Toxicon 48 (2006) 246–254 Botulinum type A toxin neutralisation by specific IgG and its fragments: A comparison of mouse systemic toxicity and local flaccid paralysis assays R.G.A. Jones a,Ã , T.-A. Alsop a , R. Hull b , R. Tierney a , P. Rigsby c , J. Holley d , D. Sesardic a a Division of Bacteriology, National Institute for Biological Standards and Control, South Mimms, Potters Bar, Hertfordshire. EN6 3QG, UK b BSS, National Institute for Biological Standards and Control, South Mimms, Potters Bar, Hertfordshire. EN6 3QG, UK c Informatics, National Institute for Biological Standards and Control, South Mimms, Potters Bar, Hertfordshire. EN6 3QG, UK d Dstl, Porton Down, Salisbury, Wiltshire, SP4 0JQ, UK Received 24 January 2006; received in revised form 12 May 2006; accepted 30 May 2006 Available online 14 June 2006 Abstract In this study, we have compared two in vivo assay methods to measure the type A botulinum toxin neutralising activity of specific immunoglobulin G (IgG) and its fragments (F(ab 0 ) 2 , Fab 0 , Fab) purified from pentavalent botulinum antisera raised in goats. Each assay method was repeated on three separate occasions in mice and relative potencies calculated with respect to a type A equine reference antitoxin. The conventional assay, which measures the number of mice surviving typically after 72 or 96 h following the intraperitoneal administration of a mixture of toxin and antitoxin, gave the following order of potency IgG4F(ab 0 ) 2 4Fab 0 4Fab (6.844.743.542.6 IU/mg). Differences in potency are likely to be due to differences in the pharmacokinetics of the antitoxins, which are related to their molecular weight. The alternative local flaccid paralysis assay, where toxin and antitoxin are injected subcutaneously into the left inguinocrural region, gave results with a narrower range of activities: IgG4Fab 0 4F(ab 0 ) 2 4Fab (6.045.945.544.6 IU/mg). Comparison of the two assay methods showed no significant differences for IgG, F(ab 0 ) 2 or Fab 0 , although the Fab fragment was significantly more potent in the non-lethal assay probably because of the reduced influence of antitoxin pharmacokinetics in this localised assay. These findings show that a local flaccid paralysis assay provides a less time consuming and more humane alternative to the lethal assay for the potency testing of botulinum IgG and F(ab 0 ) 2 antitoxins. r 2006 Elsevier Ltd. All rights reserved. Keywords: Botulism; Antitoxin; In vivo; Immunoglobulin; Toxin 1. Introduction Seven types or serotypes (A, B, C, D, E, F and G) of botulinum toxin are currently known. Each serotype is composed of a heavy and a light chain linked together by disulphide bonds. The N-terminal end of the heavy chain is responsible for binding to specific pre-synaptic neuronal cell receptors (Synaptic Vesicle protein 2 and ganglio- sides, for type A toxin) and the C-terminal end facilitates internalisation (Montecucco et al., 1994; ARTICLE IN PRESS www.elsevier.com/locate/toxicon 0041-0101/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2006.05.007 Ã Corresponding author. Fax: +44 01707 663796. E-mail address: rjones@nibsc.ac.uk (R.G.A. Jones).