作者要感谢H.奥利瓦雷斯和D如歌的有价值的讨论和建议。这项研究是由美国国家科学基金会一SBIR计划奖（IIP-1127245至BioSentinel Inc。）和美国国防部的合同（W81XWH-07-2-0045到BioSentinel公司）的部分资助。

1。测定试剂的配制<ol><li>解冻200X二硫苏糖醇（DTT），10倍的基质结合缓冲液（10×结合缓冲液下同），10倍的中和缓冲液（食物或pH失衡样品只），和15分钟的10倍BoTest反应缓冲液（10X反应缓冲下同），在室温（RT）或直到完全解冻。 见表1为在此协议中使用的缓冲液和试剂的列表。请参阅表2的本议定书所需的材料和设备清单。 </li><li>旋涡解冻缓冲5秒混匀。 10X和缓冲液，200xDTT和10x反应缓冲液应显示清晰，同时10倍的结合缓冲液将有混浊的外观。暖10X反应缓冲液5分钟，在37°C和重复涡旋如果它融化之后会出现浑浊。 </li><li>产生3.8毫升1X反应缓冲液。 <ol><li>标签15毫升锥形管“1X反应缓冲液”，并添加3.42毫升分子生物学级笏呃，380微升10X反应缓冲液和19微升200倍数码地面电视。颠倒混匀缓冲好。 </li><li>切用干净的刀片季度单EDTA蛋白酶抑制剂药片和四分之一的平板电脑添加到1X反应缓冲液（剩余药片部分可以储存在4℃下以备后用）。NB蛋白酶抑制剂可能不若使用纯化的毒素和简单的缓冲器（ 例如药物样品）是必要的。 </li><li>涡1X反应缓冲液，直到蛋白酶​​片完全溶解。 </li></ol></li><li>暖矩阵A珠（即IP-A珠下同）20分钟在室温。 </li><li>解冻BoTest A / E记者（A / E以下简称记者）在室温避光。 </li><li>标签15毫升锥形管“0.5微米的A / E记者”，并添加45微升的20微米的A / E记者股票至1.8毫升的1X反应缓冲液。吹打调匀，并储存在冰避光。 </li></ol> 2。站ARD曲线生成示例此处所述的标准曲线跨越10-30,000 MLD 50 /克食物或每毫升缓冲液中的半对数稀释（ 表3）。最终用户可以自由地使用替代浓度（如适用）。 <ol><li> 扣球和温育该矩阵与参考材料。使用相同的矩阵的稀释剂为未知的，如果可能的话，以减少基体效应产生的标准曲线。使用明胶的磷酸盐缓冲液（GPB）或磷酸盐缓冲盐水（PBS）作为稀释剂，如果附加，肉毒毒素负矩阵不可用（ 例如，字段或肉毒毒素爆发样品测试）。通过扣球肉毒毒素/ A到所选择的基质按照下面相应的协议生成标准曲线。 <ol><li> 低复杂度的样品（ 如 PBS，GPB，或药物） <ol><li>加入10 ml合适的缓冲液到15毫升锥形管中，备用。该样品将被用作稀释剂的STANDAR3D曲线和未知的稀释液（第4节）。 </li><li>添加1.2毫升缓冲液到离心管中。 </li><li> 注意：此步骤使用肉毒毒素和处理和处置，在与毒素接触任何试剂和材料时，必须特别注意使用。使用个人防护装备和妥善处置所有材料必须按照劳动OSHA准则肉毒毒素（http://www.osha.gov/SLTC/botulism/index.html）美国能源部执行。添加肉毒毒素/ A至1.2毫升样品，使终浓度为30,000 MLD 50 /毫升（36,000 MLD 50个）。 </li></ol></li><li> 液体食物样本（或其他复杂的液体样品）注：初始建议进行试验以确定从上清液澄清的样品的颗粒物质（ 如纸浆）在液体基质中回收的数量会有所不同。这个协议假定至少1.2毫升澄清上清液将会从1.4毫升SAMP恢复乐。增加样本量，如果必要的。 <ol><li>加入10 ml液体食物到15毫升锥形管，并把它放在一边。该样品将被用作稀释剂的标准曲线和未知稀释物（第4项）。 </li><li>称量一个空的1.5 ml离心管并记录其质量。 </li><li>加1.4毫升的液体食品到称重的离心管中。 </li><li>再次称重的离心管中，并通过减去空管的质量计算所添加的食品样品的质量。 </li><li> 注意：此步骤使用肉毒毒素和处理和处置，在与毒素接触任何试剂和材料时，必须特别注意使用。