Name: determination of the relative potency of an anti-tnf monoclonal antibody (mab) by neutralizing tnf using an in vitro bioanalytical method
Uploaded: 2017-09-16T14:00:00.000+00:00
Duration: 16 min 7 s
Description: Watch this Scientific Journal Video about Determination of the Relative Potency of an Anti-TNF Monoclonal Antibody mAb by Neutralizing TNF Using an In Vitro Bioanalytical Method at JoVE.com

这项工作得到了国家科学和技术委员会 (委员会) 的支持, 墨西哥授予 PEI 委员会 2015 220333, 没有参与研究的设计。

1. 媒体和解决方案的准备 <ol> <li> 准备培养基: RPMI-1640 与 10% FBS, pH 7.4. </li>
	<li> 制备测定培养基: RPMI-1640 无酚红, 1% FBS, pH 7.4. </li>
	<li> 准备细胞洗涤液: DPBS 镁和无钙溶液与 0.02% EDTA, pH 7.4. </li>
	<li> 准备细胞剥离液: 0.125% 胰蛋白酶与1毫米 EDTA. 
	<ol> <li> 解冻100毫升的0.25% 溶液胰蛋白酶-EDTA 和转移到无菌500毫升烧瓶. </li>
		<li> 混合100毫升的细胞洗涤液和配发15毫升等分到15毫升无菌管。存储在-70 到-80 和 #176; C 直到使用. </li>
		<li> 将这些解决方案通过 0.22-#181; m 膜进行过滤, 预热到37和 #176; C 至少30分钟, 然后使用. </li>
	</ol> </li> <li> 准备凋亡诱导砧木溶液 TNF 和 #945; 3.3 和 #181 的解决方案; g/毫升. 
	<ol> <li> 溶解20和 #181; g 的 TNF 和 #945; 与500和 #181; 过滤消毒水的主要容器和混合, 直到完全溶解. </li>
		<li> 转移到15毫升无菌管, 并加入5.5 毫升的 DPBS 镁和无钙溶液的这个管。使用涡流混合器轻轻搅拌. </li>
		<li> 将解决方案分为70和 #181; L 部分。免除每分0.5 毫升管内和存储在-80 和 #176; C. </li>
	</ol> </li> <li> 准备细胞凋亡诱导溶液: TNF 和 #945; 溶液在 40 ng/毫升. 
	<ol> <li> 将凋亡诱导储存液的分解冻, 在25和 #730 的水浴中孵化; C 为 10 min. </li>
		<li> 将凋亡诱导库存溶液稀释至 40 ng/毫升, 增加61和 #181; 3.3 和 #181; g/毫升 TNF 和 #945; 15 毫升无菌管中4.939 毫升的测定培养基的溶液. </li>
		<li> 混合涡旋混合器十年代; 此解决方案必须在使用前新鲜准备. </li>
		<li> 将解决方案预热到37和 #176; C 至少在 eneutralization 检测之前使用30分钟. </li>
	</ol> </li> <li> 准备基板解决方案: 酶3/7 全球解决方案 7 8 . 
	<ol> <li> 在使用前解冻酶缓冲区解决方案 (酶3/7 全球缓冲区) 12 小时. </li>
		<li> 让酶缓冲液和基板 (酶3/7 全球基板) 分别位于25和 #177; 5 和 #176; C 在混合前30分钟. </li>
		<li> 将酶缓冲液的10毫升转移到基板瓶中, 并通过反转进行混合. </li>
		<li> 保持在25和 #177; 5 和 #176; C, light-protected 直到使用. 
		注: 溶液在室温下稳定6小时. </li>
	</ol> </li> </ol> 2。细胞培养和计数 <ol> <li> 单元格解冻和第一次区域性. 
	<ol> <li> 从-80 和 #730 的冰箱中取出一个带有 WEHI 164 单元格的小瓶 9 , 然后将它们转到冰浴中. </li>
		<li> 吸管向上和向下与1毫升的5ml 培养基, 直到冷冻细胞完全解冻. </li>
		<li> 将9毫升的5ml 培养基放在15毫升无菌管上. </li>
		<li> 将电池悬浮液转移到15毫升无菌管中, 并通过反转将其轻轻混合五次. </li>
		<li> 离心机悬浮在 125 x g 为 3 min. 丢弃上清和分解细胞颗粒. </li>
		<li> 在试管中加入5毫升培养基。混合, 直到细胞完全悬浮. </li>
		<li> 用于单元计数, 传输50和 #181; l 细胞悬浮到500和 #181; l 微和混合与50和 #181; l 0.4% 台盼蓝。计算单元格并调整为 0.5 x 10 6 单元格/mL。请参见下面的步骤 2.2. </li>
		<li> 将13毫升的5ml 培养基添加到75毫升的细胞培养瓶中. </li>
		<li> 从步骤2.1.6 中分配足够的单元悬浮体积以实现 0.5 x 10 6 单元格/mL 在细胞培养烧瓶中孵育, 在37和 #176; C 和 5% CO 2 过夜。
	</ol> </li> <li> 单元格计数. 
	注意: 请参见参考 10 。
	<ol> <li> 使用从步骤2.1.6 的解决方案, 将 0.05 mL 转换为例, 并使用台盼蓝排除方法在显微镜下确定细胞密度. </li>
		<li> 量化单元格和可行单元格的总数. </li>
		<li> 将单元格悬浮液调整为 0.5 x 10 6 单元格/mL. 
		 公式 1 
		v 培养基 (ml) = <img alt="Equation 1" src="/files/ftp_upload/55376/55376eq1.jpg"/> V 培养基 (ml) = (5mL-v 单元悬浮 ) V 培养基 (ml) = 调整后的卷 WEHI 164细胞悬架 NVC = 可行的 WEHI 164 细胞/毫升 V 培养基 (ml) = 检测培养基体积增加到细胞悬浮达到 0.5 x 10 6 细胞/ml 0.5 x 10 6 和 #160; = 目标单元格密度 </li>
	</ol> </li> <li> 单元格拆分和第二和第三亚文化. 
	注: 真空系统可用于从烧瓶中去除溶液。可使用一次性或玻璃无菌管。如果吸管在顶部有一个棉花堵塞, 它必须在使用前删除。
	<ol> <li> 使用1毫升无菌吸管和真空, 从细胞培养 T 瓶中取出培养基. </li>
		<li> 将5毫升的细胞洗涤液放入培养的 T 型烧瓶中, 轻轻混合, 并丢弃溶液。重复此步骤两次. 
		注意: 完全去除培养基对于有效的细胞剥离至关重要. </li>
