Paul-Ehrlich-Institut (Federal Institute for Vaccines and Biomedicines), Paul-Ehrlich-Strasse 51-59, 63225 Langen, Germany.
Vaccine. 2013 Dec 16;31(52):6247-53. doi: 10.1016/j.vaccine.2013.10.028. Epub 2013 Oct 21.
Tetanus toxoids (i.e. chemically inactivated preparations of tetanus neurotoxin) are used for the production of tetanus vaccines. In order to exclude the risk of residual toxicity or of a "reversion to toxicity", each batch of tetanus toxoid is subject to strict safety testing. Up to now, these prescribed safety tests have to be performed as in vivo toxicity tests in guinea pigs. However, as animal tests are generally slow, costly and ethically disputable, a replacement by an in vitro method would be desirable. A suitable alternative method would have to be able to sensitively detect already low concentrations of active tetanus neurotoxin in matrices containing large amounts of inactivated toxoid molecules. We have developed a method which detects active tetanus neurotoxin molecules based on their specific receptor-binding capacity as well as their proteolytic activity. By taking into account two relevant functional characteristics, this combined "BINding And CLEavage" (BINACLE) assay more reliably discriminates between toxic and detoxified molecules than other in vitro assays which solely rely on one single toxin function (e.g. endopeptidase assays). Data from an in-house validation show that the BINACLE assay is able to detect active tetanus neurotoxin with a detection limit comparable to the in vivo test. The sensitive detection of active toxin which has been spiked into toxoid samples from different manufacturers could also be demonstrated. Specificity and precision of the method have been shown to be satisfactory. The presented data indicate that for toxoid batches from some of the most relevant European vaccine manufacturers, the BINACLE assay may represent a potential alternative to the prescribed animal safety tests. In addition, this novel method may also provide a convenient tool for monitoring batch-to-batch consistency during toxoid production.
破伤风类毒素（即破伤风神经毒素的化学失活制剂）用于生产破伤风疫苗。为了排除残留毒性或“毒性回复”的风险，每批破伤风类毒素都要经过严格的安全性测试。到目前为止，这些规定的安全性测试必须在豚鼠体内进行毒性测试。然而，由于动物试验通常较慢、成本较高且存在伦理争议，因此希望用体外方法替代。合适的替代方法必须能够在含有大量失活类毒素分子的基质中，灵敏地检测到已经很低浓度的活性破伤风神经毒素。我们已经开发了一种基于其特定受体结合能力和蛋白水解活性来检测活性破伤风神经毒素分子的方法。通过考虑两个相关的功能特性，这种组合的“结合和切割”（BINACLE）检测比其他仅依赖于单一毒素功能（如内肽酶检测）的体外检测更可靠地区分有毒和解毒的分子。来自内部验证的数据表明，BINACLE 检测法能够以与体内检测相当的检测限检测到活性破伤风神经毒素。还可以证明该方法能够灵敏地检测到已掺入来自不同制造商的类毒素样品中的活性毒素。该方法的特异性和精密度已被证明令人满意。所提供的数据表明，对于来自一些最相关的欧洲疫苗制造商的类毒素批次，BINACLE 检测法可能是规定的动物安全性测试的潜在替代方法。此外，这种新方法还可以为类毒素生产过程中的批次间一致性监测提供便利工具。
