作者没有确认。

人的矩阵是从商业来源购买的, 经其机构审查委员会批准, 但应视为潜在的传染性。确保实验室环境使用维护安全文化12。
1. 在开始化验之前
<ol><li>根据制造商的说明, 准备链亲和素磁性微珠13。</li>
	<li>
准备质量控制样品 (QCs): 在特定物种的基质 (例如, 汇集脑脊液或血清) 中, 将阳性控制抗体 (如, a) (例如, a) 钉在一起。 注: FDA 和均线指导建议准备一个高和低 QC 的化验验证和例行测试1,14。<ol>
<li>例如, 要获得10µg/毫升的 QC, 将10µL 1 毫克/毫升的股票阳性控制量增加到990µL 的相关基质 (如CSF), 总容积为1000µL。</li>
		<li>整除在一个适合单一用途的体积 (例如, 50 µL) 的 QC 样品, 并冻结整除数在-60 到-80 摄氏度1。</li>
		<li>整除一个切点控制 (CC), 物种特异矩阵没有处理的测试文章 (如汇集脑脊液或血清) 为单一用途 (例如, 50 µL) 和冻结整除数在-60 到-80 摄氏度1。</li>
	</ol>
</li>
	<li>根据制造商的15号协议, 使用蛋白质标签试剂盒, 在荧光和反应器之间进行共轭响应。整除在一个适合单一用途的体积 (例如, 50 µL) 和冻结整除数在-60 到-80 摄氏度。</li>
</ol>2. 1 天: 细胞电镀和样品制备
<ol><li>
细胞板制备
	<ol>
<li>使用组织培养罩, 并保持无菌环境的以下步骤。喷雾每一个对象, 将放置在引擎盖与70% 乙醇和保持一个干净的环境16,17,18。</li>
		<li>温暖细胞生长培养基 (例如, RPMI-1640 与10% 胎牛血清和1% 青霉素-链霉素) 在水或珠浴在37摄氏度19。</li>
		<li>将人 T 淋巴细胞 Jurkat 细胞和板100µL 每井在 7.5 x 105细胞/毫升的 96-井圆底细胞培养板, 适合使用的流动细胞仪配备了板式装载机。<ol>
<li>例如, 使用 hemocytometer 或自动单元格计数器计算单元格20、21。</li>
			<li>确保细胞在进行实验前至少有70% 的生存能力 (例如, 评估与台盼蓝染色的可行性)17。</li>
		</ol>
</li>
		<li>在一个37°c 孵化室孵化的细胞与 5% CO2和95% 湿度过夜 14–20 h。</li>
	</ol>
</li>
	<li>
筛选样品准备
	<ol>
<li>使用无血清的培养基 (如RPMI-1640) 稀释实验对象或测定对照样品。在这里的例子方法, 稀释样品 1:2. 5 在 RPMI-1640 通过增加60µL 样品到90µL 无血清的媒介。(例如, 8 条带管)。继续步骤3.1 以进行化验, 准备荧光标记的治疗方法。 注: 1:2. 5 的稀释是经过实验确定的, 对于这里提出的方法是最佳的。应为每个开发的方法确定最佳样品稀释。</li>
	</ol>
</li>
	<li>
确认珠和样品制备
	<ol>
<li>计算验证样品所需的微球共轭珠的数量。<ol>
<li>例如, 对于10个样本, 请使用10个样本 x 100 µL, 每个样本 + 30% 额外的共轭珠, 以弥补在洗涤步骤中的任何损失 = 1300 µL-共轭珠。</li>
		</ol>
</li>
		<li>把珠子彻底涡旋。将计算出的珠子数添加到15毫升锥形管中进行洗涤。</li>
		<li>通过添加1300µL 的耦合缓冲器或由制造商建议的 (例如, 磷酸盐缓冲盐水与0.1% 吐温 20), 在13以下的步骤洗涤。</li>
		<li>彻底涡流管。将管置于磁性管架中2分钟。在不干扰珠子的情况下, 小心地吸入并丢弃上清。 注意: 当珠子被拉到磁铁上时, 溶液将从暗褐色变成透明。</li>
		<li>并用重悬1300µL 耦合缓冲器的珠子。重复洗涤步骤, 共四洗涤。</li>
		<li>最后一次清洗后, 将珠子悬浮在1300µL 的耦合缓冲器中 (以前在步骤2.3.1.1 中计算)。根据板材图 (例如, 每个样品或 QC 的一个证实的好, 每井加100µL 到一个96井, 白色, 圆底, 无粘结聚丙烯板材). 当与荧光标记的应急检测表一起孵化时, 该样本将被分割为重复项。</li>
		<li>把盘子放在96井边的磁铁上, 让珠子形成小球。小心地吸清清液并丢弃。 注: 解决方案将从暗褐色转到清除后, 在侧面掠过磁铁约最小。得到的珠子被称为 "干" 珠。</li>
		<li>如果样品筛选阳性, 并被确认, 添加100µL 的每个样品与和没有反应反应-共轭珠进入适当/分配好。用塑料薄膜封住盘子, 在室温 (RT) 大约 800 rpm 的旋转振动筛上晃动至少60分钟。 注: 以前准备和冻结的积极和负 QCs 和 CC 应包括在每一个板块。</li>
		<li>孵化后, 将验证板放在96井侧掠过的磁铁上, 允许珠子形成小球。</li>
	</ol>
</li>
	<li>
滴度稀释系列制剂
	<ol>
<li>要准备一个效价系列, 连续稀释样品在汇集的矩阵足够的次数, 以跨越预定的效价削减点1。</li>
		<li>例如, 将30µL 的样本混合到1:3 稀释系列中60µL 的共用矩阵, 总共为8稀释 (例如, 8 条带管)。继续步骤3.1 以进行化验, 准备荧光标记的治疗方法。</li>
	</ol>
</li>
</ol>3. 1 天: 化验程序
<ol><li>
在无血清培养基 (例如, 60 µL, 或大约6毫升/盘) 中制备荧光标记的酶。
	<ol>
<li>例如, 要准备10毫升1µg/毫升荧光标记的酶, 添加10µL 的股票1毫克/毫升荧光标记为9.99 毫升无血清介质 (例如, RPMI-1640)。</li>
	</ol>
</li>
	<li>在一个新的孵化板 (例如, 96 井, 白色, 圆底, 无粘结聚丙烯板), 添加从步骤 2.2, 2.3, 或2.4 和一个荧光标记的, 在一个1:1 混合物中的重复井 (例如, 每个µL 转移60的样本) 的准备样品准备样品, 并添加60µL 的荧光标记的每井)。 注:图 1提供了一个示例实验板图。</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57777/57777fig1.jpg"> 图 1: 实验板布局的示例.样品和一个荧光标记的应急检测在2–8°c 过夜。对 HQC、低质量控制 (LQC) 和负质量控制 (NQC) 等控制进行了筛选, 并在板的相对角上进行了确认, 以评估板材的均匀性。<a href="https://www.jove.com/files/ftp_upload/57777/57777fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<ol start="3"><li>用多通道吸管将样品和荧光标记的吹打进行多次混合。包装在箔板和孵化他们过夜在2–8°c 为 14–20 h。</li>
