Standardized Assay for Clostridium botulinum Toxins

Abstract

To improve the accuracy and precision of the mouse assay for botulinum toxins found in foods and body fluids, a stable reference standard of botulinum toxin has been developed. It contains 100 ng crystalline type A botulinum toxin/ml 0.05M pH 4.2 sodium acetate buffer which contains 3 mg bovine serum albumin and 2 mg gelatin/ml. The albumin and gelatin stabilize the toxin. For distribution to laboratories carrying out the assay, 0.5 ml of the standard is sealed in 1 ml glass vials. The standard reference toxin, used as directed, enables the assayer to establish the response of laboratory mice, under a specific set of conditions, to a definite amount of toxin and to express the toxin content of an unknown in terms of ng toxin/g or ml food or body fluid. The mouse assay by itself is not specific for botulinum toxin, but when used in conjunction with specific antisera for identification and typing, it is the most reliable test available.

Factors Affecting Growth and Toxin Production by Clostridium Botulinum Type E on Irradiated (0.3 Mrad) Chicken Skins

“…Control toxicity was done by heating the sample as described in the Manual. The mouse L D~o was determined according to the method described by Schantz and Kautter (1978).…”

Opinion of the Scientific Panel on biological hazards (BIOHAZ) related to Clostridium spp in foodstuffs

“…Methods are not suited to routine food microbiology laboratories because it is necessary to test for the neurotoxin, and special safety precautions are necessary (CDC, 1999a(CDC, , 2000a. Furthermore the traditional test for toxin uses mice (Schantz and Kautter, 1978) and although other methods have been developed (see Table 10 in SCVPH (2002) Opinion on Honey and Microbiological Hazards", reproduced in Annex II)), no validated alternative method is yet available commercially.…”

“…The reference method for detection of botulinum neurotoxin remains the mouse bioassay (Schantz and Kautter, 1978) coupled with neutralisation with monovalent antisera. Production of those antisera has offered poor commercial returns, and, as a consequence, antisera are difficult to obtain.…”

Refinement and Validation of an Alternative Bioassay for Potency Testing of Therapeutic Botulinum Type A Toxin

“…Historically, assays for detecting neurotoxins derived from CI. hotiilinum have been developed and used largely in connection with food contamination and outbreaks of botulism (Schantz & Kautter 1978). Boroff & Fleck (1966) first described an in vivo mouse bioassay for titration of botulinum type A toxin; this was subsequently adopted as the assay of choice for the potency testing of therapeutic Author for correspondence: Dorothea Sesardic, Division of Bacteriology, NIBSC, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, United Kingdom (fax (44) 1707 646730).…”

“…Contrary to general belief, the mouse unit is not a standardised unit, even though the same biological end point is used. It is well documented that this assay is prone to interlaboratory variability (Schantz & Kautter 1978). Up to 10fold difference in results was reported in one study which involved participation by 11 laboratories to standardise a type A botulinum toxin assay for assessing the toxin in food contaminations.…”