Peptide Microarrays on Coated Silicon Slides for Highly Sensitive Antibody Detection
Peptides, with their well-established chemistry and fully automated synthesis, provide an invaluable tool for the screening of protein ligands, for epitope mapping, and for antibody diagnostics on the microarray format.
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ntroduction Classical methods of protein analysis such as electrophoresis, ELISA, and liquid chromatography are generally time-consuming, labor-intensive, and lack high-throughput capacity (1). Peptide and protein microarrays, with their high-throughput ability and low consumption of reagents, open new opportunities for the study of molecular recognition events in basic biological assays as well as for the development of new diagnostic tools in clinical applications (2–4). Mahesh Uttamchandani and Shao Q. Yao (eds.), Small Molecule Microarrays: Methods and Protocols, Methods in Molecular Biology, vol. 669, DOI 10.1007/978-1-60761-845-4_12, © Springer Science+Business Media, LLC 2010
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Synthetic peptides have some very interesting features as capture ligands in microarray experiments: they are easy to synthesize and manipulate and are usually highly stable and inexpensive. More importantly, since peptide ligands can be modeled to act as a binding site for almost any target structure of the proteome (5), they can mimic biological activities of proteins and provide a straightforward approach in a variety of applications such as the measurement of enzymatic activities (6, 7), the identification of ligands that are active in cell adhesion (8), and the mapping of epitopes (9, 10). Peptide microarrays also provide efficient information in serodiagnosis, for example, in the detection of pathogen infections (11, 12) and for antibody diagnostics (13, 14). One of the fundamental characteristics of any analytical method is the smallest concentration that can be reliably measured. A high sensitivity is an intrinsically desirable property of any analytical technique as it sets the lower concentrations of analyte that can be distinguished from background noise. A lower assay detection limit allows one to use smaller amounts or more easily obtainable samples, such as capillary blood from newborns or saliva. Moreover, the ability to detect smaller amounts of analyte drastically reduces the influence of the matrix effect (when the combined effect of all the components of sample other than the analyte influences the quantitation) allowing one to dilute the sample. In general, highly sensitive assays open new opportunities in the diagnosis of a disease. Detection methods developed for microarrays, due to the miniaturized format, provide high sensitivity and high-throughput (15). The use of fluorescent probes and signal amplification techniques with chromogenic or fluorescent labels usually leads to performances that meet such criteria. However, the increased sensitivity in microarray experiments is still a challenge and different approaches to improve detection limits are currently under development. They include alternative labeling involving the use of DNA dendrimers (16), gold (17) or silver (18) nanoparticles, quantum dots (19) or signal amplification methods through the use of tyramide precipitation (20) and the rolling circle amplification (RCA) (21) even combined to nanoparticle-based optical detection (
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