</ol>4. 2 天: 将准备好的样品添加到单元格中
<ol><li>从2–8°c 的孵化处取出样品孵化板, 并将其放在37摄氏度的珠浴中, 加热盘子, 10–15分钟。</li>
	<li>从 CO2孵化器中取出单元格。使用倒置显微镜对所镀细胞进行目视检查, 以确保细胞看起来健康, 并均匀分布在水井17,19。</li>
	<li>根据实验板图, 将先前混合样品的100µL 和荧光标记的应急传感器添加到细胞板上。</li>
	<li>将电池板与添加的样品放回 CO2孵化器在37°c。将盘子孵化3小时, 15 分钟。 注: 这一次是在实验室建立的, 作为最佳的信号-噪声反应的检测。</li>
	<li>在14–18°c 的台式离心机中, 将电池板离心6分钟, 在 320 x g处。确认在板井底部有一个细胞颗粒的存在。</li>
	<li>用30–45°的角度握住盘子。小心地从每个井中取出上清, 不干扰细胞颗粒。在每个井中添加200µL 1x DPBS 到细胞颗粒中并用重悬细胞。</li>
	<li>重复细胞洗涤步骤, 共3洗涤。立即对步骤5进行细胞活力染色。</li>
</ol>5. 2 天: 细胞活力染色
<ol><li>使用多通道吸管, 添加100µL 的工作溶液的1x 活/死染色的每一个井, 选择的发射波长不同的荧光标记的应急和准备根据制造商的指令22。</li>
	<li>在黑暗中在 RT 中孵化出15分钟的盘子。在14–18°c 的台式离心机上, 将板材离心6分钟, 在 320 x g处。</li>
	<li>小心地从每个井中取出上清, 不干扰细胞颗粒。用 1x DPBS 清洗细胞1x。</li>
</ol>6. 2 天: 用1% 多聚甲醛固定细胞
<ol><li>使用多通道吸管, 在每个井中分配100µL (在2–8°c) 1% 多聚甲醛 (粉煤灰)。</li>
	<li>密封板, 轻轻脉冲涡混合。把盘子裹在铝箔上, 放在2–8°c 至少10分钟, 以便固定。 注意: 小心避免溅到印版上。</li>
	<li>在分析之前, 使用多通道吸管在每个井中添加50µL 1x DPBS。</li>
</ol>7. 流式细胞仪
<ol><li>将盘子放在流式细胞仪上, 并将其运行。</li>
	<li>使用流式细胞仪软件获取和记录测量的中值荧光强度 (多边基金会) (见材料表)。 注: 请参见图 2作为加载程序设置的示例。本试验优化了样品流速、试样体积、混合体积、混合速度和混合数量。这些标准可以用 "向上箭头" 或 "向下" 按钮在每个标准的数字旁边调整, 具体取决于流式细胞仪采集软件23。</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57777/57777fig2.jpg"> 图 2: 流式细胞仪加载器设置的示例.加载器设置应针对每个已开发的检测进行优化。<a href="https://www.jove.com/files/ftp_upload/57777/57777fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<ol start="3"><li>创建一个细胞仪分析/闸门/地块, 如图 3所示。 注: 每个流式细胞术软件23的门的创建和应用可能会有所不同。</li>
</ol><img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57777/57777fig3.jpg"> 图 3: 流式细胞术门策略.Jurkat 细胞与总事件分离, 通过绘制正散射 (FSC)与侧向散射 (SSC) 通道并在目标种群周围绘制一个门来收集。汗衫 (单细胞) 与双峰或更大的细胞聚集使用 fsc 区域 (fsc A) 和 fsc 高度 (fsc H) 通道分离。活细胞被选择的门对单线细胞阴性的活性染色。在单、活 Jurkat 细胞中测定了中值荧光强度, 并绘制成直方图。用药物吸收阻断的细胞 (如HQC) 和具有控制摄取量的细胞 (分别为红色和蓝色) 的多药电池值显示。<a href="https://www.jove.com/files/ftp_upload/57777/57777fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<ol start="4"><li>从分析中排除死细胞, 并记录大约1万事件23。</li>
</ol>8. 数据分析
<ol><li>将数据 (原始的多边化) 导出到分析软件 (见材料表)。 注意: 根据所需的分析, 可以使用分析软件执行以下数据分析。</li>
	<li>计算重复井 (QCs 和样本) 的平均多边化和变异系数 (%CV), 以评估重复值的分布变异性。 <img alt="Equation 1" src="/files/ftp_upload/57777/57777eq1v2.jpg"> <img alt="Equation 2" src="/files/ftp_upload/57777/57777eq2v2.jpg"> <img alt="Equation 3" src="/files/ftp_upload/57777/57777eq3v2.jpg">
</li>
	<li>对于在检测中筛选的样品和 QCs, 计算出相对于 QCs 池矩阵 CC 的平均%SI 的百分比信号抑制。 <img alt="Equation 4" src="/files/ftp_upload/57777/57777eq4v2.jpg">
</li>
	<li>如果需要, 请计算已确认的 QCs 和相对于相应屏幕值的示例的恢复比率 (rr)。 <img alt="Equation 5" src="/files/ftp_upload/57777/57777eq5v2.jpg">
</li>
</ol>

