The Application of Protein Microarrays to Serum Diagnostics: Prostate Cancer as a Test Case

225 The application of protein microarrays to serum diagnostics: Prostate cancer as a test case Jeremy C. Millera , E. Brian Butlerb , Bin Sing Tehb and Brian B. Haab a,∗ a The Van Andel Research Institute, 333 Bostwick NE, Grand Rapids, MI 49503, USA b Baylor College of Medicine, Houston, TX 77030, USA 1. Introduction Reliable and specific serum disease markers have great value as non-invasive, rapid and inexpensive assays. The discovery of new disease markers is particularly necessary for diseases that are difficult to detect or diagnose at an early and curable stage. For example, the early detection of pancreatic cancer and the differentiation of malignant from benign disease are extremely difficult using current imaging and cytological methods. Improved screening tools would permit the avoidance of unnecessary pancreaticoduodenectomies and allow the opportunity to perform the procedure at a curative stage [1]. The challenge to the discovery of new serum markers lies in the difficulties of highthroughput detection and quantitation of proteins. A new tool that is potentially well suited to meet this challenge is the protein microarray. The feasibility of accurate, sensitive and specific protein microarray detection of multiple proteins in a serum background was recently demonstrated [2], and efforts are now underway to apply this technology to marker discovery. The technology as described by Haab et al. [2] was built upon the existing DNA microarray platforms that are present in many labs (see http://cmgm.stanford.edu/pbrown/), making the method practical and easy to implement. ∗ Corresponding author: Tel.: +1 616 234 5268; Fax: +1 616 234 5269; E-mail: . Disease Markers 17 (2001) 225–234 ISSN 0278-0240 / $8.00  2001, IOS Press. All rights reserved In addition, further availability of protein microarray technology is coming through the many commercial ventures that are actively working to get various types of protein chips to market. This article addresses the use of protein microarrays for serum marker detection and discovery, using prostate cancer as a model disease. We initially describe protein microarray technology and its suitability for serum analysis, then discuss the existing serum markers for prostate cancer and the potential advantages of using multiple markers, and finally describe serum protein studies using protein microarrays. 2. Protein microarray technology for highly-parallel serum protein detection The microarray format has many beneficial features for protein analysis, such as highly parallel detection, low sample consumption, and the potential for highly accurate and sensitive detection in multiple wavelength regions using scanning fluorescence microscopy, as recently demonstrated [2]. Certain aspects of the technology make it particularly well suited to the analysis and discovery of serum markers. For example, the ability to run many microarray experiments rapidly enables studies on the large populations of samples that are needed for good statistics on new markers. Additionally, the sophisticated software tools that are continually under development to analyze DNA microarray data also may be used to analyze protein microarray data. Many of these tools are specifically designed for the identification of genes or sets of genes that have diagnostic utility. It has been noted that new markers may be comprised of combinations of genes rather than individual genes [3]. Microarrays provide a highly effective tool to analyze the relationship between many genes to evaluate their combined value. Multiplexed protein detection using spotted antibodies and antigens has been demonstrated for a variety 226 J.C. Miller et al. / The application of protein microarrays to serum diagnostics: Prostate cancer as a test case of applications with diverse technological implementations. Protein arrays on poly(vinylidene fluoride) (PVDF) and nitrocellulose membranes have been used to screen binding specificities of a protein expression library [4–6] and to detect DNA, RNA, and protein binding targets [7]. Phage displayed antibodies were arrayed onto filters for high-throughput screening of their specificities [8]. Derivatized glass slides have been used to attach microarrays of antibodies and antigens for high-throughput ELISA [9], to detect autoantibodies [10], and to detect protein-protein [2] and proteinsmall molecule interactions [11]. Since the technology is relatively new, most of the published reports focus on feasibility studies and technological characterization rather than biological studies. Efforts are underway to apply the technology to biological studies and to address the issues necessary to make the method more robust and practical. The primary experimental challenge in obtaining useful protein microarray data is the acquisition of high specificity and high affinity protein capture reagents. The specificity and affinity of the capture reagents define the sensitivity and accuracy of the assay. Having many high quality capture reagents adds to usefulness of protein microarray data, but several aspects of protein chemistry make the collection of a such a set difficult. Unlike nucleic acids, for which binding interactions are well characterized and predictable, protein binding interactions must be identified empirically. Since protein-protein interactions have a wide variety in binding strengths, stabilities and specificities, finding a suitable binding partner to a particular protein may be difficult in some cases. Additionally, proteins are expensive and time-consuming to produce and purify. Several approaches have been put forth to address the generation of protein capture reagents for arrays. High-throughput protein expression and purification methods have been developed, based on recombinant baculoviruses [12] or the GatewayTM recombinant cloning system [13]. The proteins are produced in 96well microtiter plates and efficiently purified through the amino- or carboxyl-terminal attachment of an epitope tag, such as poly-histidine or Glu-Glu. An efficient method to test for proper protein expression and folding is based on the arraying of individual bacterial colonies of a cDNA library onto membranes [4]. The arrayed colonies were induced for protein expression, the cells were lysed on the membrane, and the proteins were tested for proper expression, folding, and antibody specificity by antibody staining. Highthroughput, rapid and less expensive antibody produc- tion for microarrays may be possible using phage display libraries [14]. Antibodies to specific antigens can be selected from a diverse library of antibodies displayed on the surface of phage clones, and after selection the selected clones can be amplified. The feasibility of multiplexed antigen detection using arrayed scFv phage display clones on membranes was recently shown [8]. There are other technological challenges in the development of practical protein microarrays. Because prote (...truncated)