Our observations are consistent with the hypothesis that dissociation or destruction of complement C1 eliminates the prozone effect. Addition of EDTA to serum of highly immunized patients is the easiest way to avoid false-negative results in SAB testing caused by a prozone effect.

The possible role of a 140K membrane-associated protein complex (140K) in fibronectin-cytoskeleton associations has been examined. The 140K was identifed by the monoclonal antibody JG22E. Monoclonal and polyclonal antibodies to the 140K showed identical patterns of binding to the cell membranes of fixed and permeabilized chicken embryonic fibroblasts; localization was diffuse, but with marked concentration in cell-toextracellular matrix contact sites. Correlative localization with interference reflection microscopy and double-label or triple-label immunofluorescence showed that 140K co-distributed with extracellular fibronectin fibrils and intracellular a-actinin in microfilament bundles at extracellular matrix contact sites but tended not to co-localize with tropomyosin present in bundles at sites farther from adhesion sites. In addition, binding of antibodies to 140K, aactinin, and fibronectin was excluded from vinculin-rich focal adhesion sites at the cellular periphery. A progressive development of cell surface a-actinin-140K-fibronectin associations was observed in early spreading cells. The anti-140K monoclonal antibody JG22E inhibited the attachment and spreading of both normal and Rous sarcoma virus-transformed chicken embryonic fibroblasts to a fibronectin substratum. However, the anti-140K monoclonal antibody became a positive mediator of cell attachment and spreading if it was adsorbed or cross-linked to the substratum. Our results provide the first description of a membraneassociated protein complex that co-localizes with fibronectin and microfilament bundles, and they suggest that the 140K complex may be part of a cell surface linkage between fibronectin and the cytoskeleton.Interactions of cells with extracellular materials are critically important events during embryonic development and for the maintenance of normal tissue functions. Fibronectin has been shown to promote the adhesion and spreading of cells ~ on a variety of materials including plastic, collagen, gelatin, and fibrin (for reviews, see references 15, 22, 26, 31, 37, and 47). Concomitant with the spreading induced by added fibronectin, cells often acquire highly ordered intracellular microfilament bundles (MFBs) I (1, 45). In highly spread cells, extracellular matrix (ECM) fibers that contain fibronectin are often Abbreviations used in this paper: CEF, chicken embryo fibroblasts; CEL, chicken embryonic lung; ECM, extracellular matrix; IRM, interference reflection microscopy; 140K, a three component, membrane-associated protein complex; MFB, microfilament bundle; RSV, Rous sarcoma virus.observed to correspond in their arrangement with intracellular MFBs (20,23,39). At sites where ECM fibrils appear to attach to the plasma membrane, there is a co-distribution of actin and a-actinin (and sometimes vinculin) inside cells and fibronectin outside cells (4,9,24,40, 41).Immunoelectron microscopy has shown a spatial relationship between fibronectin and a-actinin at membrane attachment sites in spread fibroblasts, which were termed ECM...

• Strong complement activation overrides the terminal pathway inhibition by the anti-C5 antibody eculizumab.• The more powerful complement is activated, the less effective is terminal pathway inhibition by diverse anti-C5 agents.Eculizumab inhibits the terminal, lytic pathway of complement by blocking the activation of the complement protein C5 and shows remarkable clinical benefits in certain complement-mediated diseases. However, several reports suggest that activation of C5 is not always completely suppressed in patients even under excess of eculizumab over C5, indicating that residual C5 activity may derogate the drug's therapeutic benefit under certain conditions. By using eculizumab and the tick-derived C5 inhibitor coversin, we determined conditions ex vivo in which C5 inhibition is incomplete. The degree of such residual lytic activity depended on the strength of the complement activator and the resulting surface density of the complement activation product C3b, which autoamplifies via the alternative pathway (AP) amplification loop. We show that at high C3b densities required for binding and activation of C5, both inhibitors reduce but do not abolish this interaction. The decrease of C5 binding to C3b clusters in the presence of C5 inhibitors correlated with the levels of residual hemolysis. However, by employing different C5 inhibitors simultaneously, residual hemolytic activity could be abolished. The importance of AP-produced C3b clusters for C5 activation in the presence of eculizumab was corroborated by the finding that residual hemolysis after forceful activation of the classical pathway could be reduced by blocking the AP. By providing insights into C5 activation and inhibition, our study delivers the rationale for the clinically observed phenomenon of residual terminal pathway activity under eculizumab treatment with important implications for anti-C5 therapy in general. (Blood. 2017;129(8):970-980)