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作者是 BioMarin 制药公司的员工和股东。

中和的抗体或其他因素, 以防止通过 CI-M6PR 的摄入量, 有可能影响治疗的安全性或功效1。因此, 使用与药物作用机制相关的健壮模型对哥们进行评价是很重要的。我们和其他人发现, 某些生物测定格式 (如受体二聚化、荧光素酶表达等) 的性能与卫生当局建议的检测精度、重现性和灵敏度不一致.29. 在这里描述的生物测定方法中, Jurkat 细胞用于表达内源性 CI-M6PR, 用于监测特定于溶酶体吸收的哥们。结合流式细胞仪的读数, 本试验采用了一种生理细胞模型, 具有适当的检测精度、灵敏度、重现性和较高的样品吞吐量。采用流式细胞术的检测方法也适用于其他适应症。例如, 已制定方法来检测前已存在的抗体抗戊型肝炎和腺相关病毒 (AAV)30,31。
该协议中的关键步骤包括选择合适的荧光, 结合一个 LQC, 监测检测灵敏度, 确保足够的细胞活力水平, 并保持严格遵守孵化时间。在这里给出的例子中, 显影 (例如, alexa 488, alexa 氟 647, 和 5e), 共轭到溶酶酶的, 在他们的表现广泛的变化。Alexa 647, 也是最亮的荧光测试了32, 被选择为进一步的化验发展。我们建议在试验的开发中及早评估一些显影, 以提供最佳的灵敏度和动态范围。样品测试中的检测灵敏度应通过包括一个 LQC 足够接近灵敏度限制, 它将失败的测试运行11%。与 HQC, LQC 还作为一个系统适合 QC 监测检测漂移的时间3。最佳的细胞生存能力和性能是通过准备一个单细胞银行的单一使用整除数和资格在新的细胞银行根据可比较的 QC 结果3,18。细胞和荧光共轭药物潜伏期也是一个一致的检测性能的核心, 因为药物摄取量随着时间的推移, 药物与细胞孵化。为了最大限度地减少药物摄取的日常变化, 样本数据可以归一化到每个板块上的共用矩阵控制样本 (例如, 上述方法中的切点控制样本)。使用此方法生成一致数据的另一个重要因素是监视板的均匀性并尽量减少可能的边缘效应。最初的实验可能包括在整个板块上使用单一 QC 进行化验, 而在化验验证 (如精确度和准确度)1中, 可以进行更健壮的实验。这说明了板块图布局33的重要性。如板块图示例所示, 在平板显示器两侧放置的质量控制样本可以随时间利维-詹宁斯图34进行跟踪。
虽然这种检测监测哥们和其他因素, 可能抑制溶酶酶的吸收, 还可以进行额外的实验, 以确认吸收抑制由于抗体。一种选择是治疗样品与蛋白质 A/克/L, 其中非专门结合免疫球蛋白35。如果样品在蛋白 A/克/升耗尽后继续检测阳性, 则非抗体抑制因子可能会导致药物摄取受阻。本发明的方法旨在测量抗体介导的吸收抑制, 如果发现具有非抗体抑制因子的高百分比样本, 则应重新考虑阳性控制和其他检测参数。
这项化验还可以进一步说明, 根据药物的作用机制, 向适当的细胞室展示荧光标记的药物流量。在这里提出的例子中, 预计将 CI-M6PR 绑定到细胞表面, 并将其与溶酶体通信。我们以前报告了几个实验的结果, 表明在该方法中所观察到的几乎所有的荧光信号都是溶酶体8中荧光标记的。通过对细胞松弛素 B 细胞的治疗, 我们发现荧光标记的检测信号被消除, 从而通过抑制肌动蛋白重组来扰乱内化。此外, 实验, 淬火外部荧光与台盼蓝, 或减缓内化通过放置细胞在4°c, 表明, 荧光标记的探针是迅速内化, 非常少的荧光是由于在细胞表面的研究。应用共焦显微术可视化 pH 敏感 lysotracker 染料与荧光共轭药物的协同定位, 证实了溶酶体靶向性。通过 CI-M6PR 吸收的特异性也可能被证实使用外源 M6P 与标记药物竞争受体结合。在其他化验中, 应进行类似的实验, 以验证荧光共轭药物的摄取动力学和细胞定位。
以细胞为基础的检测平台, 用于研究利用 CI-M6PR 受体介导吞的治疗药物哥们。我们最近报告的结果使用这个测试平台, 表明没有相关性的发展与药物疗效的 elosulfase 阿尔法36,37。为验证临床样品检测的结果, 应根据已建立的指导3,对检测灵敏度、精密度、选择性、特异性、耐药性、鲁棒性和切口点等参数进行评估,4. 还应指出的是, 对于溶酶体 ERTs, 哥们可能会有可能通过在酵素催化部位附近的结合来干扰药物活性。一般而言, 我们认为监测这种类型的抓获是一个较低的优先级, 因为溶酶体的严酷酸性和蛋白水解环境不利于抗体-反应性的相互作用2,39,40。然而, 有可能是耐蛋白水解哥们存在, 并可能抑制的催化部分药物40。这种检测监测荧光标记的酶对溶酶体的吸收, 而该检测的局限性是无法监测抗蛋白蛋白酶哥们。如果这种类型的逮捕是基于安全或有效性数据的怀疑, 应制定一个监测治疗活动的检测, 并用于测试样品。
免疫原性评估对于了解哥们对药物安全性和疗效的影响具有重要意义。哥们的鉴定可以通过CI-M6PR 抑制体外药物摄取, 为了解体内的吸收活动提供了机会。本文提出的方法是利用人类细胞系表达 CI-M6PR 来测量荧光共轭溶酶体细胞吸收的干扰。这种方法已经被用来监测哥们的几个 ERTs, 目的是治疗溶酶贮存疾病。这个检测平台可能适用于其他方法, 研究哥们的影响, 需要细胞内化的生物疗法, 其适当的功能。

