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Development of phage-based antibody fragment reagents for affinity enrichment of bacterial immunoglobulin G binding proteins
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2015 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 14, no 11, 4704-4713 p.Article in journal (Refereed) PublishedText
Abstract [en]

Disease and death caused by bacterial infections are global health problems. Effective bacterial strategies are required to promote survival and proliferation within a human host, and it is important to explore how this adaption occurs. However, the detection and quantification of bacterial virulence factors in complex biological samples are technically demanding challenges. These can be addressed by combining targeted affinity enrichment of antibodies with the sensitivity of liquid chromatography-selected reaction monitoring mass spectrometry (LC-SRM MS). However, many virulence factors have evolved properties that make specific detection by conventional antibodies difficult. We here present an antibody format that is particularly well suited for detection and analysis of immunoglobulin G (IgG)-binding virulence factors. As proof of concept, we have generated single chain fragment variable (scFv) antibodies that specifically target the IgG-binding surface proteins M1 and H of Streptococcus pyogenes. The binding ability of the developed scFv is demonstrated against both recombinant soluble protein M1 and H as well as the intact surface proteins on a wild-type S. pyogenes strain. Additionally, the capacity of the developed scFv antibodies to enrich their target proteins from both simple and complex backgrounds, thereby allowing for detection and quantification with LC-SRM MS, was demonstrated. We have established a workflow that allows for affinity enrichment of bacterial virulence factors.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2015. Vol. 14, no 11, 4704-4713 p.
Keyword [en]
affinity proteomics, in vivo biotinylation, mass spectrometry, scFv antibody fragments, selected reaction monitoring, Streptoccous pyogenes, virulence factors, immunoglobulin G, membrane protein, single chain fragment variable antibody, surface protein h, surface protein m1, unclassified drug, antibody affinity, antigen binding, Article, bacterial strain, bacterial virulence, biotinylation, in vivo study, liquid chromatography, nonhuman, phage display, priority journal, protein binding, protein targeting, selected reaction monitoring mass spectrometry, Streptococcus pyogenes, wild type
National Category
Biochemistry and Molecular Biology
URN: urn:nbn:se:kth:diva-181233DOI: 10.1021/acs.jproteome.5b00585ISI: 000364435100023ScopusID: 2-s2.0-84946867028OAI: diva2:903555

Funding Details: 621-2012-3559, Svenska Forskningsrådet Formas

QC 20160216

Available from: 2016-02-16 Created: 2016-01-29 Last updated: 2016-02-16Bibliographically approved

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Persson, Helena
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Protein TechnologyScience for Life Laboratory, SciLifeLab
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