In Vitro Models for Complement Dysregulation (360G-Wellcome-211543_Z_18_Z)

Complement is an integral component of innate immunity, and its dysregulation is associated with diseases such as C3 glomerulopathy (C3G), atypical haemolytic-uremic syndrome (aHUS) and many chronic inflammatory diseases where self-tissues are inappropriately targeted and damaged. Complement factor H (fH) is an essential inhibitor of the alternative pathway of complement activation, it downregulates complement on self-tissues. In this project, I will produce an in vitro model of fH dysregulation using the factor H-blocking antibody, IIB6. I will purify the antibody from a pre-established hybridoma cell-line and determine its integrity using SDS-page and its binding ability with a fH western blot. Normal fH function will be tested in a fluid phase co-factor assay by incubating C3b, fH and factor I and testing for cleavage and inactivation of C3b. The ability of IIB6 to block inactivation of C3b in this pure protein system will be confirmed first, before spiking IIB6 into whole serum and assessing ‘fluid phase’ dysregulation of complement by western blot analysis of activation products. Finally, cell-surface inhibition of fH by IIB6 will be confirmed in a cell-based hemolysis assay, optimised for sensitivity. This assay can subsequently be used as a platform for testing complement therapeutics.