Monoclonal antibodies against alpha toxin of Clostridium perfringens

Abstract

Ten distinct monoclonal antibodies (MAbs) against alpha toxin of Clostridium perfringens were produced by the fusion of SP2/O with spleen cells of micr immunized with alpha toxoid, and alpha toxin mixed with or without ethylenediamine‐tetraacetate (EDTA). The antibody activity was evaluated by antigen‐binding activity in an enzyme linked immunosorbent assay (ELISA), by phospholipase C (PLC)‐neutralizing activity using both egg yolk lecithin and p‐nitrophenylphosphoryl‐choline (PNPPC) hydrolysis reactions and by anti‐lethal activity in mice. Since the toxin‐neutralizing activities of each MAb were not parallel, it has been suggested that the three biological activities may not be located in the same site in the toxin molecule. This report also describes the development of a simple purification of the toxin by affinity chromatography and a sensitive immunoassay for quantitation of the toxin using the monoclonal antibody.

Enhanced Production of Phospholipase C and Perfringolysin O (Alpha and Theta Toxins) in a Gatifloxacin-Resistant Strain of Clostridium perfringens

“…Blood (100 l) was mixed with 10 l each of fluorescein isothiocyanate (FITC)-conjugated anti-human CD42b (a panplatelet marker; green; Coulter, Hialeah, FL) and phycoerythrin (PE)-conjugated anti-human CD11b (a neutrophil marker; red; Coulter) or the relevant conjugated control antibodies. One unit of rPLC (diluted in PBS containing 2.0 mM CaCl 2 and 100 M ZnCl 2 ) or each bacterial supernatant either alone or in combination with neutralizing anti-PLC antibody (clone 1C6; final concentration, 20 g/ml; kindly provided by Hiroko Sato, Japanese National Institute of Health, Tokyo, Japan [26]) was added and the tubes were incubated for 10 min at 37°C. Red blood cells were removed by formic acid lysis, and the remaining cells were fixed in 2% (vol/vol) paraformaldehyde in PBS for flow cytometric analysis on an Epics XL flow cytometer (Beckman Coulter, Fullerton, CA).…”

Progress and problems in vaccination against necrotic enteritis in broiler chickens

“…The alpha toxin is composed of two domains, which are associated with phospholipase C activity (N-domain) and membrane recognition (C-domain) (Naylor et al, 1998). Reports on monoclonal antibodies against alpha toxin led to the finding that monoclonal antibodies, which were capable of neutralizing the phospholipase C activity, were not necessarily effective in neutralizing the haemolytic and lethal activities of the toxin in gangrene models (Sato et al, 1989;Logan et al, 1991). The immune response against the C-domain provided protection against challenge with alpha toxin and also against experimental gas gangrene in mice (Stevens et al, 2004).…”

Clostridium perfringens phospholipase C-induced platelet/leukocyte interactions impede neutrophil diapedesis

“…Phospholipase activity of the stock toxin preparation, determined by p-nitrophenylphosphorylcholine hydrolysis (NPPC) assay (Stevens et al, 1987), was 31 100 units ml 21 , specific activity 19 438 units mg 21 . Neutralizing monoclonal anti-PLC antibody (clone 1C6, murine IgG 1 ) was provided by Dr Hiroko Sato, National Institute of Health, Tokyo, Japan (Sato et al, 1989).…”