		<li> 在 T 形烧瓶中添加15毫升的细胞剥离液, 并在37和 #176 的恒温箱中让3分钟站立; C 和 5% CO 2 . </li>
		<li> 验证显微镜下烧瓶内壁上是否有附着的细胞。用20毫升无菌吸管将细胞从培养液中取出, 并将其放入一个50毫升的不育管中. </li>
		<li> 离心机悬浮在 125 x g 为3分钟. 丢弃上清和重颗粒与另5毫升培养基. </li>
		<li> 根据 公式1对单元格进行计数并添加足够的培养基以达到所需的细胞浓度 . </li>
		<li> 将此悬浮液添加到 72 cm 2 t-烧瓶, 并在夜间孵育37和 #176; C 和 5% CO 2 . </li>
		<li> 在中和检测中使用这些细胞至少要两次亚文化。在接下来的两天中重复步骤 2.3. 1-2. 3.8. </li>
	</ol> </li> <li> 检测单元格挂起。
	<ol> <li> 选择至少有三次传递的 WEHI 164 子区域性。请参见步骤 2.1. </li>
		<li> 根据此协议的步骤2.2 和2.3 分离和计数单元格. </li>
		<li> 根据 公式1将单元格悬浮液稀释为 0.5 x 10 6 单元格/mL。
		<li> 使用此悬浮液进行中和检测。使用前旋流混合器混合所有的悬浮液. </li>
	</ol> </li> </ol> 3。抗体制备和稀释的 <ol> <li> 定量的抗体. 
	<ol> <li> 使用它们的质量消光系数 (1.39) 11 , 通过 280 nm 的紫外线吸收来确定参考物质、控制样品和分析样品的浓度. 
		注: 原始浓度可从药物产品标签中取出。然而, 这必须通过紫外线吸收来验证. </li>
	</ol> </li> <li> 单克隆稀释. 
	<ol> <li> 将所有样品单独稀释一式三份, 用 DPBS 镁和无钙溶液在2毫升管内中, 降低到2毫克/毫升。使用 DPBS 镁和无钙溶液作为空白, 通过紫外吸收来确认此浓度. </li>
		<li> 使用涡流混合器混合 5 s 的股票蛋白溶液. </li>
		<li> 稀释100和 #181; 每2毫克/毫升的单克隆溶液, 0.9 毫升的测定培养基. </li>
		<li> 用涡流混合器混合5秒. 
		注: 这些溶液的浓度为200和 #181;每三份必须稀释. </li>
		<li> 稀释 10 & #181;每个200和 #181 的 L. 0.99 毫升的测定培养基中的单/毫升单克隆溶液。使用涡流混合器混合5秒。这些解决方案的浓度为2和 #181; g/毫升。在中和试验中使用前, 对每三个稀释进行串联. </li>
		<li> 使抗 TNF 和 #945; 单克隆稀释在三独立板。从每一个独立副本中复制一份, 并将它们放在一个微中, 如 表 1 所示。参考物质 <table fo:keep-together.within-page="1" fo:keep-with-next.within-page="always" fo:text-align="center"> <tbody> <tr> <td colspan="2"> 板 1 </td> <td colspan="2"> 板 2 </td>
					<td colspan="2"> 板 3 </td> </tr>
				<tr> <td> 韦尔斯 </td> <td> 示例 </td> <td> 井 </td> <td> 示例 </td> <td> 井 </td> <td> 示例 </td> </tr>
				<tr> <td> B2:B11 </td> <td rowspan="2"> 参考物质 </td> <td> B2:B11 </td> <td rowspan="2"> 控件示例 </td> <td> B2:B11 </td> <td rowspan="2"> 分析示例 </td> </tr>
				<tr> <td> C2:C11 </td> <td> C2:C11 </td> <td> C2:C11 </td> </tr>
				<tr> <td> D2:D11 </td> <td rowspan="2"> 分析样例 </td> <td> D2:D11 </td> 参考物质 <td rowspan="2"> </td> D2:D11 <td> </td> 控件示例 <td rowspan="2"> </td>
				<tr> <td> E2:E11 </td> <td> E2:E11 </td> <td> E2:E11 </td> </tr>
				<tr> <td> F2:F11 </td> <td rowspan="2"> 控件示例 </td> <td> F2:F11 </td> <td rowspan="2"> 分析示例 </td> <td> F2:F11 </td> <td rowspan="2"> 参考物质 </td> </tr>
				<tr> <td> G2:G11 </td> <td> G2:G11 </td> <td> G2:G11 </td> </tr> </tbody>
		</table> 表 1: 微示例数组. 一个完整的中和化验必须运行在三板在坐标 B2 到 G11。随机分配的参考, 分析和控制样本允许研究人员验证任何偏见的化验. </li>
		<li> 执行每个引用、示例或控件的单克隆稀释, 如 表 2 所示. 
		注: 抗肿瘤坏死因子和 #945; 在本表中描述的单克隆抗体浓度不是中和测定中的最终浓度。
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"> <tbody> <tr> <td> 板列 </td> <td> 检测培养基容积 (#956; L) </td> <td> 参考物质体积, 分析样品或对照样品 (uL) </td> <td> 浓度在检测板 (ng/毫升) </td> </tr> <tr> <td> 2 </td> <td> 0 </td> <td> 230 </td> <td> </td> </tr> <tr> <td> 3 </td> <td> 150 </td> <td> 150 从行 2 </td> <td> 1000 </td> </tr>
				<tr> <td> 4 </td> <td> 75 </td> <td> 75 从行 3 </td> <td> 500 </td> </tr> <tr> <td> 5 </td> <td> 100 </td> <td> 50 从行 3 </td> <td> 333 </td> </tr> <tr> <td> 6 </td> <td> 75 </td> <td> 75 线 4 </td>>> 250 </td> </tr> <tr> <td> 7 </td> <td> 75 </td> <td> 75 从行 5 </td> <td> 166 </td> </tr> <tr> <td> 8 </td> <td> 75 </td> <td> 75 行 6 </td> <td> 125 </td> </tr> <tr> 9 <td> </td> 75 <td> </td> 75m 行 7 </td> <td> 83 </td> </tr> <tr> <td> 10 </td> <td> 75 </td> <td> 75 行 9 </td> <td> 41 </td> </tr> <tr> 11 <td> </td> 150 <td> </td> 75 行 10 <td> </td> 13 <td> </td> </tr> </tbody> </table>能 2: 抗 TNF 和 #945; 单克隆稀释。 抗 TNF 和 #945 的序列稀释; 抗体在该表中演示。本表中所描述的最终浓度不是测定中的浓度, 其中抗 TNF 和 #945; 抗体被稀释的因子为 3 (单克隆稀释 + 培养基 + 细胞悬浮液)。粗体线表示稀释来自3、5、7、9和10行;non-bolded 线代表了3、4和6行的稀释。这些串联稀释是在执行中和化验前完成的。在配发稀释之前, 必须注意移上下三次的混合. </li>
