Abstract
Abstract Systemic lupus erythematosus (SLE) is a clinically heterogeneous disease characterized by a large number of aberrations in the immune system. Studies with SLE patients have shown that heightened levels of the cytokine interferon-α (IFNα) can interfere with immune regulation and effect lupus pathogenesis. The plasmacytoid dendritic cells (pDCs) are the main producer of type I IFNs and have the capacity to synthesize up to 109 IFNα molecules per cell after activation. Crosslinking BDCA2, a typeII C-type lectin specific to pDCs, results in the inhibition of IFNα production in pDCs stimulated with TLR7/9. To develop an agonistic therapeutic antibody to BDCA2, a large number of antibody candidates have to be screened in a functional assay that detects blocking of IFNα production in pDCs isolated from peripheral blood mononuclear cells (PBMC). pDCs constitute less than 0.4% of PBMCs making pDC purification inefficient and challenging. Here we report an automated high-throughput BDCA2-agonistic functional assay using fraction 5 (F5) cells from an elutriated leukopak as substitute for purified pDCs coupled with a robust INFα detection method. The Integrated automation program in 384well format has provided a powerful tool for antibody screening and can be applied to other projects requiring a similar screening strategy.
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