Scand. J. Immunol. 22, 183-195, Monoclonal Antibodies Recognizing a Neoantigen of Poly(C9) Detect the Human Terminal Complement Complex in Tissue and Plasma T. E. MOLLNES, T. LEA. M. HARBOE & J. TSCHOPP Institute of Itnmunology and Rheumatology, Rikshospitalet. The National Hospital, Oslo. Norway, and Institute of Biochemistry, University of Lausanne. Lausanne, Switzerland Motlnes. T.E., Lea. T.. Harbne. M. & Tschopp. J. Monoelonal Antibodies Recognizing a Neoantigen of PoIy(C9) Detect the Human Terminal Complenicni Complex in Tissue and Plasma. Scund. J. Immunol. 22, 183-195. 19S5. The terminal complement complex (TCC), consisting of C5b. C(>, C7. CIS. and C9. contains neoanligens that are abseiu from Ihe individual native coniponenls. Neoaniigens are present both in the membrane-bound (MAC) and the fluid-phase (SC5b-9) complex. The present study describes produetion of monoclonal antibodies against neoantigcns of both forms of Ihe TCC. A convenient screening and detection system, based mainly on enzyme-linked immunosorbent as.says. crossed immunoelectrophoresis wilh autoradiography, and affinity chromatography with subsequent sodium dodecyl sulphate-polyacrylamide gel electrophoresis including immunoblotting. is described in detail. Two monoclonal antibodies were specific for a neoan- tigen located in the poly(C9) moiety nf the ICC. One of these antibodies. MCaEll, was used for immunohistochemical detection of MAC in tissue and for quantification of the fluid-phase TCC in ethylenediaminetctraacetic acid plasma. Tom Eirik Moltnes. Institute of ImmuiiotofivandRtieiimatntogy. Fr. Q\am.sKl. I. 0172 Oslo I. Norway The terminal complement complex (TCC) con- sists of the components C5b. C6, C7, C8, and C9. It exists in two analogous forms: the membrane attack complex (MAC) |i(I-12]. which is the mediator of complement lysis, and the fluid-phase non-lytic SC5b-y complex [20, 22). which in addition to the terminal compon- ents contains the S-protein [21| of human plasma. The MAC has been demonstrated both in vitro and In vivo, whereas the SC5b-y complex is characterized only in vitro. Recently, however, a TCC has been detected in normal human plasma by means of an assay based on antibodies against the native components [17]. This a.ssay requires separation of the native components from the complex and thus cannot be used for quantification of large series of plasma samples. Neoantigens |I3| are new antigenic determi- nants that appear in the TCC upon its assembly, whereas they cannot be detected in the individual native components. Several xenoan- tisera [1. 4] and one tnonoclonal antibody [7] against neoantigens have been described. These antibodies have been used to examine the MAC in tissue, but they could not be used for detection of the TCC in normal human plasma. The aims of the present study were to produce monoclonal antibodies highly specific for neoan- tigens of the TCC and to develop and standard- ize a convenient screening and detection system for these antibodies. We obtained two monoclo- nal atitibodies. MCaEll and MCbC5. which were specific for a neoantigen oi poly(C9). The MCaEU antibody wa.s used for immune fluores- cence to detect the MAC in tissue and for quan- tification of the fluid-phase TCC in plasma samples by an enzyme-hnked immunosorbent assay (ELISA). 183