Background: Many putative disease blood biomarkers discovered in genomic and proteomic studies await validation in large clinically annotated cohorts of patient samples. ELISA assays require large quantities of precious blood samples and are not high-throughput. The reverse phase protein microarray platform has been developed for the high-throughput quantification of protein levels in small amounts of clinical samples.
Results: In the present study we present the development of reverse-phase protein microarrays (RPPMs) for the measurement of clusterin, a mid-abundant blood biomarker. An experimental protocol was optimized for the printing of serum and plasma on RPPMs using epoxy coated microscope slides and a non-denaturing printing buffer. Using fluorescent-tagged secondary antibodies, we achieved the reproducible detection of clusterin in spotted serum and plasma and reached a limit of detection of 780 ng/mL. Validation studies using both spiked clusterin and clinical samples showed excellent correlations with ELISA measurements of clusterin.
Conclusion: Serum and plasma spotted in the reverse phase array format allow for reliable and reproducible high-throughput validation of a mid-abundant blood biomarker such as clusterin.
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| http://dx.doi.org/10.1186/1477-5956-7-15 | DOI Listing |
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