Isolation and Quantification of Botulinum Neurotoxin From Complex Matrices Using the BoTest Matrix Assays

Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample testing. Rapid BoNT testing of foodstuffs is needed during outbreak forensics, patient diagnosis, and food safety testing while accurate potency testing is required for BoNT-based drug product manufacturing and patient safety. The widely used mouse bioassay for BoNT testing is highly sensitive but lacks the precision and throughput needed for rapid and routine BoNT testing. Furthermore, the bioassay''s use of animals has resulted in calls by drug product regulatory authorities and animal-rights proponents in the US and abroad to replace the mouse bioassay for BoNT testing. Several in vitro replacement assays have been developed that work well with purified BoNT in simple buffers, but most have not been shown to be applicable to testing in highly complex matrices. Here, a protocol for the detection of BoNT in complex matrices using the BoTest Matrix assays is presented. The assay consists of three parts: The first part involves preparation of the samples for testing, the second part is an immunoprecipitation step using anti-BoNT antibody-coated paramagnetic beads to purify BoNT from the matrix, and the third part quantifies the isolated BoNT''s proteolytic activity using a fluorogenic reporter. The protocol is written for high throughput testing in 96-well plates using both liquid and solid matrices and requires about 2 hr of manual preparation with total assay times of 4-26 hr depending on the sample type, toxin load, and desired sensitivity. Data are presented for BoNT/A testing with phosphate-buffered saline, a drug product, culture supernatant, 2% milk, and fresh tomatoes and includes discussion of critical parameters for assay success.     

Efficacy of reconstituted and stored botulinum toxin type A: an electrophysiologic and visual study in the auricular muscle of the rabbit

Once botulinum toxin type A is reconstituted, the manufacturer recommends that it be used in approximately 4 hours. As a result, a significant amount of this costly drug is often discarded because it is not completely used in the recommended period. The purpose of the present study was to compare fresh versus stored reconstituted botulinum toxin type A for (1) initial potency, (2) duration of action, and (3) bacterial colonization.Using a rabbit model, 20 New Zealand White rabbits were divided into four groups (I to IV). All rabbits had an injection of 2.5 U of reconstituted botulinum toxin into the right anterior auricular muscle. The first group was injected with botulinum toxin type A that was freshly reconstituted and served as the control. The second, third, and fourth groups were injected with botulinum toxin type A that had been reconstituted and stored for 2, 6, and 12 weeks, respectively, in a conventional freezer. Each rabbit had daily visual evaluation of the ear, with the position of auricle being graded from I to III. In addition, each rabbit had a nerve conduction study performed on the right anterior auricular muscle before injection and every 2 weeks after injection. Amplitude was chosen as the principal variable in the data analysis because it is the best predictor of physiologic changes at the muscle motor unit level. The endpoint of the study was defined as the time at which the nerve conduction studies and the visual inspections returned to baseline, preinjection levels. Botulinum toxin type A was also cultured before injection into each group.Overall, the nerve conduction data revealed a trend with a faster recovery (return to baseline) with the stored botulinum toxin. Groups IV and III returned to baseline first, followed by groups II and I. However, there was no significant difference among the groups at 2 and 4 weeks after injection, indicating that initial potency was unchanged. The differences between the groups became significant (p < 0.05) at 6 weeks and onward, suggesting that the duration was affected. Group I (fresh botulinum toxin) and group II (toxin stored for 2 weeks) had comparable outcomes and were not significantly different at any time period. Under visual inspection, the mean recovery time for each group was as follows: group IV, 5.4 weeks; group III, 7.0 weeks; group II, 6.75 weeks; and group I, 7.80 weeks. The results showed significance (p < 0.05) beginning after 3 weeks among some groups. Again, there was an overall quicker trend to return to baseline with the longer storage of the botulinum toxin (groups III and IV). These results support the authors' conduction study data, which suggest that the initial potency is not affected but the duration of action is. Again, groups I and II had comparable results. Microbiology cultures showed no growth of either aerobic or anaerobic bacteria at 7 days.In conclusion, using the rabbit model, it seems that reconstituted and stored botulinum toxin type A has the same initial potency but the duration of action is affected sometime after 2 weeks of storage. No bacterial contamination was associated with storing unpreserved reconstituted botulinum toxin type A for up to 12 weeks.     