		<li> 将板材保持在25和 #177; 5 和 #176; C 直到使用. </li>
	</ol> </li> </ol> 4。中和 WEHI 164 细胞的中和含量 <ol> <li> 通过涡流所有单元格悬浮 (0.5 x 10 6 单元/mL) 混合, 然后在该协议的任何步骤中进行分配. 
	注意: 在本节中, 将每个解决方案预热到37和 #176; C 在使用前为30分钟. </li>
	<li> 传输50和 #181; L 单元悬浮到板的60口井中的每一个, 从2栏移动到11和 B 行到 G. </li>
	<li> 传输50和 #181; 单克隆参考、样本和控制稀释到板。按照 图 1 中描述的模式进行. </li>
	<li> 添加50和 #181; L 细胞凋亡诱导溶液对每一个井. </li>
	<li> 使用50和 #181 的蜂窝式控制; WEHI 164 细胞, 在三口井中配发。把每一个井的最后一卷150和 #181; L 与分析培养基. </li>
	<li> 使用50和 #181 的混合物的细胞毒性控制; WEHI 164 细胞 + 50 和 #181; 细胞凋亡诱导溶液 l。把每一个井的最后一卷150和 #181; L 与分析培养基. </li>
	<li> 用于 TNF 和 #945; 控制, 使用50和 #181; i. 细胞凋亡诱导溶液, 并将其带到150和 #181; 测定培养基. </li>
	<li> 对于空白, 请使用150和 #181; 仅适用于检测培养基的 L. </li>
	<li> 将剩余的井用150和 #181 填充培养基, 避免板的蒸发效果. </li>
	<li> 重复步骤 4.1. 1-4. 1.9 两次在其他板. 
	注: 微中的单克隆人最终浓度为: 0.666、0.333、0.167、0.111、0.083、0.056、0.042、0.028、0.014 和0.004 和 #181; g/毫升. </li>
	<li> 在板中加载示例, 如 图 1 所示. 
	<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55376/55376fig1.jpg"/> 图 1: 检测板中样品的配置。B1 对 G11 的坐标在板, 并描述了样品稀释放置的位置。缺少的坐标是充满控制和测定培养基 (A1-A12 和 H1-H12) 的水井。这种随机分布的样品 (向前和反向稀释在板) 有助于消除偏差的结果, 由于蒸发的介质或其他变量。最好的做法是, 每次微都由一位分析师完成。R: 参考, S: 样品, CS: 对照样品, Dil: 稀释。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55376/55376fig1large.jpg" target="_blank"> 请单击此处查看此图的较大版本. </a> </li>
	<li> 孵育三板在37和 #176; C 和 5% CO 2 16 和 #177; 2 h. </li>
	<li> 让酶3/7 全球试剂站在25和 #177; 5 和 #176; C 在使用前30分钟. </li>
	<li> 在所有井中添加100和 #181; L 此试剂, 包括样品和控制. </li>
	<li> 使用微涡流混合器在25和 #177 处摇动板, 5、#176; C。
	<li> 孵育2.5 和 #177 的板; 0.5 小时在25和 #177; 5 和 #176; C, 免受光照. </li>
	<li> 插入麦克风roplates 进入计, 完成下一节. </li>
</ol> 5。结果分析 <ol> <li> 使用用于发光检测的软件, 选择发光模式和端点函数. </li>
	<li> 选择96井底板及其80内部井, 不包括1和12栏. </li>
	<li> 选择1250毫秒和十年代的集成时间, 以便在阅读前混合微. </li>
	<li> 选择要放置参考物质、分析物质和控制样品的井, 并确定其相应浓度. </li>
	<li> 读取在板中放置的示例与计. </li>
	<li> 使用第四参数公式分析结果。图的剂量响应曲线, 如 图 2 所示. 
	<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55376/55376fig2.jpg"/> 图 2: 剂量-响应曲线. 抗 TNF 和 #945; 单克隆抗体浓度与发光 (细胞活力) 的描述。描述抗 TNF 和 #945 的第四参数方程; 抗体的保护模型。EC50 是单克隆体的浓度, 可以抵消肿瘤坏死因子和 #945 的数量; 在每一个化验中导致50% 细胞死亡, 在曲线图中作为斜率变化的例证。条形图描述了每个单克隆人浓度的发光标准偏差。 x 表示抗 TNF 和 #945; Ab 浓度, 在 ng/mL 中被描述为对数函数, 而 y 表示 在任意发光单元中的发光响应。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55376/55376fig2large.jpg" target="_blank"> 请单击此处查看此图的较大版本. </a> 
	注: 在第四参数方程中, C 是有效浓度 50 (EC50)。这个值将被用来比较的参考物质, 分析样本, 和控制样品的手段的效应函数. </li>
	<li> 用于计算相对效, 将参考物质固定到 100%, 并据此计算样品的效和控制. 
	注意: 这些值在 图 3 中描述. 
	<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55376/55376fig3.jpg"/> 图 3: 用于计算 EC50s 及其值的数学公式. EC50 值, 或 C 参数, 其不确定性被描述为标准错误。比较了样品的 EC50s 和相对效力的参考结果。置信区间计算与 #945; = 0.05。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55376/55376fig3large.jpg" target="_blank"> 请单击此处查看此图的较大版本. </a> </li>
</ol>

