高亲和力抗体 - 抗原的结合动力学的测定，使用四个生物传感器平台
概要
在这里，我们描述了抗原抗体的结合亲和力和使用四种常用的生物传感器平台动力学测量协议。
Abstract
无标签的光学生物传感器是在药物发现的生物分子相互作用的表征功能强大的工具。在这项研究中，我们描述了在我们的实验室进行评估的结合亲和力和对人前蛋白转化酶枯草杆菌蛋白酶Kexin 9型十大高亲和力的单克隆抗体（mAb）（PCSK9）的动力学使用四个常规使用的生物传感器平台。虽然两者的Biacore T100和的Proteon XPR36从成熟的表面等离子体共振（SPR）技术衍生，前者具有串行流构造连接的四个流动池，在平行通过一个简易6×6十字形而后者呈现36个反应斑点微流体通道的配置。的IBIS MX96也控制装置根据上述SPR传感器技术，具有一个额外的成像特征是，在空间方向提供检测。这再加上连续流动Microspotter（CFM）检测技术通过e显著扩展的吞吐量同时nabling 96个反应运动多路复用阵列印刷和检测。与此相反，八位位组RED384是基于生物层干涉测量（BLI）光学原理，使用光纤探针作为生物传感器来检测干涉图案时在尖端表面结合相互作用发生变化。不同于基于SPR的平台上，BLI系统不依赖于连续流射流;替代地，传感器尖端收集读数，同时它们的轨道搅拌期间浸没在384孔微孔板的分析物溶液。
每一种生物传感器平台都有自己的优点和缺点。提供的这些工具来提供优质的动力学数据，所描述的协议说明了使用相同的测定形式和相同的高品质的试剂来表征抗体 – 抗原动力学适合的简单的1实验能力的直接比较：1分子相互作用模型。
Introduction
The acquisition of reliable kinetic parameters for characterizing antibody-antigen interactions is an essential component of the drug discovery and development process1. Optical Surface Plasmon Resonance (SPR) biosensors, the “gold standard” for real-time detection of these interactions, have been used for approximately two decades to enable early selection of criteria-meeting therapeutic antibody candidates2,3. In addition to providing an affinity ranking of antibody candidates by the rapid binding screening of crude supernatants4, and rigorous kinetic constant determinations of purified preparations5, biosensors can further differentiate the functional activity of lead candidates via epitope binning studies6,7.
To meet the growing demand of antibody-based products for various therapeutic indications8, a wide variety of innovative biosensor instruments have been developed in recent years that increase the efficiency of candidate identification and characterization9,10. These instruments differ in microfluidic channel configuration design and/or in the optical principles involved in detecting biomolecular interactions. Specifically, the Octet RED38411, ProteOn XPR3612, and IBIS MX967 have expanded the number of interactions measured in a single binding cycle to 16, 36, and 96 respectively, representing significant throughput improvements over the traditional Biacore platform. Although these various biosensor platforms all provide essential kinetic data for characterizing antibody-antigen interactions, they differ in experimental setup and operational procedures due to variations in instrumental configurations. For example, in the IBIS MX96’s SPR imaging array platform, the multiplex ligand immobilization step is performed in an off-line mode in an external printing device separate from the detector7,13; in contrast, the other three biosensor platforms utilize an “all-in-one” setup, where the addition of component onto the sensor surface, whether it is the activation reagent, ligand, or analyte, is recorded in real time and through pre-programmed commands in sequential order. In the Octet RED384, a unique feature of this BioLayer Interferometry (BLI)-based platform is the availability of pre-coated optical fiber biosensors for immediate “dip-and-read” use14, eliminating the need for ligand surface preparation and initiation/conditioning steps, which are often required for the sensor chips in SPR-based platforms. This instrument’s fluidic-free design also simplifies the mechanics and avoids clogging and contamination concerns when dealing with crude samples. With the novel 6 x 6 crisscrossing fluidic design in the ProteOn XPR36, a “one-shot” kinetics approach can be implemented by switching the flow channels between horizontal and vertical directions to create 36 interaction spots15. Instead of assessing binding kinetics one ligand or analyte concentration at a time, as is done in the traditional serial flow Biacore T100 platform, this approach offers the ability to monitor up to 36 different interactions simultaneously in a single binding cycle.
