Development of a double-recombinant antibody sandwich ELISA for quantitative detection of epsilon toxoid concentration i
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RESEARCH ARTICLE
Open Access
Development of a double-recombinant antibody sandwich ELISA for quantitative detection of epsilon toxoid concentration in inactivated Clostridium perfringens vaccines Maryam Alibeiki1, Mehdi Golchin2 and Mohammad Tabatabaei1*
Abstract Background: Epsilon toxin (ETX) causes a commonly fatal enterotoxemia in domestic animals. Also, ETX causes serious economic losses to animal husbandry. In this study, we selected several clones against ETX using repertoires displayed on filamentous phage. Anti-ETX specific clones were enriched by binding to immobilized antigen, followed by elution and re-propagation of phage. After multiple rounds of binding selection, ELISA analysis showed that most isolated clones had high affinity and specificity for ETX. Results: Two recombinant monoclonal antibodies against ETX were isolated by phage display technology. B1 phage VH antibody isolated from DAb library and G2 soluble scFv antibody isolated from Tomlinson I + J libraries have been applied as the capture and detection antibodies for developing an ETX sandwich ELISA test, respectively. Conclusions: Designed ETX sandwich ELISA could be a valuable tool for quantitative detection of ETX in inactivated commercial vaccines against enterotoxemia. Keywords: Epsilon toxin, Clostridium perfringens, Recombinant antibody, Phage display, Sandwich ELISA
Background Epsilon toxin (ETX) is a 33 kDa protein and one of the pore-forming toxins synthesized by Clostridium perfringens type B and D strains [1]. ETX has high potency and the Centers for Disease Control and Prevention (CDC) categorized epsilon toxin as the second highest priority agents (Category B) [2]. This toxin plays a significant role in causing enterotoxemia in domestic ruminants, especially sheep, and can cause sudden death and severe economic losses [3]. Enterotoxemia begins when C. perfringens type B or D * Correspondence: [email protected] 1 Department of Pathobiology, Faculty of Veterinary Medicine, Shiraz University, Shiraz, Iran Full list of author information is available at the end of the article
strains secrete ETX prototoxin into the intestinal lumen [4]. At first, the toxin is inactive, but it is activated after cleaving the 13 N-terminal and 22 C-terminal residues by proteases [5]. After activation, ETX forms the pores across the cell membrane of ruminant cells and alters the permeability of cell monolayers, such as epithelium and endothelium, and leads to necrotic lesions and perivascular edema in different tissues, especially in kidney and brain cells [6, 7]. An effective manner to control ETX-induced enterotoxemia in domestic ruminants is vaccination. Commercially available vaccines are based on inactivated toxins isolated from formaldehyde-treated bacterial culture filtrate. Unfortunately, these vaccines usually have many production defects and have variable immunogenicity. One
© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing,
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