A Novel Panel of Rabbit Monoclonal Antibodies and Their Diverse Applications Including Inhibition of <i>Clostridium perfringens</i> Epsilon Toxin Oligomerization
The pore-forming epsilon toxin (ETX) produced by <i>Clostridium perfringens</i> 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 c...
| Main Authors: | , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2018-10-01
|
| Series: | Antibodies |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2073-4468/7/4/37 |
| _version_ | 1818574225052532736 |
|---|---|
| author | Jennifer R. Linden Kiel Telesford Samantha Shetty Paige Winokour Sylvia Haigh Ellen Cahir-McFarland Giovanna Antognetti Abhishek Datta Tao Wang Werner Meier Timothy Vartanian |
| author_facet | Jennifer R. Linden Kiel Telesford Samantha Shetty Paige Winokour Sylvia Haigh Ellen Cahir-McFarland Giovanna Antognetti Abhishek Datta Tao Wang Werner Meier Timothy Vartanian |
| author_sort | Jennifer R. Linden |
| collection | DOAJ |
| description | The pore-forming epsilon toxin (ETX) produced by <i>Clostridium perfringens</i> 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. |
| first_indexed | 2024-12-15T00:23:44Z |
| format | Article |
| id | doaj.art-3d10856f453c49769bff5bff93b3c8ab |
| institution | Directory Open Access Journal |
| issn | 2073-4468 |
| language | English |
| last_indexed | 2024-12-15T00:23:44Z |
| publishDate | 2018-10-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Antibodies |
| spelling | doaj.art-3d10856f453c49769bff5bff93b3c8ab2022-12-21T22:42:13ZengMDPI AGAntibodies2073-44682018-10-01743710.3390/antib7040037antib7040037A Novel Panel of Rabbit Monoclonal Antibodies and Their Diverse Applications Including Inhibition of <i>Clostridium perfringens</i> Epsilon Toxin OligomerizationJennifer R. Linden0Kiel Telesford1Samantha Shetty2Paige Winokour3Sylvia Haigh4Ellen Cahir-McFarland5Giovanna Antognetti6Abhishek Datta7Tao Wang8Werner Meier9Timothy Vartanian10Brain and Mind Research Institute, Weill Cornell Medical College of Cornell University, New York, NY, 10065, USABrain and Mind Research Institute, Weill Cornell Medical College of Cornell University, New York, NY, 10065, USABrain and Mind Research Institute, Weill Cornell Medical College of Cornell University, New York, NY, 10065, USABrain and Mind Research Institute, Weill Cornell Medical College of Cornell University, New York, NY, 10065, USABrain and Mind Research Institute, Weill Cornell Medical College of Cornell University, New York, NY, 10065, USABiogen, Cambridge, MA 02142, USABiogen, Cambridge, MA 02142, USABiogen, Cambridge, MA 02142, USABiogen, Cambridge, MA 02142, USABiogen, Cambridge, MA 02142, USABrain and Mind Research Institute, Weill Cornell Medical College of Cornell University, New York, NY, 10065, USAThe pore-forming epsilon toxin (ETX) produced by <i>Clostridium perfringens</i> 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.https://www.mdpi.com/2073-4468/7/4/37<i>Clostridium perfringens</i>epsilon toxinepsilon protoxinneutralizingantibodiesoligomerizationpore formation |
| spellingShingle | Jennifer R. Linden Kiel Telesford Samantha Shetty Paige Winokour Sylvia Haigh Ellen Cahir-McFarland Giovanna Antognetti Abhishek Datta Tao Wang Werner Meier Timothy Vartanian A Novel Panel of Rabbit Monoclonal Antibodies and Their Diverse Applications Including Inhibition of <i>Clostridium perfringens</i> Epsilon Toxin Oligomerization Antibodies <i>Clostridium perfringens</i> epsilon toxin epsilon protoxin neutralizing antibodies oligomerization pore formation |
| title | A Novel Panel of Rabbit Monoclonal Antibodies and Their Diverse Applications Including Inhibition of <i>Clostridium perfringens</i> Epsilon Toxin Oligomerization |
| title_full | A Novel Panel of Rabbit Monoclonal Antibodies and Their Diverse Applications Including Inhibition of <i>Clostridium perfringens</i> Epsilon Toxin Oligomerization |
| title_fullStr | A Novel Panel of Rabbit Monoclonal Antibodies and Their Diverse Applications Including Inhibition of <i>Clostridium perfringens</i> Epsilon Toxin Oligomerization |
