Abstract
BackgroundMany 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.ResultsIn 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.ConclusionSerum 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.
Highlights
Many putative disease blood biomarkers discovered in genomic and proteomic studies await validation in large clinically annotated cohorts of patient samples
In the present study we describe the development of a protocol to print serum and plasma on Reverse-phase protein microarray (RPPM) for the measurement of clusterin
The first subarray was composed of all test samples prepared in the Urea buffer spotted in triplicate, and the second subarray comprised samples prepared in protein printing buffer (PPB) spotted in triplicate
Summary
Many putative disease blood biomarkers discovered in genomic and proteomic studies await validation in large clinically annotated cohorts of patient samples. The increasing application of genomics and proteomics technologies in medical research is making possible the development of "personalized medicine", i.e. medical care characterized by the use of biomarkers for the molecular diagnosis of different disease states and for the selection of therapies tailored to the individual's disease Both tissue and blood biomarkers are being discovered, blood derived-biomarkers are attractive in the clinic since blood collection is inexpensive and relatively non-invasive and blood comes in contact with all tissues in the body. Slides containing hundreds or thousands of antibodies are incubated with the sample of interest (e.g., cell lysate or serum) allowing the simultaneous screening of a large number of putative protein biomarkers in a single test sample [1]. Cell and tissue lysates as well as biological fluids such as urine, CSF, serum and plasma can be spotted onto such arrays Slides containing these sample sets are incubated with antibodies targeted against a single protein of interest, one marker per slide. Reverse phase protein microarrays enable the high-throughput screening of thousands of clinical samples in a single microarray experiment [3]
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