A cell-based multiplex immunoassay platform using fluorescent protein-barcoded reporter cell lines.
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2021-11
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Abstract
Multiplex immunoassays with acellular antigens are well-established based on solid-phase platforms such as the Luminex® technology. Cell barcoding by amine-reactive fluorescent dyes enables analogous cell-based multiplex assays, but requires multiple labeling reactions and quality checks prior to every assay. Here we describe generation of stable, fluorescent protein-barcoded reporter cell lines suitable for multiplex screening of antibody to membrane proteins. The utility of this cell-based system, with the potential of a 256-plex cell panel, is demonstrated by flow cytometry deconvolution of barcoded cell panels expressing influenza A hemagglutinin trimers, or native human CCR2 or CCR5 multi-span proteins and their epitope-defining mutants. This platform will prove useful for characterizing immunity and discovering antibodies to membrane-associated proteins.
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Song, Shengli, Miriam Manook, Jean Kwun, Annette M Jackson, Stuart J Knechtle and Garnett Kelsoe (2021). A cell-based multiplex immunoassay platform using fluorescent protein-barcoded reporter cell lines. Communications biology, 4(1). p. 1338. 10.1038/s42003-021-02881-w Retrieved from https://hdl.handle.net/10161/26168.
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Scholars@Duke
Jean Kwun
Research interests include humoral tolerance to organ transplants in animal model and humans, developing a clinically relevant animal model to study the mechanisms of antibody-mediated rejection (AMR), and establishing a conceptual basis that will translate into therapeutic intervention of AMR.
Annette M Jackson
Stuart Johnston Knechtle
During my career as an academic surgeon, I have had the privilege of leading and/or participating in a diverse portfolio of hypothesis-driven research projects. These projects have centered on the immunology of surgery and transplantation, including both cellular and antibody-mediated immune responses. During my training I studied the response of hyper-sensitized recipients to allogeneic liver transplantation, and am currently studying means of reducing immunologic memory that might allow more successful transplantation in sensitized recipients. This immune response involves pathways of coagulation, antibody-mediated rejection, and cellular rejection and current work in my lab involves these three pathways. The other major focuses of my work have been co-stimulation blockade and immune cell depletion as approaches to immunologic unresponsiveness or tolerance. My research group has been involved in translational and clinical research to develop these mechanistic tools for the benefit of human organ transplant recipients.
Recent Publications
Knechtle SJ, Shaw JM, Hering BJ, Kraemer K, Madsen JC. Translational impact of NIH-funded nonhuman primate research in transplantation. Sci Transl Med. 2019 Jul 10;11(500). pii: eaau0143. Reprint | Full Text
Garnett H. Kelsoe
- Lymphocyte development and antigen-driven diversification of immunoglobulin and T cell antigen receptor genes.
2. The germinal center reaction and mechanisms for clonal selection and self - tolerance. The origins of autoimmunity.
3. Interaction of innate- and adaptive immunity and the role of inflammation in lymphoid organogenesis.
4. The role of secondary V(D)J gene rearrangment in lymphocyte development and malignancies.
5. Mathematical modeling of immune responses, DNA motifs, collaborations in bioinformatics.
6. Humoral immunity to influenza and HIV-1.
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