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
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?...

<ol>
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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lyophilized+formulations+of+recombinant+tumor+necrosis+factor.">Lyophilized formulations of recombinant tumor necrosis factor.</a> Pharm Res. 9 (1), 33-36 (1992).</li><li>Ponnappan, S., Ponnappan, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+and+immune+function:+molecular+mechanisms+to+interventions.">Aging and immune function: molecular mechanisms to interventions.</a> Antiox Redox Signal. 14 (8), 1551-1585 (2011).</li><li>Matsumaru, K., Ji, C., Kaplowitz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+for+sensitization+to+TNF-induced+apoptosis+by+acute+glutathione+depletion+in+murine+hepatocytes.">Mechanisms for sensitization to TNF-induced apoptosis by acute glutathione depletion in murine hepatocytes.</a> Hepatology. 37 (6), 1425-1434 (2003).</li><li>Camacho-Villegas, T., Mata-Gonzalez, T., Paniagua-Solis, J., Sanchez, E., Licea, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+TNF+cytokine+neutralization+with+a+vNAR+from+Heterodontus+francisci+shark:+a+potential+therapeutic+use.">Human TNF cytokine neutralization with a vNAR from Heterodontus francisci shark: a potential therapeutic use.</a> mAbs. 5 (1), 80-85 (2013).</li><li>M&#228;nnel, D. N., Falk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+induction+of+tumor+necrosis+factor+production+in+human+monocytes+requires+complete+S-form+lipopolysaccharide.">Optimal induction of tumor necrosis factor production in human monocytes requires complete S-form lipopolysaccharide.</a> Infect Immun. 57 (7), 1953-1958 (1989).</li><li>Lis, K., Kuzawi&#324;ska, O., Ba&#322;kowiec-Iskra, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+necrosis+factor+inhibitors-state+of+knowledge.">Tumor necrosis factor inhibitors-state of knowledge.</a> Arch Med Sci. 10 (6), 1175-1185 (2014).</li></ol>

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...

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 border="0" cellpadding="0" cellspacing="0" width="904">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">WEHI 164</td>
			<td style="width:111px;">ATCC</td>
			<td style="width:127px;">CRL-1751</td>
			<td style="width:463px;">Fibrosarcoma cells from Mus musculus</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">RPMI-1640 Medium</td>
			<td style="width:111px;">ATCC</td>
			<td style="width:127px;">30-2001</td>
			<td style="width:463px;">Store medium at 2 &#176;C to 8 &#176;C</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">RPMI 1640 Medium, no phenol red</td>
			<td style="width:111px;">GIBCO</td>
			<td style="width:127px;">11835-030</td>
			<td style="width:463px;">Store medium at 2 &#176;C to 8 &#176;C</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Trypsin-EDTA(0.25%),phenol red</td>
			<td style="width:111px;">GIBCO</td>
			<td style="width:127px;">25200-056</td>
			<td style="width:463px;">Store medium at -10 &#176;C to -20 &#176;C</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">DPBS, no calcium, no magnesium</td>
			<td style="width:111px;">GIBCO</td>
			<td style="width:127px;">14190-136</td>
			<td style="width:463px;">Store medium at 2 &#176;C to 8 &#176;C</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:203px;">Recombinant Human TNF-alpha Protein</td>
			<td style="width:111px;">R&#38;D Systems</td>
			<td style="width:127px;">210-TA-020</td>
			<td style="width:463px;">Store at -20 &#176;C to -70 &#176;C</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Fetal Bovine Serum (U.S), Super Low IgG</td>
			<td style="width:111px;">HyClone</td>
			<td style="width:127px;">SH3089803</td>
			<td style="width:463px;">Store at -10 &#176;C to -20 &#176;C</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Fetal Bovine Serum (U.S.), Characterized</td>
			<td style="width:111px;">HyClone</td>
			<td style="width:127px;">SH3007103</td>
			<td style="width:463px;">Store at -10 &#176;C to -20 &#176;C</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Caspase-Glo 3/7 Assay kit</td>
			<td style="width:111px;">Promega</td>
			<td style="width:127px;">G8093</td>
			<td style="width:463px;">Store the Caspase-Glo. 3/7 Substrate and Caspase-Glo. 3/7 Buffer at &#8211;20 &#186;C protected fromLight</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:203px;">EDTA, Disodium Salt, Dihydrate, Crystal, A.C.S. Reagent</td>
			<td style="width:111px;">J.T.Baker</td>
			<td style="width:127px;">8993-01</td>
			<td style="width:463px;">--</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:203px;">Sample mAb Adalimumab</td>
			<td style="width:111px;">Probiomed</td>
			<td style="width:127px;">NA</td>
			<td style="width:463px;">Final concentrations in the microplate are:&#160; 0.666, 0.333, 0.167, 0.111, 0.083, 0.056, 0.042, 0.028, 0.014 and 0.004 &#956;g/mL</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:203px;">Reference and Control mAb Adalimumab</td>
			<td style="width:111px;">Abbvie</td>
			<td style="width:127px;">NA</td>
			<td style="width:463px;">Final concentrations in the microplate are:&#160; 0.666, 0.333, 0.167, 0.111, 0.083, 0.056, 0.042, 0.028, 0.014 and 0.004 &#956;g/mL</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:203px;">Microplate Reader</td>
			<td style="width:111px;">Molecular Devices</td>
			<td style="width:127px;">89429-536</td>
			<td style="width:463px;">SpectraMax M3 Multi-Mode</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:203px;">Microplate reader Software&#160;</td>
			<td style="width:111px;">Molecular Devices</td>
			<td style="width:127px;">--</td>
			<td style="width:463px;">SoftMax Pro 6.3 GxP</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">Incubator&#160;</td>
			<td>Revco&#160;</td>
			<td>30482</td>
			<td style="width:463px;">Revco RNW3000TABB Forced-Air CO2</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:203px;">Laminar Flow Hood&#160;</td>
			<td style="width:111px;">The Baker Company&#160;&#160;</td>
			<td>200256</td>
			<td style="width:463px;">Baker SG603A-HE | High Efficiency, Class II Type A2&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</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。
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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。

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诱导大鼠心肌梗死的半微创方法及独立工作心脏系统对心脏功能的评估

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily ...

An In Vitro Dissolution Determination of Multi-Index Components in Tibetan Medicine Rhodiola Granules

An In Vitro Dissolution Determination of Multi-Index Components in Tibetan Medicine Rhodiola Granules

藏药红景天颗粒中多指标组分的 体外 溶出度测定

The composition of the Tibetan medicine Rhodiola granules (RG) is complex, and the overall quality of RG is difficult to determine. Therefore, ...