensitivity as the stripping process can result in denaturation or removal of proteins in the array.

SUMMARY OF THE INVENTION

The present invention relates generally to the field of monoclonal antibody microarrays, methods of designing monoclonal antibody microarrays and methods of using the arrays for protein detection and differential expression between, for example, abnormal cells and normal cells. Differential expression patterns can be useful in diagnosing disease and monitoring the progress of a patient in response to treatment or indicate whether a patient can be classified in terms of prognosis (i.e., good or bad).

More specifically, the present invention relates to monoclonal antibody arrays comprising a plurality of antibodies immobilized on a support, wherein different antibodies, each directed against a specific protein, are provided at known, predetermined positions on said support such that each of said antibodies can be identified by its predetermined position. Preferably, the microarrays of the invention include at least about 1000 antibodies, about 5000 antibodies, about 10,000 antibodies, or about 100,000 antibodies. In an embodiment of the present invention, the monoclonal antibody microarrays are designed to target a particular disease indication or disorder, such as the proteins differentially expressed by a particular cancer for comparison with the protein expression profile of normal tissue or a different grade of the same type of cancer. For example, the microarrays and methods of the invention can be used to detect differential expression of proteins associated with borderline tumors and/or adenocarcinoma. The invention includes methods for making the monoclonal antibody microarrays of the invention, and microarrays made^' by the methods of the invention. This method allows for the preparation of the high quality microarrays of the invention with a large number of antibodies without disruption of antibody function. The method includes preparing monoclonal antibody solutions at concentration of 1 —3 mg/ml in 10-20% glycerol;contacting the antibody solution with a nitrocellulose coated slide using an automated arrayer and solid pins at 60-80% humidity; and drying the slide at room temperature and humidity. hi addition, the present invention relates to a method for identifying differentially expressed proteins form an abnormal cell compared to a normal cell, said method comprising the steps of: obtaining a microarray of the instant invention comprising a plurality of antibodies immobilized on a support, wherein different antibodies, each directed against a specific protein, are provided at known, predetermined positions on said support such that each of said antibodies can be identified by its predetermined position; preparing a protein extract from an abnormal cell and a protein extract from a normal cell of corresponding type; labeling the protein extract from the abnormal cells with first detection marker and labeling the protein extract from the corresponding normal cell with a different, second detection marker; contacting equal amounts of protein from both the labeled abnormal extract and the labeled normal extract with the antibodies on the support and incubating the support under conditions to permit binding of said labeled proteins thereto; detecting, the relative quantity of labeled proteins from the labeled abnormal cell extract to the labeled proteins from the corresponding normal cell extract which bind to the antibodies at known, predetermined positions on the array, wherein the detection label (fluorescence intensity) from the protein binding to the array from one extract being greater quantity than that of the other extract indicates differential expression between the two cells of the protein corresponding to the position on the array. The invention further relates to the use of proteins found to be differentially expressed in adenocarcinomas and borderline tumors as compared to each other or normal ovarian surface epithelial cells, for the identification and diagnosis of adenocarcinoma or borderline tumor, particularly ovarian adenocarcinoma or ovarian borderline tumor tissue.

The invention further relates to identification of protein-protein interactions between a tagged protein of interest and an interacting protein using the microarrays of the instant invention. The methods include the steps of: obtaining a microarray of the instant invention, comprising a plurality of antibodies immobilized on a support, wherein different antibodies, each directed against a specific protein, are provided at known, predetermined positions on said support such that each of said antibodies can be identified by its predetermined position; expressing a tagged protein of interest in a cell, preparing an extract from the cell under conditions to maintain protein-protein interactions; contacting the extract with the antibodies on the support; detecting the presence of the tagged protein at specific a position on the solid support; and identifying the interacting protein based on the predetermined position of the antibody. The arrays and methods of the invention enable the protein profiling of different sources of samples such as tumor tissue, drug-treated cells, human cells infected with different pathogens (parasites, virus) etc. The protein expression profile of clinical samples allows the identification of disease markers for diagnosis and new drug targets to enable the development of new therapeutic drugs. Furthermore the correlation of protein expression profile with clinical symptoms may allow us to use the antibody microarray as a diagnostic and/or prognostic tool for different diseases. These examples show the importance of this technology for medicine and research.

Other aspects of the invention are discussed infra.

BRIEF DESCRIPTION OF THE FIGURES AND TABLES

Figure 1. Image of an antibody microarray slide, hybridized with labeled goat anti-mouse IgG (labeled with dye Cy3 and Cy5). All spot light up in yellow because goat anti-mouse IgG binds nonspecifically with antibodies generated in mouse.

Figure 2. Schematic drawing of the antibody microarray technology workflow for dual-labeling approach. Proteins are extracted from references and query samples and labeled with either fluorescent dyes Cy3-NHS and Cy5-NHS ester. After 1.5 h of labeling on ice, the labeling reaction is quenched and subjected to Sephadex G- 15 gel filtration columns to remove unbound dyes. The labeled proteins are mixed and hybridized to the antibody microarray overnight at 4°C. After washing, the microarray is scanned using a microarray scanner. Hybridization signals are extracted using GenePix software (Molecular Devices) and the proteins are characterized as differently expressed or not. Figure 3. The coefficient of variation (CV) was calculated for each antibody between replicate data in the three experiments sets. The CVs were averaged resulting in a CV of 7.9%.

Figure 4. Antibody microarray slide consisting of 1010 different antibodies. The slide was incubated with fluorescently labeled HL-60 and UO-31 protein extracts, a) HL-60 extract labeled with Cy3 and UO-31 extract labeled with Cy5. b) Dye swapping: HL-60 Cy5 and UO-31 Cy3.

Figure 5. MA-Scatter plot of the three independent replicates experiments. Three experiments were performed, each of which generated samples from HL-60 (leukemia cell line) and UO-31 (renal cell line) extracts. Triplicate spots representing individual up or down-regulated proteins are identified and encircled. Note the consistent pattern of regulation observed in the three array experiments.

Figure 6. Illustration of the direct labeling of free amino groups with Cyanine 3 (Cy3). Figure 7. Dendogram for clustering experiments, using centered correlation and average linkage with various grade ovarian tumors as compared to normal cells.

Figure 8. Ingenuity Pathway Analysis of a network of 77 genes that distinguish between borderline tumor tissue and adenocarcinoma tissue.

Figure 9. Schematic of altered expression of proteins in signal transduction pathways between ovary tumors and normal ovarian cell lines.

Figure 10. Schematic of altered chemokine expression between ovary tumors and normal ovarian cell lines.

Table 1. List of all of the antibodies in the microarray used in the methods herein. Table 2. List of the proteins differentially expressed in adenocarcinoma and normal cells. DETAILED DESCRIPTION OF THE INVENTION

The rapid development of genomic databases, bioinformatics tools, and enabling technologies such as cDNA and oligonucleotide microarrays have provided new insights and understanding into biological and disease processes through the global analysis of gene expression patterns. Although gene expression profiling at the mRNA level has proved to be a powerful and useful tool, this approach suffers from inherent limitations. For example, mRNA abundance does not typically correlate well with protein abundance and protein structure, activity, and function can be altered and regulated by post-translational modifications. So, although mRNA expression profiling continues to be a valuable tool, there is a growing recognition that these approaches should be complemented by profiles of the gene products or proteins themselves. The global analysis of protein expression patterns, proteomics, is a natural extension and complement to genomics and the genomics platforms that have developed over the last two decades. A major challenge during the post-genome era will be to develop protein profiling platforms and methodologies and to correlate the data sets they generate with disease, biological and environmental phenotypes.