The Intercostal NMJ Assay: a new alternative to the conventional LD50 assay for the determination of the therapeutic potency of botulinum toxin preparations

Therapeutic botulinum neurotoxin type A preparations have found an increasing number of clinical uses for a large variety of neuromuscular disorders and dermatological conditions. The accurate determination of potency in the clinical application of botulinum toxins is critical to ensuring clinical efficacy and safety, and is currently achieved by using a lethal dose (LD50) assay in mice. Ethical concerns and operational constraints associated with this assay have prompted the development of alternative assay systems that could potentially lead to its replacement. As one such alternative, we describe the development and evaluation of a novel ex vivo assay (the Intercostal Neuromuscular Junction [NMJ] Assay), which uses substantially fewer animals and addresses ethical concerns associated with the LD50 assay. The assay records the decay of force from electrically-stimulated muscle tissue sections in response to the toxin, and thus combines the important mechanisms of receptor binding, translocation, and the enzymatic action of the toxin molecule. Toxin application leads to a time-related and dose-related reduction in contractile force. A regression model describing the relationship between the applied dose and force decay was determined statistically, and was successfully tested as able to correctly predict the potency of an unknown sample. The tissue sections used were found to be highly reproducible, as determined through the innervation pattern and the localisation of NMJs in situ. Furthermore, the efficacy of the assay protocol to successfully deliver the test sample to the cellular target sites, was critically assessed by using molecular tracer molecules.     

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.     

THE BINDING OF BOTULINUM TOXIN TO MEMBRANE LIPIDS: SPHINGOLIPIDS, STEROIDS AND FATTY ACIDS

A number of lipids known to be constituents of nerve-ending membranes were tested for their ability to inactivate botulinum toxin. Inactivation of the toxin by a lipid was taken as presumptive evidence that the lipid might be the in vivo receptor for the toxin. Several sphingolipids (sphingosine, galactosylceramide, glucosylceramide, lactosylceramide, cytolipin K and cytolipin R), steroids (cholesterol and deoxycholic acid) and fatty acids (palmitic acid, stearic acid, prostaglandin E₁) did not affect the potency of botulinum toxin, and thus were discounted as potential toxin receptors. However, the gangliosides did inactivate botulinum toxin rapidly (in less than 5 min), within a temperature range of 2°-40°C, and at ionic strengths of 0.05-0.40. Inactivation diminished as pH fell below 6. The activity of gangliosides in suppressing the potency of botulinum toxin was a function of the number of sialic acid residues in the lipid. Thus, the data suggest that a molecule containing sialic acid may be the receptor for 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。     

Reduction of numbers of animals used in the quality control of biologicals

Laboratory animals are used for the quality control of vaccines. In particular, the potency testing of batches of inactivated vaccine requires large numbers of animals. The possibilities for reduction have been evaluated, and the results are summarised in this paper. Several approaches were studied, including the retrospective analysis of test data, with the objectives of determining the minimum number of animals required per vaccine dilution group, and evaluating the feasibility of a single-dose potency test. Other studies focused on the development of serology-based models and the use of genetically uniform animals. Based on the outcome of these studies, a substantial reduction in the number of animals used for the potency testing of toxoid vaccines has been achieved or will be achieved in the near future. As reduction alternatives can generally be explored in a relatively simpler and less time-consuming way than replacement alternatives, more emphasis should be placed on reduction strategies than at present.     

The use of the in vitro toxin binding inhibition (ToBI) test for the estimation of the potency of tetanus toxoid

The in vitro toxin binding inhibition (ToBI) test was used to determine antitoxin responses in mice immunized with tetanus toxoid. The ToBI test showed good correlation with the in vivo toxin neutralization (TN) test in titration of sera of mice immunized with various doses of DPT-Polio, DT-Polio and a tetanus reference preparation. Estimates of potency of tetanus toxoid obtained in mice by ToBI test correlated significantly with those obtained in mice by the lethal challenge test. In addition, potency values of the European reference preparation, succeedingly estimated by ToBI test and lethal challenge test in a single group of guinea-pigs, showed good correlation. From the study it is concluded that the ToBI test is a promising alternative to the toxic challenge procedure in the potency assay of tetanus toxoid vaccines. A substantial refinement and reduction in the use of animals can be achieved. Additional savings can be made by combining diphtheria and tetanus potency testing.     

