H. J., ed.), pp. 715-731, Grune and Stratton. New York. Fraker, P. J. & Speck, J. C. (1978) Biochem. ... immune disorders (Campbell & Luzio, 1981; Morgan et al.,.
616th MEETING, LONDON (Eberhad. H. J., ed.), pp. 715-731, Grune and Stratton. New York Fraker, P.J. & Speck, J. C. (1978) Biochem. Biophys. Res. Commun. 80. 849 -857 Hamada, N., Grimm, C.. Mori. H. & DeGroot. L. (1985) J. Clin. Endocrinol. Metah. 61, 120-128
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The function of C8 in the complement membrane attack complex investigated using monoclonal antibodies AREFAINE ABRAHA,* ELAINE M. BAILYES,* PETER J. RICHARDSON,* ANTHONY K . CAMPBELL? and J . PAUL LUZIO* *Department of Clinical Biochemistry, University of Cambridge, Addenbrookes Hospital, Hills Roud, Cambridge CB2 2QR. U . K . , and tDepartment of Medicul Biochemistry, University of Wales College of Medicine, Heath Park, Cardif CF4 4 X N , U . K . The insertion of the terminal complement component C9 into appropriate target membranes to form tubular complexes provides the principal mechanism of complementmediated cell killing (Tschopp et al., 1 9 8 2 ~ )It. may also be responsible for sublytic cell damage important in some autoimmune disorders (Campbell & Luzio, 1981; Morgan et al., 1984a,b). The target membranes must contain membranebound complement components C 5 b 8 which act as a receptor for C9 and together form the complement membrane attack complex (MAC). The C 5 b 8 complex is thought to catalyse the polymerization of C9 (Tschopp et al., 1982h, 1985) with C8 participating directly in this process (Monahan et al., 1983). Human C8 is a serum glycoprotein, with an apparent M , of 151 000, composed of three non-identical polypeptide chains: a ( M , 64000), fi ( M , 64 000) and y ( M , 22 000). These subunits exist as a covalently linked ay dimer non-covalently associated with the fi-subunit (Steckel et al., 1980). Solution binding studies using purified complement components have suggested that the fi-subunit mediates the binding of C8 to C5b-7, with the a-subunit being responsible for interaction with C9 (Stewart & Sodetz, 1985). In the present study monoclonal antibodies to C8 have been prepared with the aim of using them to purify C8, to investigate the function of C8 in the formation of the MAC, and to estimate the serum concentration of C8 in different clinical conditions. Monoclonal antibodies were prepared by standard methods (Galfre & Milstein, 198 1) as previously described for C9 (Morgan et al., 1983). BALBjC mice were immunized subcutaneously with 5 pg of human C8 (kindly donated by Dr. A. Esser) in Freund's complete adjuvant. After 6 weeks the mice were boosted intraperitoneally with 5 p g of C8 in phosphate-buffered saline. On the fourth day after boosting, spleen cells were fused with P3-NSl/I-Ag 4-1 mouse myeloma cells and the products of fusion cultured in microtitre trays. Culture supernatants were tested for antibody after 10-14 days, the assay consisting of incubating '251-C8 with aliquots of the supernatant and measuring the subsequent binding of radioactivity to a sheep anti-mouse immunoglobulin immunoadsorbent (Morgan et al., 1983). Positive cell cultures were cloned twice and then grown to large volume in tissue culture flasks before intraperitoneal injection into mice for ascites production (Goding, 1980). Four monoclonal antibodies to human C8 were prepared from a single fusion and coded A2, BI, H9 and L6. Immunoblotting 1 pg samples of C8 from SDS/polyacrylAbbreviation used: MAC, membrane attack complex.
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amide gels (Burnette, 1981) using a 1 : 50 dilution of ascites fluid showed that antibodies A2 and BI reacted with the /]-subunit and antibodies H9 and L6 with the a-subunit of human C8. Epitope mapping (Soos & Siddle, 1982) suggested that antibodies A2 and BI reacted with the same antigenic site on the [hubunit of C8. Immunoglobulin fractions containing antibodies A2 and H9 were prepared from ascites fluid and coupled to diazocellulose (Hales & Woodhead, 1980) providing immunoadsorbents (200 pg of immunoglobulin/mg of cellulose) capable of removing C8 from human plasma. C8 could be recovered by elution with freshly prepared 50 mwdiethylamine at 4 C. Using the criteria of rocket immunoassay with commercially available goat anti-C8 serum and SDS/polyacrylamide-gel electrophoresis, recovery of C8 was 30% when starting from 5ml of fresh plasma and 5 m g of immunoadsorbent. Antibodies A2 and H9 were both shown to interfere with the formation of a functional MAC. Pigeon erythrocyte ghosts loaded with obelin, a Ca2+-sensitive photoprotein, were prepared (Campbell et al.. 1979, 1981), incubated with guinea-pig antibodies then with C9-depleted human serum and washing the ghosts after each incubation. Addition of purified C9 (Morgan et al., 1983) caused an influx of Ca'+, triggering obelin luminescence. Addition of antibodies A2 and H9 before addition of C9 prevented the stimulation of obelin luminescence suggesting that both the fi- and a-subunits of C8 may play a role in the functional insertion of C9 into the MAC. We thank the W.H.O. and the Arthritis and Rheumatism Council for financial support. Burnette. W. (19x1) A n d . Biochcw. 112. 195 203 Campbell. A. K. & Luzio, J. P. (19x1) E.vper/mtiu 37, 110-1 12 Campbell. A. K.. Daw. R. A. & Luzio, J. P. (1979) FEES Lett. 107, 55 60 Campbell. A. K.. Daw. R. A,. Hallett. M . B. & Luzio. J. P. (1981) Biochem. J . 194. 551 560 Galfre. G. & Milstein, C. (1981) Mrthodc Enzyniol. 73. 3-46 Goding, J. W. (1980) J. Immunol. Mathod.s 39, 285 308 Hales. C. N . & Woodhead, J. S. (1980) Methods Enzj*mol.70, 334-354 Morgan, B. P.. Daw. R. A,. Siddle. K.. Luzio, J. P. & Campbell. A. K . (1983) J . Ininiunol. Methods 64. 269-281 Morgan. B. P.. Sewry. C. A,. Siddle. K.. Luzio. J . P. & Campbell, A. K. ( 1 9 8 4 ~ Imniunokigy ) 52. I8 1 I88 Morgan, B. P., Campbell. A. K . & Compston. A. (1984h) Lancet ii, 251 255 Monahan. J. B.. Stewart. J. L. & Sodetz, J . M . (1983) J. Bid. Cheni. 258. 5056 5062 Soos. M. & Siddle, K . (1982) J. Immunol. Methods 51. 5 7 68 ~ Steckel. E. W., York, R. G.. Monahan, J . B. & Sodetz. J. M. (1980) J. Biol. Chcv77. 255, I1997 12005 Stewart, J . L. & Sodetz, J. M . (1985) Biochemistry 24, 4598-4602 Tschopp. J.. Muller-Eberhard. H. J. & Podack. E. R. ( 1 9 8 2 ~Nature ) (London) 298, 534 539 Tschopp. J.. Podack. E. R. & Muller-Eberhard, H. J. (19826) Proc. Nut/. Acud. Sci. U.S.A. 79, 7474 7498 Tschopp, J.. Podack. E. R. & Muller-Eberhard. H. J. (1985) J . Inimunol. 134, 495- 499 ~