The present invention relates to antibody microarrays and their use. This technology comprises of a large number of regularly arranged small spots of antibodies that are spotted on a solid support using spotting robots. Protein samples to be analyzed are labeled (usually with fluorescence) and hybridized to a target immobilized on the array. Following a wash step, protein binding to individual targets (i.e., antibodies bound to defined positions on the support) are measured, quantified and identified. The antibody array contains several groups of antibodies directed to proteins such as cytokines, kinases, growth factor receptors, apoptotic proteins, tumor suppressor genes and oncogenes among others, represented by 1010 different antibodies immobilized in nitrocellulose slides (FAST slides) (Figure 1). In an embodiment, multiple labeled abnormal extracts can be mixed with a single normal extract that acts as a reference across the series of abnormal extracts. This allows for comparisons to be made across the abnormal extracts, not just to the normal extract with which the abnormal extract is mixed. Microarrays offer a number of advantages as a protein profiling platform. First, microarrays allow large numbers of pre-determined, specific target molecules to be assayed simultaneously with low sample consumption (as little as lOOμg of total protein extracted from samples). Second, large number of samples (clinical and research samples such as tumors, serum, culture cells) can be analyzed in parallel on identical arrays at low cost in a high-throughput manner. Third, microarrays also allow direct comparative analysis by differentially labeling two samples (dual color labeling) and co-hybridizing them to the same microarray. Multiple matched microarrays can be prepared for binding to a series of abnormal extracts mixed with a single, normal, common control extract to allow for comparison across extracts from a series of abnormal cell extracts. The ratio of the two differentially labeled binding targets signals directly reveals the concentration of one target in relation to the other in the samples. A schematic drawing of the dual-labeling approach appears in Figure 2. The microarrays and methods of the instant invention have been used to identify a number of proteins associated with adenocarcinoma. This information can be use to improve selection of therapeutics for the treatment of disease, to develop improved diagnostics for disease progression, and potentially new therapeutic agents. The invention includes the use of at least 5, preferably at least 10, preferably at least 15, preferably at least 20, preferably at least 25, preferably at least 35, preferably at least 40 of the proteins identified herein to be associated with the disease adenocarcinoma for the detection, or diagnosis or the disease; or to determine the prognosis of a subject suspected of or known to have adenocarcinoma, specifically ovarian adenocarcinoma. Proteins identified to be differentially expressed in carcinoma, especially cervical adenocarcinoma, are listed in Table 2 and include: CCL3, CCL5, CCL7, vascular endothelial growth factor (VEGF), interleukin (IL)-IA, IL-8, IL-18, PASLG, THBSl, CCR5, CEACAMl, TNFRSF8, MS4A1, DAF, IL-IRl, CD34, LAT, FPRl, H-RAS, AMPH, insulin like growth fractor receptor (IGFR)I, DCBKAP, mitogen activated protein kinase (MAPK)3, MAPK14, HSPDl, BAGl, ACTAl, GSTPl, MYODl, TP53, TP73, TP53BP2, CDC2, Actin, Paxillin (PXN), GRB2, ERK1/2, MCP-3, RANTES, p38 MAPK, fibrinogen, HSP60, Dual specificity protein phosphatase 8 (DUSP8), serum albumin, Chromatin-specific transcription elongation factor 140 kDa subunit (FACTpUO), CD55, CD58, CD66, CDl Ia, CD25, CD20, CDl 1, CD41b, CD15s, CD30, CD34, CDwl31, CDC121a, IKAP, Nup88, CD195, CD221, Galectin, Ras GTPase-activating protein-binding protein 1 (G3BP), VAP33, Tat-SFl, MIP-I, Keratin 19, c-erb B2A, V9-TCR, IL-Ia, CAS, Cdkl/Cdc2, CLA-I, Annexin I,

Annexin IV, p53 Ab-8, SRP54, CD178, DNA polymerase epsilon catalytic subunit A, Fl 1 receptor, Exportin-1, HPV 16 early, cytokeratin, amphiphysin, £MLP receptor, inhibitor 2, GST-p, caspase 2, NKT, SNX2, TSP, Na/K ATPase subunit beta-2, DRBP76, p53 Ab-3, p73 (AB-4), MAD2B, MCP-3, pl30 Cas, pgr Ab-2, DLPl, MUPPl, alpha actinin-1, and anti-cidea. The invention includes microarrays including at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, or at least 70 of the proteins listed above associated with carcinoma.

Mechanisms of action of chemotherapeutic agents are becoming better understood. Moreover, chemotherapeutic agents are being designed to interact with specific proteins such as kinases and growth factors, to reduce the unwanted side effects of chemotherapeutic agents. However, these agents can be most effective in forms of cancer in which the specific protein targeted by the drug is dysregulated or over-expressed. Methods such as immunofluorescence to detect specific proteins that may be over-expressed in a tumor or diseased tissue can be time consuming as each antigen must be tested separately. Also, cancer cells are typically genetically unstable; therefore, multiple mutations may exist within the cells of a single tumor. The microarray and methods of the invention allow for the detection and identification of multiple dysregulated proteins in a single sample from a tumor. This can allow for the optimization of selection of chemotherapeutic agents for the treatment of cancer. Comparison of extracts prepared from various times from the tumor can be used to detect genetic drift of the tumor, potentially revealing drug resistance prior to obviously resistant tumors becoming present.

The term "detection marker", "detection label", "detectable label" or other like term as used herein is understood as a tag such as a fluorescent, colormetric, enzymatic, or radioactive tag that can be readily observed by direct or indirect methods such as microscopy and/or exposure to film or other recording device such as a scanner. In a preferred embodiment of the invention, fluorescent tags are used. Fluorescent tags include, but are not limited to, Cy3, Cy5, Cy5.5, fluorescence, rhodamine, SYBR green, Texas Red, DyLight Reactive Dyes and Conjugates including DyLight 488, 549, 649, 680 and 800 Reactive Dyes, Alexa Dyes (Alexa 488, Alexa 546, Alexa 555, Alexa 647, Alexa 680) and IRDye 800. Proteins and cell extracts are preferably labeled with detectable labels attached to reactive groups such as reactive ester groups, specifically N-hydroxysuccinimide (NHS) esters. These reagents allow for direct labeling of free amino groups in proteins. Such reagents are commercially available from a number of sources. The term "detection", "detect," or variations thereof as used herein is understood to mean looking for a specific indicator of the presence of one or more proteins bound to a specific location on the solid support corresponding to a specific antibody. The amount of protein detected can be none, i.e., below the detection limit. In the case of application of multiple extracts to a single array, the amount of one or both proteins detected can be none, i.e., below the detection limit. The detection limit can depend on a number of factors including the efficiency and specific activity of the label, or tag used and the amount of one labeled protein bound to the antibody relative to the other. The term "identification," "identify," or variations thereof is understood as the correlation of a specific location on the solid support to a specific antibody. A protein is identified by correlating the presence of the detectable marker with the predetermined position of the corresponding antibody on the support. Due to the specificity of antibody binding, the identity of the corresponding antigen can be determined.

The term "array" or "microarray" as used herein, refers to an intentionally created collection of molecules that can be prepared either synthetically or biosynthetically. Arrays of the invention can be prepared using robotic printing methods to reproducibly place antibodies in precise, predetermined locations. The molecules in the array can be identical or different from each other. The array can assume a variety of formats, such as for example, a repeating series of the same antibodies in a single array for internal controls, or an array in which each antibody in the array is essentially unique to the array. The term "surface," "solid support," "support," and "substrate" as used herein, are used interchangeably and refer to a material or group of materials having a rigid or semi-rigid surface or surfaces such as nitrocellulose, nylon, polyvinylidene difluoride, glass, or plastics, and their derivatives. Membranes are easier to handle and monoclonal antibodies can be readily immobilized on them. Glass or plastic plates provide rigid support and are therefore preferred in some applications. The surface allows antibodies to be "immobilized" or attached to a fixed position on the support at a specified or predetermined position. In many embodiments, at least one surface of the solid support will be substantially flat, although in some embodiments it may be desirable to physically separate synthesis regions for different compounds with, for example, wells, raised regions, pins, etched trenches, or the like. According to other embodiments, the solid support(s) will take the form of beads, resins, gels, microspheres, or other geometric configurations. See, e.g., U.S. Pat. No. 5,744,305 for other exemplary substrates. Methods and techniques applicable to polymer (including protein) array synthesis have been described in U.S. Ser. No. 09/536,841, WO 00/58516, U.S. Pat. Nos. 5,143,854, 5,242,974, 5,252,743, 5,324,633,

5,384,261, 5,405,783, 5,424,186, 5,451,683, 5,482,867, 5,491,074, 5,527,681, 5,550,215, 5,571,639, 5,578,832, 5,593,839, 5,599,695, 5,624,711, 5,631,734, 5,795,716, 5,831,070, 5,837,832, 5,856,101, 5,858,659, 5,936,324, 5,968,740, 5,974,164, 5,981,185, 5,981,956, 6,025,601, 6,033,860, 6,040,193, 6,090,555, 6,136,269, 6,269,846 and 6,428,752, in PCT Applications Nos. PCT/US99/00730 (International Publication No. WO 99/36760) and PCT/US01/04285 (International Publication No. WO 01/58593), which are all incorporated herein by reference.

