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

Fredolini, ClaudiaKTH,Affinitets-proteomik,Science for Life Laboratory, SciLifeLab,KTH Royal Inst Technol, Dept Prot Sci, Div Affin Prote, Sci Life Lab, S-17121 Solna, Sweden (författare)

QC 20190624
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.

Byström, SannaKTH,Affinitets-proteomik,Science for Life Laboratory, SciLifeLab,KTH Royal Inst Technol, Dept Prot Sci, Div Affin Prote, Sci Life Lab, S-17121 Solna, Sweden(Swepub:kth)u184s19p (författare)
Sanchez-Rivera, LauraKTH,Affinitets-proteomik,Science for Life Laboratory, SciLifeLab,KTH Royal Inst Technol, Dept Prot Sci, Div Affin Prote, Sci Life Lab, S-17121 Solna, Sweden(Swepub:kth)u13bwsp9 (författare)
Ioannou, MarinaKTH,Affinitets-proteomik,Science for Life Laboratory, SciLifeLab,KTH Royal Inst Technol, Dept Prot Sci, Div Affin Prote, Sci Life Lab, S-17121 Solna, Sweden(Swepub:kth)u1kgg7p3 (författare)
Tamburro, DavideKarolinska Inst, Sci Life Lab, Dept Oncol Pathol, Canc Prote, S-17121 Solna, Sweden. (författare)
Pontén, FredrikUppsala universitet,Science for Life Laboratory, SciLifeLab,Klinisk och experimentell patologi(Swepub:uu)fredpont (författare)
Branca, Rui M.Karolinska Institutet (författare)
Nilsson, PeterKTH,Affinitets-proteomik,Science for Life Laboratory, SciLifeLab,KTH Royal Inst Technol, Dept Prot Sci, Div Affin Prote, Sci Life Lab, S-17121 Solna, Sweden(Swepub:kth)u1ws88sk (författare)
Lehtio, JanneKarolinska Institutet (författare)
Schwenk, Jochen M.KTH,Affinitets-proteomik,Science for Life Laboratory, SciLifeLab,KTH Royal Inst Technol, Dept Prot Sci, Div Affin Prote, Sci Life Lab, S-17121 Solna, Sweden(Swepub:kth)u1h7wtme (författare)
KTHAffinitets-proteomik (creator_code:org_t)

Av författaren/redakt...: Fredolini, Claud ...; Byström, Sanna; Sanchez-Rivera, ...; Ioannou, Marina; Tamburro, Davide; Pontén, Fredrik; visa fler...; Branca, Rui M.; Nilsson, Peter; Lehtio, Janne; Schwenk, Jochen ...; visa färre...

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Av lärosätet: Kungliga Tekniska Högskolan; Uppsala universitet; Karolinska Institutet

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