Homogeneous bioluminescent immunoassays detect profound synergistic regulation of IL-8 and MCP-1 release in cultured human monocytes

We have developed sensitive bioluminescent immunoassays for IL-8 and MCP-1 and applied these homogeneous assays to demonstrate synergistic regulation of their release in a culture model of human monocytes. IL-8 and MCP-1, key proinflammatory chemokines, play important roles in neutrophil and monocyte recruitment. Employing Lumit™ technology, we labeled candidate monoclonal antibodies for these chemokines with NanoBiT luciferase subunits (SmBiT or LgBiT), identifying pairs that produce strong luminescent signals upon target binding. These assays exhibited outstanding sensitivity (IL-8 at 0.4 pg/mL, MCP-1 at 0.8 pg/mL) and a broad linear range over three logs of analyte concentration, with high specificity against a panel of cytokines. Applying these assays to THP-1 monocytes in 96-well format, we observed lipopolysaccharide (LPS) induced a dose-dependent IL-8 release (max 1532 pg/mL), whereas IFN-γ triggered minimal release (169 pg/mL). However, LPS and IFN-γ together significantly increased IL-8 levels (8554 pg/mL). More profound synergy was observed for MCP-1 release; LPS or IFN-γ alone elicited modest responses (23 and 48 pg/mL, respectively), but their combination led to a dramatic increase (5902 pg/mL). This work establishes rapid, simple luminescent immunoassays for IL-8 and MCP-1 detection directly in cell culture and shows application of these assays to demonstrate synergistic regulation of chemokine release with implications for modulation of inflammatory responses.