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作者没有透露

这种特性有助于在进行昂贵且耗时的临床试验之前确定一个分子在发育过程中的生物学行为. 它也有助于批次释放一个批准的药物产品。值得一提的是, 这些化验结果对于确定分子对其作用机制是否具有足够的生物学效应是有用的。本教程中提出的生物分析方法对不同抗 TNFα分子的比较至关重要。尽管常用的物理化学方法, 这种方法可以确定, 通过生物手段, 药物的效力和功效作为一个质量属性, 从而显示了完整和充分的意义的效应函数。
通常, 细胞凋亡对 TNFα的反应可能是一个具有挑战性的任务, 研究人员执行。故障诊断可以通过对细胞库的描述, 这是在标准化这种生物方法之前每天使用的。一个示例是与单元格线老化12相关的时间段内的单元响应变异性。这一问题被排除在-80 ° c 的主细胞库冻结。工作细胞库必须足够大, 以满足由 R 和 #38;D 进行的 DOE 研究的要求, 或在质量控制实验室使用一年的时间。此外, 此响应可以使用温度稳定的解决方案来修复, 然后在本协议中的任何步骤中将它们添加到单元格中。在运行中和检测之前必须至少执行三刀路, 并限制由于适应和人口动态而在连续区域性中生长细胞的时间长度12,13。
TNFα分子必须受到保护, 免受冻融循环的影响, 因为如果不稳定, 这种蛋白质的效力就会受到极大的破坏。在其他地方引用了14用于细胞因子准备的制剂, 当重组细胞因子被储存时, TNFα的浓度对协议的成功至关重要。细胞与细胞因子的反应性取决于每个细胞的受体数量。因此, 最佳的 TNFα浓度取决于单元格线和密度15,16。我们稀释 TNFα到最后浓度为 13.3 ng/毫升和调整细胞密度使用曲线约2.5万细胞/井。因此, 每 TNFα浓度必须验证细胞密度。
卡马乔-Villegas et al.报告 TNFα最终浓度为 1.25 ng/毫升;该组还使用放线菌 D 作为应力单元因子9,17,18。我们反而改变了 FBS 从10% 到1% 从培养基到测定培养基, 给出一个强的重音信号诱导细胞凋亡在 WEHI 164 细胞与 TNFα单独, 去除另一个可变物从协议。其他组使用 MTT 法报告细胞活力。使用对酶 37敏感的发光基板的方法, 而不是分光光度法, 即在培养基中或细胞本身中存在紫外-可见吸收物质 (如,酚红吸收由细胞), 可以干扰信号。因此, 由于在培养基中存在发光物质 (背景), 因此, 使用这种交流 DEVD 肽 luminescentreactant 增强了测定的灵敏度。
这种方法的一个局限是它只能用于 TNFα敏感的细胞;其他细胞线必须进行测试, 细胞因子浓度调整, 以优化细胞反应。此外, 绝对反应不能被测量, 因为我们没有使用一个主细胞系或一个在体内化验;采用正交等温滴定量热法 (ITC) 对初始方法进行了实验调整。来自贸易中心的信息有助于建立与正在开发的新分子的 TNFα亲和性, 并用于指定生物方法的初始条件。
这种方法是有用的测试新的分子时, 研究人员有一个参考物质, 以获得一个基本的相对反应;因此, 建议对一个生物更好的或与细胞保护有关的分子反应的评价。它可以应用于其他抗 TNFα蛋白。例如, 它适用于评估普、昔、Certolizumab 或 Golimumab19的相对生物效力。然而, 所有这些抗 TNFα分子对细胞因子有不同的亲和力;因此, 它们的浓度必须单独调整。这种方法的另一个优点是在实验开始和结果之间的时间很短, 这使得它易于执行, 而且与动物模型相比价格低廉。此外, 该方法还测量了单克隆人与一个完全活跃的 TNFα分子的相互作用, 这表明单克隆抗体是识别 TNFα三的, 并且分子在储存或实验室操作过程中没有被改变。另一方面, 一个纯理化化验结果仍然是可疑的。例如, TNFα和单克隆抗体之间的相互作用, 使用传统的 ELISA 可以是一个伪影与结构变化, 由于细胞因子化学固定化;因此, 亲和性可能会受到影响, 测量也会受到损害。此外, 在贸易中心的分析, 稀释溶液可以修改 TNFα或单克隆的结构, 从而抑制 TNFα三形成, 单克隆人相互作用, 或 artifactual 表位生成。这种方法对原电池线的使用可能要求很高;然而, 对 TNFα的保护可以给出有趣的结果, 模仿在体内响应。
我们设计这种方法易于跟踪和重现。由于酚红或其他紫外-可见光吸收物质不能掩盖发光, 几乎所有培养基添加剂都可以用于培养细胞。这种改变有助于研究人员对细胞系的要求, 并在细胞因子治疗后对生存能力有充分的检测反应。在执行中和检测之前, 必须至少进行三细胞通道和细胞密度调整。此外, 稳定细胞因子及其浓度, 以及升温和均衡 CO2浓度在中的培养基和溶液是测定成功的关键步骤. 总的来说, 这篇文章显示了必要的步骤, 以消除 TNFα细胞因子与抗体使用的体外生物试验比较的参考和样本正在开发。

