Detection and Quantification of Protein Biomarkers from Fewer than 10 Cells
Open Access
- 1 May 2006
- journal article
- Published by Elsevier BV in Molecular & Cellular Proteomics
- Vol. 5 (5), 895-901
- https://doi.org/10.1074/mcp.m500350-mcp200
Abstract
The use of antibody microarrays continues to grow rapidly due to the recent advances in proteomics and automation and the opportunity this combination creates for high throughput multiplexed analysis of protein biomarkers. However, a primary limitation of this technology is the lack of PCR-like amplification methods for proteins. Therefore, to realize the full potential of array-based protein biomarker screening it is necessary to construct assays that can detect and quantify protein biomarkers with very high sensitivity, in the femtomolar range, and from limited sample quantities. We describe here the construction of ultramicroarrays, combining the advantages of microarraying including multiplexing capabilities, higher throughput, and cost savings with the ability to screen very small sample volumes. Antibody ultramicroarrays for the detection of interleukin-6 and prostate-specific antigen (PSA), a widely used biomarker for prostate cancer screening, were constructed. These ultramicroarrays were found to have a high specificity and sensitivity with detection levels using purified proteins in the attomole range. Using these ultramicroarrays, we were able to detect PSA secreted from 100 LNCaP cells in 3 h and from just four LNCaP cells in 24 h. Cellular PSA could also be detected from the lysate of an average of just six cells. This strategy should enable proteomic analysis of materials that are available in very limited quantities such as those collected by laser capture microdissection, neonatal biopsy microspecimens, and forensic samples.Keywords
This publication has 27 references indexed in Scilit:
- Biomarkers in Cancer Staging, Prognosis and Treatment SelectionNature Reviews Cancer, 2005
- Are multiple markers the future of prostate cancer diagnostics?Clinical Biochemistry, 2004
- Multiplexed sandwich assays in microarray formatJournal of Immunological Methods, 2004
- Profiling receptor tyrosine kinase activation by using Ab microarraysProceedings of the National Academy of Sciences of the United States of America, 2003
- Protein microarrays: Meeting analytical challenges for clinical applicationsCancer Cell, 2003
- Array-Based Multiplexed Screening and Quantitation of Human Cytokines and ChemokinesJournal of Proteome Research, 2002
- Multiplexed protein profiling on microarrays by rolling-circle amplificationNature Biotechnology, 2002
- Global Analysis of Protein Activities Using Proteome ChipsScience, 2001
- Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutionsGenome Biology, 2001
- Light-Scattering Submicroscopic Particles as Highly Fluorescent Analogs and Their Use as Tracer Labels in Clinical and Biological ApplicationsAnalytical Biochemistry, 1998