Calcium-dependent and calcium-independent signals in the conglutinin-binding assay (KgBa) for immune complexes. Influence of anti-collagen-antibodies.
A solid phase ELISA conglutinin-binding assay (KgBa) was evaluated for the detection of circulating immune complexes. ELISA wells were coated with purified bovine conglutinin and incubated with test sera. Bound IgG was detected with enzyme labelled anti-immunoglobulin. Heat aggregated IgG which had been "solubilized" (i.e., complement treated by incubation with serum) was employed as a reference. The binding of the complement-reacted IgG to solid phase conglutinin was found to be calcium-dependent and inhibitable with N-acetyl-D-glucosamine (GlcNAc). Prolonged incubation (4 days) of aggregated IgG with serum at 37 degrees C abolished the binding to conglutinin, a finding consistent with the complete degradation of deposited C3b to C3c and C3d. The solubilized IgG that bound to solid phase conglutinin was found by gel chromatography to be of high molecular weight (greater than 600 kDa). Binding of IgG to solid phase bovine conglutinin was also observed to a variable degree in normal and pathological sera. However, in this situation the IgG binding was largely calcium-independent, was not inhibited by GlcNAc and did not decrease after prolonged incubation of the serum at 37 degrees C. The reactive IgG eluted on gel chromatography at the position of monomeric IgG suggesting binding via the antigen binding sites. Binding of this IgG was inhibited by both collagen type II and purified conglutinin. These observations suggest that the assay detects cross-reacting autoantibodies against collagen epitopes, or, alternatively, antibodies against the dietary antigen, bovine conglutinin.
Holmskov
U
U
Department of Medical Microbiology, Odense University, Denmark.
Haas
H
H
Teisner
B
B
Andersen
O
O
Jensenius
J C
JC
D000116:Acetylgalactosamine / Q000494:pharmacology; D000117:Acetylglucosamine / Q000494:pharmacology; D000936:Antigen-Antibody Complex / Q000032:analysis*; D000937:Antigen-Antibody Reactions / Q000187:drug effects; D001323:Autoantibodies; D002118:Calcium / Q000494:pharmacology*; D002850:Chromatography, Gel; D003094:Collagen / Q000494:pharmacology; D003168:Complement Fixation Tests; D003429:Cross Reactions; D004306:Dose-Response Relationship, Immunologic; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis
D000936:Antigen-Antibody Complex; D001323:Autoantibodies; D007074:Immunoglobulin G; D003094:Collagen; D000116:Acetylgalactosamine; D002118:Calcium; D000117:Acetylglucosamine