The term "monoclonal antibody" or "MAb" refers to antibodies that are identical because they are produced by immune cells that are all clones of a single parent cell. Given (almost) any substance, it is possiblejo create monoclonal antibodies that specifically bind to that substance; MAbs can then serve to detect or purify that substance. Monoclonal antibodies are preferred as they can theoretically be prepared in infinite quantity and are more specific than polyclonal antibodies raised in animals which typically include a mixture of antibodies directed to a specific protein or antigen. For example, monoclonal antibodies can be more readily generated to bind differentially to wild type and mutated forms of proteins.

The term "proteins associated with a disease or disorder" is understood to refer to proteins currently known to be associated with a disease or disorder, or those identified using the microarrays and methods of the instant invention. Proteins associated with a disease or disorder are differentially expressed in response to the specific disease or disorder. Differentially expressed can mean that an abnormal cell expresses a version of the protein distinct from a normal cell, including a protein having a mutation in the sequence, or post-translationally modified in a manner distinct from the wild type protein.

For example, proteins known to be associated with cancer include certain oncogenes, kinases, cytokines, growth factors, and apoptotic proteins. Specific oncogenes can further be associated with specific types of cancer, for example, Bcr- AbI is associated with chronic myelogenous leukemia (CML). Increased expression or expression of a mutated form of an oncogene can be indicative of cancer. Proteins known to be associated with inflammation include, but are not limited to, cytokines, interleukins, and other inflammatory mediators. Increased expression of such proteins can be indicative of inflammation. Microarrays and methods of the invention can also be used for the detection of the presence of viral, bacterial, fungal, or parasitic pathogen contaminants or infectious agents upon selection of appropriate monoclonal antibodies. It is understood that proteins associated with cancer, inflammation, and infection are not exclusive groups. For example, both infection and cancer are typically associated with some inflammation, or have overlapping symptoms such that detection of proteins indicative of various diseases can be included in a single microarray for diagnostic or other purposes. Proteins can also be differentially expressed in response to treatment with a drug or exposure to a chemical or other insult. The microarrays and methods of the invention allowed for the identification of at least 77 proteins that are associated with differential expression in the disease adenocarcinoma include cytokines, kinases, growth factor receptors, and apoptotic proteins. The proteins include CCL3 (chemokine (C-C motif) ligand 3), CCL5 (chemokine (C-C motif) ligand 5), CCL 7 (chemokine (C-C motif) ligand 7), vascular endothelial growth factor (VEGF), interleukin (IL)-IA, IL-8, IL-18,

PASLG, THBSl (thrombospondin 1), CCR5 (chemokine (C-C motif) receptor 5), CEACAMl (carcinoembryonic antigen-related cell adhesion molecule 1), TNFRSF8 9 tumor necrosis factor receptor superfamily, member 8), MS4A1 (membrane- spanning 4-domains, subfamily A, member 1), DAF (Complement decay- accelerating factor), IL-IRl (interleukin 1 receptor, type 10, CD34 (Hematopoietic progenitor cell antigen CD340, LAT (Linker for activation of T-cells family member 1), FPRl (formyl peptide receptor 1) , H-RAS (GTPase HRas), AMPH

(Amphiphysin), insulin like growth fractor receptor (IGFR)I, DGBKAP (inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase complex -associated protein), mitogen activated protein kinase (MAPK)3, MAPK14 (mitogen activated protein kinase 140, HSPDl (Heat shock 60 kD protein 1), BAGl (Bcl2-Interacting Protein-1), ACTAl (Actin, alphal) , GSTPl (glutathione S-transferase pi gene), MYODl (myogenic differentiation 1), DUSP8 (Dual specificity phosphatase 8) , TP53 (Tumor protein 53), TP73 (Tumor protein 73), TP53BP2 (Tumor protein p53 binding protein 2), CDC2 (cell division cycle 2), Actin, Paxillin (PXN), GRB2(growth factor receptor-bound protein 2) , ERK 1/2 (extracellular signal- regulated kinase), MCP-3 (Monocyte chemotactic protein-3), RANTES (Regulated on Activation, Normal T Expressed and Secreted), p38 MAPK (MAPK, Mitogen- activated protein kinase), fibrinogen, HSP60, Dual specificity protein phosphatase 8 (DUSP8), serum albumin, Chromatin-specific transcription elongation factor 140 kDa subunit (FACTpl40), CD55, CD58, CD66, CDl Ia, CD25, CD20, CDIl, CD41b, CD15s, CD30, CD34, CDwl31, CDC121a, IKAP, Nup88, CD195, CD221, Galectin, Ras GTPase-activating protein-binding protein 1 (G3BP), VAP33, Tat- SFl, MIP-I, Keratin 19, c-erb B2A, V9-TCR, IL-Ia, CAS, Cdkl/Cdc2, CLA-I, Annexin I, Annexin IV, p53 Ab-8, SRP54, CD 178, DNA polymerase epsilon catalytic subunit A, Fl 1 receptor, Exportin-1, HPVl 6 early, cytokeratin, amphiphysin, fMLP receptor, inhibitor 2, GST-p, caspase 2, NKT, SNX2, TSP, Na/K ATPase subunit beta-2, DRBP76, p53 Ab-3, p73 (AB-4), MAD2B, MCP-3, pl30 Cas, pgr Ab-2, DLPl, MUPPl, alpha actinin-1, and anti-cidea.

As used herein "cytokine" refers to a group of proteins and peptides that are used in organisms as signaling compounds. The cytokine family consists mainly of smaller water-soluble proteins and glycoproteins with a mass of about 8-30 kDa. Cytokines include, but are not limited to, RANTES, Interleukin ILl-a and -b, ILl, IL2, IL3, IL4, IL5, IL6- IL13, a-defensϊn 1-3, AP-2 Transcription Factor (Ab-I), C3a Receptor, CCR4,CCR6, CCR7, CD 105 (Endoglin), CDl 14 (G-CSF Receptor), CDl 16 (GM-CSF Receptor a chain), CDl 16 (GM-CSF Receptor a chain, CDl 17 (c- kit), CDl 19 (IFN-g Receptor a chain), CDl lb/Mac-1 (CR3), CD120a (TNF Receptor Type I), CD120a (TNF Receptor Type I), CD120b (TNF Receptor Type π), CD121a (IL- 1 Receptor Type I), CD 122 (IL-2 Receptor b chain), CD 123 (IL-3 Receptor a chain),CD123 (IL-3 Receptor a chain),CD124 (IL-4 Receptor a chain), CD 126 (IL-6 Receptor a chain), CD 127 (IL-7R),

CD128a (CXCRl, EL-8RA), CD128b (CXCR2, IL-8RB), CD130 (gpl30), CD130 (gpl30), CD135, CD137 (4-1BB), CD137 (4-1BB), CD137 Ligand (4-1BB Ligand), CD140a (PDGF receptor a chain), CD140b (PDGF Receptor b chain), CD154

(CD40L), CD154 (CD40L), CD18 (Integrin b2 chain, CR3/CR4), CD18 (Integrin b2 chain) , CR3/CR4CD183 (CXCR3), CD184 (CXCR4, Fusin), CD195 (CCR5), CD195 (CCR5),CD21 (CR2), CD21 (CR2),CD212 (IL-12 Receptor b chain),CD212 (EL- 12 receptor bl subunit) CD221 (IGF-I receptor), CD221 (IGF-I receptor a subunit), CD25 (IL-2 Receptor a chain),CD35 (CR1),CD46 (MCP),CD55 (DAF).

As used herein, "protein kinase" refers to a protein that is responsible for phosphorylation of other proteins, typically to regulate the activity of the protein being phosphorylated. Phosphorylation usually results in a functional change of the target protein (substrate) by changing enzyme activity, cellular location, or association with other proteins. Protein kinases include, but are not limited to tyrosine kinases, including receptor tyrosine kinases, serine kinases, threonine kinases, mitogen activated protein (MAP) kinases, MEK, MEKK, ERK, Akt, JNK, Protein kinase C (PKC), and focal adhesion kinase (FAK).

The term "growth factor receptor" as used herein refers to proteins that bind specific signaling molecules. The receptors frequently interact with kinases and other proteins involved in signal transduction pathways. Growth factor receptors include, but are not limited to epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor receptor (FGFR), insulin-like growth factor receptor (IGFR), platelet derived growth factor (PDGF), transforming growth factor (TGF), and nerve growth factor receptor.

