Immunologic and structural analysis of eight novel domain-deletion β3integrin peptides designed for detection of HPA-1 antibodies
| Autor: | P, Stafford, C, Ghevaert, K, Campbell, C, Proulx, G, Smith, L M, Williamson, E, Ranasinghe, N A, Watkins, J A, Huntington, W H, Ouwehand |
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| Rok vydání: | 2008 |
| Předmět: |
Blood Platelets
Models Molecular Protein Conformation Recombinant Fusion Proteins Infant Newborn Integrin beta3 Enzyme-Linked Immunosorbent Assay Platelet Glycoprotein GPIIb-IIIa Complex Hematology Polymorphism Single Nucleotide Protein Structure Tertiary Antigen-Antibody Reactions Thrombocytopenia Neonatal Alloimmune Epitopes Dogs Isoantibodies Pregnancy Protein Interaction Mapping Animals Humans Antigens Human Platelet Female Intracranial Hemorrhages Protein Binding Sequence Deletion |
| Zdroj: | Journal of Thrombosis and Haemostasis. 6:366-375 |
| ISSN: | 1538-7836 1538-7933 |
| DOI: | 10.1111/j.1538-7836.2007.02858.x |
| Popis: | The single-nucleotide polymorphism (SNP) rs5918 in the ITGB3 gene defines the human platelet antigen-1 (HPA-1) system encoding a Leu (HPA-1a) or Pro (HPA-1b) at position 33. HPA-1 antibodies are clinically the most relevant in the Caucasoid population, but detection currently requires alpha(IIb)beta3 integrin from the platelets of HPA-genotyped donors.We set out to define the beta3 integrin domains required for HPA-1a antibody binding and produce recombinant soluble beta3 peptides for HPA-1 antibody detection.We designed two sets (1a and 1b) of four soluble beta3 domain-deletion peptides (deltaSDL, deltabetaA, PSIHybrid, PSI), informed by crystallography studies and computer modeling. The footprints of three human HPA-1a-specific phage antibodies were defined by analyzing binding patterns to the beta3 peptides and canine platelets, and models of antibody-antigen interfaces were derived. Specificity and sensitivity for HPA-1a detection were assessed using sera from 140 cases of fetomaternal alloimmune thrombocytopenia (FMAIT).Fusion of recombinant proteins to calmodulin resulted in high-level expression in Drosophila S2 cells of all eight beta3 peptides. Testing of FMAIT samples indicated that deltabetaA-Leu33 is the superior peptide for HPA-1a antibody detection, with 96% sensitivity and 95% specificity. The existence of type I and II categories of HPA-1a antibodies was confirmed by the study of HPA-1a phage antibody footprints and the reactivity pattern of clinical samples with the four beta3-Leu33 peptides, but there was no correlation between antibody category and clinical severity of FMAIT.Soluble recombinant beta3 peptides can be used for detection of clinical HPA-1a antibodies. |
| Databáze: | OpenAIRE |
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