原标题：桦木花粉变应原bet v 1定量参考抗体表位特异性特征的鉴定
① 免疫治疗变应原提取物的一致性需要通过精确的定量试验来记录；② 单克隆抗体5B4对应的肽序列与患者血清IgE的结合位点重叠；③ 单克隆抗体6H4识别与5B4结合位点相反的构象表位；④ 两种单克隆抗体与桦木花粉提取物中90%以上的Bet v 1有相互作用；⑤ 单克隆抗体5B4和6H4可以识别Bet v 1绝大多数异构体上的不同表位。
Characterization of epitope specificities of reference antibodies used for the quantification of the birch pollen allergen Bet v 1
Background: Accurate allergen quantification is needed to document the consistency of allergen extracts used for immunotherapy. Herein, we characterize the epitope specificities of two monoclonal antibodies used in an ELISA for the quantification of the major birch pollen allergen Bet v 1, established as a reference by the BSP090 European project.
Methods: The ability of mAbs 5B4 and 6H4 to recognize Bet v 1 isoforms was addressed by immunochromatography. The capacity of each mAb to compete with patients’ IgE for binding to Bet v 1 was measured by ELISA inhibition. Epitope mapping was performed by pepscan analysis, site-directed mutagenesis and hydrogen/deuterium exchange-mass spectrometry.
Results: The 5B4 epitope corresponds to a peptide sequence (I56-K68) overlapping with the binding sites of patients’ serum IgEs. Mutation of residues P59, E60 and K65 abolishes 5B4 binding to Bet v 1 and reduces the level of IgE recognition. In contrast, 6H4 recognizes a conformational epitope lying opposite to the 5B4 binding site, involving residues located in segments I44-K55 and R70-F79.Substitution of E45 reduces the binding capacity of 6H4, confirming that it is critical for the interaction. Both mAbs interact with >90% of Bet v 1 content present in the birch pollen extract,while displaying a weak cross-reactivity with other allergens of the PR-10 family.
Conclusions: MAbs 5B4 and 6H4 recognize structurally distinct epitopes present in the vast majority of Bet v 1 isoforms. These results support the relevance as a reference method of the Bet v 1-specific quantitative ELISA adopted by the European Pharmacopoeia.
Emmanuel Nony, Research Department, Stallergenes Greer
Sébastien Brier, Maxime Le Mignon, Karine Jain, Constance Lebrun, François Peurois,Christine Kellenberger, Véronique Bordas-Le Floch, Laurent Mascarell, Emmanuel Nony1 and Philippe Moingeon