免疫原性评估是任何生物治疗产品, 包括 ERTs 的安全和效能监测计划的重要组成部分。患者可能会产生免疫反应, 直接影响药物的安全性、功效和药动学/药效学的概况。这些 ADA 的一个子集, 称为哥们, 可以通过两种方式抑制疗效: 通过抑制对靶向细胞的吸收, 或抑制反应酶的催化活性。这里提出的方法是用来测量干扰哥们细胞吸收的。为了充分监测治疗性反应的安全性和有效性, 持续监测哥们是至关重要的, 以阐明任何潜在的相关性与临床结果或药效学效果1。
评估哥们对蛋白质治疗的平台包括细胞基础, 酶活性和配体结合测定1。根据各种标准选择最佳检测平台: 治疗产物的作用机理、检测平台的灵敏度、选择性、精密度, 以及其在体内模拟哥们抑制作用的能力。.配体结合化验在某些情况下可能是适当的 (例如, 当一个相关的细胞系不能被辨认, 或者如果适当的敏感性不能达到在细胞基础的化验)。然而, 在 2016 FDA 关于工业文件和其他行业接受的白皮书草案中, 建议以细胞为基础的检测方法, 因为它们可以更好地反映药物在体内1、2的生物机制。,3。
开发流式细胞术细胞的关键部件包括一个合适的细胞系, 反应药物刺激, 一个替代品阳性对照, 中和的应答, 荧光共轭反应, 和测试物种生物矩阵4,5,6。细胞系的选择依赖于反应机制的作用和多细胞系应评估在检测发展3。在这里描述的方法, 人 Jurkat T 细胞被选择为他们的内源性 CI-M6PR 表达在细胞表面和缺乏抗体片段受体 (FcRs), 不加区别地束缚多数抗体的 Fc 区域7,8.在化验的发展过程中, 重要的是建立一个阴性对照的验证研究和病人样本测试, 如血清汇集从个人没有被治疗的测试文章1。细胞系还应容忍不同物种的相关基质, 以便在药物开发1的有意、临床和后营销阶段保持连续性。另一个成分是检测的阳性控制的选择。根据其结合治疗和中和吸收通过 CI-M6PR9,1的能力, 选择阳性对照的细胞吸收试验。通常很难获得有用的或可持续的数量的中和血清从人类的对象, 以用作化验控制, 特别是在罕见的疾病患者群体2。替代品包括来自超免疫动物的血清, 或亲和纯化的多克隆或单克隆抗体, 被注射到相关基质1中。在使用一种特定于物种的基质时, 可能存在于基质中的抗体以外的抑制因子可能会抑制其吸收。该检测的另一个关键成分是荧光共轭酶。根据检测对亮度、pH 稳定性以及流式细胞仪上其他通道的潜在光谱重叠的需要, 对荧光的选择进行评估。
这里所描述的化验是一个例子, 用来测量一个治疗蛋白, 例如,通过CI-M6PR 进入细胞的药物疗法。一些 ERTs, 目的是治疗溶酶体存储紊乱 (LSDs), 利用这一途径, 以细胞吸收和溶酶体靶向, 包括 elosulfase Morquio 综合征, cerliponase 阿尔法 CLN2 条病, agalsidase 阿尔法病, 并alglucosidase 阿尔法为庞贝氏病10,11。该方法的目的是通过CI-M6PR 来测量干扰药物结合和内化的哥们的相对水平。这是在分层筛选、验证和效度步骤3中执行的。样品先筛选出的阳性, 然后确认积极的步骤。最后, 筛选和确认阳性的样品可以连续稀释以产生抗体滴度1。这种基于细胞的流式细胞仪药物摄取检测提供了一种敏感和机械化相关的体外测量药物特异性哥们的方法, 可能会影响药物的药理学特征。我们以前验证的方法和测试临床样品使用这个平台的药物 elosulfase 阿尔法8。在这里, 我们描述的详细的分步协议, 可能适用于其他治疗蛋白或 ERTs。