使用个人防护装备和妥善处置所有材料必须按照劳动OSHA准则肉毒毒素（http://www.osha.gov/SLTC/botulism/index.html）美国能源部执行。添加肉毒毒素/ A至1.4 mL样品在30,000 MLD 50 /克食物的终浓度。 </li><li>温浴忒稀释剂和掺入的样品在室温或4℃下进行2小时，得到的BoNT时间与食品基质相互作用，模仿天然污染。 </li></ol></li><li> 固体食物样本（或其他固体样品）注：初始测试被推荐来确定上清液从均质化和澄清的样品后加入1ml GPB /克食物中回收的量。这个协议假定至少1.2毫升澄清的上清液会从2克样品回收。增加样本量，如果必要的。 <ol><li>称取满分克固体样品放入50ml锥形管中，备用。这将被用来作为稀释剂。 </li><li>称出2克固体食物样品到第二50ml锥形管中。 </li><li> 注意：此步骤使用肉毒毒素和处理和处置，在与毒素接触任何试剂和材料时，必须特别注意使用。使用个人防护装备和妥善处置所有材料s必须根据劳动OSHA准则肉毒毒素（http://www.osha.gov/SLTC/botulism/index.html）美国能源部执行。加的BoNT / A至2克样品的表面，以30,000 MLD 50 /克食物（60,000总MLD 50）的终浓度。 </li><li>孵育所述稀释剂和加料样品在室温或4℃下进行2小时，得到的BoNT时间与食品基质相互作用，模仿天然污染。 </li></ol></li></ol></li><li> 样品均质和缓冲调整。过程中的加标标准曲线样品和稀释液，根据样品类型上面生成。 <ol><li> 低复杂度的样品 <ol><li>没有进一步的样品处理是必要的。 </li></ol></li><li> 液体食物样本（或其他复杂的液体样品） <ol><li>没有样本的同质化是必要的。 </li><li>加入140微升10X缓冲液中和到1.4毫升加标样品和1ml 10倍Neutralizat离子缓冲液中加入10毫升稀释样品。混合样品深受反转。 </li><li>部分在6000 XG和4℃离心10分钟澄清两个样本立即取出上清液并转移到新管。 </li></ol></li><li> 固体食物样本（或其他固体样品） <ol><li>加入2ml GPB（1毫升GPB /克食物）的2克的BoNT / A-飙升固体食物样品和10ml GPB以各10 g稀释样品。 </li><li>用杵直到充分混合使样品均质化。取决于样品的性质，可能有重大的小块，不能均匀，这是可以接受的。另外，也可以使用机械同质化的方法。 使用搅拌机的不推荐，因为它可能会失活以及雾化的毒素。 </li><li>加入10倍和缓冲液体积的十分之一基于近似总量上的样品稀释液（ 如 2毫升，如果量为20毫升）。推断在肉毒毒素/ A-飙升样品的总量和新增的10倍和缓冲液体积的十分之一。混合样品深受反转。 </li><li>部分在6000 XG和4℃离心10分钟澄清两个样本立即取出上清液并转移到新管。 </li></ol></li></ol></li><li> 制备标准曲线的连续稀释用于测试 <ol><li>使用的BoNT / A-掺入标准曲线样品作为D1和nonspiked样品作为稀释液，按表3产生在1.5ml微量离心管中剩余的标准曲线样品。 </li></ol></li></ol> 3。准备未知样本这部分可以并行第2完成。 <ol><li> 确定未知量的数目和稀释液。如果可能的未知测试，一式三份。 <ol><li>对于定性测定，比要求的要处理的样品作为记述的运行未知数没有任何进一步的稀释下面床。 </li><li>对于定量测定，准备至少2 1:10稀释的样品，如下所述，以确保一个或多个样品落入测定响应的线性范围内。使用相同的矩阵的稀释剂为未知的，如果可能的话，如第3节中所述，以减少基体效应产生的稀释。否则，用PBS或GPB作为稀释剂。 </li></ol></li><li> 生成并根据样品类型稀释未知样品。 <ol><li> 低复杂度的未知数（ 如 PBS，GPB，或药物） <ol><li>新增至少750微升未知的微量离心管中产生未知稀释1。 </li><li>加入675微升稀释至标有未知稀释2和3两的微量离心管。稀释剂将用于标准曲线的生成相同的材料。 </li><li>通过串行传输75微升稀释1到2稀释管和混合稀释稀释1。 </li><li>连续dilutË稀释2转增75微升稀释2到3稀释管和混合。 </li></ol></li><li> 液体食品未知数（或其他复杂的液体样品）注：初始建议进行试验以确定从上清液澄清的样品回收在第3节中讨论的音量。增加样本量，如果必要的。 <ol><li>添加≥875微升液体未知到微量离心管中。 </li><li>加入10倍和缓冲液体积的十分之一到样品（ 如 87.5微升为875微升的样品）。 </li><li>部分在6000 XG和4℃离心10分钟澄清样品立即将上清液转移到新管中。这是未知的稀释度。 </li><li>添加675微升稀释到两个管子标记未知稀释2和3。稀释剂将用于标准曲线的生成相同的处理物 。 </li><li>通过串行传输稀释稀释1环75微升稀释1到2稀释管和混合。 </li><li>通过串行传输75微升稀释2到3稀释管和混合稀释稀释2。 </li></ol></li><li> 固体食物未知数（或其它固体样品）注：初始测试被推荐来确定上清液澄清的样品回收作为在第3节中讨论的体积。增加样本量，如果必要的。 <ol><li>称出2克固体未知样品放入50ml锥形管中。 </li><li>加入2ml GPB（1毫升GPB /克食物），以2克固体样品。 </li><li>使样品均质化，如第3.2.3.2中所述。 </li><li>加入10倍和缓冲液体积的十分之一基于近似总量的样品（ 如 0.4毫升，如果量为4毫升）。混合样品深受反转。 </li><li>部分在6000 XG和4℃离心10分钟澄清样品立即出现Ÿ转移≥750微升上清到离心管中。这是未知的稀释度。 </li><li>加675毫升稀释剂以两个管标记为未知稀释2和3。稀释剂将用于标准曲线的生成相同的处理物 。 </li><li>通过串行传输75微升稀释1到2稀释管和混合稀释稀释1。 </li><li>通过串行传输75微升稀释2到3稀释管和混合稀释稀释2。 </li></ol></li></ol></li></ol> 4。最终样本澄清如果检测液体或固体食物样品，离心所有样品5分钟，≥14,000 XG在微量充分澄清样品。立即取出上清液并转移到新管。 5。板安装和肉毒毒素/一个下拉<ol><li>具体的板块布局是依赖于应用程序，但是，不要用外面的井，从而以避免边缘效应。每个未知样品和标准曲线样品D1-D8需要3口井，而样品D9需要6口井。一个建议的板布局如图1所示。 </li><li>加入20微升10×结合缓冲液中向每个孔中使用。 </li><li>加入200μl每个澄清稀释和未知的每个三口井（六口井的D9）为一式三份测试。混合板，持续10秒的微混合器。 </li><li>添加IP-A珠。 <ol><li>旋涡的IP-A珠，持续10秒的最高速度。继续涡旋如果珠粒不充分悬浮并均匀。 </li><li>移液管20微升的IP-A珠子每个样品孔。 </li><li>混合盘30秒的微混合器。 </li></ol></li><li>使用旋转板孵化器2小时，在750转，25℃或室温孵育板。检测性能高度依赖于生成和在所有孵育步骤保持的IP-A珠悬浮液。总重悬珠子用microp在所有孵育步骤，使用轨道微孔板摇床造粒和维护停牌后后期混音。 </li></ol> 6。板清洗和再悬浮珠<ol><li>通过手或通过使用磁珠兼容自动洗板机洗板。配置为磁珠自动洗板机大大提高测定的吞吐量。 <ol><li> 手动清洗 <ol><li>标记一个50ml锥形管中“1X洗涤缓冲液”和添加45毫升分子生物学级水和5ml 10倍矩阵洗涤缓冲液。颠倒混匀缓冲好。 </li><li>从旋转板孵化器中取出钢板。 </li><li>立即将板于96孔磁珠分离板5分钟。 </li><li>同时使板在96孔磁珠分离板，轻轻取出并丢弃从使用单或多通道移液器样品孔的上清液。不吸珠 - 视觉监测吸气缓冲枪头意外珠去除。