Role for standards in assays of botulinum toxins: international collaborative study of three preparations of botulinum type A toxin

The biological activity of therapeutic preparations of botulinum type A toxin is currently expressed in units defined on the basis of the median lethal intraperitoneal dose of that preparation in mice at 72 h, the LD50 dose. In this study we describe the comparison, by ten laboratories in five countries, of three different formulations of botulinum type A toxin using the mouse lethality test, and also using the relative activities of the preparations. The results of this study show that use of a standard preparation and expression of relative potency gives substantially greater consistency between and within laboratories than when mouse LD50 unit is used to define activity of botulinum toxin.     

Efficacy of a type C botulism vaccine in green-winged teal

We tested the efficacy of a single dose of Botumink toxoid for protecting wild green-winged teal (Anas crecca) during botulism epizootics caused by Clostridium botulinum type C. We challenged control and immunized ducks with four different doses of type C botulinum toxin to determine the LD50 for this species and to evaluate vaccine protection. Fewer immunized ducks were affected with botulism than control ducks, indicating that a single dose of Botumink toxoid could increase the survival of ducks during epizootics. However, the frequency of immunized ducks with signs of botulism increased with the challenge dose of botulinum toxin. Even at doses of botulinum toxin approximately 2 to 4 green-winged teal LD50, about 50% of the immunized ducks were affected. We believe an improved vaccine or a better delivery system is required to justify immunization of wild birds for experimental survival studies.     

Enzyme linked immunosorbent assay (ELISA) for detection of Clostridium botulinum type B toxin.

The enzyme-linked immunosorbent assay using different techniques has been applied to determine botulinum type B toxin. With the so-called "sandwich" technique, about 5,000 mouse ip LD50 of type B toxin can be detected. With the "double-sandwich" technique, about 400 mouse ip LD50 of toxin is detected and different commerical antisera are useful. For accurate quantification of botulinum toxins in culture filtrates, addition of EDTA to samples seems to be necessary. Cross-reactivity of the assay depends on the specificity of the antisera against botulinum type B toxin used and is almost eliminated with antiserum prepared against the toxic component of type B toxin.     

Growing old disgracefully: the cosmetic use of botulinum toxin

The explosive growth in the use of botulinum toxin for cosmetic purposes has undoubtedly had an impact on the number of animals used in the potency testing of this product. The test used is a classical LD50, a severe procedure during which animals experience increasing paralysis until the occurrence of death. The enthusiastic adoption by the general public of the use of botulinum toxin as an anti-wrinkle treatment has, at least in Europe, paradoxically taken place against a background of moves to stop animal testing of cosmetics and cosmetic ingredients. There appears to be a dearth of information aimed at the public concerning botulinum toxin testing. Botulinum toxin does have important medical applications; however, the question arises whether a blanket licence for the testing can be justified, when a large proportion of the product is being used cosmetically. A further question is why death continues to be the endpoint of the potency test, when a more-humane endpoint has been proposed. In addition, a number of alternative methods have been developed, which could have the potential to replace the lethal potency test altogether. These methods are discussed in this paper, and the importance of establishing a strategy for their validation is emphasised, a need that has become even more urgent in the light of the recently published draft monograph on botulinum toxin by the European Pharmacopoeia Commission.     

Enzyme-linked immunosorbent assay for detection of Clostridium botulinum toxin type A.

The enzyme linked immunosorbent assay using the so-called "double-sandwich technique" has been applied to determine botulinum toxin type A. By this assay, 50-100 mouse ip LD50 of toxin type A can be detected. No cross-reaction occurs with botulinum toxins of other types tested. In all probability this is due to the high specificity of the antiserum prepared against the toxic component of type A toxin.     

