我们要感谢美国国立卫生研究院共同基金-蛋白捕获试剂项目获得资金的重组抗体网络的抗体筛选和鉴定管道和加拿大创新基金会拨款购买机器人选择平台的发展。

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作者什么都没有透露。

体外抗体选择导通时，选择成功的两个主要因素是：1）的良好折叠的抗原靶的隔离，用于选择后和2）的高功能的多样性抗体库的可用性。在许多情况下，足量的良好的折叠，全长蛋白质的可用性可以是限制性的。一种方法来克服这种限制是使用识别从生物信息学分析22并合成用于插入域到细菌表达载体与合适的亲和标签。 有各种各样的用于生物技术的应用，?...

与后基因组时代的到来，高品质的结合试剂的可用性来表征和调节蛋白是必不可少的，打开新的研究和治疗的途径。抗体继续成为学术界和工业界研究的基础研究和诊断工具和潜在的治疗至关重要。这并不奇怪，出现了合同抗体开发公司，其中大部分依靠常规的杂交瘤技术产生的抗体定制一个令人印象深刻的增长。然而， 在体外利用噬菌体展示抗体库的选择正在成为能够提供独特的优势和成功的地方传统技术可能面临限制1，2强大的替代技术。 鉴于高品质的抗体作为研究工具，产生两个主要的挑战相当大的需求的可再生抗体是1）选用吞吐量和2）抗原AVailability。一些团体现在已经在体外筛选管道旨在提高吞吐量和抗体识别率描述。这些描述详细介绍了各种可行的方法后，无论是全长，包括针对选择3,4，或结构相关领域5,6,7，无论是使用基于微珠6,8或板为基础的3,4-抗原固定的计划。此外，越来越多地采用的基因合成技术9取得了系统的抗原的产生，特别是分离的结构域，合理的成本效益，并且可以潜在地减轻在取得足够量的纯化的，全长抗原的难度。通过使用两种技术中串联，一个独立的，可伸缩的抗原的产生和抗体的选择管道被设计，这将使抗体大集表达的抗原结构域的并行分离和促进试剂对茶的发展racterizing结构或功能上相关的蛋白质的整个类。 为了实现这一目标，夫妇表达抗原域，基因合成，抗原和可扩展的噬菌体展示抗体选择高通量细菌表达的硅片鉴定已经开发了一个集成的管道。这条管道需要提供给大多数生命科学实验室（包括抗体库，这是越来越多可以通过许可或材料转让协定），只有基本的基础设施，而且还易于自动化的工业规模使用。使用该协议，有可能产生数百亲和标记的抗原结构域，并例行隔离高度特异性抗体片段的许多这些抗原。 噬菌体展示技术已经显示与多种重组亲和试剂的格式，包括的Fab，单链抗体，和自主的Fv结构域和一个的证明兼容性小“替代框架”（设计的锚蛋白重复蛋白（DARPINS），纤维连接蛋白（FN），脂质运载蛋白域多10）不断增长的阵列。讨论被限制于这个例子的Fab抗体片段的分离，尽管可以推测这些方法可以适用于其他类型的库。采用这种技术，具有低纳摩尔亲和力小的Fab，标记的蛋白质结构域，包括转录因子结构域，SH2结构域，RNA结合蛋白和其他人已成功选择，其中许多结合全长蛋白质和有功能的免疫测定，如免疫荧光，免疫和免疫组化。通过细菌生产提供增加的一致性，再现性和成本效益重​​要的是，重组结合克隆是完全可再生的，并从表达可以重新生成构建，从而证明了严格克隆验证为代价。 在这个普罗特ocol和附带视频，基本方法使用固定的抗原结构域噬菌体展示文库的抗体的选择被证明。这个特定的方法使用通过被动吸附在微孔板固定GST标记的蛋白质结构域，尽管其他标签11,12,13和选择格式13,14,2也已成功地使用。关键的考虑因素选择与平行监测选择参数旨在确定和隔离尤其丰富克隆抗体确认的设置和行为有详细。

