title: Detection of palytoxin-like compounds by a flow cytometry-based immunoassay supported by functional and analytical methods.
abstract: Palytoxin (PLTX) is a complex marine toxin produced by zoanthids (i.e. Palythoa), dinoflagellates (Ostreopsis) and cyanobacteria (Trichodesmium). PLTX outbreaks are usually associated with Indo-Pacific waters, however their recent repeated occurrence in Mediterranean-European Atlantic coasts demonstrate their current worldwide distribution. Human sickness and fatalities have been associated with toxic algal blooms and ingestion of seafood contaminated with PLTX-like molecules. These toxins represent a serious threat to human health. There is an immediate need to develop easy-to-use, rapid detection methods due to the lack of validated protocols for their detection and quantification. We have developed an immuno-detection method for PLTX-like molecules based on the use of microspheres coupled to flow-cytometry detection (Luminex 200™). The assay consisted of the competition between free PLTX-like compounds in solution and PLTX immobilized on the surface of microspheres for binding to a specific monoclonal anti-PLTX antibody. This method displays an IC50 of 1.83 ± 0.21 nM and a dynamic range of 0.47-6.54 nM for PLTX. An easy-to-perform extraction protocol, based on a mixture of methanol and acetate buffer, was applied to spiked mussel samples providing a recovery rate of 104 ± 8% and a range of detection from 374 ± 81 to 4430 ± 150 μg kg(-1) when assayed with this method. Extracts of Ostreopsis cf. siamensis and Palythoa tuberculosa were tested and yielded positive results for PLTX-like molecules. However, the data obtained for the coral sample suggested that this antibody did not detect 42-OH-PLTX efficiently. The same samples were further analyzed using a neuroblastoma cytotoxicity assay and UPLC-IT-TOF spectrometry, which also pointed to the presence of PLTX-like compounds. Therefore, this single detection method for PLTX provides a semi-quantitative tool useful for the screening of PLTX-like molecules in different matrixes.
authors.affiliation: Departamento de Farmacología, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27002, Lugo, Spain.
authors.forename: María
authors.initials: M
authors.lastname: Fraga
authors.affiliation: Departamento de Farmacología, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27002, Lugo, Spain. Electronic address: natalia.vilarino@usc.es.
authors.forename: Natalia
authors.initials: N
authors.lastname: Vilariño
authors.affiliation: Departamento de Farmacología, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27002, Lugo, Spain.
authors.forename: M Carmen
authors.initials: MC
authors.lastname: Louzao
authors.affiliation: Departamento de Farmacología, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27002, Lugo, Spain.
authors.forename: Diego A
authors.initials: DA
authors.lastname: Fernández
authors.affiliation: Diagnostic Systems Division, U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD, USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Poli
authors.affiliation: Departamento de Farmacología, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27002, Lugo, Spain. Electronic address: luis.botana@usc.es.
authors.forename: Luis M
authors.initials: LM
authors.lastname: Botana
mesh_terms: D000178:Acrylamides / Q000032:analysis*; D003064:Cnidarian Venoms; D005434:Flow Cytometry; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D000095702:Polyether Toxins
chemical_list: D000178:Acrylamides; D003064:Cnidarian Venoms; C010272:palytoxin; D000095702:Polyether Toxins
keywords: Flow-cytometry system; Microsphere-based array; Palytoxin; Palytoxin-like molecules; Screening method