Anti-C1q column: ligand specific purification of immune complexes from human serum or plasma. Analysis of the interaction between C1q and immune complexes.
An efficient and reproducible procedure has been developed for the specific isolation of immune complexes. PEG precipitation of EDTA serum or plasma was an essential preliminary step to separate complex-bound from free C1q. PEG had no discernible effect on the molecular weight size of the extracted complexes. Redissolved complexes were incubated with a Sepharose-4B column coated with anti-human C1q antibodies and following removal of unbound material the bound complexes were sequentially eluted with 0.02 M EDTA, 0.5 M NaCl and 1 M propionic acid. Characteristics of the affinity column were established by the purification of 125I-labelled BSA-anti-BSA complexes and heat-aggregated IgG (HAGG) incubated in normal human serum (NHS). EDTA and NaCl eluted complexes were of similar molecular size and contained antigen, specific antibody, as well as human IgM, IgG, albumin, C3, C3c, C3d and C1q. Acid eluted complexes contained the highest yield of specific antigen and antibody and comprised in addition human C1q and C3d. Activation of complement components after C1q made the bond between C1q and immune complexes resistant to 0.5 M NaCl and interfered with the binding between solid phase anti-C1q and complex bound C1q. Using BSA-anti-BSA complexes and HAGG activated in NHS it was apparent that only a minority of the complexed material was isolated via the C1q ligand and this probably applies to the C1q binding assay. Most complexed material could be isolated using an anti-C3 affinity column.
D000936:Antigen-Antibody Complex / Q000276:immunology / Q000302:isolation & purification*; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D003166:Complement Activating Enzymes / Q000276:immunology*; D015922:Complement C1q; D003176:Complement C3 / Q000276:immunology; D006358:Hot Temperature; D006801:Humans; D005779:Immunodiffusion; D007074:Immunoglobulin G / Q000276:immunology; D008024:Ligands; D008722:Methods; D011092:Polyethylene Glycols
D000936:Antigen-Antibody Complex; D003176:Complement C3; D007074:Immunoglobulin G; D008024:Ligands; D011092:Polyethylene Glycols; D015922:Complement C1q; D003166:Complement Activating Enzymes