Originally Published IVD Technology July/August 2002
Laboratory Instrumentation
The role of protein-chip technology in molecular diagnosticsUsing protein biochips offers a number of alternatives and advantages in clinical diagnostics.
K. K. Jain
While technological innovations have enabled the analysis of genetic material in miniaturized test formats, the more delicate nature of protein structures has hindered the development of such devices for the analysis of proteins.
Nevertheless, protein microarrays making use of new developments in protein engineering and detection physics have recently begun to emerge. The basic construction of such protein chips has some similarities to DNA chips, such as the use of a glass or plastic surface dotted with an array of molecules. These molecules can be DNA or antibodies that are designed to capture proteins. The implementation of new surface chemistries allows the immobilization of defined quantities of proteins on each spot, while retaining some activity of the protein. With fluorescent markers or other methods of detection revealing the spots that have captured these proteins, protein microarrays are being used as powerful tools in high-throughput proteomics and drug discovery.
Currently, the major focus of protein-chip technology is understanding molecular pathways that in turn lead to useful information for drug discovery and molecular diagnostic applications. This article will focus on the role of protein biochips in the discovery of disease markers and diagnosis.
Protein Biochip Types and Makers
Glass slides are still widely used, since they are inexpensive and compatible with standard microarrayer and detection equipment. However, their limitations include multiple-based reactions, high evaporation rates, and possible cross-contamination.
Matrix slides offer a number of advantages, such as reduced evaporation and no possibility of cross-contamination, but they are expensive. Nanochips for proteomics have the same advantages, in addition to reduced cost and the capability of multiple-component reactions.
Some companies are also doing significant work in developing surface coatings, coupling agents, and instrument platforms. For example, PerkinElmer Inc. (Wellesley, MA) has introduced an integrated system for preparing protein microarrays.
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| Figure 1. An illustration of the surface-enhanced laser desorption and ionization (SELDI) process and ProteinChip array. The sample goes directly onto the array, and proteins are captured, retained, and purified directly on the chip (affinity capture). Retente Map is read by SELDI, and retained proteins can be processed directly on the chip. (click to enlarge) |
ProteinChip. The earliest and best-known protein chip is the ProteinChip by Ciphergen Biosystems Inc. (Fremont, CA). The first complete tool for disease-focused protein biology, the ProteinChip is based on the surface-enhanced laser desorption and ionization (SELDI) process (see Figure 1).
The ProteinChip system includes arrays and reagents consumed in the process, the chip reader, software to analyze results, and a proprietary database to enable comparisons between phenomic and genomic data. Known proteins are analyzed using functional assays that are on the chip. For example, chip surfaces can contain enzymes, receptor proteins, or antibodies that enable researchers to conduct protein-protein interaction studies, ligand binding studies, or immunoassays. With state-of-the-art ion optic and laser optic technologies, the ProteinChip system detects proteins ranging from small peptides of less than 1000 Da up to proteins of 300 kDa and calculates the mass based on time-of-flight (TOF).
New ProteinChip arrays have been packaged into a series of application-specific kits to enhance ease-of-use for the biologist who is conducting protein analyses. A SELDI ProteinChip interface and high-end quadruple TOF tandem mass spectrometers enable high-performance protein identification, epitope and phosphorylation mapping, and protein-interaction analyses. Such additions will complement both affinity liquid chromatography–mass spectrometry (LC-MS) and two-dimensional gel methods that are used in basic research and drug-discovery proteomics.
The ProteinChip benchtop system and the tandem MS system have several advantages over the two-dimensional gel method. These advantages include speed of detection (hours versus days), coverage of a broader region of the proteome, small sample requirement (1 ml or 500 cells), and combination of discovery and assay in a single system.
An example of the advantages that the ProteinChip offers can be seen in the discovery of prostate cancer biomarkers. With ProteinChip technology, it is possible to discriminate between benign prostatic hypertrophy with bound prostate-specific antigens (PSA) and cancer of the prostate with free PSA. In addition to having similar advantages over affinity LC-MS, the ProteinChip is a nonexpert, versatile benchtop system and is automated for high-throughput compatibility.
The ProteinChip biomarker system is the first protein biochip–based system that enables biomarker pattern recognition analysis to be done. This system allows researchers to ask and rapidly answer important clinical questions by investigating the proteome from a range of crude clinical samples (i.e., laser capture microdissected cells, biopsies, tissue, urine, and serum) and using ProteinChip array-based expression difference mapping and integrated SELDI-TOF-MS detection processes.