<table><tbody><tr><td>0.22 &amp;micro;m filter unit, 500 mL vacuum filter flasks</td><td>Corning</td><td>CLS430769</td><td></td></tr><tr><td>Round Bottom 96-well culture plates</td><td>Thermo-Nunclon</td><td>163320</td><td></td></tr><tr><td>Sterile reagent reservoirs</td><td>VistaLab</td><td>3054-1004</td><td></td></tr><tr><td>96 well white round bottom polystyrene microplate plate</td><td>Corning</td><td>3605</td><td></td></tr><tr><td>96-well Polypropylene Tubes, 8-Tube Strips</td><td>Corning</td><td>4408</td><td></td></tr><tr><td>Magnetic bead separator tube rack</td><td>V&amp;amp;P Scientific, Inc</td><td>VP 772F2M-1, VP 772F2R-2, VP 772F2R-3</td><td></td></tr><tr><td>DynaMag -2 Magnet</td><td>Thermo Fisher Scientific</td><td>12321D</td><td></td></tr><tr><td>DynaMag -96 Side Skirted Magnet</td><td>Thermo Fisher Scientific</td><td>12027</td><td></td></tr><tr><td>BD FACSCanto II Flow Cytometer</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>BD High Throughput Sampler (HTS)</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>BioRad TC20 Automated Cell Counter</td><td>BioRad</td><td>1450103</td><td></td></tr><tr><td>Microplate Shaker</td><td>VWR</td><td>12620-928</td><td></td></tr><tr><td>Galaxy MiniStar Microcentrifuge</td><td>VWR</td><td>C1413</td><td></td></tr><tr><td>tissue culture CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Nuaire</td><td>NU-4750</td><td></td></tr><tr><td>Biosafety cabinet</td><td>Labcono</td><td>Purifier Cell Logic +</td><td></td></tr><tr><td>Jurkat Cell Line</td><td>ATCC</td><td>TIB152&amp;trade;</td><td></td></tr><tr><td>Alexa Fluor 647 Protein Labeling Kit</td><td>Thermo Fisher Scientific</td><td>A20173</td><td></td></tr><tr><td>Dynabeads M270 Streptavidin</td><td>Thermo Fisher Scientific</td><td>65306</td><td></td></tr><tr><td>EZ-Link NHS-LC-LC-Biotin</td><td>Thermo Fisher Scientific</td><td>21343</td><td></td></tr><tr><td>RPMI-1640 1x Medium</td><td>Life Technologies</td><td>A10491-01-500mL</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>ATCC</td><td>30-2020</td><td></td></tr><tr><td>Pen-Strep (100x) liquid formulation</td><td>Corning</td><td>30-002-CI</td><td></td></tr><tr><td>1x DPBS without calcium and magnesium</td><td>Corning</td><td>21-031-CV</td><td></td></tr><tr><td>4% Para-formaldehyde</td><td>Electron Microscopy Science</td><td>15735-85</td><td></td></tr><tr><td>LIVE/DEAD Fixable Violet Dead Cell Stain Kit, for 405 nm excitation</td><td>Life Technologies</td><td>L34955</td><td></td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P1379-100mL</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>3059</td><td></td></tr><tr><td>Pooled human matrix (&lt;em&gt;e.g.&lt;/em&gt; human serum or cerebrospinal fluid)</td><td>Bioreclamation IVT</td><td>request a quote from website</td><td></td></tr><tr><td>VWR Polyester Plate Film</td><td>VWR</td><td>60941</td><td></td></tr><tr><td>Seal &amp;amp; Sample Aluminum Foil</td><td>Beckman Coulter</td><td>538619</td><td></td></tr></tbody></table>

detection of antibodies that neutralize the cellular uptake of enzyme replacement therapies with a cell-based assay

对患者的酶替代疗法 (ERTs) 和其他生物疗法的管理可能引起抗药物免疫应答。这些抗药物抗体 (ADA) 的特性, 特别是那些可能中和药物的生物活性的, 称为中和抗体 (哥们), 对于了解这些抗体对药物的影响至关重要。药理学资料。本协议描述了一种基于细胞的流式细胞仪方法, 用于检测与人基质中有代表性的溶酶酶的细胞吸收的因子。该协议包括三程序: 筛选, 验证步骤, 和效价测定, 以检测, 识别, 并建立相对水平的中和抗体滴度在主题样品。
在这种方法中, 样品首先混合与荧光共轭的受体产物, 然后孵化与细胞 [例如, 人 T 淋巴细胞 (Jurkat 细胞)], 表达细胞表面阳离子无关甘露糖 6-磷酸盐受体 (CI-M6PR), 并最后, 用流式细胞仪进行分析。一个没有哥们的样本将导致通过CI-M6PR 吸收荧光共轭的产品, 而哥们的存在将与药物结合, 并干扰 CI-M6PR 的束缚和吸收。用流式细胞仪测量 Jurkat 细胞内化的荧光共轭应答剂的数量, 并将其与具有代表性的药物-天真基质存在的反应相比, 评估为百分比 (%) 信号抑制。在验证性的步骤中, 样品是预先孵化的与药物治疗结合的磁性珠子, 以耗尽药物特异的因素, 绑定到药品 (如哥们) 之前, 孵化与细胞。样品的筛选和确认阳性的药物特异的哥们在化验中, 然后连续稀释, 以产生抗体滴度。半定量抗体滴度可能与药物安全性和疗效的测量相关。

