Chemiluminescent determination of reactive oxygen species using Pholasin luminophore in birds and insect phagocytes
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| Year of publication | 2014 |
| Type | Conference abstract |
| MU Faculty or unit | |
| Citation | |
| Description | Phagocytosis is one of the most important innate immunity mechanisms which prevents organism against pathogens overcoming the natural barriers. There are several methods 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 as lipopolysaccharide (LPS), zymosan, starch particles, phorbol-12-miristate-13-acetate (PMA) or N-formyl-methionyl-leucyl-phenylalanin (fMLP). Produced ROS cause oxidation of luminophore which subsequently emits the energy in form of light measured by luminometers. Luminol is the most widely used luminophore. On the other hand evaluation of respiratory burst using this method on birds or insect samples is not possible due to a low production of ROS and low sensitivity of luminol. In birds is production limited by absence of myeloperoxidase, enzyme responsible for the main production of ROS and hypochlorous acid. Purpose of this work was to find more sensitive luminophore and optimize chemiluminescent (CL) measurement of oxidative burst for birds' and insects' samples. As more sensitive (with app. thirty times higher luminescent signal than luminol) was found the luminophore Pholasin - photoprotein extracted from bioluminescent mollusc Pholas dactylus. For CL measurement of oxidative burst in birds we combined this luminophore with Salmonella enterica and Escherichia coli LPS; an ideal activators that give fast and stable enhancement of ROS production (Fig. 1). Data acquired by this assay can be subsequently compared to results of other experiments by evaluation of peak of the reaction (maximum intensity of respiratory burst in counts per second, CPS) or integral of the reaction. So far there are four studies of heterophil respiratory burst determination in birds' immunology with the use of Pholasin and the first study on great tits (Parus major). |
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