Despite their differences, these four biosensor platforms are all widely used by many laboratories worldwide. For new users with little hands-on biosensor experience, deciding which instrument to use can be a challenging task given the differences in instrumental design. To determine the most appropriate instrument for their research purposes, factors such as data quality, performance consistency, throughput, ease of operation, and material consumption need to be considered collectively. While several benchmark studies have explored the variability of kinetic rate constants obtained from multiple laboratories and biosensors5,16, a recently published head-to-head comparison study further addresses the systematic factors that influence data reliability from the instrumental performance point of view17,18. The protocols supplied in this video focus on the experimental setup and procedures in detail, and are accompanied by the research article entitled, “Comparison of biosensor platforms in the evaluation of high affinity antibody-antigen binding kinetics”17. These protocols are intended not only to help new biosensor users implement these instruments for their research purposes, but also to provide additional insights for current biosensor users regarding technical challenges and considerations in experimental designs for evaluating high-affinity antibody-antigen interactions.
Protocol
Representative Results
Discussion
我们的头对头比较研究表明，每一个传感器平台都有自己的长处和短处。尽管抗体的结合轮廓是通过视觉比较相似（ 图3 – 6），并且将获取的动力学速率常数的排列顺序是在整个工具高度一致（ 图7），我们的结果表明，基于SPR的文书连续流动流体）是在解决具有较慢解离速率高亲和力相互作用更好。在解离相向上漂移在数据集观察到（ 例如 ，单克隆抗体2，单抗5，和mAb 9; 图5）通过BLI自由流体的仪器产生的。这一发现可以在很大程度上超过在微板中，这是该系统的一个主要限制时间归因于样品蒸发。与此固有的限制，实验时间也被限制为小于12个小时;因此，实验用的sh编程orter倍相比，那些的其它平台（10分钟关联和45分钟解离）（500秒关联和30分钟解离）。然而，缩短实验时间并未以减轻对数据质量/一致性蒸发的影响，如由基于BLI-仪器所产生的速率常数显示出较少的线性度如在一些离速率的波动的结果（ 图8C ）。除了样品蒸发，在所使用的捕获试剂的差异可能也有助于在得到的结果的差异。虽然在所有三个基于流体-SPR平台使用蛋白A / G，AHC传感器在非流体BLI平台中使用。因为蛋白A / G是可能对所述的单克隆抗体并不比AHC，基于抗体的生物传感器表面，从所述蛋白A / G表面上的单克隆抗体 – 抗原复合物的解离速率可人为地出现比更快亲和力较弱从AHC表面获得。这种可能性支持By中的实验数据显示，通过BLI平台所产生的解离速率值比从其他仪器（ 图7， 红色线 ）得到的那些一致地低。尽管如此，BLI平台具有比其他平台不同的优势。例如，它是相对于传感器的选择和测定配置高度灵活由于可即时使用的各种预涂覆的传感器。在我们的实验中，使用AHC传感器，消除了配体固定步骤的需求，降低了准备时间。此外，相比于其他流体SPR平台，其特点复杂油管和值开关配置的BLI平台需要更少的维护。此功能对于涉及原油样品，可能会导致堵塞和污染问题实验的优势。