The term "apoptotic protein" as used herein refers to proteins that are involved in apoptosis or programmed cell death. Expression and/or activity of proteins involved in apoptosis can be altered in cancer or other proliferative disorders. Apoptotic proteins include, but are not limited to BAD, BcI-X, BcI-XL, Fas, JNK-I, x-linked inhibitor of apoptosis protein (XIAP), interleukins such as interleukin-3 and -7, caspases such as caspases 3, 7, and 9, and survivin.

The term "extract", "cell extract", or "extract of proteins" is understood to mean a cell-based preparation in which the cells are sufficiently disrupted to allow for interaction of the proteins in the cell with the antibodies of the microarray. The cells used can be obtained from a tissue or bodily fluid of a subject, such as an animal, mammal, or preferably a human subject. The cells can be obtained from cultured cells, including primary or immortalized cell lines. A cell extract may be a total cell extract or a fractionated extract such as a membrane, cytoplasmic, or nuclear extract. At minimum, it is preferred that the extract is treated to disrupt high molecular weight nucleic acids (e.g., by nuclease treatment or mechanical means such as sonication or shearing using a needle) and subject to centrifugation to remove insoluble components. As used herein, an "extract" can also be a bodily fluid preferably with the cells removed, such as serum.

An "equal amount" is understood to mean equal amount of labeled protein, meaning an equal protein concentration with similar specific activity of labeling. A "normal cell" is a cell or extract derived from non-diseased tissues or cells from a subject, or from a primary cell line or a cell line immortalized by expression of a non-transforming agent, e.g., SV40 large T antigen. When normal cells are to be compared with diseased (e.g., cancerous, hypoxic, or otherwise damaged) "abnormal cells" it is preferred that the normal cells are isolated from an area of tissue close to the diseased tissue/abnormal cells, but with a sufficient margin to avoid the diseased tissue. Similarly, due to the small amount of sample that is required for the method of the invention, the "abnormal cells" should be isolated from a portion of the diseased tissue such that it is not contaminated with normal cells, and preferably necrotic cells are isolated separately from viable cells in the abnormal tissue. When cells from a bodily fluid or a bodily fluid is to be tested (e.g. blood or serum), "normal cells" or "normal extract" cannot be readily isolated from a diseased subject. Therefore, "normal cells" or a "normal extract" is derived from a healthy, separate donor, preferably matched (e.g., age, gender, weight, genetic background especially with transgenic animals) as well as possible to the subject from whom the diseased cells or fluid was isolated. Alternatively, the normal tissue can be derived from a site corresponding to the abnormal area in the case of induced damage or injury (e.g., the same muscle, nerve, or other tissue from the opposite side of the body when a localized insult is administered). The terms "normal" and "abnormal" can also refer to cells or tissues not treated with a drug or agent, and cells or tissues treated with a drug or agent, respectively. The cells or tissues to be treated can include diseased or damaged cells or tissue.

The term "bodily fluid" is understood herein to mean any essentially liquid sample obtained from a subject, such as an animal, mammal, or preferably human subject, that may or may not contain cells. If the bodily fluid includes cells, the cells are preferably removed (e.g., by centrifugation or filtration) or extracted prior to contacting the bodily fluid with the microarray. Bodily fluids can include, for example, blood, serum, breast milk, semen, urine, and lymph. Bodily fluids are preferably diluted in an appropriate buffer before labeling or contacting the fluid with a microarray.

The term "conditions to allow binding" or "under conditions to allow for binding" is understood herein as buffer, salt, detergent, and temperature conditions under which antigen binding by antibodies can occur. Such conditions are well known to those skilled in the art and are discussed, for example, in Harlow and Lane (1988, infra). Typically conditions for binding are about 150 mM NaCl, 10 mM Tris, pH 8.0, 0.05% Tween with 2-5% non-specific protein (e.g., bovine serum albumin, non-fat dry milk).

The term "obtaining" as in "obtaining an antibody" or "obtaining a cell" refers to purchasing, synthesizing, removing from a subject, or otherwise procuring an agent, antibody, or other material.

The term "subject" refers to an animal, preferably a mammal including a human. A subject is a source for cells, bodily fluids, and/or tissues for the preparation of extracts for use in the methods of the invention. A subject can also be an individual suspected of having a predisposition to a disease or disorder; or suspected of having a disease or disorder. A subject can be an individual having a predisposition to a disease or disorder, or having a disease or disorder. Human subjects suspected of or known to have a disease or disorder can be referred to as "patients." Identification of subjects having or prone to a disease or disorder can be accomplished by standard diagnostic methods.

The term "diagnosis", "diagnosing", and the like are understood to mean to recognize (as a disease) by signs and symptoms a disease or condition in a subject or patient, or to analyze the cause or nature of a problem, particularly a physiological problem. Diagnosis does not require a conclusive indication of disease. Diagnosis can be a process.

The term "prognosis", "determining the prognosis", and the like as used herein relates to the evaluation of the chances of recovery or elimination of disease as anticipated from the usual course of disease or peculiarities of the case. Prognosis in cancer, for example, includes the evaluation of the chances that the cancer will become more aggressive or go into remission. A prognosis can be based on a single evaluation, or can vary based on monitoring of signs or symptoms of disease over time such as with monitoring the efficacy of treatment.

The term "plurality" is understood to mean more than one. The terms "a" and "the" are understood to be both single and plural unless otherwise indicated by context. The term "or" is understood to be inclusive unless otherwise indicated by context.

Ranges are understood to include all of the numbers within the range. For example, 1 to 50 is understood to include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 37, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.

Monoclonal antibody microarrays will become one of the most important tools in the field of high-throughput biology analysis because of their enormous potential in basic research, diagnostics and drug discovery and consequently the antibody microarray will be a very powerful tool in this field. The monoclonal antibody arrays of the instant invention provide a powerful and quantitative tool for determining protein expression patterns, protein posttranslational modifications and protein-protein interactions. Protein array is based on several principles. First, a protein can be recognized and identified unambiguously by specific molecules, monoclonal antibodies, which can specifically interact with it. Second, a monoclonal antibody can be immobilized on a solid support and the immobilized molecule still retains its ability in protein-protein interaction. Monoclonal antibodies can be immobilized on solid supports such as glass plates, agarose beads, or PVDF membranes (LeGendre, 1990, BioTechniques, Vol.9, No.6, p. 788-805) glass slides and membranes coated slide, hydrogel slides (Readypolyacrylamide coated slides), gold surfaces, and microliter 96 weel plates. Third, many different monoclonal antibodies can be immobilized at different positions on a solid support without cross interactions between them. This insures that each monoclonal antibody independently interacts with its respective target protein.

Monclonal antibodies are raised by immunizing animals (e.g., rabbit, mouse, rat, goat or chicken) with antigens (e.g., proteins or peptides). A large number of antibodies (monoclonal) are commercially available (see Table 1) and new monoclonal antibodies can be developed (see below). Many monoclonal antibodies have been conveniently expressed in a recombinant form with a tag such as glutathione-S-transferase (GST) and polyhistidine (6xHis), to facilitate purification and identification.

Monoclonal antibodies are immobilized on a solid support directly or indirectly. Monoclonal antibodies can be directly deposited at high density on a support, which can be as small as a microscopic slide. Similar technology was developed for making high density DNA microarray (Shalon et al., Genome Research, 1996 JuI; 6(7): 639645.). Monoclonal antibodies can also be immobilized indirectly on the support. For instance, protein A or G which bind antibody constant regions can be printed on a support. Antibodies are then immobilized on the support through their interactions with protein A or G. The advantage of this method is that by engaging the constant regions of antibodies with protein A or G, the variable regions of the antibodies (antigen-binding domains) will be fully exposed to interact with antigens. One most important characteristic of monoclonal antibody arrays is that all antibodies are immobilized at predetermined positions, so that each antibody, and therefore its binding partner, can be identified by its position. After antibodies are immobilized, the support can be treated with 5% non-fat milk or 5% bovine serum albumin for several hours in order to block non-specific protein binding.

