title: AFFIRM--a multiplexed immunoaffinity platform that combines recombinant antibody fragments and LC-SRM analysis.
abstract: Targeted measurements of low abundance proteins in complex mixtures are in high demand in many areas, not the least in clinical applications measuring biomarkers. We here present the novel platform AFFIRM (AFFInity sRM) that utilizes the power of antibody fragments (scFv) to efficiently enrich for target proteins from a complex background and the exquisite specificity of SRM-MS based detection. To demonstrate the ability of AFFIRM, three target proteins of interest were measured in a serum background in single-plexed and multiplexed experiments in a concentration range of 5-1000 ng/mL. Linear responses were demonstrated down to low ng/mL concentrations with high reproducibility. The platform allows for high throughput measurements in 96-well format, and all steps are amendable to automation and scale-up. We believe the use of recombinant antibody technology in combination with SRM MS analysis provides a powerful way to reach sensitivity, specificity, and reproducibility as well as the opportunity to build resources for fast on-demand implementation of novel assays.
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.forename: Fredrika
authors.initials: F
authors.lastname: Carlsson
authors.forename: Niclas
authors.initials: N
authors.lastname: Olsson
authors.forename: Christer
authors.initials: C
authors.lastname: Wingren
authors.forename: Mats
authors.initials: M
authors.lastname: Ohlin
authors.forename: Helena
authors.initials: H
authors.lastname: Persson
authors.forename: Sofia
authors.initials: S
authors.lastname: Waldemarson
mesh_terms: D000595:Amino Acid Sequence; D000915:Antibody Affinity / Q000276:immunology; D019313:BRCA1 Protein / Q000097:blood / Q000276:immunology / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D053539:Keratin-19 / Q000097:blood / Q000276:immunology / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D018396:Mucin-1 / Q000097:blood / Q000276:immunology / Q000378:metabolism; D010455:Peptides / Q000097:blood / Q000276:immunology / Q000378:metabolism; D020543:Proteome / Q000276:immunology / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D015203:Reproducibility of Results; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
chemical_list: D019313:BRCA1 Protein; D053539:Keratin-19; C060192:MUC1 protein, human; D018396:Mucin-1; D010455:Peptides; D020543:Proteome; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies
keywords: SRM; affinity proteomics; biomarker; scFv; serum