Correlation of Clostridium botulinum type C antitoxin titers in mink and guinea pigs to protection against type C intoxication in mink

In USA, the potency of commercial vaccines containing Clostridium botulinum type C toxoid is determined by a mink vaccination-challenge assay outlined in the Code of Federal Regulations, Title 9, Part 113.110. A more humane potency test is desired, and this study provides preliminary data in support of a serological assay that correlates post-vaccination antitoxin titers of guinea pigs to vaccine efficacy in mink. Mink and guinea pigs were injected with varying dilutions of a vaccine containing C. botulinum type C toxoid. Blood samples were collected from each animal prior to challenging the mink with type C toxin. Serum antitoxin titers of mink and guinea pigs were measured by a mouse protection test, and the results were compared to the outcome of the toxin challenge in mink. A dose-dependent antitoxin response was observed in guinea pigs vaccinated with the critical dilutions of vaccine bracketing the minimum protective dose in mink. These preliminary data suggest that it may be possible to correlate post-vaccination antitoxin titers in guinea pigs to vaccine efficacy in mink. This correlation could be used as the basis for a more humane potency test for C. botulinum type C toxoids.     

Detection of type A, B, E, and F Clostridium botulinum neurotoxins in foods by using an amplified enzyme-linked immunosorbent assay with digoxigenin-labeled antibodies

An amplified enzyme-linked immunosorbent assay (ELISA) for the detection of Clostridium botulinum complex neurotoxins was evaluated for its ability to detect these toxins in food. The assay was found to be suitable for detecting type A, B, E, and F botulinum neurotoxins in a variety of food matrices representing liquids, solid, and semisolid food. Specific foods included broccoli, orange juice, bottled water, cola soft drinks, vanilla extract, oregano, potato salad, apple juice, meat products, and dairy foods. The detection sensitivity of the test for these botulinum complex serotypes was found to be 60 pg/ml (1.9 50% lethal dose [LD50]) for botulinum neurotoxin type A (BoNT/A), 176 pg/ml (1.58 LD50) for BoNT/B, 163 pg/ml for BoNT/E (4.5 LD50), and 117 pg/ml for BoNT/F (less than 1 LD50) in casein buffer. The test could also readily detect 2 ng/ml of neurotoxins type A, B, E, and F in a variety of food samples. For specificity studies, the assay was also used to test a large panel of type A C. botulinum, a smaller panel of proteolytic and nonproteolytic type B, E, and F neurotoxin-producing Clostridia, and nontoxigenic organisms using an overnight incubation of toxin production medium. The assay appears to be an effective tool for large-scale screening of the food supply in the event of a botulinum neurotoxin contamination event.     

An alternative in vivo method to refine the mouse bioassay for botulinum toxin detection

Botulism is a rare, life-threatening paralytic disease of both humans and animals that is caused by botulinum neurotoxins (BoNT). Botulism is confirmed in the laboratory by the detection of BoNT in clinical specimens, contaminated foods, and cultures. Despite efforts to develop an in vitro method for botulinum toxin detection, the mouse bioassay remains the standard test for laboratory confirmation of this disease. In this study, we evaluated the use of a nonlethal mouse toe-spread reflex model to detect BoNT spiked into buffer, serum, and milk samples. Samples spiked with toxin serotype A and nontoxin control samples were injected into the left and right extensor digitorum longus muscles, respectively. Digital photographs at 0,8, and 24 h were used to obtain objective measurements through effective paralysis scores, which were determined by comparing the width-to-length ratio between right and left feet. Both objective measurements and clinical observation could accurately identify over 80% of animals injected with 1 LD(50) (4.3 pg) BoNT type A within 24 h. Half of animals injected with 0.5 LD(50) BoNT type A and none injected with 0.25 LD(50) demonstrated localized paralysis. Preincubating the toxin with antitoxin prevented the development of positive effective paralysis scores, demonstrating that (1) the effect was specific for BoNT and (2) identification of toxin serotype could be achieved by using this method. These results suggest that the mouse toe-spread reflex model may be a more humane alternative to the current mouse bioassay for laboratory investigations of botulism.     

