IgE and IgG4 epitope mapping by microarray immunoassay reveals the diversity of immune response to the peanut allergen, Ara h 2
IgE and IgG4 epitope mapping by microarray immunoassay reveals the diversity of immune response to the peanut allergen, Ara h 2
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DOI：
10.1016/j.jaci.2005.06.033
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发表时间：
2005-10-01
影响因子：
14.2
通讯作者：
Sampson, HA
中科院分区：
文献类型：
--
作者：
Shreffler, WG;Lencer, DA;Sampson, HA
背景资料：对过敏原的抗体反应的详细评估揭示了关于宿主反应和抗原结构的临床相关信息。目的：设计一种可用于花生过敏原Ara h 2的IgE和IgG（4）表位定位的冗余肽芯片。方法：合成6组重叠肽，与环氧衍生化的载玻片进行位点特异性结合，重复3次。肽的长度为10、15或20个氨基酸，偏移2或3个氨基酸。共检测了10份对照血清和45份花生过敏血清。结果：用15-mer和20-mer肽段，我们可以确定11个抗原区，而用10-mer肽段只能确定5个抗原区。对照组和患者产生的IgG 4识别相当数量的Ara h 2肽，尽管优势表位不同。正如预期的，患者IgE结合了更多数量的Ara h 2肽（9.4%对0.9%）。患者识别的IgE和IgG 4表位基本相同，IgE和IgG 4信号之间呈正相关，提示协同调节。肽结合模式的聚类分析证实了抗体-肽相互作用的特异性，并被用来定义9个核心表位，从6到16个残基的长度-其中7（78%）同意与以前的mapping.Conclusion：表位定位芯片肽免疫分析和聚类分析揭示了患者间的异质性和更详细的地图。
Background: Detailed assessment of antibody responses to allergens reveals clinically relevant information about both host response and antigen structure. Microarray technology offers advantages of scale and parallel design over previous methods of epitope mapping.Objective: We designed a redundant peptide microarray for IgE and IgG(4) epitope mapping of the previously characterized peanut allergen, Ara h 2.Methods: Six complete sets of overlapping peptides were commercially synthesized and site-specifically bound to epoxy-derivatized glass slides in triplicate. Peptides were 10, 15, or 20 amino acids in length with an offset of either 2 or 3 amino acids. A total of 10 control and 45 peanut-allergic sera were assayed. Specific IgE and IgG4 were detected by using fluorochrome-labeled monoclonal secondary antibodies.Results: By using 15-mer and 20-mer peptides, we could define 11 antigenic regions, whereas only 5 were identifiable using 10-mers. Controls and patients produced IgG4 recognizing a comparable number of Ara h 2 peptides, although the dominant epitopes were distinct. As expected, patient IgE bound a larger number of Ara h 2 peptides (9.4% vs 0.9%). IgE and IgG4 epitopes recognized by patients were largely the same, and there was a positive association between IgE and IgG4 signal, suggesting coordinate regulation. Cluster analysis of peptide binding patterns confirmed the specificity of antibody-peptide interactions and was used to define 9 core epitopes ranging from 6 to 16 residues in length-7 of which (78%) agreed with previous mapping.Conclusion: Epitope mapping by microarray peptide immunoassay and cluster analysis reveals interpatient heterogeneity and a more detailed map.