The system also utilizes biomarker pattern software that automates pattern recognition–based statistical analysis methods to correlate protein expression patterns from clinical samples with disease phenotypes. Studies conducted at several cancer research centers have shown that this approach offers significant clinical sensitivity and specificity in classifying disease phenotypes, leading to the potential for predictive diagnosis and early detection of diseases.1
The ProteinChip biomarker system can perform biomarker discovery in days and validation of large sample sets within weeks. Its advanced robotics system accessory automates sample processing, allowing hundreds of samples to be run per week and enabling a sufficient number of samples to be run, which provides high statistical confidence in comprehensive studies for marker discovery and validation. This system will facilitate the development of a clinical proteomics platform for research in medicine, toxicology, pharmaceuticals, and clinical diagnostics.
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| Figure 2. The LabChip technology by Caliper Technologies (Mountain View, CA) combined with the 2100 bioanalyzer by Agilent Technologies provides a single platform for performing DNA, RNA, protein, and cellular analyses. (click to enlarge) |
LabChip Technology. The LabChip technology by Caliper Technologies Corp. (Mountain View, CA) combined with the 2100 Bioanalyzer by Agilent Technologies (Palo Alto, CA) provides researchers with a convenient and efficient analytical platform for performing DNA, RNA, protein, and cellular analyses (see Figure 2). For example, the 2100 Bioanalyzer is used with LabChip kits for the high-resolution analysis of DNA samples, such as polymerase chain reaction (PCR) products and restriction digest fragments. The LabChip technology combines manufacturing methods from the biochip industry with expertise in fluid dynamics, biochemistry, and software and hardware engineering to develop miniature, integrated biochemical processing systems.
The Protein 200 LabChip kit contains chips and reagents for analyzing protein samples and can complete an analysis of 10 protein samples in less than half an hour, 5–10 times faster than traditional gel-based techniques. The LabChip format eliminates the laborious manual staining and destaining steps that are required with traditional sodium dodecyl sulfate polyacrylamide gel electrophoresis.
With the integration of these processes, this technology represents a step closer to a complete laboratory-on-a-chip. Compared with gels, the chip format also allows for real-time data acquisition and efficient sample comparison. This feature is especially important for proteins, as sample quality can vary widely. Automated software extracts size and protein purity from the real-time digital data, which is displayed both visually in a simulated gel image and in exportable report formats.
Trinectin Proteome Chip. The Trinectin proteome chip by Phylos Inc. (Lexington, MA) is based on the company's PROfusion technology, a process that is used to select peptides and proteins with desired properties. The fundamental advantage of PROfusion is its ability to facilitate in vitro covalent linkage of a protein (phenotype) to its encoding messenger RNA (genotype).
PROfusion can rapidly isolate large numbers of high-affinity binding proteins from in vitro libraries in excess of 1014 molecules, representing the repertoire of proteins that are naturally expressed in a given cell type or tissue source. From a library of such molecules, one can select a protein function with the benefit that the genetic material is linked to the protein for subsequent PCR amplification, enrichment, and, ultimately, identification.2 The Trinectin proteome chip will accelerate the drug discovery and development process and provide diagnostic information for drug development and patient management.
Microfluidic Chip-Based Immunoassays. Microfluidics is one of the most important innovations in biochip technology. Since microfluidic chips can be combined with mass spectrometric analysis, a microfluidic device has been devised in which an electrospray interface to a mass spectrometer is integrated with a capillary electrophoresis channel, an injector, and a protein digestion bed on a monolithic substrate.3 This chip thus provides a convenient platform for automated sample processing in proteomics applications.
These chips can also analyze expression levels of serum proteins with detection limits comparable to commercial enzyme-linked immunosorbent assays, with the advantage that the required volume sample is markedly lower compared with conventional technologies. While several technical problems associated with the use of nucleic acid chips outside the laboratory are still being worked out, microfluidic immunoassays appear likely to get to point-of-care first.
One drawback of the microscale electrophoresis systems is the electro-osmotic flow that is used to pump liquids from various ports through the micrometer-deep channels in order to inject and separate the components. Research done at Micralyne Inc. (Edmonton, AB, Canada) shows that peak height, shape, asymmetry, migration time, and baseline drift are all affected by minuscule deviations in the liquid levels of the wells on a chip. Since chip and channel design also contribute significantly to the deviations, this problem can be corrected by making sure all liquid levels on a chip and all parts of the chip itself are as uniform as possible.