Different monoclonal antibody arrays can be designed for different purposes. For example, an antibody array was designed to detect and identify the profile expression of proteins which may be up-regulated or down-regulated in various cancers. In another instance, a "Cytokine Array" can be made of antibodies directed cytokines involved in certain inflammation disorders. "Cell Cycle Array" can be made of antibodies for detecting cell cycle related factors; "Signal Transduction Array" can be made of antibodies for examining signaling proteins; and "Transcription Factor Array" can be made of antibodies for analyzing activators and suppressors of transcription. In order to reveal the broad protein expression pattern in a source (e.g. a cell line), thousands of different antibodies are immobilized in a single support. The amount of antibodies immobilized can also be different, preferably in the range of nanogram to microgram. The number of different antibodies immobilized on one solid support varies depending on the particular applications. Monoclonal arrays can be applied in studying protein expression patterns. An antibody array is incubated with a protein sample prepared under the conditions that native protein-protein interactions are minimized. After incubation, unbound or nonspecific binding proteins can be removed with washes. Proteins specifically bound to their respective antibodies on the array are then detected. Because the antibodies are immobilized in a predetermined pattern, the identity of the protein captured at each predetermined position is therefore known. Measurement of protein amount at all positions on the array thus reflects the protein expression pattern in the sample. The quantities of the proteins bound to the array can be measured in several ways. In a preferred embodiment of the invention, identification and relative quantification of proteins is performed by labeling the protein extracts (abnormal versus normal) with different detection makers, such as Cy3 (fluorescent red) for proteins from an abnormal cell and Cy5 (fluorescent green) for proteins from a normal cell. If a particular antibody binds more of a protein labeled with Cy3, then the abnormal cell is differentially expressing that protein at a higher or greater level, and vice versa.

Monoclonal antibody array can be applied in studying protein-protein interactions, for example, between a specific protein of interest and its interacting proteins within a cell. In an embodiment, the proteins of interest is expressed as a fusion protein in a cell wherein a protein tag such as GFP, 6xHis, or myc tag is fused to the protein of interest. The cell may be, for example, a tissue culture cell or a cell in a transgenic animal. An extract is prepared under conditions to preserve protein- protein interactions. The extract is then applied to the microarray of the invention under conditions to allow binding of the proteins in the extract to the antibodies of the array. The array is washed and the presence of the tag on the protein of interest is detected. By determining the location of the tagged protein, the identity of the interacting protein can be determined by the location of the tagged protein in the array. The antibody microarray method includes a protein extraction step, a protein detection labeling step, binding step, and a detection step.

The protein extraction from cellular samples can be carried out by a number of methods currently known to one of ordinary skill in the art and are commercially available, such as the BD Clontech™ Protein Extraction and Labeling Kit by Becton Dickinson, or a number of protein extraction kits from Pierce. Other methods for preparation of extracts for use with antibody binding methods are provided, for example, in Harlow and Lane (1988, infra). The exact methods and conditions for preparation of extracts depends on a number of considerations well known to those skilled in the art, including source of cells (e.g., tissues or unattached cells) and the fraction of the cell to be analyzed (e.g., total cell extract or cell fraction). Once total cellular protein has been extracted, all proteins from a particular cellular sample (i.e., abnormal or normal) are labeled with a fluorescent dye, such as DyLight Reactive Dyes and Conjugates including DyLight 488, 549, 649, 680 and 800 Reactive Dyes, Alexa Dyes (Alexa 488, Alexa 546, Alexa 555, Alexa 647, Alexa 680) and IRDye 800, and CyeDyes (Cy3, Cy5, Cy5.5). Dyes should be selected such that they have sufficiently distinct excitation and emission spectra to allow the dyes to be distinguished. Such considerations are well understood by those skilled in the art. A preferred pair of dyes for use in the methods of the invention are Cy3 and Cy5. Dyes can be attached, preferably covalently attached, to proteins by any of a number of linking groups including reactive NHS-ester linkages. In a preferred embodiment, each protein extract is labeled individually with each label, and each pair of extracts is applied to a single array. For example, an extract from normal cells labeled with Cy3 is mixed with an extract from abnormal cells labeled with Cy5 and incubated with a microarray. A second identical microarray is contacted with a mixture of normal cells labeled with Cy5 and abnormal cells labeled with Cy3. Such methods provide a control for variations in detection of the two dyes.

In the binding step, the test sample containing protein is incubated with the monoclonal microarray. If a protein in the extract binds to at least one of the plurality of monoclonal antibodies immobilized on the monoclonal microarray, it is bound to the microarray via that target antibody. In this case, binding between the antibody microarray and protein is detected in the subsequent detection step. The specific location of the binding is correlated with a specific antibody present at that predetermined position, and the color is indicative of the presence of the protein in each of the extract, and their relative concentration of the proteins in each of the extracts. On the other hand, if neither extract contains sufficient protein corresponding to a specific location on the microarray to generate a signal from the detectable label, binding between the antibody microarray and protein is not detected in the subsequent detection step (i.e., no signal is detected). If a protein from each the two differently labeled extracts is present in both the extracts, the detection read out will be a color that is the combination of the two dyes. For example, the combination of red (Cy5) and green (Cy3) is visualized as yellow. In a preferred embodiment, the amounts of red and green are quantitated in separate channels to determine the relative amount of protein in each of the extracts. If a protein is present in only one of the extracts, the specific predetermined position on the microarray is either red or green depending on the extract from which the protein was derived.

In the detection step, binding of proteins from the labeled extracts to the protein microarray is detected. This detection uses known detection means that can be applied to a microarray method, particularly fluorescence spectroscopy. Apparatuses and methods for detection and quantitation of fluorescence based on the location of the spot within the microarray are well known. Alternatively, quantitation can be performed manually using, for example, fluorescence photomicroscopy.

The antibody detection method of the present invention can be applied to the measurement of proteins in bodily fluids including, but not limited to blood, serum, urine, semen, breast milk, and lymph. If a bodily fluid containing cells is to be tested, it is preferred that the cells are separated from the fluid, and an extract is prepared from the cells if they are to be tested. The fluids/ extracts are labeled using the methods and dyes set forth above, and mixed for the competition assay set forth above. The contacting and detection steps are performed as set forth above.

To increase the range of detection and potentially quantitative nature of the arrays and methods of the invention, serial dilutions (e.g., multiple 10-fold dilutions to make samples that are diluted 10-, 100-, 1000-, etc. fold) of the labeled extracts or bodily fluids can be made prior to contacting the microarray with the fluid or extract. The array is first incubated with the most dilute (i.e., lowest protein concentration) extract and the detection step is performed. After the detection step is performed, a less dilute extract is applied. The detection step is repeated. Progressively more concentrated (less dilute) extracts are applied to the array and detected. After the detection step, the array can be stripped to remove the antibody bound proteins or photo-bleached to eliminate signal from the previously applied extracts. Such a step can be useful if some areas of the microarray become too bright and obscure detection of adjacent spots. The presence, resistance, or susceptibility of a subject or patient to a disease such as an infectious disease, allergy, or cancer is then determined from the relative level of various proteins bound to various locations in the microarray.

In measuring protein levels, an antibody microarray (single array) is used on which a plurality of antibodies are respectively spotted at a plurality of locations. More specifically, monoclonal antibodies are spotted at a plurality of locations on a single antibody microarray at the same concentration and in the same amount. The array can include each antibody essentially once, optionally with control antibodies repeated at various portions of the array to act as controls.

The antibody testing method is used to identify pathogens, tumor antigens, and/or allergens and so forth that are the cause of diseases including infectious diseases, cancer, allergies and the like (e.g., diseases associated with activation of the immune system). In addition, the antibody testing method is used to predict the sensitivity or resistance (e.g., carriers that become sick compared to those who do not) of a subject or patient (e.g., human or animal) to these diseases.

In the case of identifying the cause of an infectious disease, cancer or allergy, a physician or other health professional predicts the pathogen, tumor antigen or allergen based on symptoms of a subject, and then selects an antibody microarray having an antigen corresponding to the predicted pathogen, tumor antigen or allergen, namely the target protein (i.e., antigen). Preferably, an antibody microarray is selected that has spots of antibodies targeted to all of the predicted pathogens, cancer cells markers, inflammatory markers, or allergens associated with the potential symptoms, disease, or disorder of the subject. It should be noted for common diseases or group of symptoms, a plurality of antibody microarrays can be prepared in advance in accordance with the testing pattern for diagnosing those diseases. Moreover, a single microarray can have markers from multiple classes of proteins provided. For example, cancer and infection typically include an inflammatory aspect.

