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Pine wood nematode (PWN)-secretory antigen 571 as a biomarker for the PWN and its monoclonal antibodies for detecting PWN and its infected pine tree
Journal of Asia-Pacific Entomology ( IF 1.1 ) Pub Date : 2020-07-21 , DOI: 10.1016/j.aspen.2020.07.009
Angelina F. Osabutey , A-Young Kim , Phoung Nguyen , Kyu Ha Oh , Hyerim Han , Young Ho Koh

The biochemical characteristics of pine wood nematode (PWN)-secretory antigen 571 (PWN-SA571) identified from the PWN secretome and PWN-infected pine tree extracts (PWNIPT) were investigated. PWN-SA571 has homologs in various nematodes in diverse families and a signal peptide at the amino-terminal end. Since it was not possible to express the full-length PWN-SA571 protein, two peptides showing high antigenicity were synthesized and used as antigens for generating monoclonal antibodies (Mabs). Mab-secreting fusion hybridoma cell lines were selected based on immunoreactivity to free peptide and BSA-conjugated peptide (BSA pep) antigens by enzyme-linked immunosorbent assay for establishing Mab-secreting hybridoma lines. Ascites containing PWN-SA571-specific Mabs showed stronger immunoreactivity to PWNIPT than to healthy pine tree extracts. In addition, PWN-SA571-specific Mabs could recognize less than 20 ng of BSA pep in lateral flow assays. Furthermore, purified biotin-conjugated PWN-SA571-specific Mab IgG or IgM were able to detect BSA pep (<15 ng) and PWN extracts (<600 ng). Taken together, these results demonstrate that PWN-SA571-specific Mabs could detect PWN or PWNIPT extracts by ELISA, LFA, or dot blot analysis.



中文翻译:

松木线虫(PWN)分泌抗原571作为PWN的生物标志物及其单克隆抗体,用于检测PWN及其感染的松树

研究了从PWN分泌组和感染PWN的松树提取物(PWNIPT)中鉴定的松木线虫(PWN)分泌抗原571(PWN-SA571)的生化特性。PWN-SA571在不同家族的各种线虫中具有同源物,并且在氨基末端具有信号肽。由于不可能表达全长PWN-SA571蛋白,因此合成了两个显示出高抗原性的肽,并将其用作产生单克隆抗体(Mabs)的抗原。根据对游离肽和BSA结合肽(BSA pep)抗原的免疫反应性,通过酶联免疫吸附法选择单克隆抗体分泌型融合杂交瘤细胞系,以建立单克隆抗体分泌型杂交瘤细胞系。含有PWN-SA571特异性单克隆抗体的腹水对PWNIPT的免疫反应性强于对健康松树提取物的免疫反应性。此外,PWN-SA571特异性单克隆抗体在侧向流动检测中可识别不到20 ng的BSA pep。此外,纯化的结合生物素的PWN-SA571特异性Mab IgG或IgM能够检测BSA pep(<15 ng)和PWN提取物(<600 ng)。综上所述,这些结果表明,PWN-SA571特异性单克隆抗体可以通过ELISA,LFA或斑点印迹分析检测PWN或PWNIPT提取物。

更新日期:2020-07-21
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