生物效力是根据与相关生物特性有关的检测产品属性而进行的生物活性的定量测量, 而数量 (以质量表示) 是蛋白质含量的物理化学量度。效力测试, 连同其他分析方法, 是作为产品一致性、稳定性和可比性研究的一部分进行的。在这个意义上, 效力测量是用来证明产品批次符合关键质量属性 (CQAs) 或验收标准在所有阶段的临床试验和经市场批准。
凋亡是程序性细胞死亡, 当细胞被病毒感染时自然发生, 或者当细胞被环境因素所胁迫时, 会危及细胞的生存能力和功能1,2。除其他以外, 凋亡抑制, 或生物中和, 是一个主要已知的治疗机制的抗体, 特别是在治疗慢性疾病, 如免疫介导的炎症性疾病。抗 TNFα分子通过阻断肿瘤坏死因子α (TNFα) 与 p55 和 p75 细胞表面受体3的相互作用而发挥其治疗特性, 从而阻止最终导致细胞凋亡的信号通路。
TNFα在一些慢性病症可能导致炎症4。TNFα是地分泌入细胞外环境由巨噬细胞, 是固有免疫系统的哨兵和主要演员在这种疾病5。作为一条共同的道路, TNFα放松管制与这些疾病的发病机制有关。不受控制, 并在恒定诱导和细胞应力, TNFα诱导细胞死亡和组织变性, 最终导致类风湿关节炎, 克罗恩病, 和其他病理概况6。
tnf 拮抗剂阻断肿瘤坏死因子及其受体之间的相互作用, 被越来越多地用来作为一种有效的治疗方法来减少症状和阻碍这些疾病的进展。目前, 抗 TNFα药物产品被广泛用于控制这种细胞因子的系统集中, 从而防止受累组织的进一步变性。在这个意义上, 提供一个重现性和健壮的生物测定来描述药物的具体能力, 以达到其生物学效应是当务之急。
在本议定书中, 着重强调了执行生物分析方法所需的技能, 这是在发展中和化验过程中确定的关键步骤, 以成功测量生物效力。这种生物分析方法提供了不同批次或抗 TNFα药物产品的有用的可比性信息, 与临床试验的参考物质进行比较。

<table><tbody><tr><td>WEHI 164</td><td>ATCC</td><td>CRL-1751</td><td>Fibrosarcoma cells from Mus musculus</td></tr><tr><td>RPMI-1640 Medium</td><td>ATCC</td><td>30-2001</td><td>Store medium at 2 &amp;deg;C to 8 &amp;deg;C</td></tr><tr><td>RPMI 1640 Medium, no phenol red</td><td>GIBCO</td><td>11835-030</td><td>Store medium at 2 &amp;deg;C to 8 &amp;deg;C</td></tr><tr><td>Trypsin-EDTA(0.25%),phenol red</td><td>GIBCO</td><td>25200-056</td><td>Store medium at -10 &amp;deg;C to -20 &amp;deg;C</td></tr><tr><td>DPBS, no calcium, no magnesium</td><td>GIBCO</td><td>14190-136</td><td>Store medium at 2 &amp;deg;C to 8 &amp;deg;C</td></tr><tr><td>Recombinant Human TNF-alpha Protein</td><td>R&amp;amp;D Systems</td><td>210-TA-020</td><td>Store at -20 &amp;deg;C to -70 &amp;deg;C</td></tr><tr><td>Fetal Bovine Serum (U.S), Super Low IgG</td><td>HyClone</td><td>SH3089803</td><td>Store at -10 &amp;deg;C to -20 &amp;deg;C</td></tr><tr><td>Fetal Bovine Serum (U.S.), Characterized</td><td>HyClone</td><td>SH3007103</td><td>Store at -10 &amp;deg;C to -20 &amp;deg;C</td></tr><tr><td>Caspase-Glo 3/7 Assay kit</td><td>Promega</td><td>G8093</td><td>Store the Caspase-Glo. 3/7 Substrate and Caspase-Glo. 3/7 Buffer at &amp;ndash;20 &amp;ordm;C protected fromLight</td></tr><tr><td>EDTA, Disodium Salt, Dihydrate, Crystal, A.C.S. Reagent</td><td>J.T.Baker</td><td>8993-01</td><td>--</td></tr><tr><td>Sample mAb Adalimumab</td><td>Probiomed</td><td>NA</td><td>Final concentrations in the microplate are:&amp;nbsp; 0.666, 0.333, 0.167, 0.111, 0.083, 0.056, 0.042, 0.028, 0.014 and 0.004 &amp;mu;g/mL</td></tr><tr><td>Reference and Control mAb Adalimumab</td><td>Abbvie</td><td>NA</td><td>Final concentrations in the microplate are:&amp;nbsp; 0.666, 0.333, 0.167, 0.111, 0.083, 0.056, 0.042, 0.028, 0.014 and 0.004 &amp;mu;g/mL</td></tr><tr><td>Microplate Reader</td><td>Molecular Devices</td><td>89429-536</td><td>SpectraMax M3 Multi-Mode</td></tr><tr><td>Microplate reader Software&amp;nbsp;</td><td>Molecular Devices</td><td>--</td><td>SoftMax Pro 6.3 GxP</td></tr><tr><td>Incubator&amp;nbsp;</td><td>Revco&amp;nbsp;</td><td>30482</td><td>Revco RNW3000TABB Forced-Air CO2</td></tr><tr><td>Laminar Flow Hood&amp;nbsp;</td><td>The Baker Company&amp;nbsp;&amp;nbsp;</td><td>200256</td><td>Baker SG603A-HE | High Efficiency, Class II Type A2&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td></tr></tbody></table>