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| Figure 3. The Triage cardiac panel by Biosite Inc. (San Diego) is a rapid, quantitative blood test designed to aid in the diagnosis of acute myocardial infarction. |
Biosite (San Diego) manufactures the Triage protein chip that simultaneously measures 100 different proteins by immunoassays (see Figure 3). The Triage protein chip immunoassays are performed in a microfluidic plastic chip, and the results are achieved in 15 minutes with picomolar sensitivities. Microfluidic fluid flow is controlled passively in the protein chip by the surface architecture and surface hydrophobicity in the microcapillaries. The immunoassays utilize high-affinity antibodies and a near-infrared fluorescent label, which is read by a portable, battery-powered fluorometer. The first chip to be commercialized by Biosite is the Triage Cardiac, which employs 10 antibodies arrayed in six zones. The test identifies three cardiac protein markers (myoglobin, troponin I, and creatinine kinase) that are released in the blood and rise during an acute myocardial infarction.
The microfluidic eTag assay system by Aclara Biosciences (Hayward, CA) contains eTag reporters that are fluorescent labels with unique and well-defined electrophoretic mobilities, and each label is coupled to biological or chemical probes via cleavable linkages (see Figure 4). When an eTag reporter– labeled probe binds to its target, the coupling linkage is cleaved, and the eTag is released. The distinct mobility address of each eTag reporter allows mixtures of these tags to be rapidly deconvoluted and quantitated by capillary electrophoresis. Since Aclara has synthesized eTag reporter libraries in spectrally distinct colors, it is possible to use both mobility and color to increase the degree of multiplexing, thus making it possible to perform concurrent gene expression, protein expression, and protein function analyses using the same sample. Multiplexed assays can be configured to monitor various types of molecular recognition events, such as protein-protein interactions and protein-small molecule binding. With these features, this technology is suited for automated, high-throughput applications in drug discovery.
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| Figure 4. eTag reporters are detectable molecules, typically fluorescent molecules, each with a unique and defined mobility that provides for their separation from one another. Each eTag group is coupled to a biological or chemical probe via a cleavable linkage to form an eTag probe. When an eTag probe binds to its target, the cleavable linkage is broken, releasing the corresponding eTag reporter. (click to enlarge) |
Tissue Microarray Technology. Tissue microarray technology provides a new high-throughput approach for linking genes and gene products with normal and disease tissues at the cellular level in a parallel fashion. Compared with classical in situ technologies in molecular pathology that are very time-consuming, tissue microarrays provide increased throughput in two ways: up to 1000 tissue specimens can be analyzed in a single experiment, either at the DNA, RNA, or protein level; and tens of thousands of replicate tissue microarrays can be generated from a set of tissues. This process provides a template for analyzing many more biomarkers than has ever been possible previously in a clinical setting, even using archival, formalin-fixed specimens. It is now in use at the National Human Genome Research Institute (Bethesda, MD).
With large-scale cDNA expression arrays, tissue microarray analysis also provides a rapid and powerful method to determine the prevalence and prognostic significance of novel candidate genes that are discovered to be involved in cancer development. The tissue array can be adapted as a routine tool in research laboratories for analyses of large tumor series at the DNA, RNA, or protein level. With such a tool, cancer researchers can study vast numbers of tumor samples in a short time and can generate a wealth of data on the application of tumor markers.
An example of the application of this approach is high-throughput tissue microarray analysis of cyclin E gene (CCNE) amplification and overexpression, which is a characteristic of a subset of bladder carcinomas, especially at the early invasion stage. An analysis of the prognostic impact of CCNE gene amplification and protein expression in more than 1500 arrayed bladder cancers was accomplished in a period of two weeks, illustrating how the tissue microarray technology facilitates the evaluation of the clinical relevance of molecular alterations in cancer.4
Nanoscale Protein Analysis. Most current protocols including protein purification and automated identification schemes yield unacceptably low recoveries, thus limiting the overall process in terms of sensitivity and speed, and requiring more starting material. Such low protein yields and proteins that can only be isolated from limited source material (e.g., biopsies) can be subjected to nanoscale protein analysis: a nanocapture of specific proteins and complexes, and optimization of all subsequent sample-handling steps, leading to a mass analysis of peptide fragments. This focused approach, also termed targeted proteomics, involves examining subsets of the proteome (e.g., those proteins that are specifically modified, bind to a particular DNA sequence, or exist as members of higher-order complexes or any combination thereof). This approach is used to identify genetic determinants of cancer that alter cellular physiology and respond to agonists.
