Binding of high-avidity anti-beta2-glycoprotein I antibodies.
OBJECTIVE
We studied the influence of different binding conditions on the interaction between high- or low-avidity IgG anti-beta2-glycoprotein I antibodies (anti-beta2-GPI) and beta2-GPI, which was either free in solution or bound to microtitre plates or nitrocellulose membranes.


METHODS
Sera from 30 patients with antiphospholipid syndrome and/or systemic lupus erythematosus were selected on the basis of anti-beta2-GPI positivity. Avidity of IgG anti-beta2-GPI was determined by chaotropic ELISA, using increased NaCl concentration during the antibody binding. Immunodetection on nitrocellulose membrane followed reducing or non-reducing sodium dodecyl sulphate-polyacrylamide gel electrophoresis (PAGE) of beta2-GPI. In converted, non-reducing PAGE, the preparation with high-affinity Fab fragments (obtained by papain digestion) was subjected to electrophoresis, while purified beta2-GPI was used as the sample in immunodetection.


RESULTS
Anti-beta2-GPI antibodies of high, low or heterogeneous (low and high) avidity were found in 5/30, 9/30 and 16/30 sera, respectively. The density of beta2-GPI, which was 20-30 times higher on the nitrocellulose membrane than on the surface of ELISA plates, was not sufficient for the recognition of the antigen by anti-beta2-GPI: 2/5 high-avidity samples reacted only with non-reduced beta2-GPI, 3/9 low-avidity samples recognized only denatured and reduced beta2-GPI, and 1/16 samples with heterogeneous-avidity antibodies reacted with reduced and non-reduced beta2-GPI.


CONCLUSIONS
Our results suggest that neither high density of the antigen nor high avidity of the antibodies (or Fab fragments) alone was sufficient for the binding of anti-beta2-GPI to beta2-GPI. Some conformational modifications and, consequently, exposed neo-epitopes are required for the recognition of beta2-GPI by polyclonal anti-beta2-GPI antibodies.
Cucnik
S
S
Department of Rheumatology, University Medical Centre, Vodnikova 62, 1000 Ljubljana, Slovenia.
Kveder
T
T
Rozman
B
B
Bozic
B
B
D000915:Antibody Affinity; D000937:Antigen-Antibody Reactions; D016736:Antiphospholipid Syndrome / Q000276:immunology*; D001323:Autoantibodies / Q000097:blood*; D001324:Autoantigens / Q000276:immunology; D006023:Glycoproteins / Q000276:immunology*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D007074:Immunoglobulin G / Q000097:blood; D008180:Lupus Erythematosus, Systemic / Q000276:immunology*; D053482:beta 2-Glycoprotein I
D001323:Autoantibodies; D001324:Autoantigens; D006023:Glycoproteins; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D053482:beta 2-Glycoprotein I