determination of the relative potency of an anti-tnf monoclonal antibody (mab) by neutralizing tnf using an in vitro bioanalytical method

本协议显示了使用抗 TNFα单克隆抗体对小鼠成纤维细胞模型 (WEHI 164) 中 TNFα的凋亡活性中和的测量。此外, 该协议可用于评估其他抗 TNFα分子, 如融合蛋白。细胞模型在这里使用的是敏感的 TNFα介导的细胞凋亡时, 额外的应力因子诱导的单元培养条件 (例如,血清剥夺)。这一过程说明了如何执行这一分析化验, 突出的关键操作与样品制备, 细胞稀释, 诱导细胞凋亡, 和分光光度测量, 是至关重要的, 以确保成功的结果。该协议揭示了与细胞凋亡诱导和有效信号记录有关的 best-performance 条件, 导致低不确定性值。

剂量-反应图 (带控制) 
图 1表示发光响应与单克隆浓度。这 sigmoidal 功能的例证酶3和7释放在分析培养基由于细胞裂解。通过血清饥饿加 TNFα信号诱导, 细胞死亡增加。因此, 抗 TNFα分子 (单克隆抗体) 与细胞因子相互作用, 抑制 (由空间位阻) 与 TNF 细胞受体的互动。这导致细胞存活在更高的单克隆浓度。
该方法所采用的控制方式为: 细胞与测定培养基、细胞加 TNFα加测定培养基、测定培养 mediumalone。细胞单独与化验培养基 FBS 饥饿没有经历细胞凋亡, 从而发展发光。此外, 细胞暴露在 TNFα单独, 但培养与培养基确实生存。
另一个重要的控制是单独测定培养基。这种控制有助于理解分子干扰有关的媒介, 特别是蛋白质, 可以消化酶基板, 从而表明假阳性发光信号。表 3中描述了 EC50 和相对效能的结果。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>示例</td>
			<td>EC50</td>
			<td>相对效力 (%)</td>
			<td>置信区间 (%)</td>
			<td>RDS (%)</td>
		</tr>
		<tr>
			<td rowspan="3">参考物质</td>
			<td>241。5</td>
			<td rowspan="3">100</td>
			<td rowspan="3">--</td>
			<td rowspan="3">--</td>
		</tr>
		<tr>
			<td>243。6</td>
		</tr>
		<tr>
			<td>234。2</td>
		</tr>
		<tr>
			<td rowspan="3">分析样本</td>
			<td>225。2</td>
			<td rowspan="3">99。7</td>
			<td rowspan="3">86.0-115。1</td>
			<td rowspan="3">8。5</td>
		</tr>
		<tr>
			<td>240。3</td>
		</tr>
		<tr>
			<td>258。8</td>
		</tr>
		<tr>
			<td rowspan="3">控件示例</td>
			<td>230。5</td>
			<td rowspan="3">97。1</td>
			<td rowspan="3">86.5-108。7</td>
			<td rowspan="3">6。9</td>
		</tr>
		<tr>
			<td>264</td>
		</tr>
		<tr>
			<td>248。4</td>
		</tr>
	</tbody>
</table>
表 3: 相对效力结果.分析样品与已知的效力样品进行比较, 在表中描述为参考, 具有固定的100% 效力。该关系的计算与每个样本的 EC50。间隔计算在95% 信心 (α = 0.05)。RSD: 相对标准偏差。
表 3显示了参考单克隆和正在调查的样本之间的相对效力 (百分比)。可比性在参考的80-120% 范围之内假设。然而, 有时参考在批次之间有大可变性;因此, 接受的范围可以改变。因此, 需要在短时间内选择参考批次, 收紧理化性能和验收间隔。
该表显示该参考的效力为 100%, 而分析样本的效力为99.7%。这一结果意味着样品的 TNFα中和能力可与参考文献相媲美。这是预期的疾病与过度表达的细胞因子可以控制这些抗体。

Watch this Scientific Journal Video about Determination of the Relative Potency of an Anti-TNF Monoclonal Antibody mAb by Neutralizing TNF Using an In Vitro Bioanalytical Method at JoVE.com

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

本文介绍了一种利用 WEHI 164 细胞中和机制测定抗 TNFα单克隆人的相对亡活性的协议。本协议对于比较不同分子的中和强度具有相同的生物功能是有用的。

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 ...

determination-relative-potency-an-anti-tnf-monoclonal-antibody-mab

Universidad San Sebastian

Research

JoVE Journal

Medicine

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

9.2K 次观看.  Probiomed. 本文介绍了一种利用 WEHI 164 细胞中和机制测定抗 TNFα单克隆人的相对亡活性的协议。本协议对于比较不同分子的中和强度具有相同的生物功能是有用的。

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

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by pharmaceutical companies is a market opportunity, but it is also a strategy to increase drug accessibility and reduce therapy-associated costs. The protocols detailed here describe the evaluation of target binding and CDC induction by rituximab in Daudi cells. These two functions require different structural regions of the antibody and are relevant to the clinical effect induced by rituximab. The protocols allow the side-to-side comparison of a reference rituximab and a marketed rituximab biosimilar. The evaluated products showed differences both in target binding and CDC induction, suggesting that there are underlying physicochemical differences and highlighting the need to analyze the impact of those differences in the clinical setting. The methods reported here constitute simple and inexpensive in vitro models for the evaluation of the activity of rituximab biosimilars. Thus, they can be useful during biosimilar development, as well as for quality control in biosimilar production. Furthermore, the presented methods can be extrapolated to other therapeutic mAbs.

