Using structural analysis to generate parasite-selective monoclonal antibodies.
Using structural analysis to generate parasite-selective monoclonal antibodies.
复制标题
使用结构分析生成寄生虫选择性单克隆抗体。
DOI：
10.1110/ps.073429808
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发表时间：
2008
期刊：
影响因子：
--
通讯作者：
Kuhn,LeslieA
中科院分区：
文献类型：
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作者：
Kron,MichaelA;Cichanowicz,Sam;Hendrick,Angela;Liu,Aizhuo;Leykam,Joseph;Kuhn,LeslieA
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 fromB. malayi.B. malayiand human AsnRS amino acid sequences were aligned to identify regions that are relatively unconserved, and a 1.9 Å crystallographic structure ofB. malayiAsnRS 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 IgG1mAbs 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.