Development of New Monoclonal Antibodies I. Production of hybridoma cells

Hybridoma cell lines producing a desired antibody may be produced by conventional methods such as the well-known methods of Kohler and Milstein. Briefly, an animal, preferably a rodent such as a Balb/c mouse is immunized and later re-immunized (boosted) with the desired immunogen, with an adjuvant as desired, as is well known in the art. Assaying the serum of the animal by conventional methods such as a specific ELISA reveals whether the animal is producing an antibody of the desired affinity and avidity. An immunized animal having an appropriate titer of the desired antibody is sacrificed and its spleen removed. The spleen cells are then carefully separated and fused with a suitable myeloma cell line by conventional procedures or otherwise immortalized, as is also well known in the art. The immortalized cells producing the desired antibody are then identified by routine, conventional screening and are then subcloned as desired.

II. Cloning Heavy and Light Chain-encoding DNAs

Methods for cloning immunoglobulin heavy and light chains are well known in the art. See e.g. Beidler et al, 1988, J. Immunol. 141 :4053 (genomic) and Liu et al, 1987, Proc. Natl. Acad. Sd. USA 84:3439 (cDNA). Briefly, cDNA or genomic libraries are constructed for the RNA or genomic DNA, respectively, from hybridomas producing a specific antibody of interest, as is known in the art. The immunoglobulin clones from such libraries can be identified by hybridization to DNA or oligonucleotide probes specific for J_H or C_H sequences for the heavy chain clones, or J_L or C_L sequences for the light chain clones. The positive clones are then further characterized by conventional restriction endonuclease site mapping and nucleotide sequencing.

in. Expression Vector Construction Any conventional eukaryotic, preferably mammalian, expression vectors designed for high expression levels, of which many are known in the art, may be used in the practice of this invention. However, in the practice of this invention the expression vector for the light chain antibody DNA contains or is cotransfected with a first selectable, amplifiable marker gene while the expression vector for the heavy chain antibody DNA contains or is cotransfected with a second selectable, amplifiable marker. The two selectable, amplifiable markers must be differentially amplifiable, i.e. must each be susceptible to amplification under conditions which do not result in amplification of the other.

The eukaryotic cell expression vectors described herein may be synthesized by techniques well known to those skilled in this art. The components of the vectors such as the bacterial replicons, selection genes, enhancers, promoters, and the like may be obtained from natural sources or synthesized by known procedures. See Kaufinan et al., J. MoI. Biol, 159:601-621 (1982); Kaufinan, Proc Natl. Acad. ScL 82:689-693 (1985). Eucaryotic expression vectors useful in practicing this invention may also contain inducible promoters or comprise inducible expression systems as are known in the art. pMT2 and pMT3SVA are exemplary expression vectors which are described below. Both vectors contain an SV40 origin of replication and enhancer, adenovirus major late promoter and tripartite leader sequence, a cloning site followed by an SV40 polyadenylation site, the adenovirus VA I gene, E. coli origin of replication and an ampicillin resistance gene for bacterial selection. PMT2 further contains a DHFR gene between the cloning site and the polyadenylation signal, while pMT3SVA contains an adenosine deaminase (ADA) gene under the expression control of the SV40 early promoter. While both of these vectors contain appropriate selectable, amplifiable markers, it should be understood that separate vectors containing the markers may be cotransfected or cotransformed by conventional means with the respective heavy and light chain DNAs.

IV. Production of Transformed Cell Lines Established cell lines, including transformed cell lines, are suitable as hosts.

Normal diploid cells, cell strains derived from in vitro culture of primary cells, as well as primary explants (including relatively undifferentiated cells such as hematopoietic stem cells) are also suitable. Candidate cells need not be genotypically deficient in the selection gene so long as the selection gene is dominantly acting.

The host cells preferably will be established mammalian cell lines. For stable integration of the vector DNA into chromosomal DNA, and for subsequent amplification of the integrated vector DNA, both by conventional methods, CHO (Chinese Hamster Ovary) cells are currently preferred. Other usable mammalian cell lines include HeLa, human 293 cells, COS-I monkey cells, melanoma cell lines such as Bowes cells, mouse L-929 cells, 3T3 lines derived from Swiss, Balb/c or NIH mice, BHK or HaK hamster cell lines and the like, as well as lymphocyte derived cell lines such as the murine hybridoma SP2/0-Agl4 or murine myeloma cells such as P3.653 and J558L or Abelson murine leukemia virus transformed pre-B lymphocytes.

The expression vectors may be introduced into the host cells by purely conventional methods, of which several are known in the art. Electroporation has been found to be particularly useful.

Stable transformants may then be screened for the presence and relative amount of incorporated antibody DNA and corresponding mRNA and polypeptide synthesis by standard methods. For example, the presence of the DNA encoding the desired antibody chain may be detected by standard procedures such as Southern blotting, the corresponding mRNA by Northern blotting and the protein thereby encoded by Western blotting. It should be appreciated that the two antibody genes may be introduced serially into the same host cells, or may be introduced in parallel into separate host cells. In the former case, the antibody genes would be transfected separately, and the transfectants after the first of the two transfections, may or may not be selected in iteratively increasing amounts of the appropriate selective agent, prior to the second transfection. In the latter case, the two transfectants may be fused by conventional means to produce a cell containing and capable of expressing both antibody chains, as well as both selectable markers to facilitate isolation of hybrid cells. One of the parental cells of a fusion may be exposed to ionizing radiation before the fusion event. In addition, both heavy and light chain DNAs may be co-transfected with a single selectable, amplifiable marker, and the transfectants then passaged in iteratively increasing amounts of the selective agent. Once the relative levels of the heavy and light chains expressed in such a transfectant has been determined, a DNA encoding the chain found in limiting amounts can then be transfected into the cell, linked to a different selectable, amplifiable marker. The expression level for that chain can then be increased by iterative amplification as previously described. V. Specific Amplification

Specific and independent amplification of the two DNAs may be readily accomplished using conventional amplification procedures appropriate for each of the respective markers. See e.g. published International Application WO 88/08035 for an exemplary description of independently amplifying a first gene linked to a DHFR gene and a second gene linked to an ADA gene. Other selectable, amplifiable markers can also be used, and examples are reviewed in Kaufman, R. J., Genetic Engineering, 9:155, J. K. Setlow, ed. (Plenum Publishing Corp.) 1987.

VI. Characterization of monoclonal antibodies

The monoclonal antibodies so produced by the amplified cell lines can be characterized by standard immunochemical techniques, including SDS-PAGE, Western blotting and immunoprecipitation of intrinsically ³⁵ S-methionine-labeled proteins. The levels of heavy and light chains produced can be quantitated by ELISAs, and binding to solid-phase antigens can be demonstrated by ELISA. The binding characteristics of the antibodies can also be studied in similar antigen- binding ELISAs in the presence of varying concentrations of free antigen. The effector functions of the antibodies can be characterized by standard techniques, e.g. for complement fixation and antibody-dependent cellular cytotoxicity. As evident in the above description, monoclonal arrays have broad applications. It allows one to compare protein expression patterns, protein posttranslational modification, and protein-protein interactions between two types of cell, tissue, or patient specimen. Therefore, it will be an extremely useful tool in clinical diagnosis and new drug search. Only time will reveal its full potential. All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Exemplification Example 1; Microarrav Features and Fabrication

Antibodies for use in a specific microarray are selected based on the disease or condition to be identified or characterized. Antibodies are characterized for all possible antigens that they bind, and this information including database access numbers, such as SWISS-PROT access number, are associated with each antigen. This facilitates use of the Ingenuity Program to analyze data generated using the method.

All antibodies were tested at least by western blot prior to use to determine sensitivity and specificity. Antibodies were prepared in PBS solution at the concentration of 2mg/ml containing 15% glycerol in a 384-well plate (Genetix) and arrayed on nitrocellulose coated slides (Fast Slides-Schleicher & Schuell

BioScience) using a Microgrid π arrayer and solid pins at 70% humidity using manufacturer's instructions. This approach allows us to print great number of antibodies with very good quality and without affect protein function. The antibodies features were distributed in 16 subarrays, containing 64 spots each. After arraying the FAST slides with AB, they were left in Room Temperature for 1 hour. Slides were blocked with 0.1% PVP/0.05% Tween 20 for 1 h prior to experiments.