在该方法的第一天, 解冻和电镀 Jurkat 细胞的冷冻整除, 并制备了试验样品。图 1显示了一个例子板块图。第二天, 样品与 Jurkat 细胞混合, 在37摄氏度处孵化约3小时和15分钟。这些细胞被洗涤, 固定与粉煤灰, 并分析了流式细胞仪。图 2显示了流式细胞仪设置示例。
采用流式细胞术门策略, 测定活单 Jurkat 细胞24 (图 3) 中荧光共轭药物的含量。这些细胞是通过绘制一个不包括细胞碎片 [通常, 低正散射 (FSC)] 和死细胞 [通常, 高侧散射 (SSC)] 的门与碎片分离的, 只留下活细胞进行分析25。通过绘制一个不包括高 fsc 区域和低 fsc 高度26的门, 将单个细胞与细胞簇分离。细胞被治疗的生存能力染色标记任何死亡或不健康的细胞没有完整的膜, 并创建了一个额外的门, 以排除死亡的细胞22。利用活单细胞的荧光, 对其进行了分析。作为潜在筛查结果的一个例子, 基质中有大量的替代药物抗体阳性控制 (例如, 高质量的控制 (HQC)) 抑制了荧光共轭的吸收, 导致了低的多边化大约 300 (图 3)。相比之下, 在没有阳性控制抗体的情况下, 与基质一起孵化的细胞应该有一个更高的多边基金会 (如图3所示的 37830), 表明了荧光共轭反应的吸收。
以该药物的作用机制为例, 选择了中和阳性对照抗体 (即通过 CI-M6PR 吸收)。还对显影进行了测试, 并对最佳检测灵敏度和动态范围1进行了比较。由于其在酸性 pH 值上的荧光增加, 对5e 进行了测试, 这可能与某些 ERTs 相关, 作为对药物27的溶酶体靶向的补充措施。还研究了一种绿色荧光染料 (例如, alexa 488) 和一种远红色荧光染料 (例如, alexa 氟 647)。图 4a和4b显示了不同显影可能执行的示例。在这个例子中, Alexa 的647的研究是最好的性能, 由于其优越的灵敏度 (例如, 最高的%SI 在最低的 PC 浓度) 和广泛的动态范围 (3 级的数量)。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57777/57777fig4v2.jpg"> 图 4: 不同荧光的例子结果.(a) 在分析开发过程中, 应使用不同的荧光共轭 ERTs 评价阳性控制 (PC) 稀释曲线。在这里所示的例子中, 两个荧光共轭 ERTs (alexa 488 和 5e) 在6.25 µg/毫升和一个更明亮的荧光共轭反应 (alexa 氟 647) 在1.56 µg/毫升的存在增加浓度的积极控制哥们。(B) 本小组用一幅图表显示了面板中的曲线, 利用多边基金会显示了使用 Alexa 的647共轭应变器的动态范围的显著增加。<a href="https://www.jove.com/files/ftp_upload/57777/57777fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
所描述的方法是检测、确认和插值3的准定量水平的分层方法。为确定该检测的有效性, 应根据已建立的指导1、3 等参数对检测灵敏度、精密度、选择性、特异性、耐药性、鲁棒性和切口点进行评估..
当%SI 值大于筛切点 (SCP) 时, 样品被认为可能是阳性的。通过对来自代表性人群的药物天真样本进行测试, 并测量信号强度的相对下降 (SI)3, 对 SCP 进行了统计学测定。指导 (例如 FDA) 建议 scp 建立在正常分布数据集的95个百分点上 , 并且在其他地方1详细描述了用于计算 scp 的方法。任何减少检测信号的样品 (测量为%SI 中的增加) 在 scp 或以上被确定是潜在的阳性, 并且结果的样品以高于 scp (在%SI 没有变动或减退) 被认为阴性。
在验证性试验中测试了筛选阳性的样本 (%SI 以上的 SCP), 以确定哥们对反应酶的特异性。验证性的实验是通过预先孵化的样品与预结合的磁性珠子去除药物特异抗体或抑制因素。评估的 RR (验证性的多边化的比例, 以筛选多边基金会), 以确定从样本中删除的哥们的数量。高于计算出的阳性阈值的 RR (即证实性切口点 (CCP)) 表明存在抗药物哥们。在筛查试验中, 应首先通过对有代表性人群中的治疗-天真样本进行验证性试验, 建立中国共产党。中国共产党以统计确定的1% 假阳性率为基础, 是指定一个正确认样本 (图 5)3的阈值。
样品, 筛选和确认阳性被连续稀释和测试的效价测定, 以确定哥们的相对水平或效价在每个样品。当样品在越过指定阈值时测试阳性的最高稀释度 [例如,滴度切点 (TCP)] 是样品滴度 (图 5)1,3。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57777/57777fig5.jpg"> 图 5: 示例测试结果.这些面板显示的例子, 测试阳性或阴性的筛查以上或低于 17.51%si SCP。用药物共轭磁珠在验证性试验中测试阳性样品, 在化验前从样品中消耗药物特异抗体。具有回收率 (rr) 大于验证性切口点 (即rr = 1.315) 的样品被认为是阳性的。证实阳性的样品被稀释, 直到信号越过滴度切点 (TCP), 以建立的效价 (稀释因子) 的样品结果等于滴度切点。<a href="https://www.jove.com/files/ftp_upload/57777/57777fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
检测重复性和变异性在几天内和一个以上的分析师测试。QCs 最初是在一个批次和亚 aliquoted 准备1x 使用。在3维的过程中, 两位分析家用几组 QCs 进行了化验, 以证明化验的精确度。在所示的示例数据中, QCs 的%CV 和内检测精度低于 FDA 指导建议的%CV < 20% (图 6)1。
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57777/57777fig6.jpg"> 图 6: 与两位分析员三天内生成的 QC 精度数据示例.用 DeSilva等公式对方差 (方差分析) 进行了计算, 得出了精度数据。28. 批内 (在运行中) 和批间 (运行之间) 统计数据被报告为%CV.<a href="https://www.jove.com/files/ftp_upload/57777/57777fig6large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>

Watch this Scientific Journal Video about Detection of Antibodies That Neutralize the Cellular Uptake of Enzyme Replacement Therapies with a Cell-based Assay at JoVE.com

Detection of Antibodies That Neutralize the Cellular Uptake of Enzyme Replacement Therapies with a Cell-based Assay

在这里, 我们提出了一种基于细胞的流式细胞仪方法来检测中和抗体或其他因素, 干扰细胞吸收酶替代疗法在人类基质, 如脑脊髓液 (CSF) 或人血清。

细胞法检测酶替代疗法中细胞吸收的抗体

The administration of enzyme replacement therapies (ERTs) and other biologic therapies to patients may elicit an anti-drug immune response. The ...

detection-antibodies-that-neutralize-cellular-uptake-enzyme

Research

JoVE Journal

Immunology and Infection

8.8K 次观看.  BioMarin Pharmaceutical Inc. 在这里, 我们提出了一种基于细胞的流式细胞仪方法来检测中和抗体或其他因素, 干扰细胞吸收酶替代疗法在人类基质, 如脑脊髓液 (CSF) 或人血清。

视频: Detection of Antibodies That Neutralize the Cellular Uptake of Enzyme Replacement Therapies with a Cell-based Assay

Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal1. Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis1-4. However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric&#160;detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation.

Here we describe two assays for measuring complement activation induced by antibodies against red blood cells. The major advantage over the current assays is their quantitative and easy-to-interpret nature.