C型肉毒毒素的制备及类毒素保护力初探

将C型肉毒梭菌经适宜条件的产毒培养后纯化,并进行相关鉴定。制备的C型肉毒毒素用分段脱毒法脱毒,并进行类毒素保护力的初步研究。以不同蛋白含量C型肉毒类毒素免疫小鼠后攻毒,结果显示,蛋白含量为0.625μg的类毒素免疫2针或蛋白含量为1.25μg的类毒素免疫1针均可保护50LD50的C型肉毒毒素攻击。蛋白含量为5μg的C型肉毒类毒素与福氏不完全佐剂配制的抗原免疫小鼠3次所得抗血清的保护力(Anti LD50/ml)为4.3×104。说明用该纯化工艺制备的C型肉毒类毒素具有很好的免疫原性,作为抗原成分用于C型肉毒疫苗和C型肉毒抗毒素的研究和生产具有较好的应用潜力。     

Outbreaks of type C botulism in waterfowl in Japan

Four outbreaks of botulism in waterfowl were encountered over a five-year period of 1973 to 1977 in Japan. In all the outbreaks toxin was detected from all 12 sera, twenty-three of 24 gizzard contents from diseased or dead birds and one of three maggots. It was neutralized with Clostridium botulinum type C antitoxin serum, regardless of its origin. By using CO2 gas jet method, C. botulinum was isolated from four of 11 gizzards from diseased birds, five of 7 ones from dead birds, one of one maggot and one of one sludge sample, that is, eleven of 20 specimens in total. All 20 strains were identical with C. botulinum type C in biological properties. Most of the isolates showed a toxin titer ranging from 1,000 to 200,000 LD50 for mice. Four of them were identified as type C by mouse neutralization tests with antitoxin sera. The toxic suspensions of a strain 1-15 were administered orally to Chinese spot-billed ducks, which died when more than 200,000 LD50 mouse toxin was administered. Environmental conditions for occurrences of waterfowl botulism were discussed.     

The enzymes of the metabolism of exogenous compounds in the comparative assessment of the toxicity of trichothecene mycotoxins

Moderate clinical, biochemical and hematologic signs of intoxication were observed in mice after single administration of HT-2 toxin (deacetylated derivative of T-2 toxin) in LD50 of 12.75 mg/kg or in 1/5 of LD50 for 7 days. The toxin produced no toxic effect when 1/10 and 1/50 of LD50 were administered for 14 days. With the dose exceeding 1/50 of LD50 a reduction in cytochrome P-450 content, carboxylesterase activity and increased activity of UDP-glucuronosyltransferase and glutathione transferase were recorded. T-2 toxin produced a more pronounced toxic effect, inhibited UDP-glucuronosyltransferase and insignificantly stimulated glutathione transferase activity. Lower HT-2 toxin toxicity is believed to depend on its ability to activate conjugation reactions to a greater extent than T-2 toxin.     

Persistence of Clostridium botulinum type C toxin in blow fly (Calliphoridae) larvae as a possible cause of avian botulism in spring

Diverse samples were examined at a site of water-bird mortality, caused by Clostridium botulinum type C toxin in southern Moravia (Czechoslovakia). The toxin was detected in high concentrations in mute swan (Cygnus olor) carcasses (less than or equal to 1 x 10(6) LD50/g) as well as in necrophagous larvae and pupae of the blow flies Lucilia sericata and Calliphora vomitoria (less than or equal to 1 x 10(5) LD50/g) collected from them. It was detected in lower concentrations (less than or equal to 1 x 10(3) LD50/g) in other invertebrates (ptychopterid fly larvae, leeches, sow-bugs) associated with these carcasses, and occasionally in water samples (8 LD50/ml) close to the carrion. The toxin was not detected in the samples of water, mud or invertebrates collected at a distance greater than or equal to 5 m from the carcasses. The toxin-bearing larvae of L. sericata and C. vomitoria, containing 80,000 LD50/g of type C toxin, were exposed in the mud at the study site for 131 days from November to March. Although the toxin activity decreased 25-fold and 40-fold in the two samples of maggots exposed during this period, it remained very high (less than or equal to 3,200 LD50/g). Birds ingesting a relatively low number of these toxic larvae (or pupae) in the spring could receive a lethal dose of the toxin.     

