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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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>

<ol>
	<li>CDER. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Assay Development and Validation for Immunogenicity Testing of Therapeutic Protein Products (Draft). , (2016).</li><li>Gupta, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recommendations+for+the+design,+optimization,+and+qualification+of+cell-based+assays+used+for+the+detection+of+neutralizing+antibody+responses+elicited+to+biological+therapeutics.">Recommendations for the design, optimization, and qualification of cell-based assays used for the detection of neutralizing antibody responses elicited to biological therapeutics.</a> Journal of Immunological Methods. 321, 1-18 (2007).</li><li>Gupta, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recommendations+for+the+validation+of+cell-based+assays+used+for+the+detection+of+neutralizing+antibody+immune+responses+elicited+against+biological+therapeutics.">Recommendations for the validation of cell-based assays used for the detection of neutralizing antibody immune responses elicited against biological therapeutics.</a> Journal of Pharmaceutical and Biomedical Analysis. 55, 878-888 (2011).</li><li>Pedras, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NAb+me+well-+FDA+Regulatory+Perspectives+on+Neutralizing+Antibody+Assays.">NAb me well- FDA Regulatory Perspectives on Neutralizing Antibody Assays.</a> BEBPA Forum. , (2015).</li><li>Stovold, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NAB+Assay+Development+and+Validation+Immunogencity+Workflow.">NAB Assay Development and Validation Immunogencity Workflow.</a> Annual BEBPA Bioassay Meeting. , (2013).</li><li>Kromminga, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neutralizing anti-drug antibodies Emerging Trends and Clinical Impact. Symposium. , (2013).</li><li>Davis, R. S., Wang, Y. H., Kubagawa, H., Cooper, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+family+of+Fc+receptor+homologs+with+preferential+B+cell+expression.">Identification of a family of Fc receptor homologs with preferential B cell expression.</a> Proceedings of the National Academy of Sciences of the United States of America. 98, 9772-9777 (2001).</li><li>Melton, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibodies+that+neutralize+cellular+uptake+of+elosulfase+alfa+are+not+associated+with+reduced+efficacy+or+pharmacodynamic+effect+in+individuals+with+Morquio+A+syndrome.">Antibodies that neutralize cellular uptake of elosulfase alfa are not associated with reduced efficacy or pharmacodynamic effect in individuals with Morquio A syndrome.</a> Journal of Immunological Methods. 440, 41-51 (2017).</li><li>U.S. Department of Health and Human Services. Food and Drug Administration. Center for Drug Evaluation and Research (CDER). Center for Biologics Evaluation and Research (CBER). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guidance for Industry. S6 Addendum to Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals. , (2012).</li><li>Lee, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+biochemical+and+pharmacological+comparison+of+enzyme+replacement+therapies+for+the+glycolipid+storage+disorder+Fabry+disease.">A biochemical and pharmacological comparison of enzyme replacement therapies for the glycolipid storage disorder Fabry disease.</a> Glycobiology. 13, 305-313 (2003).</li><li>Kirkegaard, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+therapies+and+therapeutic+concepts+for+lysosomal+storage+diseases.">Emerging therapies and therapeutic concepts for lysosomal storage diseases.</a> Expert Opinion on Orphan Drugs. 1, 385-404 (2013).</li><li>Guidelines for Safe Work Practices in Human and Animal Medical Diagnostic Laboratories. Morbidity and Mortality Weekly Report Available from: <a target="_blank" href="https://www.cdc.gov/mmwr/preview/mmwrhtml/su6101a1.htm">https://www.cdc.gov/mmwr/preview/mmwrhtml/su6101a1.htm</a> (2012)</li><li>Invitrogen by ThermoFisher Scientific. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> DynabeadsTM M-270 Streptavidin. , (2015).</li><li>European Medicines Agency. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guideline on Immunogenicity assessment of biotechnology-derived therapeutic proteins. , (2017).</li><li>ThermoFisher Scientific. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular ProbesTM Alexa FluorTM 647 Protein Labeling Kit (A20173). , (2004).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Introduction+to+Working+in+the+Hood.">An Introduction to Working in the Hood.</a> Journal of Visualized Experiments. , (2018).</li><li>Invitrogen and Gibco. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell Culture Basics Handbook. , (2010).</li><li>Coecke, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidance+on+Good+Cell+Culture+Practice.">Guidance on Good Cell Culture Practice.</a> Alternatives to Laboratory Animals. 33, 261-287 (2005).