High-throughput screening of hybridoma supernatants using multiplexed fluorescent cell barcoding on live cells

摘要

Abstract With current available assay formats using either immobilized protein (ELISA, enzyme-linked immunosorbent assay) or immunostaining of fixed cells for primary monoclonal antibody (mAb) screening, researchers often fail to identify and characterize antibodies that recognize the native conformation of cell-surface antigens. Therefore, screening using live cells has become an integral and important step contributing to the successful identification of therapeutic antibody candidates. Thus the need for developing high-throughput screening (HTS) technologies using live cells has become a major priority for therapeutic mAb discovery and development. We have developed a novel technique called Multiplexed Fluorescent Cell Barcoding (MFCB), a flow cytometry-based method based upon the Fluorescent Cell Barcoding (FCB) technique and the Luminex fluorescent bead array system, but is applicable to high-through mAb screens on live cells. Using this technique in our system, we can simultaneously identify or characterize the antibody-antigen binding of up to nine unique fluorescent labeled cell populations in the time that it would normally take to process a single population. This has significantly reduced the amount of time needed for the identification of potential lead candidates. This new technology enables investigators to conduct large-scale primary hybridoma screens using flow cytometry. This in turn has allowed us to screen antibodies more efficiently than before and streamline identification and characterization of lead molecules. Highlights ? A multiplexed fluorescent cell barcoding (MFCB) technique has been developed to enable HTS antibody screening on live cells ? The MFCB technique relies on dye combinations at varying concentrations to develop spectrally distinct barcoded populations ? Data is highly reproducible and accurate comparing multiplexed vs single population screens ? MFCB can be applied to primary screening and binding characterization to assess binding profiles of large antibody panels