Characterization of an anti-Bla g 1 scFv: epitope mapping and cross-reactivity.
Characterization of an anti-Bla g 1 scFv: epitope mapping and cross-reactivity.
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DOI：
10.1016/j.molimm.2014.02.003
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发表时间：
2014-06
影响因子：
3.6
通讯作者：
London RE
中科院分区：
文献类型：
--
作者：
Mueller GA;Ankney JA;Glesner J;Khurana T;Edwards LL;Pedersen LC;Perera L;Slater JE;Pomés A;London RE
Bla g 1是德国布拉特氏菌的一种主要过敏原，也是用于评估蟑螂过敏原暴露的主要过敏原之一。为了更好地了解与其他组1蟑螂变应原和患者IgE表位的交叉反应，绘制了抗Bla g1单链抗体的表位图。用X射线结晶学确定了单链抗体的结构。通过丙氨酸扫描定点突变和酶联免疫吸附试验确定了Bla g1上的单链抗体表位。与野生型变应原相比，26个rBla g 1-GST丙氨酸突变体与ScFv结合的变异被评估。6个突变体的ScFv结合亲和力有显著差异。这些突变聚集在一起形成一个不连续的表位，主要由Bla g 1的两个螺旋组成。根据结果对变应原-单链抗体复合体进行建模，发现该表位区域与Per a 1序列相似性较低，特别是在被确定为与Bla g 1结合功能重要的残基中。事实上，在美国蟑螂提取物中，ScFv未能结合Per A 1。ScFv不能抑制高度蟑螂过敏患者的IgE抗体与Bla g1的结合。根据scFv封闭的Bla g1的表面积，可以推测患者IgE与Bla g1相互作用的区域。该单链抗体可作为捕捉抗体用于检测IgE，或在环境暴露试验中用于区分Bla g 1和Per A 1。
Bla g 1 is a major allergen from Blatella germanica and one of the primary allergens used to assess cockroach allergen exposure. The epitope of an anti-Bla g 1 scFv was mapped in order to better understand cross reactivity with other group 1 cockroach allergens and patient IgE epitopes. X-ray crystallography was used to determine the structure of the scFv. The scFv epitope on Bla g 1 was located by alanine scanning site-directed mutagenesis and ELISA. Twenty-six rBla g 1-GST alanine mutants were evaluated for variations in binding to the scFv compared to the wild type allergen. Six mutants showed a significant difference in scFv binding affinity. These mutations clustered to form a discontinuous epitope mainly comprising two helices of Bla g 1. The allergen-scFv complex was modeled based on the results, and the epitope region was found to have low sequence similarity with Per a 1, especially among the residues identified as functionally important for the scFv binding to Bla g 1. Indeed, the scFv failed to bind Per a 1 in American cockroach extract. The scFv was unable to inhibit the binding of IgE antibodies from a highly cockroach allergic patient to Bla g 1. Based on the surface area of Bla g 1 occluded by the scFv, putative regions of patient IgE–Bla g 1 interactions can be inferred. This scFv could be best utilized as a capture antibody in an IgE detection ELISA, or to differentiate Bla g 1 from Per a 1 in environmental exposure assays.
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DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
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通讯作者：
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影响因子：
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作者：
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影响因子：
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通讯作者：
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影响因子：
4.8
作者：
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通讯作者：
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影响因子：
14.2
作者：
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通讯作者：
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