Chemiluminescent determination of reactive oxygen species using Pholasin luminophore in birds and insect phagocytes
Abstract
Phagocytosis is one of the most importantinnate immunity mechanisms which prevents organism againstpathogens overcoming the natural barriers. There are severalmethods widely used for evaluation of phagocytosis efficiency.One of them is to measure the level of reactive oxygen species(ROS) produced by phagocytes - respiratory burst of phagocytes.ROS production can be elicited by addition of activators such aslipopolysaccharide (LPS), zymosan, starch particles,phorbol-12-miristate-13-acetate (PMA) orN-formyl-methionyl-leucyl-phenylalanin (fMLP).
Produced ROScause oxidation of luminophore which subsequently emits theenergy in form of light measured by luminometers. Luminol is themost widely used luminophore.
Purpose of this work was to findmore sensitive luminophore and optimize chemiluminescent (CL)measurement of oxidative burst for birds' and insects' samples.As more sensitive (with app. thirty times higher luminescentsignal than luminol) was found the luminophore Pholasin - photoprotein extracted from bioluminescent mollusc Pholasdactylus. For CL measurement of oxidative burst in birds wecombined this luminophore with Salmonella enterica andEscherichia coli LPS; an ideal activators that give fast andstable enhancement of ROS production.
So far there are fourstudies of heterophil respiratory burst determination in avianimmunology with the use of Pholasin and this is the first studyon great tits (Parus major). Furthermore, we tried to optimizethis assay also for insect haemolymph, where the level of ROS isvery low and luminol as luminophore is not sensitive enough.
Wefound out that haemocyte suspension must be diluted 100x or1000x to reduce antioxidant capacity of Galleria mellonellahaemolymph. Taken together we proved that Pholasin can be usedas luminophore for detection of ROS produced by insects andavian blood cells.