Development of reverse phase protein microarrays for the validation of clusterin, a mid-abundant blood biomarker.
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.


CONCLUSIONS
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.
Aguilar-Mahecha
Adriana
A
Lady Davis Institute for Biomedical Research, Department of Oncology, Montréal, Québec, H3T 1E2, Canada.
Cantin
Christiane
C
Biotechnology Research Institute, National Research Council of Canada, Montréal, Québec, H4P 2R2, Canada.
O'Connor-McCourt
Maureen
M
Biotechnology Research Institute, National Research Council of Canada, Montréal, Québec, H4P 2R2, Canada.
Nantel
Andre
A
Biotechnology Research Institute, National Research Council of Canada, Montréal, Québec, H4P 2R2, Canada.
Basik
Mark
M
Lady Davis Institute for Biomedical Research, Department of Oncology, Montréal, Québec, H3T 1E2, Canada.