</li><li>. Introduction to Cell Culture Available from: <a target="_blank" href="https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/introduction-to-cell-culture.html">https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/introduction-to-cell-culture.html</a> (2018)</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+a+Hemacytometer+to+Count+Cells.">Using a Hemacytometer to Count Cells.</a> Journal of Visualized Experiments. , (2018).</li><li>. Counting Cells with Bio-Rad&#39;s TC20(TM) Automated Cell Counter Available from: <a target="_blank" href="https://www.youtube.com/watch?v=sDHOL7UEL1M">https://www.youtube.com/watch?v=sDHOL7UEL1M</a> (2012)</li><li>ThermoFisher Scientific. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> User Guide LIVE / DEADTM Fixable Dead Cell Stain Kits. , (2016).</li><li>Biosciences. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Getting Started with BD FACSDiva Software. , (2007).</li><li>Biosciences. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> BD FACS Canto II Instructions For Use. , (2007).</li><li>Quirke, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+to+Flow+Cytometry:+A+Learning+Guide.">Introduction to Flow Cytometry: A Learning Guide.</a> Journal of Clinical Pathology. 45 (3), (2002).</li><li>. A guide to gating in flow cytometry Available from: <a target="_blank" href="https://www.bio-rad-antibodies.com/blog/a-guide-to-gating-in-flow-cytometry.html">https://www.bio-rad-antibodies.com/blog/a-guide-to-gating-in-flow-cytometry.html</a> (2016)</li><li>Minor, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Assay Development in Drug Discovery. , (2006).</li><li>Desilva, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recommendations+for+the+Bioanalytical+Method+Validation+of+Ligand-binding+Assays+to+Support+Pharmacokinetic+Assessments+of+Macromolecules.">Recommendations for the Bioanalytical Method Validation of Ligand-binding Assays to Support Pharmacokinetic Assessments of Macromolecules.</a> Pharmaceutical Research. 20, (2003).</li><li>Hu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+cell-based+and+non-cell-based+assay+platforms+for+the+detection+of+clinically+relevant+anti-drug+neutralizing+antibodies+for+immunogenicity+assessment+of+therapeutic+proteins.">Comparison of cell-based and non-cell-based assay platforms for the detection of clinically relevant anti-drug neutralizing antibodies for immunogenicity assessment of therapeutic proteins.</a> Journal of Immunological Methods. 419, 1-8 (2015).</li><li>Cai, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+neutralizing+assay+for+antibodies+and+sera+against+hepatitis+E+virus.">A high-throughput neutralizing assay for antibodies and sera against hepatitis E virus.</a> Scientific Reports. 6, (2016).</li><li>Charles River. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Flow+Cytometric+Method+to+Assess+Neutralizing+Antibodies+to+AAV+Gene-Therapy+Vectors.">A Flow Cytometric Method to Assess Neutralizing Antibodies to AAV Gene-Therapy Vectors.</a> Charles River Researcher. , (2015).</li><li>Waritani, T., Chang, J., McKinney, B., Terato, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ELISA+protocol+to+improve+the+accuracy+and+reliability+of+serological+antibody+assays.">An ELISA protocol to improve the accuracy and reliability of serological antibody assays.</a> MethodsX. 4, 153-165 (2017).</li><li>Iversen, P. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HTS+Assay+Validation.">HTS Assay Validation.</a> Assay Guidance Manual. , 1-30 (2004).</li><li>Thermo Fisher Scientific. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Tech Tip #34. Binding characteristics of antibody-binding proteins: Protein A, Protein G, Protein A/G and Protein L. , (2013).</li><li>Long, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+Immunogenicity+of+Elosulfase+Alfa+in+the+Treatment+of+Morquio+A+Syndrome:+Results+From+MOR-005,+a+Phase+III+Extension+Study.">Long-term Immunogenicity of Elosulfase Alfa in the Treatment of Morquio A Syndrome: Results From MOR-005, a Phase III Extension Study.</a> Clinical Therapeutics. 39, (2017).</li><li>Schweighardt, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunogenicity+of+Elosulfase+Alfa,+an+Enzyme+Replacement+Therapy+in+Patients+with+Morquio+A+Syndrome:+Results+from+MOR-004,+a+Phase+III+Trial.">Immunogenicity of Elosulfase Alfa, an Enzyme Replacement Therapy in Patients with Morquio A Syndrome: Results from MOR-004, a Phase III Trial.</a> Clinical Therapeutics. 37, (2015).</li><li>Mellman, I. R. A., Plutner, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Internalization+and+Degradation+of+Macrophage+Fc+Receptors+Bound+to+Polyvalent+Immune+Complexes.">Internalization and Degradation of Macrophage Fc Receptors Bound to Polyvalent Immune Complexes.</a> The Journal of Cell Biology. 98, 1170-1177 (1984).</li><li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutralizing+antibodies+to+the+therapeutic+enzymes:+considerations+for+testing,+prevention+and+treatment.">Neutralizing antibodies to the therapeutic enzymes: considerations for testing, prevention and treatment.</a> Nature Biotechnology. 26, 901-908 (2008).</li></ol>