<table border="0" cellpadding="0" cellspacing="0" width="470">
	<tbody>
		<tr height="22">
			<td colspan="4" height="22" style="height:22px;width:471px;">MATERIALS</td>
		</tr>
		<tr height="43">
			<td colspan="2" height="43" style="height:43px;width:249px;">Name</td>
			<td style="width:135px;">Company</td>
			<td style="width:87px;">Catalog Number</td>
		</tr>
		<tr height="40">
			<td colspan="2" height="40" style="height:40px;width:249px;">New Brunswick Innova44 stackable incubator shaker</td>
			<td style="width:135px;">Eppendorf</td>
			<td style="width:87px;">M1282-0004</td>
		</tr>
		<tr height="40">
			<td colspan="2" height="40" style="height:40px;width:249px;">Liquidator 96 channel manual benchtop pipetting system</td>
			<td style="width:135px;">Anachem Ltd</td>
			<td style="width:87px;">LIQ-96-200</td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;width:249px;">Microplate shaker</td>
			<td style="width:135px;">VWR</td>
			<td style="width:87px;">12620-926</td>
		</tr>
		<tr height="40">
			<td colspan="2" height="40" style="height:40px;width:249px;">ThermoScientific Sorvall ST-40 benchtop centrifuge</td>
			<td style="width:135px;">Thermo Product</td>
			<td style="width:87px;">75004525</td>
		</tr>
		<tr height="40">
			<td colspan="2" height="40" style="height:40px;width:249px;">ELx405 Select Deep Well Microplate Washer</td>
			<td style="width:135px;">Biotek</td>
			<td>ELX405USD</td>
		</tr>
		<tr height="41">
			<td colspan="2" height="41" style="height:41px;width:249px;">Custom High Throughput Automated Phage Selection Robot</td>
			<td style="width:135px;">S&#38;P Robotics</td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:249px;">Plastic conical Falcon tube: 50 mL</td>
			<td style="width:135px;">VWR</td>
			<td style="width:87px;">21008-178</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:249px;">Plastic conical Falcon tube: 15 mL</td>
			<td style="width:135px;">VWR</td>
			<td style="width:87px;">89039-668</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:249px;">Axygen 1.7 mL microcentrifuge tubes</td>
			<td style="width:135px;">Corning</td>
			<td style="width:87px;">MCT-175-L-C</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:249px;">96-well/384-well Maxisorp plate</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:87px;">M9410-1CS</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:249px;">Corning 96-well V-bottom deep-well block</td>
			<td style="width:135px;">Corning</td>
			<td style="width:87px;">3960</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:249px;">Axygen Mini-tube 96-well sterile microplate blue box</td>
			<td style="width:135px;">Corning</td>
			<td style="width:87px;">AXY-MTS-06-C-R-S</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:249px;">Breathable adhesive plate sealing film</td>
			<td style="width:135px;">VWR</td>
			<td style="width:87px;">60941-084</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:22px;width:471px;">REAGENTS</td>
		</tr>
		<tr height="43">
			<td colspan="2" height="43" style="height:43px;width:249px;">Name</td>
			<td style="width:135px;">Company</td>
			<td style="width:87px;">Catalog Number</td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;width:249px;">polyethylene glycol</td>
			<td style="width:135px;">Bioshop</td>
			<td style="width:87px;">PEG800.1</td>
		</tr>
		<tr height="18">
			<td colspan="2" height="18" style="height:18px;">yeast extract</td>
			<td>Bioshop</td>
			<td>YEX401.1</td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;width:249px;">bio-tryptone</td>
			<td style="width:135px;">Bioshop</td>
			<td style="width:87px;">TRP402.1</td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;width:249px;">N-Z amine</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:87px;">C7290</td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;width:249px;">glucose</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:87px;">G8270-1KG</td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;width:249px;">lactose</td>
			<td style="width:135px;">Bioshop</td>
			<td style="width:87px;">LAC234</td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;width:249px;">glycerol</td>
			<td style="width:135px;">Bioshop</td>
			<td>GLY001.500</td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;width:249px;">carbenicillin&#160;</td>
			<td style="width:135px;">Bioshop</td>
			<td style="width:87px;">CAR544.10</td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;width:249px;">kanamycin</td>
			<td style="width:135px;">Bioshop</td>
			<td style="width:87px;">KAN201.25</td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;width:249px;">tetracycline&#160;</td>