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

This protocol describes the in vitro comparison of two key functional characteristics of rituximab: target binding and complement-dependent cytotoxicity (CDC) induction. The methods were employed for a side-to-side comparison between reference rituximab and a rituximab biosimilar. These assays can be employed during biosimilar development or as a quality control in their production.

体外方法比较标的结合和CDC诱导治疗性抗体之间:应用在Biosimilarity分析

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by ...

科研

免疫与感染

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

The administration of enzyme replacement therapies (ERTs) and other biologic therapies to patients may elicit an anti-drug immune response. The characterization of these anti-drug antibodies (ADA), especially those that may neutralize the biological activity of the drug, termed neutralizing antibodies (NAbs), is crucial in understanding the effects of these antibodies on the drug&#39;s pharmacological profile. This protocol describes a cell-based flow cytometry method to detect factors that neutralize the cellular uptake of a representative lysosomal ERT in human matrix. The protocol consists of three procedures: screening, a confirmatory step, and titer assays to detect, identify, and establish the relative level of neutralizing antibody titer in subject samples.
In this method, samples are first mixed with the fluorophore-conjugated ERT product, then incubated with cells [e.g., human T lymphocytes (Jurkat cells)] that express a cell-surface cation-independent mannose 6-phosphate receptor (CI-M6PR), and finally, analyzed with a flow cytometer. A sample without NAbs will result in the uptake of the fluorophore-conjugated ERT product via CI-M6PR, whereas, the presence of NAbs will bind to the drug and interfere with the CI-M6PR binding and uptake. The amount of the fluorophore-conjugated ERT internalized by the Jurkat cells is measured by flow cytometry and evaluated as the percentage (%) signal inhibition compared to the response obtained in the presence of a representative drug-na&#239;ve matrix. In the confirmatory step, the samples are pre-incubated with ERT-conjugated magnetic beads to deplete drug-specific factors that bind to the drug (such as NAbs) prior to an incubation with cells. Samples that screen and confirm positive for drug-specific NAbs in the assay are then serially diluted to generate an antibody titer. Semi-quantitative antibody titers may be correlated with measurements of drug safety and efficacy.

Here we present a cell-based flow cytometry method to detect neutralizing antibodies or other factors that interfere with the cellular uptake of enzyme replacement therapies in a human matrix, such as cerebral spinal fluid (CSF) or human serum.

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 ...

A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological malignancies such as childhood acute lymphocytic leukemia. Efforts are currently underway to extend this promising therapy to solid tumors in addition to other hematological cancers. Here, we describe the development and production of potent CAR T cells targeting antigens with unique or preferential expression on solid and liquid tumor cells. The in vitro potency of these CAR T cells is then evaluated in real-time using the highly sensitive impedance-based xCELLigence assay. Specifically, the impact of different costimulatory signaling domains, such as glucocorticoid-induced tumor necrosis factor receptor (TNFR)-related protein (GITR), on the in vitro potency of CAR T cells is examined. This report includes protocols for: generating CAR T cells for preclinical studies using lentiviral gene transduction, expanding CAR T cells, validating CAR expression, and running and analyzing xCELLigence potency assays.

We describe a quantitative real-time in vitro cytolysis assay system to evaluate the potency of chimeric antigen receptor T cells targeting liquid and solid tumor cells. This protocol can be extended to assess other immune effector cells, as well as combination treatments.

用于嵌合抗原受体 T 细胞的实时效力测定，用于定位固体和血液癌细胞

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological ...

Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life science research and clinical diagnostics. However, ELISA has limitations. It is typically performed in a 96-well microplate, and the wells are coated with capture antibody, requiring a relatively large amount of sample to capture an antigen of interest . The large surface area of the wells and the hydrophobic binding of capture antibody can also lead to non-specific binding and increased background. Additionally, most ELISAs rely upon enzyme-mediated amplification of signal in order to achieve reasonable sensitivity. Such amplification is not always linear and can thus skew results.
In the past 15 years, a new technology has emerged that offers the benefits of the ELISA, but also enables higher throughput, increased flexibility, reduced sample volume, and lower cost, with a similar workflow 1, 2. Luminex xMAP Technology is a microsphere (bead) array platform enabling both monoplex and multiplex assays that can be applied to both protein and nucleic acid applications 3-5. The beads have the capture antibody covalently immobilized on a smaller surface area, requiring less capture antibody and smaller sample volumes, compared to ELISA, and non-specific binding is significantly reduced. Smaller sample volumes are important when working with limiting samples such as cerebrospinal fluid, synovial fluid, etc. 6. Multiplexing the assay further reduces sample volume requirements, enabling multiple results from a single sample.
Recent improvements by Luminex include: the new MAGPIX system, a smaller, less expensive, easier-to-use analyzer; Low-Concentration Magnetic MagPlex Microspheres which eliminate the need for expensive filter plates and come in a working concentration better suited for assay development and low-throughput applications; and the xMAP Antibody Coupling (AbC) Kit, which includes a protocol, reagents, and consumables necessary for coupling beads to the capture antibody of interest. (See Materials section for a detailed list of kit contents.)
In this experiment, we convert a pre-optimized ELISA assay for TNF-alpha cytokine to the xMAP platform and compare the performance of the two methods 7-11. TNF-alpha is a biomarker used in the measurement of inflammatory responses in patients with autoimmune disorders.
We begin by coupling four candidate capture antibodies to four different microsphere sets or regions. When mixed together, these four sets allow for the simultaneous testing of all four candidates with four separate detection antibodies to determine the best antibody pair, saving reagents, sample and time. Two xMAP assays are then constructed with the two most optimal antibody pairs and their performance is compared to that of the original ELISA assay in regards to signal strength, dynamic range, and sensitivity.