A new detection technique called multiphoton detection, by Biotrace Inc. (Cincinnati), can quantify subzeptomole amounts of proteins and will be used for diagnostic proteomics, particularly for cytokines and other low-abundance proteins. Biotrace is also developing supersensitive protein biochips to detect concentrations of proteins as low as 5 fg/ml (0.2 attomole/ml), thereby permitting sensitivity that is 1000 times greater than current protein biochips.
Molecular Diagnostics and Antigen-Antibody Interactions. It is possible to design protein biochips that are based on specially constructed, small recombinant antibody fragments, using nanostructure surfaces with biocompatible characteristics and resulting in sensitive detection. The assay readout enables the determination of single or multiple antigen-antibody interactions.5 Brian Haab, a special program investigator at the Van Andel Institute (Grand Rapids, MI), has developed microarrays for highly parallel quantitation of multiple proteins and antigens in complex solutions.6 Protein and antibody solutions are robotically spotted onto the surface of derivatized microscope slides and probed with fluorescently labeled protein mixes. Specific binding interactions (e.g., antibody-antigen interactions) result in localizing specific individual components of the complex mixtures to the corresponding specific spots in the array.
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| Figure
5. LabMap by Luminex Corp. (Austin, TX) combines microsphere-based assays
with small lasers, advanced digital signal processors, and proprietary software. (click to enlarge) |
Capture-molecule arrays are excellent research tools as well as potential diagnostic tools to identify tumor marker proteins in cancer tissues or surrogate markers of diseases in patient serum. Antigen arrays can act as targets of the autoimmune response in arthritis and other autoimmune diseases and have been screened by microarray technologies. Protein array–based diagnostics can be used for the parallel detection of autoantibodies involved in human autoimmune diseases, such as thyroiditis, rheumatic diseases, and insulin-dependent diabetes mellitus. Protein array–based assays will significantly facilitate and accelerate autoimmune diagnostics and can be adapted easily to any other kind of immunoassay.
Antibodies are used extensively as diagnostic tools in a wide selection of arrays for different analyses. Monoclonal and recombinant antibodies provide a never-ending source of molecules and can produce endless possibilities for novel genetic constructs. Antibodies are still very much in vogue and are also being used in microarray analysis of the proteome using protein biochips.
Substrate choice is another major consideration for researchers conducting protein microarray experiments, such as antibody screening, protein-protein interaction studies, protein expression profiling, and immunoassays. In contrast to nucleic acids, proteins are especially sensitive and prone to inactivation by their environments, such as the glass and plastic surfaces on which they can be printed. Such inactivation can impair the structure and functionality of a protein, making research problematic. Aware of these pitfalls, Packard Biosciences, now part of PerkinElmer (Wellesley, MA), formulated and manufactured its HydroGel substrate for use in protein microarray applications. Once printed onto the HydroGel substrate, protein is captured within the substrate's three-dimensional structure and is accessible for binding reactions and suitable for a variety of assays.
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| Figure 6. A mixture of Qbead microparticles, 10-µm-diam beads encoded with quantum dots, from Quantum Dot Corp. (Hayward, CA). |
LabMap combines microsphere-based assays with small lasers, advanced digital signal processors, and proprietary software to offer greater speed, precision, and flexibility compared with current bioassay technologies. The Luminex100, which is based on this technology, is a sensitive, benchtop flow analyzer that is capable of performing 100 bioassays simultaneously. Its simple operation allows targeted tests to be conducted quickly and efficiently. The versatility of the technology also allows protein, RNA, or DNA testing to be performed on the same system, while its throughput capabilities can be applied to any size laboratory.
Diagnostic Applications
Several diagnostic applications of protein biochip technology are currently available and others are in development (see Table I).The most widely used proteomic tool is two-dimensional protein gel electrophoresis (2-DE), which can display protein expression patterns to a high degree of resolution. As an alternative to 2-DE, a preliminary study used a new technique to generate protein expression patterns from whole-tissue extracts. SELDI allowed the retention of proteins on a solid-phase chromatographic surface (or ProteinChip array) with direct detection of retained proteins by TOF-MS.
