Robert J. Caiazzo

Microelectrical sensors as emerging platforms for protein biomarker detection in point-of-care diagnostics.

Current methods used to measure protein expression on microarrays, such as labeled fluorescent imaging, are not well suited for real-time, diagnostic measurements at the point of care. Studies have shown that microelectrical sensors utilizing silica nanowire, impedimetric, surface acoustic wave, magnetic nanoparticle and microantenna technologies have the potential to impact disease diagnosis by offering sensing characteristics that rival conventional sensing techniques. Their ability to transduce protein binding events into electrical signals may prove essential for the development of next-generation point-of-care devices for molecular diagnostics, where they could be easily integrated with microarray, microfluidic and telemetry technologies. However, common limitations associated with the microelectrical sensors, including problems with sensor fabrication and sensitivity, must first be resolved. This review describes governing technical concepts and provides examples demonstrating the use of various microelectrical sensors in the diagnosis of disease via protein biomarkers.

Protein microarrays as an application for disease biomarkers.

Protein microarrays are an increasingly powerful technology in the hunt for new and novel diagnostic and prognostic biomarkers. Lending credit to the highly established DNA microarray, protein microarrays are versatile tools that utilize a variety of formats to facilitate the discovery of new biomarkers and our understanding of disease pathways. The aims of this review are: to detail a variety of protein microarray technologies currently used, including forward‐phase technologies and reverse‐phase technologies useful in both the discovery and validation of candidate biomarkers; to explore the strengths and weaknesses of various proteomic microarray platforms; to explain how bioinformatics helps compare data between microarray data sets; and to discuss the downstream applications of such technologies as they relate to the development of a highly personalized approach to medicine.

A native antigen "reverse capture" microarray platform for autoantibody profiling of prostate cancer sera.

Cancer sera contain antibodies that react with autologous cellular antigens. Here, we report the use of a novel native antigen‐based platform, the “reverse capture” autoantibody microarray, for identification of autoantigens against which autoantibody expression may be used to differentiate between patients with prostate cancer and benign prostate hyperplasia (BPH). Serum samples were collected at our institution from patients with BPH and patients with prostate carcinoma with similar blood prostate‐specific antigen levels. IgG was purified from individual prostate cancer sera, differentially labeled with fluorescent dyes, and competitively hybridized against purified IgG from a group of well‐characterized BPH control patients on our reverse capture microarray platform. For each experiment, we performed a two‐slide dye‐swap. Using this platform, we identified 28 unique antigen‐autoantibody reactivities that have the potential to discriminate prostate cancer from BPH. These autoantigens, with p‐values ≤0.01, can be placed into the following general categories: protein kinases, cell‐cycle regulators, cancer‐growth factors, apoptosis mediators, and transcription factors. In addition, only 1 of the 28 autoantigens remained differentially targeted by autoantibodies in post‐surgical prostate cancer patients after a minimum 1‐year follow‐up. Autoantibody profiling using this platform may be a useful tool for differentiating between malignant and benign diseases of the prostate.

Autoantibody microarrays for biomarker discovery

Identification of autoantigens and the detection of autoantibody reactivity are useful in biomarker discovery and for explaining the role of important biochemical pathways in disease. Despite all of their potential advantages, the main challenge to working with autoantibodies is their sensitivity. Nevertheless, proteomics may hold the key to overcoming this limitation by providing the means to multiplex. Clearly, the ability to detect multiple autoantigens using a platform such as a high-density antigen microarray would improve sensitivity and specificity of detection for autoantibody profiling. The aims of this review are to: briefly describe the current status of antigen–autoantibody microarrays; provide examples of their use in biomarker discoveries; address current limitations; and provide examples and strategies to facilitate their implementation in the clinical setting.

Native Antigen Fractionation Protein Microarrays for Biomarker Discovery

In this protocol, we used the T24 human bladder cancer cell line as a source of native antigens to construct fractionated lysate microarrays. Subsequently, these microarrays were used to compare the autoantibody responses of individuals with interstitial cystitis/painful bladder syndrome (IC/PBS) to those of normal female controls. To accomplish this, T24 cells were lysed under nondenaturing conditions to obtain native antigens. These native antigens were then fractionated in 2D using a PF-2D liquid chromatography; the first dimension separated the proteins by their isoelectric points, and the second separated them according to hydrophobicity. The resulting protein fractions were printed onto nitrocellulose-coated glass slides (PATH slides) to create a set of fractionated lysate microarrays. To compare the autoantibody responses of IC/PBS patients with normal controls, the fractionated lysate arrays were competitively hybridized with fluorescently labeled IgG samples purified from both IC/PBS and control sera. This protocol presents a detailed description of the creation and use of native antigen fractionated lysate microarrays for autoantibody profiling.

Immunoregulomics : a serum autoantibody-based platform for transcription factor profiling.

Gene expression is regulated by a group of proteins known as transcription factors (TFs). By binding to specific DNA cis-elements, each TF contributes a different functional role in gene expression. Panomics has developed a TranSignal TF-TF Interaction Array, which enables the user to determine TF complexes of interest with multiple other TFs. The process works by immunoprecipitating cis-elements bound to native cell nuclear extract TFs using specific antibodies to the TFs, and hybridizing the corresponding cis-elements to a membrane array spotted with different consensus sequences. In this protocol, we adapt this technology to characterize and compare autoantibody reactivity to TFs between patients with and without disease. Using Panomics combination DNA/protein arrays with over 300 different cis-elements spotted on the membrane, we can monitor the differences in autoimmune-targeted regulatory TFs, a process we termed immunoregulomics. This method allows for a qualitative analysis of the interactions with some quantifiable data. The findings can then be verified with the use of gel-shift experiments. Autoantibodies; interactome microarrays; transcription factors.