title: Development of Phage-Based Antibody Fragment Reagents for Affinity Enrichment of Bacterial Immunoglobulin G Binding Proteins.
abstract: 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.
authors.affiliation: Department of Immunotechnology, Lund University , Medicon Village (House 406), SE 223 81, Lund, Sweden.
authors.forename: Anna
authors.initials: A
authors.lastname: Säll
authors.affiliation: Department of Immunotechnology, Lund University , Medicon Village (House 406), SE 223 81, Lund, Sweden.
authors.forename: Kristoffer
authors.initials: K
authors.lastname: Sjöholm
authors.affiliation: Department of Immunotechnology, Lund University , Medicon Village (House 406), SE 223 81, Lund, Sweden.
authors.forename: Sofia
authors.initials: S
authors.lastname: Waldemarson
authors.affiliation: Division of Infection Medicine, Department of Clinical Sciences, Lund University , SE 221 00, Lund, Sweden.
authors.forename: Lotta
authors.initials: L
authors.lastname: Happonen
authors.affiliation: Division of Infection Medicine, Department of Clinical Sciences, Lund University , SE 221 00, Lund, Sweden.
authors.forename: Christofer
authors.initials: C
authors.lastname: Karlsson
authors.affiliation: Department of Immunotechnology, Lund University , Medicon Village (House 406), SE 223 81, Lund, Sweden.|Science for Life Laboratory, Royal Institute of Technology , 17121 Stockholm, Sweden.
authors.forename: Helena
authors.initials: H
authors.lastname: Persson
authors.affiliation: Division of Infection Medicine, Department of Clinical Sciences, Lund University , SE 221 00, Lund, Sweden.
authors.forename: Johan
authors.initials: J
authors.lastname: Malmström
mesh_terms: D000595:Amino Acid Sequence; D000915:Antibody Affinity; D000918:Antibody Specificity; D000942:Antigens, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D002853:Chromatography, Liquid; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D018604:Epitope Mapping; D015870:Gene Expression; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D008222:Lymphokines / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D019151:Peptide Library; D011485:Protein Binding; D011933:Reagent Kits, Diagnostic; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D013297:Streptococcus pyogenes / Q000737:chemistry / Q000276:immunology; D013491:Suppressor Factors, Immunologic / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D053719:Tandem Mass Spectrometry / Q000379:methods; D037521:Virulence Factors / Q000737:chemistry* / Q000235:genetics / Q000276:immunology
chemical_list: D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D007074:Immunoglobulin G; D008222:Lymphokines; D019151:Peptide Library; D011933:Reagent Kits, Diagnostic; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D013491:Suppressor Factors, Immunologic; D037521:Virulence Factors; C040330:histone-like protein HU, bacteria; C013059:immunoglobulin-binding factors; C014689:streptococcal M protein
keywords: Streptoccous pyogenes; affinity proteomics; in vivo biotinylation; mass spectrometry; scFv antibody fragments; selected reaction monitoring; virulence factors