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Disease
biomarker discovery |
| Table I: Diagnostic applications of protein biochip technology. |
An innovative protein biochip immunoassay was used to quantitate and compare serum prostate-specific membrane antigen (PSMA) levels in healthy men and patients with either benign or malignant prostate disease.9 PSMA was captured from serum by an anti-PSMA antibody bound to ProteinChip arrays. The captured PSMA was detected by SELDI-MS and quantitated by comparing the mass signal integrals to a standard curve that was established using purified recombinant PSMA. Initial results suggested that serum PSMA may be a more effective biomarker than prostate-specific antigen for differentiating benign from malignant prostate disease, and warrants additional evaluation of the SELDI PSMA immunoassay to determine its diagnostic utility.
Approximately 40 companies are involved in protein biochip technology, and
a number of them actively participate in molecular diagnostics (see Table II)
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Company/Institute
|
Method
|
Applications
|
| Aclara (Mountain View, CA) | eTag reporter detection by microfluidic capillary electrophoresis. | Protein expression profiling |
| Adaptive Screening (Cambridge, UK) | Surrogate Proteome on a chip measures the complex binding relationships of compounds or proteins. | Prediction of adverse drug reactions |
| Biacore (Uppsala, Sweden) | Biomolecular Interaction Analysis (BIA) and Surface Plasmon Resonance (SPR). BIA-Mass Spectrometry (BIA-MS). | Immunodiagnostics, biomedical research and drug discovery |
| Biosite Diagnostics (San Diego, CA) | Microfluidics protein biochip with antibody arrays. | Diagnostic proteomics |
| BioTraces Inc. (Herndon, VA) | Multi Photon Detection permits quantitation of sub-zeptomole amounts of proteins. | Diagnostic proteomics |
| Bruker Daltonics (Billerica, MA) | MALDI targets are now available in patented automated AnchorChip technology. | SNP genotyping |
|
Cambridge Antibody Technology (Cambridge, UK) |
Protein biochip using antibody-based microarrays. | Research, diagnostics and drug discovery |
| Caliper/Agilent Technologies (Palo Alto, CA) LabChip | Lab-on-a-chip: a miniaturized and integrated liquid handling and biochemical processing device for computer-aided analytical laboratory procedures that can be performed automatically in seconds. | DNA amplification, automated nucleic acid analysis, genome sequencing, proteomics. |
| Ciphergen (San Diego, CA) | ProteinChip for monitoring of differential protein expression. Rapid and sensitive analysis of proteins directly from crude biological samples. | Discovery and characterization of disease marker proteins and drug target proteins |
| GenoSpectra Inc. (Fremont, CA) | High-density protein chips/fiber optics to address protein denaturation and poor binding kinetics. | Drug discovery and molecular diagnostics |
| Intrinsic Bioprobes (Tempe, AZ) | Biosensor chip mass spectrometry. | Diagnostic proteomics |
| Metrigenix Inc (Gaithersburg, MD) | Flow-through chip technology/ 4-D assay system. | Cardiovascular diagnosis and drug discovery |
| NextGen Sciences (Cambridgeshire, UK) | Microfluidic multiprotein biochip: a novel method of protein attachment by protein-protein interaction. | Breast
cancer protein biochip in development |
| Phylos Inc. (Lexington, MA) | Trinectin Proteome chip: based on automated protein selection platform (PROfusion) for production of high-affinity capture proteins. | A protein biochip for high-throughput applications |
| Protiveris (Rockville, MD) | A high-density, disposable protein chip that has the ability to detect and quantify both known and unknown proteins. | Drug discovery and molecular diagnostics |
| Quantum Dot Corp. (Hayward, CA) | QBEAD system, using microspheres that are bar coded with different colors of quantum dots. | Proteomics and gene expression. |
| SomaLogic (Boulder, CO) | PhotoSELEX process: aptamer arrays enable a large number of proteins—ultimately tens of thousands—to be measured simultaneously. | To identify protein signatures associated with disease and likely response to therapeutics |
| SurroMed (Mountain View, CA) | Detection of proteins captured on nanoparticles. | To improve 2-D microarrays for protein profiling |
| Zyomyx Inc. (Hayward, CA) | High-density
protein microarrays for quantification of proteins. Detection limits are
equal to or lower than commercial ELISA tests and reduce the sample volume.
Immobilization of exactly defined quantities of proteins on each spot while
retaining the full activity of the protein. |
Multiplexed immunoassay to analyze expression levels of multiple serum proteins
in complex mixtures |
Advantages and Limitations
DNA biochips are the most exciting genomic tools that have been developed within the last few years. However, it is evident that knowledge of the gene sequence or the quantity of gene expression is not sufficient to predict the biological nature and function of a protein. This factor can be particularly important in cancer research, where posttranslational modifications of a protein can specifically contribute to the disease. To address this problem, several proteomic tools have been developed.
