Studies have continued on methods to quantitate formation of the membrane attack complex of complement. Antibody to neoantigenic determinants within polymerized C9 (anti: poly C9) has been extensively characterized and used to develop a sensitive ELISA for the fluid phase SC5b-9 complement complex. Cerebrospinal fluids from patients with a variety of central nervous system disorders were tested. SC5b-9 was detected in over 90% of spinal fluids from patients with Guillain Barre Syndrome and over 80% of spinal fluids from patients with multiple sclerosis but in 10% or less of spinal fluids from patients with non-inflammatory central nervous system disorders. Preliminary studies also document SC5b-9 in the CSF of several patients with systemic lupus erythematosis and central nervous system involvement. Anti-poly C9 antibody is currently being used in tissue immunofluorescence studies of diseased tissue from patients with rheumatoid arthritis, osteoarthritis and autoimmune dermatologic disease. Finally, anti-poly C9 was used to quantitate formation of C5b-9 complexes on complement-treated bacteria, in studies which demonstrated that multimeric C9 within C5b-9 was necessary for killing of a rough strain of E. coli. Experiments have been initiated to study functional domains within the C9 molecule. Purified C9 has been biotinylated and then cleaved at a single site with alpha thrombin to produce a functionally active molecule with biotinylated hydrophobic and hydrophilic domains. Exposure of biotin within these domains to Avidin was tested for the monomeric C9 molecule and for C9 incorporated into C5b-9 in the fluid phase and on erythrocyte and bacterial membrane. We are now attempting to purify tryptic peptide fragments of C9 bearing biotin by reverse phase high performance liquid chromatography. These studies should provide a better understanding of the conformational changes within C9 which accompany formation of C5b-9.
