Botulinum Neurotoxins: Qualitative and Quantitative Analysis Using the Mouse Phrenic Nerve Hemidiaphragm Assay (MPN).
Botulinum Neurotoxins: Qualitative and Quantitative Analysis Using the Mouse Phrenic Nerve Hemidiaphragm Assay (MPN).
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DOI：
10.3390/toxins7124855
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发表时间：
2015-11-25
期刊：
影响因子：
4.2
通讯作者：
Rummel A
中科院分区：
文献类型：
--
作者：
Bigalke H;Rummel A
历史上检测肉毒杆菌神经毒素（BoNT）的方法以小鼠生物测定法（MBA）为代表，测定动物存活率。由于MBA的终点是小鼠因呼吸肌肉麻痹而死亡，因此一种体外动物替代方法，称为小鼠膈神经（MPN）试验，采用分离的膈神经-半膈组织。在这里，BoNT引起间接刺激肌肉收缩幅度的剂量依赖性下降。在EQuATox BoNT熟练度测试中，13个测试样品使用MPN分析，通过连续稀释至浴液浓度，分别在BoNT/ a， B和E标准生成的校准曲线范围内产生瘫痪时间。为进行血清型鉴定，将稀释后的样品与多克隆抗bont /A、B或E抗毒素或各抗毒素的组合预孵育。与LFA、ELISA和LC-MS/MS等单一体外方法相比，所有13个样品都得到了正确的定性鉴定，从而获得了更好的结果。鉴定出BoNT血清型后，使用校准曲线计算最终液浓度，然后乘以相应的稀释系数得到样品浓度。根据所使用的BoNT标准的来源，含有10个BoNT/A样本的定量的平均z分数分别为7，含有3个BoNT/B或BoNT/E样本的z分数<2。
The historical method for the detection of botulinum neurotoxin (BoNT) is represented by the mouse bioassay (MBA) measuring the animal survival rate. Since the endpoint of the MBA is the death of the mice due to paralysis of the respiratory muscle, an ex vivo animal replacement method, called mouse phrenic nerve (MPN) assay, employs the isolated N. phrenicus-hemidiaphragm tissue. Here, BoNT causes a dose-dependent characteristic decrease of the contraction amplitude of the indirectly stimulated muscle. Within the EQuATox BoNT proficiency 13 test samples were analysed using the MPN assay by serial dilution to a bath concentration resulting in a paralysis time within the range of calibration curves generated with BoNT/A, B and E standards, respectively. For serotype identification the diluted samples were pre-incubated with polyclonal anti-BoNT/A, B or E antitoxin or a combination of each. All 13 samples were qualitatively correctly identified thereby delivering superior results compared to single in vitro methods like LFA, ELISA and LC-MS/MS. Having characterized the BoNT serotype, the final bath concentrations were calculated using the calibration curves and then multiplied by the respective dilution factor to obtain the sample concentration. Depending on the source of the BoNT standards used, the quantitation of ten BoNT/A containing samples delivered a mean z-score of 7 and of three BoNT/B or BoNT/E containing samples z-scores <2, respectively.