A rapid and reproducible method for the analysis of immune complexes using affinity chromatography and Western blotting

• Published in: Journal of immunological methods Vol. 84; no. 1-2; p. 125
• Main Authors: Smith, A J, Cawston, T E, Hazleman, B L
• Format: Journal Article
• Language: English
• Published: Netherlands 28.11.1985
• Subjects: Antigen-Antibody Complex - analysis; Arthritis, Rheumatoid - immunology; Chemical Precipitation; Chromatography, Affinity - methods; Complement Activating Enzymes - immunology; Complement C1q; Complement C3 - immunology; Complement C3c; Complement System Proteins - analysis; Electrophoresis, Polyacrylamide Gel; Humans; Immunoelectrophoresis; Immunoglobulins - analysis; Polyethylene Glycols; Sepharose
• ISSN: 0022-1759
• DOI: 10.1016/0022-1759(85)90421-1

A new procedure which couples different analytical techniques in a format permitting the rapid analysis of immune complex components is described. Complexes obtained from sera by polyethylene glycol (PEG) precipitation were resuspended and then added, using a batch method, to antibody coupled to Sepharose beads. Antibody directed against either human C1q or human C3c were used in the present study. Bound immune complexes were washed and then eluted from the Sepharose by sodium dodecyl sulphate (SDS) treatment and simultaneously reduced with dithiothreitol. Individual components were separated by SDS gradient polyacrylamide gel electrophoresis and then transferred to nitrocellulose by Western blotting. Individual strips of nitrocellulose were investigated using specific antisera and a radiolabelled probe. Immune complexes (IC) isolated from the sera of 7 rheumatoid arthritis (RA) patients were analysed using this method and the results obtained for both affinity adsorbents compared.