Rheumatology Advance Access published online on July 27, 2004
Rheumatology, doi:10.1093/rheumatology/keh349
Rheumatology © British Society for Rheumatology 2004; all rights reserved
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1 Department of Rheumatology, University Medical Centre, Ljubljana, Slovenia
* To whom correspondence should be addressed. E-mail: borut.bozic{at}guest.arnes.si.
Objectives. We studied the influence of different binding conditions on the interaction between high- or low-avidity IgG anti- Methods. Sera from 30 patients with antiphospholipid syndrome and/or systemic lupus erythematosus were selected on the basis of anti- Results. Anti- 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-
Accepted June 29, 2004
Concise report
Binding of high-avidity anti-
2-glycoprotein I antibodies
u
nik 1,
i 2*
2 Department of Rheumatology, University Medical Centre, Ljubljana, Slovenia; Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia
Abstract 
2-glycoprotein I antibodies (anti-2-GPI) and 2-GPI, which was either free in solution or bound to microtitre plates or nitrocellulose membranes.2-GPI positivity. Avidity of IgG anti-2-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 2-GPI. In converted, non-reducing PAGE, the preparation with high-affinity Fab fragments (obtained by papain digestion) was subjected to electrophoresis, while purified 2-GPI was used as the sample in immunodetection.2-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 2-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-2-GPI: 2/5 high-avidity samples reacted only with non-reduced 2-GPI, 3/9 low-avidity samples recognized only denatured and reduced 2-GPI, and 1/16 samples with heterogeneous-avidity antibodies reacted with reduced and non-reduced 2-GPI.2-GPI to 2-GPI. Some conformational modifications and, consequently, exposed neo-epitopes are required for the recognition of 2-GPI by polyclonal anti-2-GPI antibodies.2-glycoprotein I antibodies; Fab fragments; Avidity; Affinity; Antiphospholipid syndrome; Systemic lupus erythematosus.
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