Complexes prepared from protein A and human serum, IgG, or Fc gamma fragments: characterization by immunochemical analysis of ultracentrifugation fractions and studies on their interconversion.
Protein A of Staphylococcus aureus is an Fc receptor for IgG that has been used as a therapeutic reagent to treat cancer in humans and experimental animals. We used ultracentrifugation combined with analysis of isolated fractions by radioimmunoprecipitation and competitive radioimmunoassay with chicken antibodies that bind free protein A or protein A in complexes but do bind free immunoglobulin reagents to localize and characterize the types of complexes formed with different molar ratios of 125I-protein A and human 131I-IgG alone or in serum, and 131I-Fc gamma fragments. This approach offers a distinct advantage over direct counting of radioactivity in the fractions because resolution of complexes and free reagents is much improved. With excess 131I-IgG or 131I-Fc, all the 125I-protein A is present only in complexes that contained 4 molecules of immunoglobulin reagent and 2 molecules of protein A (4:2 complexes), whereas with excess 125I-protein A the stoichiometry of the complexes was 1:1. We have also shown the preformed 4:2 and 1:1 complexes will interconvert in the presence of added excess protein A or IgG, respectively, and that fresh IgG will exchange with IgG or Fc gamma in preformed complexes. Because protein A has been found to elute from an immobilized reagent used in serotherapy of human cancer and is present in a large excess of IgG, the 4:2 complexes may play an active role in the tumoricidal or toxic reactions observed.
Langone
J J
JJ
Das
C
C
Mainwaring
R
R
Shearer
W T
WT
D000936:Antigen-Antibody Complex / Q000302:isolation & purification; D006801:Humans; D007120:Immunochemistry; D007141:Immunoglobulin Fc Fragments; D007128:Immunoglobulin Fragments; D007074:Immunoglobulin G / Q000378:metabolism*; D011961:Receptors, Fc / Q000378:metabolism*; D017452:Receptors, IgG; D012995:Solubility; D013205:Staphylococcal Protein A / Q000097:blood / Q000302:isolation & purification / Q000378:metabolism*; D014461:Ultracentrifugation
D000936:Antigen-Antibody Complex; D007141:Immunoglobulin Fc Fragments; D007128:Immunoglobulin Fragments; D007074:Immunoglobulin G; D011961:Receptors, Fc; D017452:Receptors, IgG; D013205:Staphylococcal Protein A