定量检测抗体诱导补体激活的方法对红血细胞

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver ...

科研

免疫与感染

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

高通量流式细胞仪检测细胞为基础的试验检测抗体，以N-甲基-D-天冬氨酸受体或多巴胺2受体在人血清

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

医学

High-Content Screening Assay for the Identification of Antibody-Dependent Cellular Cytotoxicity Modifying Compounds

Immunotherapy with antigen-specific antibodies or immune checkpoint inhibitors has revolutionized the therapy of breast cancer. Breast cancer cells expressing the epidermal growth factor receptor HER2 can be targeted by the anti-HER-2 antibody trastuzumab. Antibody-dependent cellular cytotoxicity (ADCC) is an important mechanism implicated in the antitumor action of HER-2. Trastuzumab bound to cancer cells can be recognized by the Fc receptors of ADCC effector cells (e.g., natural killer (NK) cells, macrophages, and granulocytes), triggering the cytotoxic activity of these immune cells leading to cancer cell death. We set out to develop an image-based assay for the quantification of ADCC to identify novel ADCC modulator compounds by high-content screening. In the assay, HER2 overexpressing JIMT-1 breast cancer cells are co-cultured with NK-92 cells in the presence of trastuzumab, and target cell death is quantified by automated microscopy and quantitative image analysis. Target cells are distinguished from effector cells based on their EGFP fluorescence. We show how compound libraries can be tested in the assay to identify ADCC modulator drugs. For this purpose, a compound library test plate was set up using randomly selected fine chemicals off the lab shelf. Three microtubule destabilizing compounds (colchicine, vincristine, podophyllotoxin) expected to interfere with NK cell migration and degranulation were also included in the test library. The test screen identified all three positive control compounds as hits proving the suitability of the method to identify ADCC-modifying drugs in a chemical library. With this assay, compound library screens can be performed to identify ADCC-enhancing compounds that could be used as adjuvant therapeutic agents for the treatment of patients receiving anticancer immunotherapies. In addition, the method can also be used to identify any undesirable ADCC-inhibiting side effects of therapeutic drugs taken by cancer patients for different indications.

This protocol presents an automated, image-based high-throughput technique to identify compounds modulating natural killer cell-mediated breast cancer cell killing in the presence of a therapeutic anti-HER-2 antibody.

用于鉴定抗体依赖性细胞毒性修饰化合物的高内涵筛选检测

Immunotherapy with antigen-specific antibodies or immune checkpoint inhibitors has revolutionized the therapy of breast cancer. Breast cancer cells ...

癌症研究

Validated Immunochemical Assay for Comprehensive Determination of the Human Epidermal Growth Factor Receptor 2 Released from and Bound to Cells

Human epidermal growth factor receptor 2 (HER2) is a well-established cancer marker. It became a very successful diagnostic and therapeutic target, especially in breast cancer and other HER2-expressing cancer types. In the clinic, the gold-standard immunohistochemical diagnostic methods employing the specific anti-HER2 antibodies are used to measure the expression level of the membrane-bound receptor. The soluble extracellular domain (ECD) of HER2 that is released from the overexpressing cells circulates in the blood and can reflect the tissue expression of the receptor. There is a need for accurate and validated assays to correlate the concentration of the circulating HER2 protein with disease clinical manifestations.
Our team has developed and validated the novel sandwich enzyme-linked immunosorbent assay (ELISA) for quantification of the membrane-bound and the released from cells ECD domain of HER2. The assay uses two unique monoclonal antibodies specific to HER2 developed previously. The quantitation range includes HER2 concentration from 1.56-100 ng/mL, which is expected for cancer cells cultured in vitro and shows sensitivity at the level of 0.5 ng/mL. The satisfactory intra- and inter-assay precision and accuracy of the method make it applicable for HER2 quantification in various types of biological samples, including cell culture medium, serum, and solid tumor tissue. Here, we focus on the comprehensive determination of the receptor-associated and secreted by the in vitro cultured cancer cells. The paper presents a step-by-step protocol for the quantification of HER2 protein that can be employed for testing a variety of cell lines, blood, and tissues.

We present a validated sandwich ELISA assay using novel anti-HER2 monoclonal antibodies. This assay enables precise quantification of cell-bound and released HER2 protein from in vitro cultured cells and other samples, including blood and tissues.

经验证的免疫化学检测试剂盒，用于全面测定从细胞中释放并结合到细胞的人表皮生长因子受体 2

Human epidermal growth factor receptor 2 (HER2) is a well-established cancer marker. It became a very successful diagnostic and therapeutic target, ...

Determination of the Relative Potency of an Anti-TNF Monoclonal Antibody (mAb) by Neutralizing TNF Using an In Vitro Bioanalytical Method

This protocol shows the measurement of the apoptotic activity neutralization of TNF&#945; in a mouse fibroblast cell model (WEHI 164) using an anti-TNF&#945; mAb. In addition, this protocol can be used to evaluate other anti-TNF&#945; molecules, such as fusion proteins. The cellular model employed here is sensitive to TNF&#945;-mediated apoptosis when an additional stress factor is induced in cell culture conditions (e.g., serum deprivation). This procedure exemplifies how to execute this analytical assay, highlighting the key operations relating to the sample preparation, cell dilution, apoptosis induction, and spectrophotometric measurements that are critical to ensure successful results. This protocol reveals the best-performance conditions relating to apoptosis induction and efficient signal recording, leading to low uncertainty values.

Determination of the Relative Potency of an Anti-TNF Monoclonal Antibody mAb by Neutralizing TNF Using an In Vitro Bioanalytical Method

A protocol for the determination of the relative anti-apoptotic activity of an anti-TNF&#945; mAb using a neutralization mechanism with WEHI 164 cells is presented here. This protocol is useful for comparing the neutralization strength of different molecules with the same biological functionality.