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 &#181;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&#38;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 CO2 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&#8482;</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 (e.g. 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 &#38; 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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

8.7K 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

Enzyme-linked Immunospot Assay (ELISPOT):  Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens

Adaptive immunity is an important component to clearance of intracellular pathogens. The ability to detect and quantify these responses in humans is an important diagnostic tool. The enzyme-linked immunospot assay (ELISPOT) is gaining popularity for its ability to identify cellular immune responses against microbial antigens, including immunosuppressed populations such as those with HIV infection, transplantation, and steroid use. This assay has the capacity to quantify the immune responses against specific microbial antigens, as well as distinguish if these responses are Th1 or Th2 in character. ELISPOT is not limited to the site of inflammation. It is versatile in its ability to assess for immune responses within peripheral blood, as well as sites of active involvement such as bronchoalveolar lavage, cerebral spinal fluid, and ascites. Detection of immune responses against a single or multiple antigens is possible, as well as specific epitopes within microbial proteins. This assay facilitates detection of immune responses over time, as well as distinctions in antigens recognized by host T cells. Dual color ELISPOT assays are available for detection of simultaneous expression of two cytokines. Recent applications for this technique include diagnosis of extrapulmonary tuberculosis, as well as investigation of the contribution of infectious antigens to autoimmune diseases.

Enzyme-linked Immunospot Assay ELISPOT:  Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens

Identification of microbial targets of adaptive immunity in idiopathic diseases can be accomplished by the use of the enzyme-linked immunospot assay.

酶联免疫斑点法（ELISPOT）:TH - 1细胞对微生物抗原的免疫应答的定量

Adaptive immunity is an important component to clearance of intracellular pathogens.  The ability to detect and quantify these responses in humans is an ...