However, techniques to enable efficient and highly parallel identification, measurement, and analysis of proteins remain a bottleneck. A platform technology that makes collection and analysis of proteomic data as accessible as genomic data has yet to be developed. Sensitive and highly parallel technologies analogous to the nucleic acid biochip, for example, do not exist for protein analysis.
Conclusion
Protein array technology allows high-throughput screening for gene expression and molecular interactions. Protein arrays appear as new and versatile tools in functional genomics, enabling the translation of gene expression patterns of normal and diseased tissues into protein product catalog. Protein function, such as enzyme activity, antibody specificity, and other ligand–receptor interactions and binding of nucleic acids or small molecules can be analyzed on a whole-genome level.
As the array technology develops, an ever-increasing variety of formats become available (e.g., nanoplates, patterned arrays, three-dimensional pads, flat-surface spot arrays, microfluidic chips), and proteins can be arrayed onto different surfaces (e.g., membrane filters, polystyrene film, glass, silane, gold). Various techniques are being developed for producing arrays, and robot-controlled, pin-based, or ink-jet printing heads are the preferred tools for manufacturing protein arrays. CCD cameras or laser scanners are used for signal detection; atomic force microscopy and mass spectrometry are upcoming options. The emerging future array systems will be used for high-throughput functional annotation of gene products. In addition, their involvements in molecular pathways and their response to medical treatment will become the doctor's indispensable diagnostic tools.
Of all the applications for protein microarrays, molecular diagnostics is most clinically relevant, and would fit in with the emerging trend in individualized treatment, or personalized medicine. Different proteins such as antibodies, antigens, and enzymes can be immobilized within protein microchips. Miniaturized and highly parallel immunoassays will greatly improve efficiency by increasing the amount of information acquired with single examination and will reduce cost by decreasing reagent consumption. Application of protein biochips and microarrays in molecular diagnostics has good commercial prospects.10
References
1. HJ Issaq et al., "The SELDI-TOF MS Approach to Proteomics: Protein Profiling and Biomarker Identification," Biochemical and Biophysical Research Communications 292 (2002): 587–592.
2. BL Kreider, "PROfusion: Genetically Tagged Proteins for Functional Proteomics and Beyond," Medical Care Research and Review 20 (2000): 212–215.
3. C Wang et al., "Integration of Immobilized Trypsin Bead Beds for Protein Digestion within a Microfluidic Chip Incorporating Capillary Electrophoresis Separations and an Electrospray Mass Spectrometry Interface," Rapid Communication in Mass Spectrometry 14 (2000): 1377–1383.
4. J Richter et al., "High-Throughput Tissue Microarray Analysis of Cyclin E Gene Amplification and Overexpression in Urinary Bladder Cancer," American Journal of Pathology 157 (2000): 787–794.
5. CA Borrebaeck et al., "Protein Chips Based on Recombinant Antibody Fragments: A Highly Sensitive Approach as Detected by Mass Spectrometry," Biotechniques 30 (2001): 1126– 1132.
6. BB Haab, MJ Dunham, and PO Brown, "Protein Microarrays for Highly Parallel Detection and Quantitation of Specific Proteins and Antibodies in Complex Solutions," in Genome Biology 2: [on-line] 2001: [cited 25 June 2002]; available from Internet: http://genomebiology.com.
7. P Arenkov et al., "Protein Microchips: Use for Immunoassay and Enzymatic Reactions," Analytical Biochemistry 278 (2000): 123– 131.
8. F von Eggeling et al., "Mass Spectrometry Meets Chip Technology: A New Proteomic Tool in Cancer Research?" Electrophoresis 22 (2001): 2898–2902.
9. Z Xiao et al., "Quantitation of Serum Prostate-Specific Membrane Antigen by a Novel Protein Biochip Immunoassay Discriminates Benign from Malignant Prostate Disease," Cancer Research 61 (2001): 6029–6033.
10. KK Jain, Proteomics: Technologies, Markets and Companies, 8th ed., (Basel, Switzerland: Jain PharmaBiotech, 2002).
K. K. Jain, MD, is the chief executive officer at Jain PharmaBiotech (Basel, Switzerland). He can reached via e-mail at jain@pharmabiotech.ch.
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