使用体外生物法测定抗 tnf 单克隆抗体 (抗体) 的相对效力

This protocol shows the measurement of the apoptotic activity neutralization of TNF&#945; in a mouse fibroblast cell model (WEHI 164) using an ...

Detection of SARS-CoV-2 Neutralizing Antibodies using High-Throughput Fluorescent Imaging of Pseudovirus Infection

As the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, it has become evident that the presence of neutralizing antibodies against the virus may provide protection against future infection. Thus, as the creation and translation of effective COVID-19 vaccines continues at an unprecedented speed, the development of fast and effective methods to measure neutralizing antibodies against SARS-CoV-2 will become increasingly important to determine long-term protection against infection for both previously infected and immunized individuals. This paper describes a high-throughput protocol using vesicular stomatitis virus (VSV) pseudotyped with the SARS-CoV-2 spike protein to measure the presence of neutralizing antibodies in convalescent serum from patients who have recently recovered from COVID-19. The use of a replicating pseudotyped virus eliminates the necessity for a containment level 3 facility required for SARS-CoV-2 handling, making this protocol accessible to virtually any containment level 2 lab. The use of a 96-well format allows for many samples to be run at the same time with a short turnaround time of 24 h.

The protocol described here outlines a fast and effective method for measuring neutralizing antibodies against the SARS-CoV-2 spike protein by evaluating the ability of convalescent serum samples to inhibit infection by an enhanced green fluorescent protein-labeled vesicular stomatitis virus pseudotyped with spike glycoprotein.

使用假病毒感染的高通量荧光成像检测SARS-CoV-2中和抗体

As the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, it has become evident that the ...

<meta charset="utf8"></head><body>甲状腺癌细胞作为癌症免疫治疗的 ADCC 生物测定中的效应子

Assessing Antibody-dependent, Cell-mediated Cytotoxicity in Cancer Cells using Antibody-Dependent Cell-Mediated Cytotoxicity Reporter Bioassay

The method for antibody-dependent, cell-mediated cytotoxicity (ADCC) represents an important tool to assess the efficacy of therapeutic antibodies in cancer immunotherapy. Evaluating ADCC activity in cancer cells is essential for the development and optimization of antibody-based treatments. Here, we propose a methodological approach of utilizing an ADCC bioassay kit for quantitative assessment of ADCC reaction using thyroid cancer cells as effector cells. The protocol involves the co-culture of effector cells with target cancer cells in different ratios in the presence of a therapeutic antibody. The ADCC bioassay kit used in this experiment includes the genetically engineered effector cells expressing a luciferase reporter gene under the control of Nuclear Factor of Activated T-cell (NFAT) response elements. Upon the binding of the surface antigen on the target cells with the antibodies and effector cells, the effector cells release luciferase, enabling quantification of cytotoxicity through measurement of luminescence signal. In contrast to conventional ADCC assays, this method proved the binding of target antigen with antibodies and effector cells, which can produce reliable results in a short period.

<meta charset="utf8"></head><body>作者聚焦:通过抗体依赖性细胞介导的细胞毒性测定优化基于抗体的癌症治疗

Here, we present a protocol for an antibody-dependent, cell-mediated cytotoxicity (ADCC) assay using an ADCC bioassay kit. This method offers a valuable tool for elucidating the ADCC mechanism and evaluating the therapeutic potential of antibodies in cancer immunotherapy.

使用抗体依赖性细胞介导的细胞毒性报告基因生物测定评估癌细胞中抗体依赖性、细胞介导的细胞毒性

The method for antibody-dependent, cell-mediated cytotoxicity (ADCC) represents an important tool to assess the efficacy of therapeutic antibodies in ...

A Step-By-Step Method to Detect Neutralizing Antibodies Against AAV using a Colorimetric Cell-Based Assay

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health conditions in both the laboratory and the clinic. However, pre-existing neutralizing antibodies (NAbs) against AAV capsids pose an ongoing challenge for the successful administration of gene therapies in both large animal experimental models and human populations. Preliminary screening for host immunity against AAV is necessary to ensure the efficacy of AAV-based gene therapies as both a research tool and as a clinically viable therapeutic agent. This protocol describes a colorimetric in vitro assay to detect neutralizing factors against AAV serotype 6 (AAV6). The assay utilizes the reaction between an AAV encoding an alkaline phosphatase (AP) reporter gene and its substrate NBT/BCIP, which generates an insoluble quantifiable purple stain upon combination. 
In this protocol, serum samples are combined with an AAV expressing AP and incubated to permit potential neutralizing activity to occur. Virus serum mixture is subsequently added to cells to allow for viral transduction of any AAVs that have not been neutralized. The NBT/BCIP substrate is added and undergoes a chromogenic reaction, corresponding to viral transduction and neutralizing activity. The proportion of area colored is quantitated using a free software tool to generate neutralizing titers. This assay displays a strong positive correlation between coloration and viral concentration. Assessment of serum samples from sheep before and after administration of a recombinant AAV6 led to a dramatic increase in neutralizing activity (125 to &gt;10,000-fold increase). The assay displayed adequate sensitivity to detect neutralizing activity in &gt;1:32,000 serum dilutions. This assay provides a simple, rapid, and cost-effective method to detect NAbs against AAVs.

A comprehensive laboratory protocol and analysis workflow are described for a rapid, cost-effective, and straightforward colorimetric cell-based assay to detect neutralizing elements against AAV6.

使用基于比色细胞的测定法检测针对 AAV 的中和抗体的分步方法

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health ...

细胞法检测酶替代疗法中细胞吸收的抗体

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使用体外生物法测定抗 tnf 单克隆抗体 (抗体) 的相对效力

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使用抗体依赖性细胞介导的细胞毒性报告基因生物测定评估癌细胞中抗体依赖性、细胞介导的细胞毒性

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使用基于比色细胞的测定法检测针对 AAV 的中和抗体的分步方法