Depletion efficiency and recovery of trace markers from a multiparameter immunodepletion column

The selective removal of high-abundance proteins is considered to be an important prerequisite for a sensitive proteome analysis in plasma. In this study, we examined the "multiaffinity removal system", an immunoaffinity depletion column targeted against six plasma proteins. As determined by sandwich ELISA, the depletion rate for each target protein is >99% over 200 cycles of regeneration. Our data give evidence that two column antibodies are slowly inactivated during the repeated use of the column; however, the individual depletion rate meets the specification of the manufacturer. To estimate a potential loss of analytes after the immunodepletion, we performed spiking/recovery experiments with a selection of tumor markers at concentrations in the lower to medium ng/mL range. The average recovery of 9 out of 11 markers is 78%. A significant proportion of two other markers binds to the column. Based on the average marker recovery and a depletion of ;85% of the total protein we estimate a five-fold enrichment of a potential biomarker by the use of this depletion column. We conclude that the selective depletion of plasma proteins by immunoaffinity chromatography is a valid strategy for the enrichment of potential biomarkers sought by proteomics methodologies.