			<td style="width:135px;">Bioshop</td>
			<td style="width:87px;">TET701.25</td>
		</tr>
		<tr height="43">
			<td colspan="2" height="43" style="height:43px;width:249px;">Tween 20</td>
			<td style="width:135px;">Bioshop</td>
			<td style="width:87px;">TWN510.500</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:249px;">monobasic potassium phophate</td>
			<td style="width:135px;">Bioshop</td>
			<td style="width:87px;">PPM302.1</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:249px;">dibasic sodium phosphate</td>
			<td style="width:135px;">Anachemia</td>
			<td style="width:87px;">84486-440</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:249px;">sodium chloride</td>
			<td style="width:135px;">Bioshop</td>
			<td style="width:87px;">SOD001.10</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:249px;">potassium chloride</td>
			<td style="width:135px;">Bioshop</td>
			<td style="width:87px;">POC308.1</td>
		</tr>
		<tr height="20">
			<td colspan="2" height="20" style="height:20px;width:249px;">calcium chloride</td>
			<td style="width:135px;">Bioshop</td>
			<td style="width:87px;">CCL302.500</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:249px;">magnesium sulphate</td>
			<td style="width:135px;">EMD</td>
			<td style="width:87px;">MX0070-1</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:249px;">magnesium chloride</td>
			<td style="width:135px;">Bioshop</td>
			<td style="width:87px;">MAG510.500</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:249px;">NH4Cl</td>
			<td style="width:135px;">Amresco</td>
			<td style="width:87px;">0621-1KG</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:249px;">Na2SO4</td>
			<td style="width:135px;">Bioshop</td>
			<td style="width:87px;">SOS513.500</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:249px;">agar</td>
			<td style="width:135px;">Bioshop</td>
			<td style="width:87px;">AGR001.500</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:249px;">SybrSafe DNA gel stain</td>
			<td style="width:135px;">Invitrogen</td>
			<td style="width:87px;">S33102</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:249px;">phosphoric acid</td>
			<td style="width:135px;">Acros Organics</td>
			<td style="width:87px;">201140010</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:249px;">lysozyme</td>
			<td style="width:135px;">Bioshop</td>
			<td style="width:87px;">LYS702.25</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:249px;">benzonase</td>
			<td style="width:135px;">Novagen</td>
			<td style="width:87px;">71205</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:139px;">Triton X-100</td>
			<td style="width:111px;"></td>
			<td style="width:135px;">Bioshop</td>
			<td style="width:87px;">TRX506.500</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:249px;">protease inhibitor cocktail tablets</td>
			<td style="width:135px;">Roche</td>
			<td style="width:87px;">11 836 170 001</td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;width:249px;">Ni-NTA resin&#160;</td>
			<td style="width:135px;">Qiagen</td>
			<td style="width:87px;">1018240</td>
		</tr>
		<tr height="42">
			<td colspan="2" height="42" style="height:43px;width:249px;">1000x helper phage (M13K07) stock (1013 phage per mL)</td>
			<td style="width:135px;">NEB</td>
			<td style="width:87px;">N0315S</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="39" rowspan="2" style="height:40px;width:249px;">HRP conjugated M13-specific antibody (anti-M13-HRP).</td>
			<td rowspan="2" style="width:135px;">GE Healthcare</td>
			<td rowspan="2" style="width:87px;">27-9241-01</td>
		</tr>
		<tr height="18">
		</tr>
		<tr height="44">
			<td colspan="2" height="44" style="height:44px;width:249px;">TMB substrate: mix equal volumes of TMB and H2O2 peroxidase substrate&#160;</td>
			<td style="width:135px;">KPL</td>
			<td style="width:87px;">50-76-00</td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;width:249px;">dNTPs</td>
			<td style="width:135px;">Biobasic</td>
			<td style="width:87px;">DD0056</td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;width:249px;">Taq polymerase</td>
			<td style="width:135px;">Genscript</td>
			<td style="width:87px;">E00007</td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;width:249px;">Exonuclease</td>
			<td style="width:135px;">GE Healthcare&#160;</td>
			<td style="width:87px;">EZ0073X-EZ</td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;width:249px;">Shrimp alkaline phosphatase</td>
			<td style="width:135px;">GE Healthcare</td>
			<td style="width:87px;">E70092Z-EZ</td>
		</tr>
	</tbody>
</table>

