Preparation and Application for Immunocytochemistry of Antibody to Gallinacin-3, an Antimicrobial Peptide, in Chicken

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The aim of this study was to develop an antibody to gallinacin-3 which is one of the antimicrobial peptides in chickens. The antibody was raised in rabbit using a synthetic peptide of gallinacin-3 (Gal-3), and purified through an affinity column. Specificity of antibody was examined by competitive ELISA using Gal-3-HRP and synthetic Gal-3, or the tissue peptides isolated from the uropygial gland and the uterus. Immunostaining was performed on sections of the trachea, uterus, vagina, cloacal gland and uropygial gland. In the competitive ELISA, co-incubation of Gal-3-HRP with Gal-3 and tissue peptides extracted from uropygialis and uterus caused a clear decline of optical density (A450) value in a dose dependent manner. The epithelial cells in the trachea, uterus, vagina, cloacal gland and uropygial gland stained positive using the prepared gallinacin-3 antibody. Sections incubated with antibody absorbed with Gal-3 (control staining) resulted in negative staining. These results suggest that the antibody to gallinacin-3 prepared in the current study is useful for immunocytochemical identification of gallinacin-3 in chickens. The current immunocytochemical results suggest the presence of gallinacin-3 in the epithelial cells of trachea, oviduct, cloacal gland and uropygialis.