Diagnosis of eukaryotic parasitic infection using antibody-based tests such as ELISAs (enzyme-linked immunosorbent assays) is often problematic because of the need to differentiate between homologous host and pathogen proteins and to ensure that antibodies raised against a peptide will also bind to the peptide in the context of its three-dimensional protein structure. Filariasis caused by the nematode, Brugia malayi, is an important worldwide tropical disease in which parasites disappear from the bloodstream during daylight hours, thus hampering standard microscopic diagnostic methods. To address this problem, a structural approach was used to develop monoclonal antibodies (mAbs) that detect asparaginyl-tRNA synthetase (AsnRS) secreted from B. malayi. B. malayi and human AsnRS amino acid sequences were aligned to identify regions that are relatively unconserved, and a 1.9 A crystallographic structure of B. malayi AsnRS was used to identify peptidyl regions that are surface accessible and available for antibody binding. Sequery and SSA (Superpositional Structural Analysis) software was used to analyze which of these peptides was most likely to maintain its native conformation as a synthetic peptide, and its predicted helical structure was confirmed by NMR. A 22-residue peptide was synthesized to produce murine mAbs. Four IgG(1) mAbs were identified that recognized the synthetic peptide and the full-length parasite AsnRS, but not human AsnRS. The specificity and affinity of mAbs was confirmed by Western blot, immunohistochemistry, surface plasmon resonance, and enzyme inhibition assays. These results support the success of structural modeling to choose peptides for raising selective antibodies that bind to the native protein.

译文

使用基于抗体的测试 (例如ELISAs (酶联免疫吸附测定)) 诊断真核寄生虫感染通常是有问题的,因为需要区分同源宿主和病原体蛋白,并确保针对肽产生的抗体也会结合肽在其三维蛋白质结构的背景下。由线虫Brugia malayi引起的丝虫病是一种重要的世界性热带疾病,其中寄生虫在白天从血液中消失,从而阻碍了标准的显微镜诊断方法。为了解决这个问题,使用了一种结构方法来开发单克隆抗体 (mab),该单克隆抗体可检测马来亚芽胞杆菌分泌的天冬酰基-tRNA合成酶 (AsnRS)。对马来亚杆菌和人AsnRS氨基酸序列进行比对以鉴定相对不保守的区域,并且使用马来亚杆菌AsnRS的1.9晶体结构来鉴定表面可及且可用于抗体结合的肽基区域。Sequery和SSA (叠加结构分析) 软件用于分析这些肽中的哪一种最有可能保持其天然构象为合成肽,并通过NMR确认了其预测的螺旋结构。合成了22个残基的肽以产生鼠mab。鉴定出四种IgG(1) mab,它们识别合成肽和全长寄生虫AsnRS,但不识别人类AsnRS。通过蛋白质印迹,免疫组织化学,表面等离子体共振和酶抑制试验证实了mab的特异性和亲和力。这些结果支持结构建模的成功,以选择用于产生与天然蛋白质结合的选择性抗体的肽。

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