scalable high throughput selection from  phage-displayed synthetic antibody libraries

为满足这两个基础和临床研究应用中的需要的抗体的需求很高，将在未来的显着增加。然而，显而易见的是传统的单克隆技术并不单单达此任务。这导致了替代方法的发展，以满足对高品质的和可再生的亲和性试剂与蛋白质的所有可访问的元素的需求。为此，高通量的方法用于进行从噬菌体展示的合成抗体文库的选择已经设计出用于涉及多种抗原的应用和快速的吞吐量和成功进行了优化。在这里，协议进行了详细的视频演示说明对数百使用手动96道液体处理器或自动机器人系统目标并行选择的Fab噬菌体克隆的高度多样性库描述。使用此协议，一个用户可以产生数百抗原，选择抗体，它们在并行和验证抗体6-8周内约束力。高亮是：i）一个可行的抗原格式，ⅱ）预选抗原的表征，ⅲ）影响的特异性和高亲和性的克隆的选择关键步骤，以及iv）监测选择有效性和早期抗体克隆的表征方法。使用这种方法，我们已获得的合成抗体片段（Fab的），以许多目标类包括单通膜受体，分泌蛋白激素，和多域的胞内蛋白。这些片段被容易地转化为全长抗体并且已经验证，以表现出高的亲和性和特异性。此外，它们已被证明是功能性的各种标准的免疫测定，包括Western印迹，ELISA，细胞免疫荧光，免疫沉淀和相关测定法。这种方法将加速抗体的发现，并最终使我们更接近实现目标Ø˚F产生可再生能源，高品质的抗体的蛋白质组。

本文所描述的协议已被用于分离抗体片段的各种从并联组合噬菌体展示的Fab文库既structurally-和功能上相关的抗原结构域。相关抗原可用性问题可以回避，在许多情况下，通过在表达抗原结构域适合于抗体选择23的硅片鉴定。通过偶联抗原表达抗体的选择在相同的实验室（ 图1）它来构造一个集成的管线，在其中抗原参数来选择关键，可以更容易地控制变得可行。在大多数情况?...

Watch this Scientific Journal Video about Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries at JoVE.com

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

一种方法是描述视觉伴奏用于同时进行可扩展，高吞吐量选择从噬菌体展示的组合合成的抗体文库针对数百抗原。使用这种平行的方式，我们已经分离，表现出对于不同的抗原有功能在标准免疫高亲和力和特异性的抗体片段。

可扩展的高通量选择从噬菌体展示合成抗体库

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

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JoVE Journal

Immunology and Infection

18.4K 次观看.  The Recombinant Antibody Network. 一种方法是描述视觉伴奏用于同时进行可扩展，高吞吐量选择从噬菌体展示的组合合成的抗体文库针对数百抗原。使用这种平行的方式，我们已经分离，表现出对于不同的抗原有功能在标准免疫高亲和力和特异性的抗体片段。 