如果去除见证，轻轻向后添加提示内容到井，reseparate，并重复删除。 </li><li>添加300μl的1X洗涤缓冲液到每个样品孔。 </li><li>通过混合盘30秒的微混合器充分重悬珠。 </li><li>将培养板在96孔磁珠分离板2分钟。 </li><li>删除和丢弃之前从样品孔的上清液。 </li><li>总共4次洗涤，重复步骤6.1.1.5-6.1.1.8三次以上。 </li><li>与板的96孔磁珠分离板，目视检查井，以确认即使除去上清液;用移液器除去必要的过量残留的缓冲液。一些缓冲将留在孔中，必须小心以避免除去珠或珠干燥。 </li><li>加50μl的1X反应缓冲液，以每个样品井和混合板30本身C对微孔板混合器充分重悬珠。如果需要，使用吸管，充分重悬珠。 </li></ol></li><li> 自动洗板 <ol><li>设置和程序根据表4的垫圈。清洁和冲洗垫圈高品质（ 如毫微纯）水。 </li><li>总理洗衣机通过运行程序“总理”。 </li><li>从旋转板孵化器中取出钢板。 </li><li>立即将板在96孔磁珠分离板对板垫圈。 </li><li>运行链接程序“主洗”。程序的第一步是5分钟孵育步骤，其中洗衣机将是静止的。 </li><li>下面的程序完成后，从洗衣机取出板，加50微升1×反应缓冲液到每个样品孔，并且混合的板进行30秒的微板混合器充分重悬珠。如果需要，使用吸管，充分重悬珠。 </li></醇&gt; </li></ol></li></ol> 7。试验启动和孵化<ol><li>加入50微升0.5微米的A / E记者（见第1节），以每个样品孔中，混合30秒的微混合器充分重悬珠。 </li><li>加入100微升水，每个未使用的以及在平板上，以防止边缘效应。 </li><li>密封板与板密封胶带，并用旋转板孵化器在750转，25℃或室温孵育板。孵化期间由轻保护板。 </li></ol> 8。数据收集和分析注意：此法是一种可以直到获得所需的灵敏度被测量多次，实时测定中，也有不需要停止的试剂。推荐初始读取时间为2，4，并与孵育时间24小时培养时间与检测的灵敏度增加。 <ol><li>数据收集<ol><li>在每个读出时间，从旋转板培养箱取出板，取下密封ING胶带，并立即放置在盘上的96孔磁珠分离板。允许珠来分离2分钟。 </li><li>将板在酶标仪和测量排放〜470和〜434 nm的激发下〜526纳米。 </li><li>如果需要额外的读取时间是理想的，重悬珠子，持续30秒的微混合器，重新密封板，并在板返回到旋转板孵化器。 </li></ol></li><li>数据分析<ol><li>通过在470 nm处的RFU值除以相对荧光单位（RFU）的值在526 nm处计算各样品的发射率。 </li><li>绘制排放比与日志[肉毒毒素/ A]进行标准曲线的数据点。根据测试的BoNT / A的效力范围，S形剂量 - 反应曲线，将获得（见代表性成果）。 </li><li>适合与可变斜率剂量-反应曲线Y =底+（顶-底）/标准曲线数据（1 +10 ^（（logEC 50-X）*坡地）），其中X是浓度的对数，Y是反应，和Y在底部开始，去到上面用S形的形状。 </li><li>确定检测限（LOD），定量限（LOQ）和半最大有效浓度（EC 50）的限制。检测限被定义为具有低于空白对照的发射率小于3个标准偏差（SDS）的样本（N = 6）。定量限被定义为具有一个样品的发射率小于10 SD中下面的空白对照组（n = 6）。 EC 50从S形的剂量-反应曲线拟合来确定。 </li><li>插任何未知样品对S形剂量 - 响应标准曲线的效力。 <ol><li>对于定量的结果，未知样品应理想落在标准曲线的线性部分内。通过计算20-80％的总测定响应窗口接近标准曲线的线性部分（ 例如 ，如果标准曲线r的发射率安格斯从0.5-2.5，线性范围将是标准曲线，从0.9-2.1范围内的部分）。 </li><li>对于定性结果，与之比较的标准曲线的LOD和LOQ未知样品。 </li><li>不推断未知样品超出标准曲线的范围。 </li></ol></li></ol></li></ol>

