A Novel Panel of Rabbit Monoclonal Antibodies and Their Diverse Applications Including Inhibition of Clostridium perfringens Epsilon Toxin Oligomerization.

Jennifer Linden,Sylvia Haigh,Giovanna Antognetti,Paige Winokour,Ellen Cahir-Mcfarland,Kiel Telesford,Werner Meier,Timothy Vartanian,Abhishek Datta,Samantha Shetty,Tao Wang

doi:10.3390/antib7040037

Jennifer Linden, Sylvia Haigh + Show 9 more

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https://doi.org/10.3390/antib7040037

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Abstract

The pore-forming epsilon toxin (ETX) produced by Clostridium perfringens is among the most lethal bacterial toxins known. Sensitive antibody-based reagents are needed to detect toxin, distinguish mechanisms of cell death, and prevent ETX toxicity. Using B-cell immuno-panning and cloning techniques, seven ETX-specific monoclonal antibodies were generated from immunized rabbits. ETX specificity and sensitivity were evaluated via western blot, ELISA, immunocytochemistry (ICC), and flow cytometry. ETX-neutralizing function was evaluated both in vitro and in vivo. All antibodies recognized both purified ETX and epsilon protoxin via western blot with two capable of detecting the ETX-oligomer complex. Four antibodies detected ETX via ELISA and three detected ETX bound to cells via ICC or flow cytometry. Several antibodies prevented ETX-induced cell death by either preventing ETX binding or by blocking ETX oligomerization. Antibodies that blocked ETX oligomerization inhibited ETX endocytosis and cellular vacuolation. Importantly, one of the oligomerization-blocking antibodies was able to protect against ETX-induced death post-ETX exposure in vitro and in vivo. Here we describe the production of a panel of rabbit monoclonal anti-ETX antibodies and their use in various biological assays. Antibodies possessing differential specificity to ETX in particular conformations will aid in the mechanistic studies of ETX cytotoxicity, while those with ETX-neutralizing function may be useful in preventing ETX-mediated mortality.

Highlights

Epsilon toxin (ETX), produced by Clostridium perfringens (C. perfringens) toxinotypes B and D, is responsible for causing enterotoxaemia, an economically devastating disease in sheep and other ruminant livestock [1,2]
ETX is listed as a category B bioterrorism agent by the Center for Disease Control (CDC)
We describe generation of seven anti-ETX rabbit monoclonal antibodies and identify which of these antibodies are suitable for various immunoassays including: western blot, immunocytochemistry (ICC), and flow cytometry for detection of ETX and pETX on the ETX-susceptible CHO cell line expressing a rat MAL fusion protein [15]

Summary

Introduction

Epsilon toxin (ETX), produced by Clostridium perfringens (C. perfringens) toxinotypes B and D, is responsible for causing enterotoxaemia, an economically devastating disease in sheep and other ruminant livestock [1,2]. ETX is listed as a category B bioterrorism agent by the Center for Disease Control (CDC). ETX is secreted by C. perfringens type B and D during exponential growth as a relatively weak, 33 kDa protoxin (pETX). Enzymatic activation by the proteases trypsin, chymotrypsin, and lambda toxin increases its potency one thousand-fold. Each enzyme cleaves at distinct amino acid residues at both the C and N termini, producing active toxin approximately 27 kDa in size. Maximum potency is achieved when pETX is activated with both trypsin and chymotrypsin [11,12,13]

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