| title_full_unstemmed | A Novel Panel of Rabbit Monoclonal Antibodies and Their Diverse Applications Including Inhibition of <i>Clostridium perfringens</i> Epsilon Toxin Oligomerization |
| title_short | A Novel Panel of Rabbit Monoclonal Antibodies and Their Diverse Applications Including Inhibition of <i>Clostridium perfringens</i> Epsilon Toxin Oligomerization |
| title_sort | novel panel of rabbit monoclonal antibodies and their diverse applications including inhibition of i clostridium perfringens i epsilon toxin oligomerization |
| topic | <i>Clostridium perfringens</i> epsilon toxin epsilon protoxin neutralizing antibodies oligomerization pore formation |
| url | https://www.mdpi.com/2073-4468/7/4/37 |
| work_keys_str_mv | AT jenniferrlinden anovelpanelofrabbitmonoclonalantibodiesandtheirdiverseapplicationsincludinginhibitionoficlostridiumperfringensiepsilontoxinoligomerization AT kieltelesford anovelpanelofrabbitmonoclonalantibodiesandtheirdiverseapplicationsincludinginhibitionoficlostridiumperfringensiepsilontoxinoligomerization AT samanthashetty anovelpanelofrabbitmonoclonalantibodiesandtheirdiverseapplicationsincludinginhibitionoficlostridiumperfringensiepsilontoxinoligomerization AT paigewinokour anovelpanelofrabbitmonoclonalantibodiesandtheirdiverseapplicationsincludinginhibitionoficlostridiumperfringensiepsilontoxinoligomerization AT sylviahaigh anovelpanelofrabbitmonoclonalantibodiesandtheirdiverseapplicationsincludinginhibitionoficlostridiumperfringensiepsilontoxinoligomerization AT ellencahirmcfarland anovelpanelofrabbitmonoclonalantibodiesandtheirdiverseapplicationsincludinginhibitionoficlostridiumperfringensiepsilontoxinoligomerization AT giovannaantognetti anovelpanelofrabbitmonoclonalantibodiesandtheirdiverseapplicationsincludinginhibitionoficlostridiumperfringensiepsilontoxinoligomerization AT abhishekdatta anovelpanelofrabbitmonoclonalantibodiesandtheirdiverseapplicationsincludinginhibitionoficlostridiumperfringensiepsilontoxinoligomerization AT taowang anovelpanelofrabbitmonoclonalantibodiesandtheirdiverseapplicationsincludinginhibitionoficlostridiumperfringensiepsilontoxinoligomerization AT wernermeier anovelpanelofrabbitmonoclonalantibodiesandtheirdiverseapplicationsincludinginhibitionoficlostridiumperfringensiepsilontoxinoligomerization AT timothyvartanian anovelpanelofrabbitmonoclonalantibodiesandtheirdiverseapplicationsincludinginhibitionoficlostridiumperfringensiepsilontoxinoligomerization AT jenniferrlinden novelpanelofrabbitmonoclonalantibodiesandtheirdiverseapplicationsincludinginhibitionoficlostridiumperfringensiepsilontoxinoligomerization AT kieltelesford novelpanelofrabbitmonoclonalantibodiesandtheirdiverseapplicationsincludinginhibitionoficlostridiumperfringensiepsilontoxinoligomerization AT samanthashetty novelpanelofrabbitmonoclonalantibodiesandtheirdiverseapplicationsincludinginhibitionoficlostridiumperfringensiepsilontoxinoligomerization AT paigewinokour novelpanelofrabbitmonoclonalantibodiesandtheirdiverseapplicationsincludinginhibitionoficlostridiumperfringensiepsilontoxinoligomerization AT sylviahaigh novelpanelofrabbitmonoclonalantibodiesandtheirdiverseapplicationsincludinginhibitionoficlostridiumperfringensiepsilontoxinoligomerization AT ellencahirmcfarland novelpanelofrabbitmonoclonalantibodiesandtheirdiverseapplicationsincludinginhibitionoficlostridiumperfringensiepsilontoxinoligomerization AT giovannaantognetti novelpanelofrabbitmonoclonalantibodiesandtheirdiverseapplicationsincludinginhibitionoficlostridiumperfringensiepsilontoxinoligomerization AT abhishekdatta novelpanelofrabbitmonoclonalantibodiesandtheirdiverseapplicationsincludinginhibitionoficlostridiumperfringensiepsilontoxinoligomerization AT taowang novelpanelofrabbitmonoclonalantibodiesandtheirdiverseapplicationsincludinginhibitionoficlostridiumperfringensiepsilontoxinoligomerization AT wernermeier novelpanelofrabbitmonoclonalantibodiesandtheirdiverseapplicationsincludinginhibitionoficlostridiumperfringensiepsilontoxinoligomerization AT timothyvartanian novelpanelofrabbitmonoclonalantibodiesandtheirdiverseapplicationsincludinginhibitionoficlostridiumperfringensiepsilontoxinoligomerization |