调频邓宁，以旧换新广场，楼Zeytin，和厕所塔克是BioSentinel公司BioSentinel员工或业主目前生产并商业化了一些本报告中的试剂。

本协议描述的量化肉毒毒素/一个复杂的，全毒素，或梭状芽孢杆菌培养上清复杂基质中的程序。该协议是一样的，但是，与他们各自的矩阵分析56,60测试其他血清型肉毒毒素（ 如肉毒毒素/ B，E和F）时，虽然检测灵敏度将跨越血清型和分析有所不同。这个协议没有考虑所有类型的样品的可能和一些修改可以根据具体的样品组合物和期望的应用是必需的。矩阵可以包括食物样?...

肉毒杆菌神经毒素毒素（BoNTs）是已知的最致命的物质，估计在1-3纳克静注人致死剂量/公斤1,2。七结构相似的肉毒毒素血清型，至G标记为A，存在，每个由负责细胞结合，摄取和易位到细胞质和轻链编码一种锌内肽酶3-5的重链结构域。肉毒毒素的毒性精美的结果，在某种程度上，在神经肌肉接头6的特异性结合并进入运动神经元。神经元一旦进入，所述轻链肽酶特异性切割1的可溶性N-乙基马来酰亚胺-敏感因子附着蛋白受体（SNARE）所需的囊泡融合蛋白的一个或多个，抑制神经递质的释放，并导致弛缓性麻痹7-14。俗称疾病“肉毒杆菌”的膈肌和肋间肌由肉毒毒素麻痹，最终导致呼吸衰竭而死亡，除非早期诊断和治疗被接收。 人类食源性肉毒中毒是最常见的肉毒毒素血清型A，B，E关联，和F（肉毒毒素/ A，肉毒毒素/ B 等 ），通常从污染的食物15,16摄入导致，虽然，伤口肉毒中毒的几种情况据报道其中静脉吸毒者17,18。在美国，从梭状芽孢杆菌孢子的一岁以下儿童的摄入导致婴儿肉毒中毒是肉毒杆菌19-21的最常见形式。然而，不当家罐头和食品加工产生的食源性肉毒毒素暴发的报道同时在美国和海外。与2000-2009年至少 ​​有338例食源性肉毒中毒的报道世界各地，包括六人死亡22。快速和灵敏地检测食源性肉毒中毒爆发的能力是可能帮助早期诊断的重要指标23,24。此外，检测方法，使成本效益和常规食品检测将导致改善粮食安全。 肉毒毒素的神经元特异性和长的生物半衰期也使得它成为有效的治疗。在美国，肉毒毒素类药物被美国食品与药物管理局的化妆品条件和神经肌肉有关的疾病，包括眉间纹，颈部肌张力障碍，偏头痛，膀胱过度活动症，以及斜视治疗通过。众多的“关闭标签”的应用被记录，包括高剂量治疗重症肌功能不全25-28。准确的毒素定量是正确的剂量至关重要，因为剂量不足可能导致治疗无效，而超剂量将患者置于潜在的有害副作用的风险。不幸的是，没有标准的效价测定的协议不同制造商之间共享，从而导致肉毒毒素为基础的药物机生产线设备之间的不平等定义仙29-31。 标准测试肉毒毒素是小鼠生物法，其中含肉毒毒素样品腹腔注射到小鼠和死亡的人数录得超过1-7天16,32,33。小鼠生物检测与检测5-10皮克的BoNT / A 34限（LOD）非常敏感，但道德问题了动物的使用，培养人才的成本高，维护动物的设施，长期的分析时间，而且缺乏标准化的协议导致的呼叫建立标准化，无动物肉毒毒素检测和定量方法35-39。最近，一些替代肉毒毒素定量方法的发展，提供鼠标或近小鼠生物检测的灵敏度40-49。这些方法通常使用的荧光，质谱，或免疫学方法，并提供检测时间比不使用动物的小鼠生物法大大缩短。质谱办法结合免疫techniq的UE却显示出了检测和量化的BoNT包含在食品和其他复杂的样品，但是，人员的培训要求，专门的设备限制这些测定50-55。大多数其他替代分析是不容易适用于复杂样品测试或缺乏所需的日常肉毒毒素测试的吞吐量。试图开发在体外试验方法的敏感性，以配合肉毒毒素的极端效力时的食品样品的粘度，pH值，含盐量和基质成分的高度可变的性质提出了一个特别困难的挑战。此外，即使是简单和相对良性的缓冲系统，如那些从肉毒毒素为基础的药物产品的再悬浮产生的，含有盐，白蛋白，糖和稳定剂（ 即赋形剂），该显著影响的体外效力的BoNT 56。毒素净化所需的所有准确的活性测试，但最简单的样品56-59的。 该BoTest矩阵分析被设计用于快速，高通量，和肉毒毒素的使用设备在研究实验室56,60通常发现高度复杂样品一致的定量。这些测定中使用的顺磁珠共价连接到血清型特异性抗肉毒毒素的抗体结合和隔离的BoNT出样品，然后除去洗涤干扰矩阵的化合物。洗涤后，肉毒毒素结合蛋白水解活性，然后量化使用的肉毒毒素血清型进行测试兼容记者优化的反应缓冲液。这些记者是荧光蛋白质组成的N-末端青色荧光蛋白（CFP）部分和C-末端的黄色荧光蛋白衍生物（金星）部分由一个基底的BoNT，SNAP25残基141-206或残基突触33-94相连构成的BoTest A / E或B / D / F / G记者，分别为45。记者裂解肉毒毒素是用福斯特共振能量转移（FRET）进行监测。当t他记者是完整的，CFP的激发导致FRET金星，淬火CFP排放和令人兴奋的金星发射。记者通过肉毒毒素的裂解防止荧光共振能量转移，导致增加CFP排放和减少排放的金星。肉毒毒素活性可再使用CFP和金星排放量的比例来定量测定。 LOD低于3皮克有可能从广泛的使用高通量的96孔板格式56的食品。更高的灵敏度可以使用更大的样本量来获得，因为试验允许在磁珠表面毒素的浓度。 对于BoNTs A，B，E和F的BoTest矩阵分析被开发，并与食品，制药，环境样品56,60测试。在这里，我们描述了执行这些分析的肉毒毒素在低复杂度的检测（ 如制药，肉毒毒素在缓 ​​冲液）和高复杂性（ 如食物，环境）的样本程序。具体的处理方法几个样品类型，将在本协议并在这里没有描述的样品类型通常可以使用的呈现方法的组合调整。该协议的开发和测试了肉毒毒素/ A，但适应于其他血清型肉毒毒素使用各自的试验为证明别处56,60。

