Evaluating the Fc-function of intravenous immunoglobulin products by flow cytometry

Intravenous Immunoglobulin products (IVIG) are indicated as the treatment of choice in a number of immune-mediated disorders, including inflammatory diseases and autoimmune diseases. Interactions with Fc-receptors have been described as an important mechanism of action of IVIG. Therefore, we have evaluated a recently described flow-cytometric method for the quantification of the Fc-function of IVIG products. Human monocytic THP-1 cells were incubated with IVIG. The IgG bound to the cells was detected using an anti-human IgG antibody conjugated with FITC. Cells were analyzed by flow cytometry. The specificity of IVIG binding via the Fc-part was evaluated by competition experiments using F(ab)2 fragments of IVIG. Furthermore, the release of cytokines into the supernatants of THP-1 cells was analyzed using a Bio-Plex array system and intracellular cytokines were analyzed by flow-cytometry. A dose-dependent binding of IVIG to THP-1 cells was found. Maximum binding was observed at 10 μg/ml IVIG. The results obtained were highly reproducible and showed little variation between different passages of THP-1 cells. Competition experiments using F(ab)2 fragments and the analysis of cytokine production by THP-1 cells are currently ongoing. The flow-cytometric method that was recently described for the quantification of the Fc-function of IVIG preparations offers a fast and highly reproducible assay. Results of competition and validation experiments will show whether this new methodology is suitable for the quality control of IVIG products.