科研

免疫与感染

In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 (Ehmeth) Enzyme

Protozoan parasites are among the most devastating infectious agents of humans responsible for a variety of diseases including amebiasis, which is one of the three most common causes of death from parasitic disease. The agent of amebiasis is the amoeba parasite Entamoeba histolytica that exists under two stages: the infective cyst found in food or water and the invasive trophozoite living in the intestine. The clinical manifestations of amebiasis range from being asymptomatic to colitis, dysentery or liver abscesses. E. histolytica is one of the rare unicellular parasite with 5-methylcytosine (5mC) in its genome. 1, 2 It contains a single DNA methyltransferase, Ehmeth, that belongs to the Dnmt2 family. 2 A role for Dnmt2 in the control of repetitive elements has been established in E. histolytica, 3 Dictyostelium discoideum 4,5 and Drosophila. 6 Our recent work has shown that Ehmeth methylates tRNAAsp, and this finding indicates that this enzyme has a dual DNA/tRNAAsp methyltransferase activity. 7 This observation is in agreement with the dual activity that has been reported for D. discoideum and D. melanogaster. 8 The functional significance of the DNA/tRNA specificity of Dnmt2 enzymes is still unknown. To address this question, a method to determine the tRNA methyltransferase activity of Dnmt2 proteins was established. In this video, we describe a straightforward approach to prepare an adequate tRNA substrate for Dnmt2 and a method to measure its tRNA methyltransferase activity.

In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 Ehmeth Enzyme

This protocol describes the preparation of a synthetic tRNA substrate for the Entamoeba histolytica DNA/tRNA methyltransferase 2 (Dnmt2) homolog Ehmeth and the measure of its methyltransferase activity. This experimental approach can be used for investigating the activity of other Dnmt2 proteins.

在体外的tRNA与阿米巴 DNA和tRNA甲基Dnmt2（Ehmeth）酶甲基化含量

Protozoan parasites are among the most devastating infectious agents of humans responsible for a variety of diseases including amebiasis, which is one of ...

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

所有流感的定量分析类型使用通用抗体的一种病毒Hemagglutinins和Neuraminidases，在简单的狭缝杂交分析

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or localization of a target molecule in cells and tissues. Immunofluorescence relies on the specificity of fluorescent-labelled antibodies against their corresponding antigens within a cell. Both direct and indirect immunofluorescence approaches can be used which rely on the use of antibodies linked with a fluorochrome. Direct immunofluorescence is less frequently used because it provides lower signal, involves higher cost and less flexibility. In contrast, indirect immunofluorescence is more commonly used because of its high sensitivity and provides an amplified signal since more than one secondary antibody can attach to each primary antibody. In this manuscript, both epifluorescence microscopy and confocal microscopy were used to monitor the internalization of human lactoferrin, an important component of the immune system, into hepatic cells. Moreover, we monitored the inhibitory potential of hLF on the intracellular replication of the Hepatitis C virus using immunofluorescence. Both the advantages and disadvantages associated with these approaches are discussed.

The human lactoferrin (hLF) is a component of the immune system. In this study, immunofluorescence assays are used to demonstrate both the hepatocellular uptake of hLF and a qualitative reduction in the hepatitis C virus replication upon treatment with hLF.

免疫监视人乳铁蛋白的细胞摄取及其相关抗病毒活性抗丙型肝炎病毒

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

病毒 DNA 的纯化及相关病毒和细胞蛋白的鉴定

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

报告酶荧光法检测小鼠结核分枝杆菌的影像学

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...

Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a formidable defense against invading pathogens via diverse mechanisms. One major goal of vaccination is to induce protective antigen-specific antibodies to prevent life-threatening infections. Both thymus-dependent (TD) and thymus-independent (TI) antigens can elicit robust antigen-specific IgM responses and can also induce the production of isotype-switched antibodies (IgG, IgA and IgE) as well as the generation of memory B cells with the help provided by antigen presenting cells (APCs). Here, we describe a protocol to characterize TD and TI Ig isotype responses in mice using enzyme-linked immunosorbent assay (ELISA). In this protocol, TD and TI Ig responses are elicited in mice by intraperitoneal (i.p.) immunization with hapten-conjugated model antigens TNP-KLH (in alum) and TNP-polysaccharide (in PBS), respectively. To induce TD memory response, a booster immunization of TNP-KLH in alum is given at 3 weeks after the first immunization with the same antigen/adjuvant. Mouse sera are harvested at different time points before and after immunization. Total serum Ig levels and TNP-specific antibodies are subsequently quantified using Ig isotype-specific Sandwich and indirect ELISA, respectively. In order to correctly quantify the serum concentration of each Ig isotype, the samples need to be appropriately diluted to fit within the linear range of the standard curves. Using this protocol, we have consistently obtained reliable results with high specificity and sensitivity. When used in combination with other complementary methods such as flow cytometry, in vitro culture of splenic B cells and immunohistochemical staining (IHC), this protocol will allow researchers to gain a comprehensive understanding of antibody responses in a given experimental setting.

