Isolation and purification of O and H antigens from Salmonella Enteritidis as diagnostic tool

Abstract

Lipopolysaccharide (LPS) and flagellin are the major antigenic structures of Salmonella Enteritidis. Purified antigens are essential components for the diagnosis of and research on Salmonella Enteritidis. In this study, LPS were isolated and purified with modified hot phenol-water procedure in a yield of 6%. The flagellin isolation and purification were done by strong shaking with glass pearl beads with 3.9 mg/ml protein content. Purity was demonstrated on SDS-PAGE, ELISA and with colorimetric assay for both isolated antigens. The antigen-mediated ELISA results exhibited immunoreactive properties for both LPS and flagellar antigen specific for Salmonella Enteritidis O:9 and H:g,m, respectively. The findings demonstrated low reactivity with Mab O:9 for isolated LPS due to their monoepitopic specificity. However, isolated flagellin reactivity was high with Mab H:g,m related to their cross-reactivity potential between H:g and H:m. Additionally in this study.the results indicated that similar antigenic structures have alternative potential for antigenic isolation of Salmonella Enteritidis, such as Salmonella Gallinarum for LPS antigen and Salmonella Adeyo for flagellar antigen isolation.

Evaluation of the immunomodulatory properties in mice and in vitro anti-inflammatory activity of cycloartane type saponins from Astragalus species

“…Goat antimouse IgG, IgG1 and IgG2b peroxidase conjugate were from Southern Biotech. Assoc., Birmingham, AL, USA; lipopolysaccharide (LPS) was prepared from Salmonella Enteritidis 64K by hot water phenol extraction method (Nalbantsoy et al, 2010). Anti IL-2R␣ receptor (CD25) and CD69 antibodies were from R&D, USA; Histostain-Plus Bulk Kits was from Zymed, South San Francisco, USA; Mayer's hematoxylin was from Zymed Laboratories, USA; paraformaldehyde was from Sigma Chemical Co., St. Louis, USA.…”

“…Mice were injected with Salmonella Enteritidis derived LPS (Nalbantsoy et al, 2010) via single intraperitoneal injection (ip) of 12.5 g/mouse (100 l total injection volume) mixed with 60 g/mouse Ast VII or Mac B. The LPS dose was based on the results of two preliminary studies (data not shown).…”

Adjuvant effects of Astragalus saponins Macrophyllosaponin B and Astragaloside VII

“…Goat anti-mouse IgG, IgG1 and IgG2b peroxidase conjugate were from Southern Biotech. Assoc., Birmingham, AL, USA; lipopolysaccharide (LPS); prepared from Salmonella Enteritidis 64 K by hot water phenol extraction method (Nalbantsoy et al, 2010). All other chemicals were of AR grade.…”

Determination ofin vivotoxicity andin vitrocytotoxicity of lipopolysaccharide isolated fromSalmonellaEnteritidis and its potential use for production of polyclonal antibody

“…The LPS isolation was done by modified Westphal and Jann procedure as lyophilised powder (Westphal & Jann, 1965;Nalbantsoy, Karaboz, & Deliloglu-Gurhan, 2010a). Estimation of the Kdo residues was performed by a modified thiobarbituric acid (TBA) assay as reported by Toman and Skultety (1996).…”