An immune complex selective affinity column utilizing site-specific attachment of bovine conglutinin.
A simple method to isolate immune complexes by column chromatography is described. The immune complex affinity column was constructed by the site-specific attachment of bovine conglutinin to agarose. Covalent attachment of conglutinin to agarose was achieved via hydrazone chemistry, which reacts aldehydes of oxidized oligosaccharides in the collagenous domain of conglutinin with hydrazide functional groups in the solid support. The constructed conglutinin affinity column captured complement-fixed model immune complexes of heat-aggregated human IgG and more classical complexes of chicken ovalbumin-anti-chicken ovalbumin. Neither the nonfixed immune complexes nor their individual components were retained by the column. The captured material was specifically eluted with EDTA without the use of low pH. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot analysis, and protein sequence analysis of the eluates revealed the presence of the expected individual components, verifying that both antibody and antigen used to prepare the soluble immune complexes were recovered from the conglutinin column. The advantages of this approach over traditional methods of immune complex isolation and characterization are discussed.
O'Donoghue
G V
GV
Department of Protein Biochemistry, Applied Immune Sciences Inc., Santa Clara, California 95054.
Okarma
T B
TB
Lee
Y M
YM
D000595:Amino Acid Sequence; D000818:Animals; D000936:Antigen-Antibody Complex / Q000032:analysis / Q000302:isolation & purification*; D000941:Antigens / Q000032:analysis; D015153:Blotting, Western; D002417:Cattle; D002645:Chickens; D002846:Chromatography, Affinity / Q000379:methods; D037222:Collectins; D003165:Complement System Proteins; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis; D007164:Immunosorbents; D008969:Molecular Sequence Data; D010047:Ovalbumin / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D012685:Sepharose; D017421:Sequence Analysis; D012712:Serum Globulins
D000936:Antigen-Antibody Complex; D000941:Antigens; D037222:Collectins; D007074:Immunoglobulin G; D007164:Immunosorbents; D012712:Serum Globulins; C024014:conglutinin; D010047:Ovalbumin; D003165:Complement System Proteins; D012685:Sepharose