Systematic assessment of antibody selectivity in plasma based on a resource of enrichment profiles

Abstract

There is a strong need for procedures that enable context and application dependent validation of antibodies. Here, we applied a magnetic bead assisted workflow and immunoprecipitation mass spectrometry (IP-MS/MS) to assess antibody selectivity for the detection of proteins in human plasma. A resource was built on 414 IP experiments using 157 antibodies (targeting 120 unique proteins) in assays with heat-treated or untreated EDTA plasma. For each protein we determined their antibody related degrees of enrichment using z-scores and their frequencies of identification across all IP assays. Out of 1,313 unique endogenous proteins, 426 proteins (33%) were detected in >20% of IPs, and these background components were mainly comprised of proteins from the complement system. For 45% (70/157) of the tested antibodies, the expected target proteins were enriched (z-score ≥ 3). Among these 70 antibodies, 59 (84%) co-enriched other proteins beside the intended target and mainly due to sequence homology or protein abundance. We also detected protein interactions in plasma, and for IGFBP2 confirmed these using several antibodies and sandwich immunoassays. The protein enrichment data with plasma provide a very useful and yet lacking resource for the assessment of antibody selectivity. Our insights will contribute to a more informed use of affinity reagents for plasma proteomics assays.

Sodium dodecyl sulfate decorated Legionella pneumophila for enhanced detection with a GaAs/AlGaAs nanoheterostructure biosensor

“…After removal of unbound SDS by washing, detection would be carried out in a PBS solution. Detailed research has yet to be carried out to verify the selectivity of the biosensor, as well as its sensitivity, reproducibility and reusability [65,66]. All of these parameters are the subject of intensive research carried out in our laboratory and they will be covered in future reports, but this exceeds the scope of the present manuscript.…”

An artificial neural network approach integrating plasma proteomics and genetic data identifies PLXNA4 as a new susceptibility locus for pulmonary embolism

“…This work started with the implementation of an ANN methodology on antibody based affinity proteomics data in relation to PE risk. This ANN was not developped as a tool to be used in clinic for predicting PE risk as 1/one is not 100% certain about the identity of the identified tagged proteins [31] (further experimental validation would be needed to assess this) and 2/ plasma protein levels determined with the antibody suspension bead array are not absolute but relative values depending on the current set of studied antibodies. Rather, our ANN based predictor for PE was aimed at serving as an intermediate surrogate biomarker that could generate new knowledge about the (genetics) mechanisms involved in PE.…”

Systematic Development of Sandwich Immunoassays for the Plasma Secretome

“… The assay specific controls included affinity purified rabbit IgG (P120‐301, Bethyl laboratories) in order to control for background binding to rabbit IgG molecules and a blocked bare bead (without coupled Ab) to monitor background binding to the beads. These two will from now on be referred to as “assay controls.” In addition, a set of ten monoclonal mouse Abs from BioSystems International, targeting plasma proteins commonly enriched by immuno‐capture assays were included . These will be referred to as “internal controls.” All Abs used are listed in (, Supporting Information).…”