Abstract
There is a great need for targeted protein assays with the capacity of sensitive measurements in complex samples such as plasma or serum, not the least for clinical purposes. Proteomics keeps generating hundreds of biomarker candidates that need to be transferred towards true clinical application through targeted verification studies and towards clinically applicable analysis formats. The immunoaffinity assay AFFIRM (AFFInity sRM) combines the sensitivity of recombinant single chain antibodies (scFv) for targeted protein enrichment with a specific mass spectrometry readout through selected reaction monitoring (SRM) in an automated workflow. Here we demonstrate a 100 times improved detection capacity of the assay down to pg/ml range through the use of oriented antibody immobilization to magnetic beads. This was achieved using biotin-tagged scFv coupled to streptavidin coated magnetic beads, or utilizing the FLAG tag for coupling to anti-FLAG antibody coated magnetic beads. An improved multiplexing capacity with an 11-plex setup was also demonstrated compared to a previous 3-plex setup, which is of great importance for the analysis of panels of biomarker targets.
Highlights
The human proteome is greatly affected during disease and is a rich source of potential protein biomarkers for disease diagnostics
The affinity selected reaction monitoring (SRM) platform AFFIRM allows for capture and detection of low abundant target proteins in complex samples such as serum [11]
The platform makes use of recombinant scFv antibodies coupled to magnetic beads for enrichment of target proteins from the complex mixture
Summary
The human proteome is greatly affected during disease and is a rich source of potential protein biomarkers for disease diagnostics. A non-covalent coupling allows for antigen capture in-solution allowing free scFv and target protein to first interact before addition of the magnetic beads. This potentially provides a more efficient antibody-antigen interaction by alleviating the negative effects associated with a random covalent coupling approach [23]. We have previously developed the AFFIRM platform and demonstrated that immobilized scFv can be used for targeted enrichment of low abundant proteins from complex mixtures such as serum followed by targeted SRM analysis for a specific readout of target peptides in single- and multiplexed assays [5,9,10,11]. The streptavidin-biotin system was used for evaluating the performance of antigen capture in-solution, allowing the scFv-antigen binding to take place in-solution before adding magnetic beads for antigen isolation
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