<table border="0" cellpadding="0" cellspacing="0" width="648">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:192px;">BoTest Matrix A Botulinum Neurotoxin Detection Kit</td>
			<td style="width:107px;">BioSentinel</td>
			<td style="width:104px;">A1015</td>
			<td style="width:245px;">Detection kits for BoNT/B and F are also available.</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:192px;">Varioskan Flash fluorescence microplate reader</td>
			<td style="width:107px;">Thermo-Fisher Scientific</td>
			<td style="width:104px;">5250040</td>
			<td style="width:245px;">Most monochromator- or filter-based units with 434 nm excitation and 470 and 526 nm emission capability can be used.</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:192px;">96-well Magnetic Bead Separation Plate</td>
			<td style="width:107px;">V&#38;P Scientific&#160;</td>
			<td style="width:104px;">VP771H</td>
			<td style="width:245px;">Other magnetic plates may be used, but the plate should be designed to separate the beads to the side of the well.</td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:192px;">Magnetic Bead-Compatible Plate Washer</td>
			<td style="width:107px;">BioTek&#160;</td>
			<td style="width:104px;">ELx405 VSRM</td>
			<td style="width:245px;">Optional, only required for automated plate washing.&#160; Other magnetic bead-compatible plate washers may also be used, but should be tested before use.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:192px;">Microcentrifuge</td>
			<td style="width:107px;">Various</td>
			<td style="width:104px;">N/A</td>
			<td style="width:245px;">Optional, only required for samples needing centrifugation.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:192px;">MixMate plate mixer</td>
			<td style="width:107px;">Eppendorf</td>
			<td style="width:104px;">22674200</td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:192px;">Orbital Shaker</td>
			<td style="width:107px;">Various</td>
			<td style="width:104px;">N/A</td>
			<td style="width:245px;">Used at room temperature or at 25 &#176;C If temperature control is available</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:192px;">EDTA-free Protease Inhibitor Tablets</td>
			<td style="width:107px;">Roche</td>
			<td style="width:104px;">4693132001</td>
			<td style="width:245px;">Only required for food or environmental testing.&#160; Protease inhibitors must be EDTA-free.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:192px;">BoNT/A&#160;</td>
			<td style="width:107px;">Metabiologics</td>
			<td style="width:104px;">N/A</td>
			<td style="width:245px;">Optional, only required for standardization and quantification purposes&#160;</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:192px;">Black, Flat-bottomed 96-well Plates</td>
			<td style="width:107px;">NUNC</td>
			<td style="width:104px;">237105</td>
			<td style="width:245px;">Plates should not be treated</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:192px;">96-well Plate Sealing Tape</td>
			<td style="width:107px;">Thermo Scientific</td>
			<td style="width:104px;">15036</td>
			<td style="width:245px;"></td>
		</tr>
	</tbody>
</table>

isolation and quantification of botulinum neurotoxin from complex matrices using the botest matrix assays

准确的检测和肉毒杆菌神经毒素（肉毒毒素）对复杂基质中的量化是必需的药品，环境和食物样本测试。期间爆发取证，病人的诊断，食品安全检测是必要的食品快速检测肉毒毒素准确而有力测试是必需的肉毒毒素为基础的药物产品制造和病人安全。被广泛使用的小鼠生物法进行肉毒毒素测试是高度敏感，但缺乏必要的快速和常规检测肉毒毒素的精度和吞吐量。此外，生物测定的使用动物导致了药品监管部门和动物权利支持者在美国和国外的电话，以取代小鼠生物法进行肉毒毒素测试。几种体外测定法更换已开发工作以及与纯化的肉毒毒素在简单缓冲器，但大部分都没有被证明是适用于检测在高度复杂的矩阵。这里，一个协议，用于检测肉毒毒素在使用BoTest矩阵分析复杂基质中呈现。该测定由三部分组成：第一部分涉及制备样品进行测试的，第二部分是使用抗肉毒毒素抗体包被的顺磁珠从基质纯化肉毒毒素免疫沉淀步骤，和第三部分量化了孤立的BoNT的蛋白水解活动采用荧光记者。该协议是用于高通量试验在96孔板上使用液体和固体的矩阵写入，并且需要大约2个小时手工制备具有4-26小时的总的分析时间取决于样品的类型，毒素负载和所需的灵敏度。数据表示为用磷酸缓冲盐水中，药物产品，培养物上清液，2％牛奶，鲜番茄的BoNT / A的测试，并且包括用于检测成功的关键参数的讨论。

的图表总结了在所描述的协议的步骤示于图2。 4-26小时之间的检测需要根据样品的类型和所需的检测灵敏度来完成，但只〜2小时的手工操作时间。该测定在96 - 孔板中进行，并根据所执行的测试的类型，允许多达20个样品，包括每盘标准的一式三份测试。 图3显示了使用肉毒毒素代表性的化验结果/ A全毒素飙升至PBS及与所描述的协议下2，4，和24小时?...

