A Sensitive Specific Hemolytic Assay for Proenzyme C1

Abstract

The traditional hemolytic assay of the functional activity of C1, the first component of the classical complement pathway, was modified to permit differentiation between proenzyme (unactivated) C1 and the activated state of the enzyme (C1). A two-step assay was developed to quantitate proenzyme C1. The C1 sample to be assayed was first preincubated with C1 inhibitor, a process that specifically inhibits the enzymatic activity of C1 without affecting the subsequent activtion of proenzyme C1 by EAC4, a model immune complex. Since the rate of reaction between C1 inhibitor, a serum regulatory protein, and C1 is concentration-dependent, this step is performed at high C1 and C1 inhibitor concentrations. Subsequent dilutions of the sample prevents C1 inhibitor-mediated inactivation of the C1 that is activated during the C1 hemolytic assay. Thus, in the presence of C1 inhibitor, the level of C1 hemolytic activity specifically reflects the activity of proenzyme C1, while in the absence of C1 inhibitor, the hemolytic activity reflects the total activity of C1. Both the absolute and the relative amounts of the proenzyme (unactivated) and activated C1 can thereby be quantitated in most samples. Furthermore, a partially purified C1 inhibitor reagent, easily prepared from serum, was shown to function identically to the purified C1 inhibitor, obviating the need for a multistep isolation procedure for this protein. Using this simple yet sensitive assay to investigate the efficiency of reconstitution of C1 activity from the purified components C1q, C1r, and C1s, we also find evidence for temperature- and concentration-dependent reaction steps in the formation of functional C1.

The Complement System: An Unexpected Role in Synaptic Pruning During Development and Disease

“…In the immune system, the activated C3 fragment C3b (iC3b) opsonizes the surface of cells/debris and tags them for elimination by phagocytic macrophages that express C3 receptors (C3R/cd11b) (Carroll 2004, Gasque 2004, van Lookeren Campagne et al 2007). Microglia, the resident phagocytes of the CNS, are the only resident brain cells to express CR3 (Tenner & Frank 1987, Guillemin & Brew 2004, Ransohoff & Perry 2009, Graeber 2010. Indeed, process-bearing activated microglia and synaptically localized C3 have been observed in the dLGN and several other postnatal brain regions, including hippocampus, cerebellum, and olfactory bulb, undergoing active synaptic remodeling (Perry et al 1985, Dalmau et al 1998, Fiske & Brunjes 2000, Schafer et al 2011; until recently, however, the function of microglia in a normal brain has remained a relative mystery.…”