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

Abstract 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 (