Watch this Scientific Journal Video about Isolation and Quantification of Botulinum Neurotoxin From Complex Matrices Using the BoTest Matrix Assays at JoVE.com

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

该BoTest矩阵A型肉毒毒素（肉毒毒素）检测方法快速净化，并从各种样品基质的量化肉毒毒素。在这里，我们提出了一个协议，用于肉毒毒素，从固体和液体基质的检测和定量，并证明有肉毒杆菌，番茄，牛奶的检测。

隔离和肉毒杆菌神经毒素定量从复杂的基质使用BoTest矩阵测定

Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample ...

isolation-quantification-botulinum-neurotoxin-from-complex-matrices

Research

JoVE Journal

Neuroscience

15.9K 次观看.  BioSentinel Inc., Madison, WI. 该BoTest矩阵A型肉毒毒素（肉毒毒素）检测方法快速净化，并从各种样品基质的量化肉毒毒素。在这里，我们提出了一个协议，用于肉毒毒素，从固体和液体基质的检测和定量，并证明有肉毒杆菌，番茄，牛奶的检测。 

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

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

成年鼠神经干细胞的分离和扩大使用神经干含量

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

科研

神经科学

Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. Reciprocally, medium spiny neurons in the striatum that give rise to the striatonigral (direct) pathway innervate the substantia nigra2. The development of these axon tracts is dependent upon the combinatorial actions of a plethora of axon growth and guidance cues including molecules that are released by neurites or by (intermediate) target regions3,4. These soluble factors can be studied in vitro by culturing mdDA and/or striatal explants in a collagen matrix which provides a three-dimensional substrate for the axons mimicking the extracellular environment. In addition, the collagen matrix allows for the formation of relatively stable gradients of proteins released by other explants or cells placed in the vicinity (e.g. see references 5 and 6). Here we describe methods for the purification of rat tail collagen, microdissection of dopaminergic and striatal explants, their culture in collagen gels and subsequent immunohistochemical and quantitative analysis. First, the brains of E14.5 mouse embryos are isolated and dopaminergic and striatal explants are microdissected. These explants are then (co)cultured in collagen gels on coverslips for 48 to 72 hours in vitro. Subsequently, axonal projections are visualized using neuronal markers (e.g. tyrosine hydroxylase, DARPP32, or &beta;III tubulin) and axon growth and attractive or repulsive axon responses are quantified. This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of mesostriatal and striatonigral axon growth and guidance during development. Using this assay, it is also possible to assess other (intermediate) targets for dopaminergic and striatal axons or to test specific molecular cues.

Explants from the midbrain dopamine system and striatum are used in a collagen matrix assay for the in vitro analysis of mesostriatal and striatonigral pathway development. In this assay axonal outgrowth and guidance can be manipulated and quantified. It can also be modified for assessing other regions or molecular cues.

小鼠多巴胺和三维胶原基质检测纹状体外植体的解剖和文化

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. ...

Wholemount Immunohistochemistry for Revealing Complex Brain Topography

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its relatively uniform cytoarchitecture is a complex array of parasagittal domains of gene and protein expression. The molecular compartmentalization of the cerebellum is mirrored by the anatomical and functional organization of afferent fibers. To fully appreciate the complexity of cerebellar organization we previously refined a wholemount staining approach for high throughput analysis of patterning defects in the mouse cerebellum. This protocol describes in detail the reagents, tools, and practical steps that are useful to successfully reveal protein expression patterns in the adult mouse cerebellum by using wholemount immunostaining. The steps highlighted here demonstrate the utility of this method using the expression of zebrinII/aldolaseC as an example of how the fine topography of the brain can be revealed in its native three-dimensional conformation. Also described are adaptations to the protocol that allow for the visualization of protein expression in afferent projections and large cerebella for comparative studies of molecular topography. To illustrate these applications, data from afferent staining of the rat cerebellum are included.

Neural circuits are topographically organized into functional compartments with specific molecular profiles. Here, we provide the practical and technical steps for revealing global brain topography using a versatile wholemount immunohistochemical staining approach. We demonstrate the utility of the method using the well-understood cytoarchitecture and circuitry of cerebellum.

为揭示复杂的大脑地形wholemount免疫组化

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

从胚胎前脑内皮细胞的分离和培养

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is required to shape functional neuronal circuits. The protocol described here explains how to use cell culture inserts bearing a porous membrane (filter) to obtain large amounts of pure axonal preparations suitable for examination by conventional biochemical or immunocytochemical techniques. The functionality of these filter inserts will be demonstrated with models of developmental pruning and Wallerian degeneration, using explants of embryonic dorsal root ganglion. Axonal integrity and function is compromised in a wide variety of neurodegenerative pathologies. Indeed, it is now clear that axonal dysfunction appears much earlier in the course of the disease than neuronal soma loss in several neurodegenerative diseases, indicating that axonal-specific processes are primarily targeted in these disorders. By obtaining pure axonal samples for analysis by molecular and biochemical techniques, this technique has the potential to shed new light into mechanisms regulating the physiology and pathophysiology of axons. This in turn will have an impact in our understanding of the processes that drive degenerative diseases of the nervous system.

This article describes a neuronal culture system that can be used to obtain large quantities of pure axonal samples for biochemical and immunocytochemical analyses. This technique will allow an improved understanding of normal axonal physiology and signaling pathways leading to neurodegeneration.

生产和轴突隔离从感觉神经元的生化分析使用多孔过滤器

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is ...

