The antigenic binding site(s) of antibodies to factor XII associated with the antiphospholipid syndrome
| Autor: | Peter Nicholls, M. J. Gallimore, David Wynne Jones, Mark Winter, S.L. Harris |
|---|---|
| Rok vydání: | 2005 |
| Předmět: |
Silver Staining
Macromolecular Substances Protein Conformation Molecular Sequence Data Enzyme-Linked Immunosorbent Assay Antibodies Antigen Affinity chromatography Catalytic Domain Humans Biotinylation Amino Acid Sequence Antigens Binding site Peptide sequence Factor XII Binding Sites biology Chemistry Prekallikrein Reproducibility of Results Hematology Kallikrein Antiphospholipid Syndrome Molecular biology Immunoglobulin M Biochemistry Immunoglobulin G Multiprotein Complexes biology.protein Electrophoresis Polyacrylamide Gel Antibody Peptides |
| Zdroj: | Journal of Thrombosis and Haemostasis. 3:969-975 |
| ISSN: | 1538-7836 |
| DOI: | 10.1111/j.1538-7836.2005.01334.x |
| Popis: | Phospholipid binding proteins, including factor XII (FXII), are known to be targeted by antiphospholipid antibodies (aPA). Factor XII antibodies (FXIIab) have been described in some patients with the antiphospholipid syndrome (APS) and have been shown to lead to reduced levels of FXII. The antigenic binding site(s) and the pathophysiological effects of FXIIab are unknown. In an attempt to elucidate the binding site of these antibodies, immobilized plasma kallikrein was used to cleave FXII into its 52-kDa heavy-chain (HCFXII) and 28-kDa light-chain (LCFXII) components. Plasma samples from 12 female patients with definite APS and FXIIab were investigated for the presence of antibodies to FXII, HCFXII and LCFXII. All but one patient's plasma reacted to FXII, HCFXII and LCFXII in a similar manner. One patient gave markedly reduced positivity to HCFXII and LCFXII, suggesting that the FXIIab in this patient had a higher affinity for the intact FXII molecule. To further investigate the antigenic binding site(s) of FXII, 150 biotinylated peptides of the known FXII sequence were synthesized using a Multipin(TM) peptide synthesis procedure. The IgG and IgM fractions of the 12 patients' plasma were purified by affinity chromatography. The synthesized peptides were captured on streptavidin plates and individual patients' purified FXIIab assayed against the peptides in a modified enzyme-linked immunosorbent assay (ELISA). Two regions were identified as possible antigenic binding site(s) for FXIIab: one in the growth factor domain and the other in the catalytic domain. |
| Databáze: | OpenAIRE |
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