Advancing the immunoaffinity platform AFFIRM to targeted measurements of proteins in serum in the pg/ml range
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 th...
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| Veröffentlicht in: | PloS one 2018-02, Vol.13 (2), p.e0189116-e0189116 |
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| creator | Säll, Anna Corbee, Daniel Vikström, Sara Ottosson, Filip Persson, Helena Waldemarson, Sofia |
| description | 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. |
| doi_str_mv | 10.1371/journal.pone.0189116 |
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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. 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| subjects | Antibodies Antigens Assaying Basic Medicine Beads Bioassay Biology and Life Sciences Biomarkers Biotin Cellular proteins Engineering and Technology Immobilization Immune response Immunoassay Immunoglobulins Immunologi inom det medicinska området Immunology in the medical area Innovations Libraries Mass spectrometry Mass spectroscopy Medical and Health Sciences Medical Engineering Medical Laboratory and Measurements Technologies Medicin och hälsovetenskap Medicinsk laboratorie- och mätteknik Medicinska och farmaceutiska grundvetenskaper Medicinteknik Multiplexing Physical Sciences Plasma Proteins Proteomics Research and Analysis Methods Scientific imaging Sensitivity analysis Streptavidin Teknik Testing Workflow |
| title | Advancing the immunoaffinity platform AFFIRM to targeted measurements of proteins in serum in the pg/ml range |
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