A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors

Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic neurotransmitter release. Botulinum neurotoxin serotype A (BoNT/A) is a zinc metalloprotease that blocks exocytosis of neurotransmitter by cleaving the SNAP-25 component of the SNARE complex. Currently there are no licensed medicines to treat BoNT/A poisoning after internalization of the toxin by motor neurons. The development of effective therapeutic measures to counter BoNT/A intoxication has been limited, due in part to the lack of robust high-throughput assays for screening small molecule libraries. Here we describe a high content imaging (HCI) assay with utility for identification of BoNT/A inhibitors. Initial optimization efforts focused on improving the reproducibility of inter-plate results across multiple, independent experiments. Automation of immunostaining, image acquisition, and image analysis were found to increase assay consistency and minimize variability while enabling the multiparameter evaluation of experimental compounds in a murine motor neuron system.

Botulinum neurotoxin is one of the most potent toxins among Category-A biothreat agents, yet a post-exposure therapeutic is not available. The high content imaging approach is a powerful methodology for identifying novel inhibitors as it enables multiparameter screening using biologically relevant motor neurons, the primary target of this toxin.

高含量的成像检测方法的肉毒毒素抑制剂的鉴定

Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic ...

External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result of chemical inputs to its synapses. However, neurons can also be excited by an imposed electric field. In particular, recent clinical applications activate neurons by creating an electric field externally. It is therefore of interest to investigate how the neuron responds to the external field and what causes the action potential. Fortunately, precise and controlled application of an external electric field is possible for embryonic neuronal cells that are excised, dissociated and grown in cultures. This allows the investigation of these questions in a highly reproducible system.
In this paper some of the techniques used for controlled application of external electric field on neuronal cultures are reviewed. The networks can be either one dimensional, i.e. patterned in linear forms or allowed to grow on the whole plane of the substrate, and thus two dimensional. Furthermore, the excitation can be created by the direct application of electric field via electrodes immersed in the fluid (bath electrodes) or by inducing the electric field using the remote creation of magnetic pulses.

Neuronal cultures are a good model for studying emerging brain stimulation techniques via their effect on single neurons or a population of neurons. Presented here are different methods for stimulation of patterned neuronal cultures by an electric field produced directly by bath electrodes or induced by a time-varying magnetic field.

使用电动和一维和二维文化磁场神经元的外部激励

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result ...

Enumeration of Neural Stem Cells Using Clonal Assays

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. NSCs and neural progenitors (NPs) are commonly cultured in vitro as neurospheres. This protocol describes in detail how to determine the NSC frequency in a given cell population under clonal conditions. The protocol begins with the seeding of the cells at a density that allows for the generation of clonal neurospheres. The neurospheres are then transferred to chambered coverslips and differentiated under clonal conditions in conditioned medium, which maximizes the differentiation potential of the neurospheres. Finally, the NSC frequency is calculated based on neurosphere formation and multipotency capabilities. Utilities of this protocol include the evaluation of candidate NSC markers, purification of NSCs, and the ability to distinguish NSCs from NPs. This method takes 13 days to perform, which is much shorter than current methods to enumerate NSC frequency.

Neural stem cells (NSCs) refer to cells&#160;which can self-renew and differentiate into the three neural lineages. Here, we describe a protocol to determine NSC frequency in a given cell population using neurosphere formation and differentiation under clonal conditions.

使用克隆测定神经干细胞的计数。

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

分离和成人神经干细胞的培养从鼠标Subcallosal区

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Light-Induced Molecular Adsorption of Proteins Using the PRIMO System for Micro-Patterning to Study Cell Responses to Extracellular Matrix Proteins

Cells sense a variety of extracellular cues, including the composition and geometry of the extracellular matrix, which is synthesized and remodeled by the cells themselves. Here, we present the method of Light-Induced Molecular Adsorption of Proteins (LIMAP) using the PRIMO system as a patterning technique to produce micro-patterned extracellular matrix (ECM) substrates using a single or combination of proteins. The method enables printing of ECM patterns in micron resolution with excellent reproducibility. We provide a step-by-step protocol and demonstrate how this can be applied to study the processes of neuronal pathfinding. LIMAP has significant advantages over existing micro-printing methods in terms of the ease of patterning more than one component and the ability to generate a pattern with any geometry or gradient. The protocol can easily be adapted to study the contribution of almost any chemical component towards cell fate and cell behavior. Finally, we discuss common issues that can arise and how these can be avoided.

Our overall aim is to understand how cells sense extracellular cues that lead to directed axonal growth. Here, we describe the methodology of Light-Induced Molecular Adsorption of Proteins, used to produce defined micro-patterns of extracellular matrix components in order to study specific events that govern axon outgrowth and pathfinding.

使用 PRIMO 微模式系统对蛋白质的光诱导分子吸附，研究细胞对细胞外基质蛋白的反应

Cells sense a variety of extracellular cues, including the composition and geometry of the extracellular matrix, which is synthesized and remodeled by the ...