Example 2; Preparation of Extracts from Serum. Tissue Samples, and Cultured Cells NCI-60 cells were grown in culture under standard conditions. IOSE 7576 normal ovarian surface epithelial cells, immortalized with SV40 large T antigen, obtained from the University of British Columbia, were grown in combination of 199 (Sigma M5017) and MCDB105 (Sigma M6395) media with 5% FBS and 50 ug/ml gentamicin. Cells were washed in PBS and protein extraction was performed using the Clontech Extraction and Label Kit. Human serum samples were obtained from Sigma. Serum proteins were labeled without further extraction or purification. Ovarian tissue samples, including normal and abnormal tissue, were obtained from Cytomyx Inc. Samples were collected according to approved standards and protocols with informed consent. Protein extractions from tissue were done by the Cytomyx company with proprietary protocols. Information relevant to the samples such as disease, patient age and gender, sample type, tissue origin pathology report, and tumor grade were known for each sample. Protein concentrations were determined by Pierce BCA assay per manufacturer's instructions.

Example 3: Protein Labeling

Samples were equalized for protein concentration and labeled with fluorescent dye N-hydroxysuccinimide (NHS) ester-linked Cyanine 3 (Cy3) or Cyanine 5 (Cy5) (Amersham). Labeling reactions were carried out with minimal exposure to light, essentially per the manufacturer's instructions. In brief, 90 μl samples of cell extract prepared having a protein concentration of 1.1 mg/ml in 0.1 M sodium bicarbonate/carbonate buffer pH 9.0 were prepared. Alternatively, samples were prepared using the Clontech Extraction/Labeling kit. For human serum 6 μl of sample diluted to 90 μl in buffer was used for the labeling reaction. The samples were mixed with 10 μl of either 2.3 mM of Cy 3 or Cy5 in sodium bicarbonate/carbonate buffer or labeled using the Extraction/Labeling kit (Clontech). Samples were incubated for 1.5 hours on ice. The reaction was stopped by the addition of lOμl of IM Tris pH. 8.0. Samples were incubated on ice for an additional 30 min with additional mixing approximately every 10 min. The unconjugated dye was removed by passing each solution through a size-exclusion chromatography spin column (Sephadex G- 15- spin columns).

Molar concentration for labeled protein and dye were calculated using manufacturer's instructions. Only extracts with similar amounts of label incorporation were used together. The Cy5- labeled extract A was mixed with equal amount (protein/protein) of the Cy3-labeled extract and dissolved in the array buffer (0.1% PVP/0.05% Tween 20). The array was incubated with the two of the labeled extracts simultaneously in an incubation chamber at 4 ⁰C with gentle rocking for at least 16 h. After incubation, slides were washed three times for 5 min each in PBS/0.05% Tween 20 (Sigma- Aldrich), followed by one wash in PBS for one min. AU washes were performed at 4 ⁰C. Slides were dried by centrifugation and subjected to fluorescence detection.

Example 4: Analysis of Antibody Binding of Extract Proteins

Dried arrays were scanned with Axon GenePix 4000 scanner (Union City, CA), and fluorescence data were collected and evaluated with the GenePix Pro 5.0 software. For the microarray imaging, the Axon GenePix 4000 scanner was set at 100% laser power and 350-400% PMT gain. To verify natural variability present in .spotted protein arrays data we carried out a self-self hybridization in which aliquots of the same human serum are labeled with Cy3 and Cy5 dyes and co-hybridized to a single array. All spots should be uniformly yellow, or show no binding of detectable label above background.

Example 5: Reproducibility of the Microarrays

A monoclonal antibody microarray comprised of monoclonal antibodies listed in Table 1 (i.e., 1010 monoclonal antibodies) was used for testing the differential expression of proteins to which these monoclonal antibodies were directed. The antibodies were selected for this microarray based on their association with neoplastic tissues and disease. Each sample was contacted with the monoclonal antibody array using a dye-swap setup with a common reference (i.e., common normal extract), an ovarian cancer cell line. The antibody microarray performance was tested through proteomic profiling of two NCI-60 cancer cell lines (Renal UO-31 and Leukemia HL-60). The validity of the reproducible antibody microarray measurements were established through application of mathematical model in the ratios of triplicates comparative experiments between HL-60 and UO-31 cell extracts (Figure 4 shows one replicate of the experiment; average coefficient of variation= 7.9%; Figure 3). The triplets corresponding to several regulated proteins are evident and show similar regulation and scatter graph location in all the three independent experiments (Figure 5). These strikingly similar results obtained in independent experiments reflect the high level of reliability and reproducibility of these microarrays. Our data demonstrate that the antibody arrays and methods are useful tools for proteomics.

The reproducibility (coefficient of variation, CV) of the antibody microarray was established through application of a mathematical model (Average (SD/mean*100) analyzing the log² feature intensity of triplicate comparative experiments using NCI-60 cancer cell lines, Renal UO-31 cell extracts and Leukemia HL-60 cell extracts.

Example 6: Identification of DifferentaiHy Expressed Proteins in Borderline Adenocarcinomas and High Grade Level Adenocarcinomas

Using the microarrays and methods described above, a number of tumor tissues were analyzed for the differential expression of proteins to allow correlation of marker expression with prognosis. Extracts were prepared or obtained from 12 samples identified by histopathological analysis as borderline ovarian tumors and 9 samples identified adenocarcinoma ovarian tumors by the same method. Borderline ovarian tumor is a low malignant potential ovarian epithelial tumor type, hystologically classified as Serous and Mocinous. It presents some evidence of cellular proliferation with no signal of invasion behavior. However, it is not normal tissue. It is intermediate between benign tumor and adenocarcinoma. Normal ovarian surface epithelial cells were used as a reference cell line. The extracts were labeled, mixed, contacted with a monoclonal microarray, washed, and dried essentially per the methods set forth above.

After scanning, images were gridded and linked to a protein print list to allow for identification of differentially expressed proteins. Local background in each color channel was subtracted from the intensity value of each spot on the array. Normalization of antibody arrays was performed through dye swapping (according to Yang, YH 2002). Briefly, two slides were hybridized in the reverse color labeling (dye swap) Thus, sample A (reference) was labeled with Cy5 and sample B (query) was labeled with Cy3, then mixed and added to slide 1. In the reverse labeling, sample A, Iabeled-Cy3, and sample B, labeled- Cy5, were mixed and added to slide 2. The ratios Cy5/Cy3 was calculated for each of the spots in the array and a normalized ratio (NR) was calculated where: NR = V Ratio 1/Ratio2 (ratios 1 and 2 correspond to slides 1 and 2). NR is the value that represents the abundance of the protein A in the sample A relative to protein A of sample B. Spots were manually examined to assess their quality and those that exhibited poor quality and negative signal were excluded from analysis. Ratios were transformed in Log2(NR) and plotted in the MA plot (reference) where A = log 2(Cy3)/2 + log 2(Cy5)/2 and the log-ratios M = log 2(probed strain/VLlO), according to Baptista et al., 2006.

The t-test revealed differentially expressed proteins between two groups, adenocarcinoma group and Bot (borderline tumor) group. Differentially expressed probes were defined as those that presented a P- values <0.05. Seventy-seven (77) proteins were identified to be differentially expressed between borderline tumors and adenocarcinomas with a p value <0.05 (see Table 2). The dendogram analysis, clusters and p values were performed using the BRB array tools developed by the Biometric Research Branch of the US National Cancer Institute (http://linus.nci.nih.gov/BRB-ArrayTools.html). The list was sorted according to fold changes between two groups. Markers for borderline adenocarcinoma indicate a good prognosis for a patient and markers for high-grade adenocarcinoma indicate a poor prognosis.

Protein expression profiles for the borderline and adenocarcinoma ovarian tissues were compared, and dendogram analysis was performed for clustering experiments using centered correlation and average linkage. These data demonstrated that the protein profiles for two of the samples from the borderline group were indistinguishable from the adenocarcinoma group (Figure 7). These data demonstrate that it is possible to distinguish between borderline and disease tissue using the arrays and methods of the invention, and that the arrays and methods of the invention can be used for as both diagnostic and prognostic indicators, especially for ovarian cancers. Moreover, the microarrays and methods of the invention can be used for the identification of modulation of protein expression to identify genes associated with cancer or other disorders. Also, the changes in protein expression can be detected prior to histological changes.

Example 7: Analysis of Pathways Involved in Ovarian Borderline Tumors and Adenocarcinoma

The microarrays and methods of the invention were used to identify a large number of proteins associated with the disorders of borderline tumors and adenocarcinoma as compared to normal (i.e., non-diseased, normal ovarian surface epithelial cells) tissue. The proteins were characterized using the Ingenuity Pathways software (http://www.ingenuity.com/products/pathways_analysis.html), a web-based software for modeling and analysis of biological systems using a generated data set or a list of genes or generated data sets to propose interactions between proteins in the list. Results from the analysis using the proteins identified in the above example is shown in Figure 8. The proteins identified using the microarrays and methods of the invention are shaded in the VEGF and chemokine signal transduction pathways provided in Figures 9 and 10.