In this paper, we describe a protocol to characterize T-dependent and T-independent immunoglobulin (Ig) isotype responses in mice using ELISA. This method used alone or in combination with flow cytometry will allow researchers to identify differences in B cell-mediated Ig isotype responses in mice following T-dependent and T-independent antigen immunization.

酶联免疫吸附法表征小鼠胸腺依赖性和胸腺独立免疫球蛋白同种反应

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

沙门氏菌筛选的高通量平台.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Dynamic Adhesion Assay for the Functional Analysis of Anti-adhesion Therapies in Inflammatory Bowel Disease

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a crucial step in this process and therapeutic strategies interfering with adhesion have been successfully established. The anti-&#945;4&#946;7 integrin antibody, vedolizumab, is used for the clinical treatment of Crohn&#39;s disease (CD) and ulcerative colitis (UC) and further compounds are likely to follow.
The details of the adhesion procedure and the action mechanisms of anti-integrin antibodies are still unclear in many regards due to the limited available techniques for the functional research in this field.
Here, we present a dynamic adhesion assay for the functional analysis of human cell adhesion under flow conditions and the impact of anti-integrin therapies in the context of IBD. It is based on the perfusion of primary human cells through addressin-coated ultrathin glass capillaries with real-time microscopic analysis. The assay offers a variety of opportunities for refinements and modifications and holds potentials for mechanistic discoveries and translational applications.

Dynamic adhesion of immune cells to the vessel wall is a prerequisite for gut homing. Here, we present a protocol for a functional in vitro assay for the impact analysis of anti-integrin antibodies, chemokines or other factors on the dynamic cell adhesion of human cells using addressin-coated capillaries.

动态粘附法在炎症性肠病抗粘连治疗功能分析中的作用

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a ...

Merkel Cell Polyomavirus Infection and Detection

Merkel cell polyomavirus (MCPyV) infection can lead to Merkel cell carcinoma (MCC), a highly aggressive form of skin cancer. Mechanistic studies to fully investigate MCPyV molecular biology and oncogenic mechanisms have been hampered by a lack of adequate cell culture models. Here, we describe a set of protocols for performing and detecting MCPyV infection of primary human skin cells. The protocols describe the isolation of human dermal fibroblasts, preparation of recombinant MCPyV virions, and detection of virus infection by both immunofluorescent (IF) staining and in situ DNA-hybridization chain reaction (HCR), which is a highly sensitive fluorescence in situ hybridization (FISH) approach. The protocols herein can be adapted by interested researchers to identify other cell types or cell lines that support MCPyV infection. The described FISH approach could also be adapted for detecting low levels of viral DNAs present in the infected human skin.

Here, we present a protocol to infect primary human dermal fibroblast with MCPyV. The protocol includes isolation of dermal fibroblasts, preparation of MCPyV virions, virus infection, immunofluorescence staining, and fluorescence in situ hybridization. This protocol can be extended for characterizing MCPyV-host interactions and discovering other cell types infectable by MCPyV.

默克尔细胞多瘤病毒感染与检测

Merkel cell polyomavirus (MCPyV) infection can lead to Merkel cell carcinoma (MCC), a highly aggressive form of skin cancer. Mechanistic studies to fully ...