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.     

Treatment of facial wounds with botulinum toxin A improves cosmetic outcome in primates

Surgeons have constantly sought to achieve the most aesthetic scar. A major factor determining the final cosmetic appearance of a cutaneous scar is the tension acting on the wound edges during the healing phase. Since Theodor Kocher pioneered the alignment of skin incisions with Langer's lines in 1892, surgical techniques that attempt to overcome closing tension have become standard. Yet, no treatment has been available to minimize underlying muscle contractions, which are the major cause of this tension. Botulinum toxin A is a potent drug that produces temporary muscular paralysis when injected locally. It has proven to be safe and effective in the treatment of a variety of disorders, including hyperkinetic facial lines. The objective of this randomized, double-blind, placebo-controlled primate study was to investigate the efficacy of a single injection of botulinum toxin A to improve the cosmetic appearance of cutaneous scars. Symmetric pairs of standardized excisions were performed on either side of the forehead of six primates. The half foreheads were randomized to the botulinum toxin A treatment side versus the placebo injection side. A panel of three blinded facial surgeons assessed the cosmetic appearance of the mature scars 3 months postoperatively. The wounds that had been immobilized with botulinum toxin A were rated as significantly better in appearance than the control wounds (p < 0.01). Histologic examination confirmed that all scars were mature. Blinded, randomized, placebo-controlled human clinical trials are presently under way at the Mayo Clinic.     

Development of an in vitro activity assay as an alternative to the mouse bioassay for Clostridium botulinum neurotoxin type A

Currently, the only accepted assay with which to detect active Clostridium botulinum neurotoxin is an in vivo mouse bioassay. The mouse bioassay is sensitive and robust and does not require specialized equipment. However, the mouse bioassay is slow and not practical in many settings, and it results in the death of animals. Here, we describe an in vitro cleavage assay for SNAP-25 (synaptosome-associated proteins of 25 kDa) for measuring the toxin activity with the same sensitivity as that of the mouse bioassay. Moreover, this assay is far more rapid, can be automated and adapted to many laboratory settings, and has the potential to be used for toxin typing. The assay has two main steps. The first step consists of immunoseparation and concentration of the toxin, using immunomagnetic beads with monoclonal antibodies directed against the 100-kDa heavy chain subunit, and the second step consists of a cleavage assay targeting the SNAP-25 peptide of the toxin, labeled with fluorescent dyes and detected as a fluorescence resonance energy transfer assay. Our results suggest that the sensitivity of this assay is 10 pg/ml, which is similar to the sensitivity of the mouse bioassay, and this test can detect the activity of the toxin in carrot juice and beef. These results suggest that the assay has a potential use as an alternative to the mouse bioassay for analysis of C. botulinum type A neurotoxin.     

C型肉毒梭菌毒素的稳定性研究

报道了保存肉毒毒素经常会遇到的某些因素对Ｃ型肉毒梭菌Ｃ６５１４培养滤液稳定性的影响观察结果。结果表明,提高环境温度及介质酸碱度、日光照射、激烈震荡都能使毒素的毒力下降。甘油或明胶磷酸盐缓冲剂对于保存Ｃ型肉毒毒素都是较好的活性稳定剂。含甘油或明胶磷酸盐缓冲剂的毒素在４℃下保存１２个月后,毒力的变动甚微。此二法都不需要特殊设备,材料易得,操作简便,比较实用。     

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.     

