A Combination of Biochemical and Proteomic Analyses Reveals Bx-LEC-1 as an Antigenic Target for the Monoclonal Antibody 3-2A7-2H5-D9-F10 Specific to the Pine Wood Nematode

Abstract

Pine wilt disease (PWD) is one of the most devastating forest diseases in Asia and Europe. The pine wood nematode, Bursaphelenchus xylophilus, has been identified as the pathogen underlying PWD, although the pathology is not completely understood. At present, diagnosis and confirmation of PWD are time consuming tasks that require nematode extraction and microscopic examination. To develop a more efficient detection method for B. xylophilus, we first generated monoclonal antibodies (MAbs) specific to B. xylophilus. Among 2304 hybridoma fusions screened, a hybridoma clone named 3-2A7-2H5 recognized a single protein from B. xylophilus specifically, but not those from other closely related nematodes. We finally selected the MAb clone 3-2A7-2H5-D9-F10 (D9-F10) for further studies. To identify the antigenic target of MAb-D9-F10, we analyzed proteins in spots, fractions, or bands isolated from SDS-PAGE, two-dimensional electrophoresis, anion exchange chromatography, and immunoprecipitation via nano liquid chromatography electrospray ionization quadrupole ion trap mass spectrometry (nano- The pinewood nematode, Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle (1), is a serious pathogen of the forest, particularly of Pinus species, causing pine wilt disease (PWD) 1 . Even though PWD was first reported in Japan in 1905 (2), the relationship between B. xylophilus and PWD was not elucidated until 1971 (3). Currently, PWD has spread to Far East Asian countries including Korea (4), China (5), and Taiwan (6) as well as to Portugal in the European Union (7). The distribution of B. xylophilus is currently extensive, explaining the seriousness of PWD. LC-ESI-Q-IT-MS). Peptides of galactose-bindingDistribution and development of PWD within pine trees have been described (8 -10). The Japanese pine sawyer beetle, Monochamus alternatus, is the known vector for B. xylo-

The pathogenic human Torsin A in Drosophila activates the unfolded protein response and increases susceptibility to oxidative stress

“…The resulting peptide extracts were pooled, lyophilised in a vacuum centrifuge and stored at 4°C for subsequent protein identification. The protocol for protein identification was published by Lee et al [ 32 ]. A nano LC/MS system consisting of a Surveyor HPLC system (Thermo Scientific, Waltham, MA, USA) and ESI-quadruple IT MS (LCQ Deca XP-Plus, Thermo Scientific, Waltham, MA) equipped with a nano-ESI source was used to perform the MS/MS experiments for protein identification.…”

“…For western blot analysis, 20.0 μg of total protein from each sample was separated on an 10% tri-glycine polyacrylamide SDS gel and then transferred to a nitrocellulose membrane (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The membranes were probed with rat-anti-HSC3 antibody (1:5,000; Babraham Bioscience Technologies Limited, Cambridge, UK) [ 32 ] and mouse monoclonal anti-α-tubulin antibody (1:2,000; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) and were developed using the peroxidase-conjugated goat anti-rat IgG or goat anti-mouse-IgG (Thermo Fisher Scientific) and Supersignal West Pico (Thermo Fisher Scientific). The intensities of the bands were semi-automatically quantified using a wander tool and a histogram function in the Adobe Photoshop program (Adobe, San Jose, CA), as previously published [ 33 ].…”

Development of two alternative Loop‐mediated isothermal amplification tools for detecting pathogenic pine wood nematodes

“…In addition, we have generated and characterized B. xylophilus ‐specific monoclonal antibodies and antigens (Lee et al. ). However, these methods are limited in application for on‐site diagnosis of PWD, requiring sophisticated equipment for performing PCRs in the field or further investments for developing antigen/antibody‐based rapid diagnosis kits for B. xylophilus .…”

Characterization of the molecular features and expression patterns of two serine proteases in Hermetia illucens (Diptera: Stratiomyidae) larvae

“…Three-dimensional (3-D) homology models of Hi-SP1 and Hi-SP2 were generated using the Swiss Model workspace (http://swissmodel.expasy.org/workspace; (8,17)). Hi-SP1 and Hi-SP2 3-D homology models were generated based on a 3-D structure of salmon trypsin (PDB ID = 1HJ8) containing 36.3% aa sequence identity with SP1 and a 3-D structure of Fusarium oxysporum Trypsin (PDB ID = 1PQ7; (6)) containing 49.1% aa sequence identity with SP2, respectively.…”