Utilizing a resource of enrichment profiles in plasma for the systematic assessment of antibody selectivity

There is a strong need for procedures that enable context and application dependent validation of antibodies. Here we describe the foundation for a resource aiding more detailed assessment antibody selectivity for capturing endogenous proteins from human plasma. In 414 immunoprecipitation (IP) experiments with EDTA plasma, data was generated by mass spectrometry (LC-MS) with 157 antibodies (targeting 120 unique proteins). Out of a total of 1,313 unique proteins, 426 proteins (33%) were detected in > 20% of the assays and indicate a background comprised of mainly proteins from the complement system. For all proteins identified either in heat-treated or untreated EDTA plasma, frequencies of occurrence were derived. We determined z-scores for each IP as a measure of enrichment to annotate the antibodies into four categories (ON-target, CO-target, OFF-target and NO-target). For 45% (70/157) of the tested antibodies, the expected target proteins were enriched (z-score ≥3) above background. There were 84% (59/70) of binders that co-enriched other proteins beside the intended target, either due to OFF-target binding or predicted interactions. Comparing several antibodies raised against IGFBP2, the established library allowed us to describe protein complexes in plasma, and we employed multiplexed sandwich immunoassays to confirm these. In summary, the generated resource of plasma enrichment profiles and background proteins adds a very useful and yet lacking starting point for the assessment of antibody selectivity in this clinically important body fluid. The provided insights will contribute to a more informed use of validated affinity reagents and may lead to further advancements of plasma proteomics assays. * ### ABBREVIATIONS HPA : Human Protein Atlas LFQ : label free quantification PrEST : Protein Epitope Signature Tags Z : z-score f : frequency of occurrence