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Immune complexes in Hodgkin's disease: isolation, immunochemical and physico-chemical analysis
Immune complexes (IC) present in sera from patients with Hodgkin's disease (HD) were isolated using three different affinity columns: C1q-degalan, anti-C1q sepharose and conglutinin (K)-degalan. The isolated IC were analysed by immunoprecipitation, SDS-PAGE and sucrose density gradients and compared with IC similarly isolated from patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and in vitro prepared BSA-anti-BSA complexes. Isolated material from each disease, and BSA-anti-BSA complexes contained proteins compatible with true immune complexes--IgM, IgG, C1q and C3 breakdown components. Albumin, fibronectin and CRP, whose affinity for IgM, C1q and C3 are known, were co-isolated along with IC material. The size of isolated IC in HD ranged from 8-40S on sucrose density gradients. Despite the operational difference in detecting and isolating HD complexes via the C1q ligand (C1q-degalan or anti-C1q column) and C3bi (K-degalan), material purified by both methods showed remarkable similarity on SDS-PAGE and immunoprecipitation analysis. Although IC isolated from different diseases showed disparate banding patterns on SDS-PAGE this was attributed to a variation in the relative concentrations of constituent proteins--IgM, IgG and C3 breakdown products. IgM, IgG and C3 bind loosely, and non-specifically, to macromolecular aggregates formed around immune complexes. Using the anti-C1q column, most of this material could be eluted using 0.02M EDTA. Least protein, yet the most specific for antigen and antibody was eluted at pH 3.0.
Immune complexes (IC) present in sera from patients with Hodgkin's disease (HD) were isolated using three different affinity columns: C1q-degalan, anti-C1q sepharose and conglutinin (K)-degalan. The isolated IC were analysed by immunoprecipitation, SDS-PAGE and sucrose density gradients and compared with IC similarly isolated from patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and in vitro prepared BSA-anti-BSA complexes. Isolated material from each disease, and BSA-anti-BSA complexes contained proteins compatible with true immune complexes--IgM, IgG, C1q and C3 breakdown components. Albumin, fibronectin and CRP, whose affinity for IgM, C1q and C3 are known, were co-isolated along with IC material. The size of isolated IC in HD ranged from 8-40S on sucrose density gradients. Despite the operational difference in detecting and isolating HD complexes via the C1q ligand (C1q-degalan or anti-C1q column) and C3bi (K-degalan), material purified by both methods showed remarkable similarity on SDS-PAGE and immunoprecipitation analysis. Although IC isolated from different diseases showed disparate banding patterns on SDS-PAGE this was attributed to a variation in the relative concentrations of constituent proteins--IgM, IgG and C3 breakdown products. IgM, IgG and C3 bind loosely, and non-specifically, to macromolecular aggregates formed around immune complexes. Using the anti-C1q column, most of this material could be eluted using 0.02M EDTA. Least protein, yet the most specific for antigen and antibody was eluted at pH 3.0.