Abstract | BACKGROUND:
Antibodies to human neutrophil antigen (HNA)-2a are responsible for a number of immune-mediated neutropenia disorders. Although several methods exist for the identification of anti-HNA-2a, all these methods have several limitations. In this study, a solid-phase enzyme-linked immunosorbent assay (ELISA) using recombinant HNA-2a antigen (rHNA-2a) allowing rapid detection of HNA-2a antibodies was developed. STUDY DESIGN AND METHODS: Soluble rHNA-2 was generated by transfection of insect cells with CD177 vector. Purified rHNA-2a was immobilized on microtiter wells coated with anti-CD177 and was applied to analyze 10 sera containing HNA-2a antibodies. For the evaluation of the ELISA method, results were compared with the standard assay, MAIGA ( monoclonal antibody antigen capture assay) for detection of neutrophil antibodies. RESULTS: The specificity of HNA-2a antibodies in all sera was confirmed by immunoblotting. Sera were then tested simultaneously in ELISA and MAIGA assays. Nine of 10 sera showed positive reactions in ELISA, whereas only 9 of 10 sera reacted in the standard MAIGA assay. All HNA-2a antibodies were detectable in MAIGA when diluted sera were applied. No reaction was observed with different sera containing neutrophil-reactive antibodies (6 anti-HNA-1a, 4 anti-HNA-1b, and 20 anti-HLA Class I and II) in ELISA. All HNA-2a antibodies were detectable in MAIGA when diluted sera were applied. Notably, sera containing anti- proteinase 3 (PR3) from patients with Wegener's granulomatosis reacted in MAIGA. In contrast, this antibody showed no reaction in ELISA with purified rHNA-2a. CONCLUSIONS: These results demonstrated that ELISA with rHNA-2a provides a good method for detecting HNA-2a antibodies in human serum. This assay enables to exclude the presence of autoantibody against PR3 in patient's sera, which cannot be differentiated from anti HNA-2a with current serologic methods.
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Authors | Behnaz Bayat, Silke Werth, Ulrich J H Sachs, Sentot Santoso |
Journal | Transfusion
(Transfusion)
Vol. 49
Issue 9
Pg. 1819-24
(Sep 2009)
ISSN: 1537-2995 [Electronic] United States |
PMID | 19497055
(Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
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Chemical References |
- Antibodies
- CD177 protein, human
- GPI-Linked Proteins
- Isoantigens
- Membrane Glycoproteins
- Receptors, Cell Surface
- neutrophil-specific antigen NA1, human
- neutrophil-specific antigen NA2
- Myeloblastin
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Topics |
- Antibodies
(immunology)
- Enzyme-Linked Immunosorbent Assay
(methods)
- GPI-Linked Proteins
- Humans
- Isoantigens
(immunology)
- Membrane Glycoproteins
(immunology)
- Myeloblastin
(immunology)
- Neutrophils
(immunology)
- Receptors, Cell Surface
(immunology)
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