anterior kidney macrophages [3]. easily detected in fish. While several Rabbit Polyclonal to Cytochrome P450 2A7 antibodies against lysozyme are commercially available for use in higher vertebrates, neither reacts with lysozyme in fish. In this study, a monoclonal antibody (mAb) for detecting and quantifying lysozyme was developed from mummichog,Fundulus heteroclitus, myeloid cells that also recognizes hen egg lysozyme (HEL), then tested for cross-reactivity in different species of teleosts, A single protein of 1415 kDa mass was identified by the mAb in fish cell lysates and plasma samples, as well as denatured HEL. Total circulating lysozyme protein was compared to lysozyme activity using standard ELISA procedures and was found to correlate with enzymatic activity. Using mAb M24-2, intracellular lysozyme protein was detected in formalin-fixed and permeabilized lymphoid cells adhered to glass cover slips. Moreover, mAb M24-2 localizes lysozyme to myeloid cells. Finally, it was demonstrated that mAb M24-2 is suitable for immunohistochemistry in that lysozyme could be detected AK-1 in plastic-embedded lymphoid tissues. Keywords:fish lysozyme, mAb M24-2, phagocytes, lysozyme quantification, mummichog,Fundulus heteroclitus == Introduction == Lysozyme is one of several anti-microbial proteins associated with front line, innate immunity in all vertebrates. This enzyme (muramidase) breaks the -1,4 glycosidic bond betweenN-acetylmuramic acid andN-acetylglucosamine in the cell wall of gram positive bacteria, and in association with complement components gram negative bacteria may be affected as well [13]. In higher vertebrates, lysozyme is an abundant component of primary and secondary granules of granulocytes [4], is secreted by macrophages [57], and circulating enzymatic activity is a biomarker for systemic inflammation [8]. Recent studies show that lysozyme has anti-oxidant properties [9], while others suggest it enhances the effects of complement [10] and may act as an opsonin to enhance phagocytosis by neutrophils and macrophages [1112]. In fish, lysozyme genes are expressed in cells of myeloid origin [1314] and very early in development [14], while enzymatic activity of the protein can be found in mucus, serum, and eggs [1518]. There are also seasonal variations in circulating lysozyme activity [19]. Lysozymes of vertebrates are classified as either c-type or g-type, as both types are expressed in all vertebrates, with several subtypes of each being found among the taxa [20]. The relative contribution of each lysozyme type to overall enzymatic activity is unclear at this point, but gene expression profiles seem to vary among tissues and between species in response to pathogenic challenge [20]. Virtually all studies to date suggest that monocytes, macrophages and granulocytes are AK-1 the main source of lysozyme. For example, exposure to -glucan and lipopolysaccharide (LPS) increase lysozyme gene expression and enzyme activity AK-1 in Atlantic salmon,Salmo salarL. anterior kidney macrophages [3]. The kinetics of lysozyme gene expression and enzyme activity in this study were both time and dose dependent, with LPS being a stronger inducer. Other studies show that immunization of adult female fish with potential bacterial pathogens leads to increased circulating lysozyme, which may be passed to the eggs after spawning [18]. Furthermore, exposure to chemical contaminants, including mercury, selenium, copper, herbicides, and co-planar PCBs modulate circulating lysozyme activity in fish [2124]. Lysozyme activity in a biological sample can be quantified using simple techniques [2526]. Typically, a volume of fish serum, plasma, mucus, cell lysate, or egg yolk is mixed with a suspension of heat killedMycococcus lysodieticusat an acidic pH. The optical density AK-1 of the solution is then measured in the beginning and at a later time point for spectrophotometric analysis, or 18 24 hr later for agar-based systems that quantify a zone of lysis. The rate of decrease in optical density or diameter of the zone of lysis is compared to a known lysozyme standard such as hen egg lysozyme (HEL), and enzymatic activity is calculated as units of HEL activity [25] or HEL equivalents [27]. The primary draw back for measuring lysozyme activity is that a minimum.
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