用皂土法制造型特异性肉毒诊断血清

用皂土为载体与类毒素结合方法及破伤风类毒素抗原抗体絮状反应方法去除A、B、C、D、E、F型肉毒抗血清原料中的异型和异种抗毒素(破伤风抗毒素)。制备的A、B、C、D、E、F型肉毒诊断血清每1m l均能中和相应型的肉毒毒素10000LD50以上,而中和异型肉毒毒素或破伤风毒素均低于5 LD50;A、B、C、D、E、F各型混合后的混合型血清每1m l能中和各型肉毒毒素亦大于10000 LD50,中和破伤风毒素低于5 LD50,即效价和特异性符合规程要求。     

Up and Down法与寇氏法测定破伤风毒素LD50的比较

目的比较研究Up and Down法与寇氏法测定破伤风毒素LD50值。方法 NIH小鼠皮下注射破伤风毒素,分别使用寇氏法和Up and Down法计算LD50值。结果同一破伤风毒素经寇氏法计算得LD50值为4.97ng/Kg体重,Up and Down法计算LD50值为4.93 ng/Kg体重,95%置信区间为（4.71～5.13）ng/Kg体重。结论应用Up and Down法可测出与寇氏法相同结果的LD50值,且动物使用数量仅用8只。     

Detection of Clostridium botulinum type G toxin by enzyme-linked immunosorbent assay.

Clostridium botulinum type G toxin was detected and quantified readily with the enzyme-linked immunosorbent assay. With the double-sandwich technique and alkaline phosphatase as the enzyme indicator, C. botulinum toxin type G was detected in quantities equaling those required for one mouse intraperitoneal median lethal dose. The time required for the procedure was approximately 6.5 h, but this requirement could have been reduced to 5.5 h or less with the use of precoated plates stored at -70 degrees C. Cross-reactions did not occur with culture extracts of C. sporogenes of C. botulinum types B, C, D, E, and F. Acidic preparations of C. botulinum type A exhibited nonspecific reactivity. Likewise, 50% of the C. subterminale isolates tested were cross-reactive in the assay. These latter isolates express similar metabolic and physiological characteristics with C. botulinum type G.     

Collaborative study to assess the suitability of a candidate International Standard for yellow fever vaccine

Yellow fever vaccines are routinely assayed by plaque assay. However, the results of these assays are then converted into mouse LD(50) using correlations/conversion factors which, in many cases, were established many years ago. The minimum required potency in WHO Recommendations is 10(3) LD(50)/dose. Thirteen participants from 8 countries participated in a collaborative study whose aim was to assess the suitability of two candidate preparations to serve as an International Standard for yellow fever vaccine. In addition, the study investigated the relationship between the mouse LD(50) test and plaque forming units with a view to updating the WHO recommendations. Plaque assays were more reproducible than mouse assays, as expected. Differences in sensitivities of plaque assays were observed between laboratories but these differences appear to be consistent within a laboratory for all samples and the expression of potency relative to the candidate standard vaccine improved the reproducibility of assays between laboratories. However, the use of potencies had little effect on the between laboratory variability in mouse LD(50) assays. There appears to be a consistent relationship between overall mean LD(50) and plaques titre for all study preparations other than sample E. The slope of the correlation curve is >1 and it would appear that 10(3) LD(50) is approximately equivalent to 10(4) plaque forming units (PFU), based on the overall means of all laboratory results. The First International Standard for yellow fever vaccine, NIBSC Code 99/616, has been established as the First International Standard for yellow fever vaccine by the Expert Committee of Biological Standards of the World Health Organisation. The International Standard has been arbitrarily assigned a potency of 10(4.5) International Units (IU) per ampoule. Manufacturers and National Control Laboratories are including the First International Standard for yellow fever vaccine in routine assays so that the minimum potency in IU of vaccines released for use and which meet the current minimum potency of 10(3) LD(50) in mouse assays, can be determined. These data will be analysed before a review of the WHO requirements, including the minimum potency per dose, is undertaken.     

