Detection of Cytokine Autoantibodies Using LIPS

The detection of autoantibodies against cytokines with the use of Luciferase Immunoprecipitation Systems has been reported previously.^{4 (link)} Autoantibodies were evaluated against 41 targets: interferons γ, α1, β1, ε, λ1, λ3, and ω; interleukins 1α and 1β; the interleukin-1 receptor antagonist; interleukins 2, 3, 4, 6, 7, 8, 10, 12p35, 12p40, 15, 17A,17F, 18, 21, 22, 23p19, 27p28, 32, and 33; Epstein–Barr virus–induced gene 3 protein (interleukin-27b); granulocyte colony-stimulating factor (G-CSF); granulocyte–macrophage colony-stimulating factor (GM-CSF); TNF-α; tumor necrosis factor β; B-cell–activating factor; a proliferation inducing ligand; the Fas ligand (FasL); the CD40 ligand; erythropoietin; transforming growth factor β; and the extracellular domain of the CD4 receptor. Additional methodological details are described in the Supplementary Appendix; a detailed protocol and video describing the Luciferase Immunoprecipitation Systems technique are also included in an article by Burbelo et al.²¹ Anti–interferon-γ–specific autoantibody isotype and IgG subclasses were determined with the use of a particle-based assay, as described previously ^{22 (link)}; total IgG subclasses were determined with the use of the Bio-Plexisotype kit (Bio-Rad Laboratories)according to the manufacturer’s instructions. Interferon-γ–specific IgG was purified by fractionating total IgG on protein G columns (Ab SpinTrap, GE Healthcare) and applying the total IgG fraction to an interferon-γ column.