title: Development of ELISA based detection system for lethal toxin of Clostridium sordellii.
abstract: OBJECTIVE
  Clostridium sordellii and its toxins are associated with diseases in animals as well as human. C. sordellii produces two protein toxins (lethal toxin and haemorrhagic toxin). Lethal toxin has gained more importance due its high toxicity. The present study was carried out to develop a sandwich ELISA for detection of lethal toxin of C. sordellii.
  
  
  METHODS
  The catalytic domain (1.6 kb) of lethal toxin of C. sordellii was PCR amplified, cloned into pQE30 UA vector and transformed into Escherichia coli SG 13009. Expression conditions were optimized and the recombinant protein was purified under native condition using Ni-NTA affinity chromatography, confirmed by SDS-PAGE and Western blot. Antibody was generated against the purified recombinant protein using Freund's complete and incomplete adjuvants (FCA and FIA) in BALB/c mice and rabbit. A sandwich ELISA was optimized for the detection of lethal toxin.
  
  
  RESULTS
  The maximum recombinant protein expression was achieved at 0.5 mM IPTG (isopropylthiogalactoside) induction 4.0 h of post-induction. The polyclonal antibody raised in mice and rabbit showed a titre up to 1:512000. The produced antibody was highly sensitive with the detection limit of 0.3 ng/ml of lethal toxin at 1:4000 dilutions of mice (capturing) and rabbit (revealing) antibody.
  
  
  CONCLUSIONS
  An ELISA based detection system was developed for the detection of lethal toxin of C. sordellii. The developed detection system was found to be specific as there was no cross-reactivity with any other clostridial toxins. It will be useful for the detection of lethal toxin of C. sordellii in clinical and environmental samples.
authors.affiliation: Biotechnology Division, Defence Research & Development Establishment, Gwalior, India.
authors.forename: Preetika
authors.initials: P
authors.lastname: Arya
authors.forename: S
authors.initials: S
authors.lastname: Ponmariappan
authors.forename: Lokendra
authors.initials: L
authors.lastname: Singh
authors.forename: Om
authors.initials: O
authors.lastname: Kumar
mesh_terms: D000818:Animals; D001427:Bacterial Toxins / Q000032:analysis* / Q000276:immunology; D020134:Catalytic Domain; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003015:Clostridium Infections / Q000175:diagnosis / Q000276:immunology; D047989:Paraclostridium sordellii / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D057230:Limit of Detection; D051379:Mice; D008807:Mice, Inbred BALB C; D016133:Polymerase Chain Reaction; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry; D013997:Time Factors
chemical_list: D001427:Bacterial Toxins; D011994:Recombinant Proteins; C072292:lethal toxin LT, Clostridium sordellii