Opportunities for reduction in acute toxicity testing via improved design

The conventional method for assessing acute oral toxicity (OECD Test Guideline 401) was designed to identify the median lethal dose (LD50), using the death of animals as an endpoint. Introduced as an alternative method (OECD Test Guideline 420), the Fixed Dose Procedure (FDP) relies on the observation of clear signs of toxicity, uses fewer animals and causes less suffering. More recently, the Acute Toxic Class method and the Up-and-Down Procedure have also been adopted as OECD test guidelines. Both of these methods also use fewer animals than the conventional method, although they still use death as an endpoint. Each of the three new methods incorporates a sequential dosing procedure, which results in increased efficiency. In 1999, with a view to replacing OECD Test Guideline 401, the OECD requested that the three new test guidelines be updated. This was to bring them in line with the regulatory needs of all OECD Member Countries, provide further reductions in the number of animals used, and introduce refinements to reduce the pain and distress experienced by the animals. This paper describes a statistical modelling approach for the evaluation of acute oral toxicity tests, by using the revised FDP for illustration. Opportunities for further design improvements are discussed.     

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。     

皮肤刺激实验替代模型的研究新进展

皮肤刺激性是日常使用化妆品最常见的不良反应之一。人类健康相关产品危险性评价常做皮肤刺激性实验,皮肤刺激性试验是化妆品原料及产品安全性评价的主要项目。传统皮肤刺激试验采用实验动物进行,2013年3月11日欧盟已经禁止销售基于动物实验研发的化妆品原料及产品.随着组织工程技术和现代生物技术的发展,多种替代动物试验的体外模型被开发和应用,新的的皮肤刺激物陆续被发现。欧盟多采纳重组人表皮实验方法作为新体外皮肤实验指南(包括模型Episkin和模型Epiderm),随着体外模型重建技术的不断改善,不仅拓展了皮肤模型的临床应用范围,也必然推动新的敏感而特异的皮肤标志物的发现和应用。     

Failure to Inhibit In Vitro or In Vivo Acetycholinesterase with Botulinum Toxin Type A

An attempt has been made to replicate an earlier finding that type A botulinum toxin can inhibit the in vitro activity of acetylcholinesterase. Two methods of enzyme assay were employed, but with neither technique were we able to reproduce the finding of in vitro enzyme inhibition. In fact, an examination of the data from the previous report leads us to question the possibility of the observations that were given. Furthermore, an investigation was carried out to determine if botulinum toxin can exert an inhibiting effect on acetylcholinesterase that is situated in the biological tissue. The answer again is negative. The experimental observations, coupled with several mathematical computations, do not support the notion that botulinum toxin is an acetylcholinesterase inhibitor.     

THE BINDING OF BOTULINUM TOXIN TO MEMBRANE LIPIDS: PHOSPHOLIPIDS AND PROTEOLIPID

A number of phospholipids known to be constituents of nerve endings were tested for their ability to inactivate botulinum toxin. Substances tested included phosphatidylcholine, phosphatidalcholine, phosphatidylethanolamine, phosphatidalethanolamine, β-acyl lysolecithin, sphingomyelin, phosphatidylserine, phosphatidic acid, phosphatidylinositol and cardiolipin. Proteolipid from bovine white matter was also tested. Neutral phospholipids potentiated the toxicity in vivo of botulinum toxin, but they had no effect on the toxicity in vitro. Some, but not all, acidic phospholipids caused loss of toxicity of botulinum toxin in solutions at low pH both in vivo and in vitro. However, none of these substances when incubated with toxin under physiological conditions of temperature, pH and ionic strength, caused loss of toxin potency. The data suggest that none of these phospholipids is likely to be a toxin receptor.     

The rapid fluorescent focus inhibition test is a suitable method for batch potency testing of inactivated rabies vaccines

The European Pharmacopoeia proposes two methods for potency determination of inactivated rabies vaccines for veterinary use: The first one is a classical mouse challenge test, which is imprecise, time-consuming, and causes severe distress to the test animals. Alternatively, the potency may be determined serologically by measuring the neutralizing antibody titers induced after vaccination of mice by using a rapid fluorescent focus inhibition test (RFFIT). Although this method is faster and less painful for the animals, it is not widely used yet, and only little data exist concerning the comparability of both methods.We have therefore performed a comparative study, in which we demonstrated a good correlation between the challenge test results and the mean titers determined by RFFIT. Furthermore, all vaccine batches failing the challenge test were also recognized as insufficient in the serological assay. This publication further describes the influence of different vaccine administration routes on the resulting antibody titers, and it proposes various modifications to the serological assay protocol which could improve its overall practicability. Finally, we recommend that the serological assay be used for the potency testing of inactivated rabies vaccines.