Detection of neutral sugars in purified type G botulinum progenitor toxin and the effects of some glycolytic enzymes on its molecular dissociation and oral toxicity

Arabinose and galactose were detected in purified type G botulinum toxin (Mr about 500,000) of Clostridium argentinense. The i.p. LD50/mg N of type G progenitor toxin was one-tenth, but the oral LD50/mg N twice that of type A-L toxin. The lysozyme-, endo-beta-galactosidase-, and N-glucanase-treated toxins each had a molecular mass of about 300,000. The oral toxicity of the endo-beta-galactosidase or N-glucanase-treated toxin was one-fifth that of untreated progenitor toxin. On DEAE-Sephadex chromatography, the N-glucanase-treated toxin dissociated into two fractions, nontoxic and toxic. SDS-PAGE of the toxic fraction showed a single band with a Mr of about 150,000, and after dithiothreitol treatment, two bands with Mr of 100,000 and 50,000.     

Radioimmunoassay for Type A Toxin of Clostridium botulinum

A preparation of pure type A toxin of Clostridium botulinum was labeled with (131)I in the presence of chloramine-T and carrier-free isotope. The radioactive toxin ((131)I Tox) was used in a radioimmunoassay procedure similar to that of Berson and Yalow. Dilutions of antibody to the toxin, capable of binding 50% of the added (131)I Tox, were mixed with a sample of the labeled toxin and various concentrations of unlabeled toxin ('Cold' Tox). When concentration of Cold Tox were plotted against the ratio (131)I bound/(131)I not bound, a standard curve was established that could be used to estimate the concentration of Cold Tox in a test mixture. This assay was sensitive to as little as 100 mouse minimum lethal dose and was highly specific for the serological type of the toxin used.     

Development of an in vitro bioassay for Clostridium botulinum type B neurotoxin in foods that is more sensitive than the mouse bioassay.

A novel, in vitro bioassay for detection of the botulinum type B neurotoxin in a range of media was developed. The assay is amplified by the enzymic activity of the neurotoxin's light chain and includes the following three stages: first, a small, monoclonal antibody-based immunoaffinity column captures the toxin; second, a peptide substrate is cleaved by using the endopeptidase activity of the type B neurotoxin; and finally, a modified enzyme-linked immunoassay system detects the peptide cleavage products. The assay is highly specific for type B neurotoxin and is capable of detecting type B toxin at a concentration of 5 pg ml(-1) (0.5 mouse 50% lethal dose ml(-1)) in approximately 5 h. The format of the test was found to be suitable for detecting botulinum type B toxin in a range of foodstuffs with a sensitivity that exceeds the sensitivity of the mouse assay. Using highly specific monoclonal antibodies as the capture phase, we found that the endopeptidase assay was capable of differentiating between the type B neurotoxins produced by proteolytic and nonproteolytic strains of Clostridium botulinum type B.     

Progress in applying the Three Rs to the potency testing of Botulinum toxin type A

Botulinum toxin type A (BTA) is being increasingly used for a range of therapeutic purposes and also for cosmetic reasons. For many years, the potency of BTA has been measured by using an LD50 assay in mice. This assay is a cause for concern due to its unpleasant nature and extreme severity, and the requirement for high numbers of mice to be used. Alternatives to this potency assay are presently reviewed with particular reference to the work at the National Institute for Biological Standards and Control (NIBSC), and to recent work by the UK manufacturer of the substance. An in vivo local paralysis assay with considerably less severity has been developed and is in use at the NIBSC. Alternative, ex vivo functional assays in use include the measurement of BTA-induced paralysis of neurally-stimulated rodent diaphragm or rat intercostal muscle. The latter method has the advantage of allowing more preparations to be derived from one animal. However, these ex vivo methods have not yet been fully validated and accepted by regulatory agencies as potency assays. Endopeptidase assays, although not measuring muscle paralysis directly, may provide a very useful consistency test for batch release and may replace the routine use of the LD50 test for that purpose. These assays measure the cleavage of the SNAP-25 protein (the final stage of BTA action), and have been validated for batch release by the National Control Laboratory (NIBSC), and are in regular use there. ELISA assays, used alongside the endopeptidase assay, also provide useful confirmatory information on the amounts of functional (and non-functional) BTA present. The UK manufacturer is further validating its endopeptidase assay, an ex vivo muscle assay and an ELISA. It is anticipated that their work will lead to a change in the product license, hopefully within the next two years, and will form a critical milestone towards the end of the LD50 potency test.