Detection and quantitation of twenty-seven cytokines, chemokines and growth factors pre- and post-high abundance protein depletion in human plasma

AbstractCytokines, chemokines and growth factors (CCGFs) in human plasma are analyzed for identification of biomarkers. However concentrations of CCGFs are very low; it is difficult to identify and quantify low abundance proteins in the presence of the high abundance proteins (HAPs) unless HAPs are removed prior to analysis. However, there is a concern that the low abundance proteins such as CCGFs may also be removed during the HAP depletion process. In this study, we have examined whether or not depletion of the HAPs enhances detection of the CCGFs by immuno-assays. Top 14 HAPs were depleted from 10 healthy volunteers’ plasma using MARS-14 immuno-depletion column and a total of 27 CCGFs were analyzed by bead-based multiplexed immuno-assay. All 27 CCGFs were detected in neat plasma (NP), 25 were detected in flow through fraction (FT) and 21 were detected in bound protein (BP) fraction. Concentrations of 22 CCGFs were significantly higher in NP compared to FT and BP. Only one CCGF had higher concentration in FT compared to NP. The remaining 2 CCGFs were not different between NP and FT. It was counter-productive for the detection of 24 CCGFs after HAP removal, primarily due to post-depletion protein precipitation and/or re-suspension of pellets