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

:由于需要区分同源宿主蛋白和病原体蛋白,并确保针对肽的抗体也能与抗体结合,因此使用基于抗体的测试(例如ELISA)(酶联免疫吸附测定)对真核寄生虫感染的诊断通常存在问题。肽在其三维蛋白质结构的背景下。由线虫引起的丝虫病是马来亚布鲁吉(Brugia malayi),是一种重要的世界性热带病,在白天,其寄生虫会从血液中消失,从而妨碍了标准的微观诊断方法。为了解决该问题,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芽孢杆菌分泌的天冬酰胺基-tRNA合成酶(AsnRS)。将马来芽孢杆菌和人AsnRS氨基酸序列进行比对以鉴定相对不保守的区域,并且使用马来芽孢杆菌AsnRS的1.9 A晶体学结构鉴定表面可及且可用于抗体结合的肽基区域。使用Sequery和SSA(叠加结构分析)软件分析这些肽中的哪一种最有可能作为合成肽保持其天然构象,并通过NMR证实了其预测的螺旋结构。合成22个残基的肽以产生鼠mAb。鉴定出四个IgG(1)mAb,它们可识别合成肽和全长寄生虫AsnRS,但不能识别人AsnRS。 mAb的特异性和亲和力已通过Western印迹,免疫组织化学,表面等离振子共振和酶抑制试验得以证实。这些结果支持结构建模成功地选择了肽,以产生与天然蛋白结合的选择性抗体。

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