Enzyme-linked immunosorbent assay (ELISA)

Enzyme-Linked Immunosorbent Assay (ELISA):

            GPNMB protein levels within human plasma and CSF samples were measured with ELISA kits (R&D systems, https://www.rndsystems.com/products/human-osteoactivin-gpnmb-duoset-elisa_dy2550) according to manufacturer’s instructions (we did not modify from instructions). CSF and plasma samples were diluted by factors of 1 in 2 (CSF) and 1 in 30 (plasma) to obtain optical density measurements within the standard range. All samples were run in duplicates and absorbance at 450nm was determined by a microplate reader (Berthold Technologies, Tristar LB 941). Only duplicate samples with a coefficient of variation (CV) <25% were retained for analysis, and the average CV across all samples used was 3.3%. Moreover, replicate samples assayed by ELISA on different days, by different operators, across multiple freeze-thaw cycles, demonstrated excellent reproducibility (Pearson r=0.97). 

All samples are from our central biobank, whose protocols can be found here: https://sites.google.com/site/chenplotkinlab/biobank.