title: Development of an immunodetection test for a botulinum-like neurotoxin produced by Clostridium sp. RKD.
abstract: OBJECTIVE
  Clostridial neurotoxins are among the most toxic substances known and cause severe illnesses in both humans and animals. A neurotoxigenic Clostridium sp. (strain RKD) isolated from intestine of decaying fish produced a novel, botulinum type B like neurotoxin as suggested by mouse bioassay, protection with anti-botulinum antibodies and PCR. The aim of the present investigation was to develop a laboratory based detection assay as an alternative to the mouse bioassay without compromising sensitivity and specificity.
  
  
  METHODS
  Growth and toxin production were carried out in trypticase peptone yeast-extract glucose (TPYG) broth. Toxicity was estimated in terms of minimum lethal dose (MLD) by mouse bioassay. The toxin was partially purified by acid precipitation. It was used for toxoid preparation by formaldehyde treatment. This purified IgG was used for detection of neurotoxin using indirect ELISA. The culture supernatant was concentrated using a stirred cell with a 50 kDa cut-off membrane at 4 degrees C. Further purification was carried out using Prep cell. Fractions showing toxicity and sufficient purity were pooled, concentrated and analyzed on sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE).
  
  
  RESULTS
  The toxin was purified with a recovery of 8.56 per cent. Polyclonal antiserum was raised in mice using partially purified toxin with a titre of 1: 80000. A detection assay with sensitivity of approximately 15 and 300 ng/ml for partially purified and crude toxins, respectively were achieved using an indirect ELISA method.
  
  
  CONCLUSIONS
  The Clostridium sp. RKD produced a potent neurotoxin earlier shown to have novelties. A specific detection assay for the neurotoxin has been developed that may be useful both from food safety and clinical point of view.
authors.affiliation: Biotechnology Division, Defence Research and Development Establishment, Gwalior, India.
authors.forename: Aparna
authors.initials: A
authors.lastname: Dixit
authors.forename: S I
authors.initials: SI
authors.lastname: Alam
authors.forename: R K
authors.initials: RK
authors.lastname: Dhaked
authors.forename: Lokendra
authors.initials: L
authors.lastname: Singh
mesh_terms: D000818:Animals; D001681:Biological Assay; D001905:Botulinum Toxins / Q000302:isolation & purification* / Q000633:toxicity; D019274:Botulinum Toxins, Type A; D003013:Clostridium / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005399:Fishes / Q000382:microbiology*; D007074:Immunoglobulin G / Q000378:metabolism; D051379:Mice; D009498:Neurotoxins / Q000302:isolation & purification* / Q000633:toxicity
chemical_list: D007074:Immunoglobulin G; D009498:Neurotoxins; C096323:rimabotulinumtoxinB; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A