An ELISA can be easily converted to a Luminex xMAP assay and, through the benefits of multiplexing, several antibodies can be screened simultaneously to identify an optimum antibody pair, resulting in increased sensitivity and dynamic range, while reducing assay cost.

转换到一个点阵仪XMAP含量，使用多重抗体筛查方法的捕获ELISA

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life ...

生物学

Development of Recombinant Proteins to Treat Chronic Pain

Chronic pain is difficult to treat and new approaches to resolve persistent pain are urgently needed. Anti-inflammatory cytokines are promising candidates for treating debilitating pain conditions due to their capacity to regulate aberrant neuro-immune interactions. However, physiologically they work in a network of various cytokines, and therefore their therapeutic effect may not be optimal when used as stand-alone drugs. To overcome this limitation, we developed a fusion protein of the anti-inflammatory cytokines IL4 and IL10. Here, we describe the methods for production and quality control of IL4-10 recombinant fusion protein and we test the effectiveness of the IL4-10 fusion protein to resolve pain in a mouse model of persistent inflammatory pain.

In this paper we provide details for the methods of production and quality control of an IL4-10 recombinant fusion protein. We also show how to test the effectiveness of this protein to resolve pain in a mouse model of inflammatory pain.

重组蛋白治疗慢性疼痛的研究进展

Chronic pain is difficult to treat and new approaches to resolve persistent pain are urgently needed. Anti-inflammatory cytokines are promising candidates ...

医学

Using Reference Reagents to Confirm Robustness of Cytokine Release Assays for the Prediction of Monoclonal Antibody Safety

New immunostimulatory antibody drugs designed to either directly stimulate specific immune cells or indirectly enhance the immune response by blocking or activating an endogenous regulator of the immune system have the potential to cause serious immune-related adverse events such as cytokine release syndrome (CRS). It is, therefore crucial to assess the safety profile of such drugs with a combination of in vivo and in vitro experiments before first-in-human dose administration. Cytokine release assays (CRAs), where the proposed antibody therapeutic is co-cultured with human immune cells (such as peripheral blood mononuclear cells (PBMCs), whole blood, or otherwise) and the amount of inflammatory cytokine produced is measured, are critical for hazard identification. However, different labs using different control antibodies can threaten the harmonization of CRAs, and clinically relevant controls (such as TGN1412) can be difficult to source, which can lead to less accurate or reliable results or data which are difficult to compare between laboratories. The inclusion of positive and negative controls in a CRA can ensure the accuracy and reliability of the results. The National Institute for Biological Standards and Control (NIBSC) has produced a panel of lyophilized antibody controls intended for use in various CRA platforms to harmonize results across various laboratories and assay methods. A set of three different positive control antibodies include anti-CD52, anti-CD3, and anti-CD28 superagonist (SA), which are known to induce dose-dependent CRS in patients. Each antibody is provided with an isotype-matched negative control antibody. This panel of reference reagents has previously been shown to have good inter-lab reproducibility and are suitable controls to increase the confidence and robustness of safety data from a variety of CRA platforms.

The use of cytokine release assay reference reagents allows for more reproducible and standardized in vitro safety profiles of immunotherapeutic monoclonal antibodies. Here we describe how cytokine release assays can be used alongside a reference reagent panel to predict the safety of some therapeutic monoclonal antibodies.

New immunostimulatory antibody drugs designed to either directly stimulate specific immune cells or indirectly enhance the immune response by blocking or ...

Monitoring PD-1-Blocking Antibodies Bound to T Cells Derived from a Drop of Peripheral Blood

Immune checkpoint inhibitors, including PD-1&#8211;blocking antibodies, have significantly improved treatment outcomes in various types of cancer. The pharmacological efficacy of these immunotherapies is long lasting, extending even beyond the discontinuation of their injections, due to persistent blood concentrations. Here we developed a simple flow cytometry assay to evaluate the T cell binding status of the PD-1&#8211;blocking antibodies nivolumab and pembrolizumab. Like a glucose test, this assay requires just a single drop of peripheral blood. Visualizing antibody binding on T cells is more reliable than measuring antibody blood concentrations. In addition, if necessary, we can potentially analyze many distinctive immune-related markers on T cells bound to PD-1&#8211;blocking antibodies. Thus, this is a simple and minimally invasive strategy to analyze the pharmacological effect of PD-1&#8211;blocking antibodies in cancer patients.

We developed a simple flow cytometry assay for evaluating the binding of PD-1&#8211;blocking antibodies to T cells, requiring only a drop of peripheral blood from cancer patients.

监测PD-1-阻断抗体绑定到T细胞派生从外周血滴

Immune checkpoint inhibitors, including PD-1&#8211;blocking antibodies, have significantly improved treatment outcomes in various types of cancer. 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 ...

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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体外方法比较标的结合和CDC诱导治疗性抗体之间:应用在Biosimilarity分析

细胞法检测酶替代疗法中细胞吸收的抗体

用于嵌合抗原受体 T 细胞的实时效力测定，用于定位固体和血液癌细胞

转换到一个点阵仪XMAP含量，使用多重抗体筛查方法的捕获ELISA

重组蛋白治疗慢性疼痛的研究进展

Using Reference Reagents to Confirm Robustness of Cytokine Release Assays for the Prediction of Monoclonal Antibody Safety

监测PD-1-阻断抗体绑定到T细胞派生从外周血滴

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

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

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