An improved understanding of proteins differentially regulated in cancer cells as compared to normal cells provides a basis for the development of improved therapeutics or selection of currently available therapeutics for a specific subject, and/or diagnostics for the treatment of specific cancers.

Claims

CLAIMS:

1. A microarray comprising a solid support with a plurality of monoclonal antibodies immobilized on the solid support at known predetermined positions.

2. The microarray of claim 1, wherein the plurality of monoclonal antibodies is comprised of the antibodies listed in Table 1.

3. The microarray of claim 1 or 2, wherein a plurality of antibodies comprises about 1000 to about 100,000 antibodies.

4. The microarray of any of claims 1 to 3, wherein a plurality of antibodies comprises about 1000 to about 10, 000 antibodies.

5. The microarray of any of claims 1 to 4, wherein a plurality of antibodies comprises about 5000 to about 10,000 antibodies.

6. The microarray of claim 1 or 2, comprising a plurality of monoclonal antibodies directed to proteins known to be associated with or differentially expressed in a particular disease or disorder.

7. The microarray of claim 6, wherein the particular disease or disorder is cancer.

8. The microarray of claim 6 or 7, wherein the particular disease or disorder is adenocarcinoma.

9. The microarray of any of claims 6 to 9, wherein the particular disease or disorder is ovarian adenocarcinoma.

10. The microarray of any of claims 6 to 9, wherein the array includes at least 10 antibodies targeted to at least 10 proteins selected from the group consisting of the antibodies targeted to proteins associated with differential expression in carcinoma.

11. The microarray of any of claims 6 to 9, wherein the array includes at least 20 antibodies targeted to at least 20 proteins selected from the group consisting of the antibodies targeted to proteins associated with differential expression in carcinoma

12. The microarray of any of claims 6 to 9, wherein the array includes at least 30 antibodies targeted to at least 30 proteins selected from the group consisting of the antibodies targeted to proteins associated with differential expression in carcinoma

13. The microarray of any of claims 6 to 9, wherein the array includes at least 40 antibodies targeted to at least 40 proteins selected from the group consisting of the antibodies targeted to proteins associated with differential expression in carcinoma

14. The microarray of any of claims 1 to 13, wherein said support is made of materials selected from the group consisting of nitrocellulose, nylon, polyvinylidene difluoride, glass, or plastics, and their derivatives.

15. A method of preparing an antibody monoclonal microarray comprising: preparing monoclonal antibody solutions at concentration of 1 —3 mg/ml in 10-20% glycerol; contacting the antibody solution with a nitrocellulose coated slide using an automated arrayer and solid pins at 60-80% humidity; and drying the slide at room temperature and humidity.

16. A monoclonal antibody array prepared by the method of claim 15.

17. A method for identifying a differentially expressed protein form an abnormal cell, said method comprising the steps of: obtaining an array of any of claims 1 to 14 or 16; preparing a first mixture containing extracted proteins from an abnormal cell and a second mixture of extracted proteins form a normal cell of corresponding type; labeling the first mixture of proteins form the abnormal cells with first detection marker and labeling the second mixture of extracted proteins from the corresponding normal cell with a different, second detection marker applying equal amounts of protein from both first and second mixtures to said solid support with immobilized antibodies and incubating under conditions to permit binding of said labeled proteins thereto; detecting the relative quantity of labeled proteins from the first mixture of abnormal cells to the labeled proteins from the second mixture of corresponding normal cells which bind to the antibodies at known, predetermined positions, wherein the detection label from the protein binding in greater quantity indicates differential expression.

18. The method of claim 17, wherein said support is made of materials selected from the group consisting of nitrocellulose, nylon, polyvinylidene difluoride, glass, or plastics, and their derivatives.

19. The method of claim 17 or 18, wherein said mixture is cell protein extract prepared from eukaryotic cells.

20. The method of any of claims 17 to 19, wherein the first mixture of extracted protein from an abnormal cell is obtained from a tumor cell and the second mixture of extracted protein from a normal cell is obtained from the same type of cell as the tumor cell.

21. The method of any of claims 17 to 20, wherein the first mixture of extracted protein from an abnormal cell is obtained from a drug treated cell and the second mixture of extracted protein from a normal cell is obtained from the same type of cell which is not drug treated.

22. The method of any of claims 17 to 21 , wherein the number of said plurality of antibodies immobilized on said solid support ranges from 100 to 100,000 different kinds.

23. The method of any of claims 17 to 22, wherein the number of said plurality of antibodies immobilized on said solid support ranges from 500 to 5,000 different kinds.

24. The method of any of claims 17 to 23, wherein the detected protein is a cytokine.

25. The method of any of claims 17 to 23, wherein the detected protein is a protein kinase.

26. The method of any of claims 17 to 23, wherein the detected protein is a growth factor receptor.

27. The method of any of claims 17 to 23, wherein the detected protein is a apoptotic protein.

28. A method of determining the prognosis of a patient diagnosed with adenocarcinoma comprising the detection and identification of a differentially expressed proteins associated with carcinoma.

29. The method of claim 28, wherein the differentially expressed protein is up-regulated.

30. The method of claim 28, wherein the differentially expressed protein is down-regulated.

31. A method of determining the prognosis of a patient diagnosed with adenocarcinoma comprising the detection and identification of a differentially expressed protein associated with carcinoma.

32. The method of claim 31, wherein the differentially expressed protein is up-regulated.

33. The method of claim 31 , wherein the differentially expressed protein is down-regulated.

34. A method of diagnosing a patient suspected of having adenocarcinoma comprising analysis of differential expression of at least 5 proteins associated with differential expression in adenocarcinoma are analyzed.

35. The method of claim 23, wherein at least 10 proteins associated with differential expression in adenocarcinoma are analyzed.

36. The method of claim 23, wherein at least 15 proteins associated with differential expression in adenocarcinoma are analyzed.

37. The method of claim 23, wherein at least 20 proteins associated with differential expression in adenocarcinoma are analyzed.

38. The method of claim 23, wherein at least 25 proteins associated with differential expression in adenocarcinoma are analyzed.

39. The method of claim 23, wherein at least 30 proteins associated with differential expression in adenocarcinoma are analyzed.

40. The method of claim 23, wherein at least 35 proteins associated with differential expression in adenocarcinoma are analyzed.

41. The method of any of claims 1-40 wherein the proteins associated with differential expression in carcinoma are selected from the group consisting of: CCL3, CCL5, CCL7, vascular endothelial growth factor (VEGF), interleukin (IL)- IA, IL-8, IL-18, PASLG, THBSl, CCR5, CEACAMl, TNFRSF8, MS4A1, DAF, IL-IRl, CD34, LAT, FPRl, H-RAS, AMPH, insulin like growth fractor receptor (IGFR) 1 , πCBKAP, mitogen activated protein kinase (MAPK)3 , MAPKl 4, HSPD 1 , BAGl, ACTAl, GSTPl, MYODl, TP53, TP73, TP53BP2, CDC2, Actin, Paxillin (PXN), GRB2, ERKl /2, MCP-3, RANTES, ρ38 MAPK, fibrinogen, HSP60, Dual specificity protein phosphatase 8 (DUSP8), serum albumin, Chromatin-specific transcription elongation factor 140 kDa subunit (FACTpl40), CD55, CD58, CD66, CDl Ia, CD25, CD20, CDl 1, CD41b, CD15s, CD30, CD34, CDwl31, CDC121a, KAP, Nup88, CD 195, CD221, Galectin, Ras GTPase-activating protein-binding protein 1 (G3BP), VAP33, Tat-SFl, MIP-I, Keratin 19, c-erb B2A, V9-TCR, IL-Ia, CAS, Cdkl/Cdc2, CLA-I, Annexin I, Annexin IV, p53 Ab-8, SRP54, CD178, DNA polymerase epsilon catalytic subunit A, Fl 1 receptor, Exportin-1, HPVl 6 early, cytokeratin, amphiphysin, fMLP receptor, inhibitor 2, GST-p, caspase 2, NKT, SNX2, TSP, Na/K ATPase subunit beta-2, DRBP76, p53 Ab-3, _P73 (AB-4), MAD2B, MCP-3, pl30 Cas, pgr Ab-2, DLPl, MUPPl, alpha actinin-1, and anti- cidea.