ABSTRACT
Botulinum neurotoxins induce a prolonged muscle paralysis by specifically blocking the release of neuronal transmitters from peripheral nerve junctions. Potency testing of toxin and antitoxin therapies is entirely dependent on mouse lethality bioassay which is associated with extreme suffering of large numbers of animals to ensure high precision. The mouse phrenic nerve-diaphragm assay is an ex vivo assay that closely mimics in vivo respiratory paralysis offering substantial refinement and reduction in the number of animals used. A range of botulinum antitoxin standards, one licenced product and experimental antitoxins were tested for neutralising potency using ex vivo hemidiaphragm assay and compared with in vivo determined activities. Overall, there was an excellent agreement between neutralising activity detected by the two assay systems and for each toxin serotype using only 4-7 replicates for each product (almost perfect concordance for type A antitoxins: ρ = 0.997, and substantial concordance for type B antitoxins: ρ = 0.991 and type E antitoxins: ρ = 0.964, respectively). These findings confirm that the mouse nerve-diaphragm preparation can provide a functional ex vivo replacement assay for specific, sensitive and precise assessment of toxin and antitoxin activity.
Subject(s)
Botulinum Antitoxin/analysis , Diaphragm/drug effects , Phenytoin/pharmacology , Phrenic Nerve/drug effects , Toxicology/methods , Animals , Botulinum Antitoxin/classification , Botulinum Antitoxin/immunology , Immunoassay/methods , Immunologic Factors/analysis , Immunologic Factors/classification , Immunologic Factors/immunology , Male , Mice , Mice, Inbred BALB C , Sensitivity and SpecificityABSTRACT
Serum levels of equine-botulism antitoxin to toxin types A, B, and E were measured in four type-A botulism patients who had received equine-botulism antitoxin. High circulating levels capable of neutralizing in excess of 1 X 10(8), 9 X 10(7), and 6 X 10(6) 50% mouse lethal doses of toxin of types A, B, and E, respectively, were detected. There was little depletion of type-A antitoxin even though two of the patients had circulating type-A toxin before treatment. The half-life for antitoxin persistence for one patient was calculated as being 6.5, 7.6, and 5.3 days for antitoxin types A, B, and E, respectively. Antitoxin levels were not proportionate to the amount (range, 2-4 vials) injected and did not appear to be affected by whether the route of administration was iv or im. Peak serum levels of antitoxin were 10-1,000 times higher than amounts needed to neutralize the toxin measured in the serum of these and other patients with botulism.
