Searchlight chemiluminescent protein array platform

Serum were taken from patients before and after CHC treatment, and stored at −80°C till analysis. Serum OPN was measured using the commercially available OPN ELISA kit (R&D; DY1433) and in accordance with the manufacturer’s instructions. All samples were run in duplicate, and expressed as pg/ml.
Selected biomarkers were assayed without access to clinical or demographic data using a mulitiplex ultrasensitive SearchLight Chemiluminescent Protein Array platform (Pierce Biotechnology, c/o Thermo Fisher Scientific Inc., Rockford, IL). Briefly, this array includes a 96 well-plate allowing upto16 capture antibodies per well. Addition of ≤10 μL of serum to the well results in capture of the arrayed antibody. This is followed by addition of biotinylated antibodies that bind to captured antibodies, streptavidin conjugated to horseradish peroxidase, and a chemiluminescent substrate. Signal intensity is captured by SearchLight Plus CCD Imaging and analyzed through the SearchLight Array Analyst software for standard curve comparisons and custom data reporting. Rapid throughput was facilitated by using Tecan Genesis (Tecan group Inc, RTP, NC), Caliper Mini-Staccato (Caliper Life Sciences, Hopkinton, MA) and rapid plate transfer pre-analytical automated sample processing.

Candidate biomarkers selected for evaluation in this study were in part based on expert opinions from an International Fibrosis Group Meeting^{16 (link)}, and availability on the SearchLight assay platform (Supplementary Table 1). Selected biomarkers were assayed by laboratory staff without access to clinical or demographic data using a mulitiplex ultrasensitive SearchLight Chemiluminescent Protein Array platform (Pierce Biotechnology, c/o Thermo Fisher Scientific Inc., Rockford, IL) (see supplementary data for methodology). All serum samples were also independently evaluated for the fibrosis marker panel HCV FibroSURE (LabCorp, Burlington, NC).