至于治疗的候选人增加高效，快速，准确鉴定的需求，需要对BIθ传感器吞吐量也不断提高。在四个生物传感器平台上，从能够96-配体阵列打印的生物传感器的吞吐量是最高，其次由耦合到一个十字交叉36 – 配体格式和与16信道同时读出所述基于BLI的生物传感器的生物传感器，其最终增加在单一结合循环中测量到96，分别为36和16，交互的数量。这些可以通过能力比传统SPR平台，这是由仅具有4通过单个串行流连接流动池限定的显著更高。由于我们的实验中所涉及的在与长的解离时间的多个表面密度评价10个单克隆抗体相对较小的样本集，器乐吞吐量在确定实验的效率发挥适度的作用。有在三个高通量平台的实验时间没有显著差异，在所有情况下，实验是在一天内完成。在另一方面，传统的串行流SPR实验需要3天完成，尽管设置之后，数据采集的步行路程自动化。在涉及（在数百或数千IE）大量样品的，对于离率排名/动力学筛选或表位分级的目的等的研究，吞吐量成为一个关键因素。
虽然在IBIS MX96吞吐量比其他生物传感器的高几个数量级，因此对于这些目的的最佳选择，它有几个缺点。特别地，由CFM阵列印刷示出大的表面的不一致（ 图1）和降低的数据再现性（ 图8D和图8E）。为了精确动力学测量，在生物传感器表面上的配体的量是需要被控制，以确保结合反应不是由次要因素如干扰的关键参数传质或空间位阻。对于T100的Biacore和的Proteon XPR36，基于如在研究第17条中所述集合R 最大值的标准计算确定了最佳R L的水平。在另一方面，对于八比特组RED384和IBIS MX96平台，被获得的单克隆抗体捕获水平凭经验利用在恒定时间进行一系列2倍连续稀释的抗体。缺乏知识或捕获步骤的控制的导致了高密度的表面，并且可能已受到损害的动力学速率常数的精度高的结合的响应信号（ 图2）。此外，从SPR检测器的打印机的分离进行涉及再生的多个结合周期测量时也提出了挑战。执行多周期动力学设置的唯一方法是通过mAb的直接胺偶联，而不是被固定化的蛋白A / G通过的mAb的Fc来捕获表面在其他三个生物传感器平台。其结果是，需要一个额外的再生侦察实验，以确定最佳的再生条件。这种设置的结果是有联系的一个〜低90％观察到的表面活性相比于Fc捕获方法（ 图9）中，除了较长的实验时间。要执行的Fc捕获方法，另一种单周期动力学的方法获得通过。这种方法涉及分析物在每次注射（ 图2）之间的增加的浓度没有再生的顺序注射。这非常方便，但较不常见施加方法不仅缩短实验时间和减少的试剂消耗，而且也产生了动力学速率常数高度相似于那些来自其它生物传感器（ 图7）。因此，在应用该单周期动力学方法克服了仪器的固有构造限制和提供了一个机会在高通量的方式获得高分辨率的动力学速率常数。
尽管吞吐量是在T100的Biacore一个主要的限制，我们的结果共同表明它产生的最一致的数据以最高的质量。这之后通过的Proteon XPR36，其具有约10倍更高的吞吐量。表征高亲和力的抗体 – 抗原相互作用，当仪器的检测限达到可技术上具有挑战性当其以产生高质量的数据能力变得具有优势。而在八位RED384的仪器的系统的限制阻碍解离速率常数的精确测量（ 即 ，低于灵敏度的解决慢解离速率足够的信号衰减），两者的Biacore T100和的Proteon XPR36可以提供敏感和可靠检测分化。
開示
The authors have nothing to disclose.
Acknowledgements
作者感谢诺厄·迪托和亚当·迈尔斯有关IBIS MX96技术援助。
Materials
| Biacore T100 System | GE Healthcare | 28975001 | The T100 system has been upgraded to T200 |
| CM5 Sensor Chip | GE Healthcare | BR100012 | |
| BIAevaluation | GE Healthcare | Version 4.1 | |
| Biacore T100 Control Software | GE Healthcare | The T100 control software has been upgraded to T200 | |
| Amine Coupling Kit | GE Healthcare | BR100050 | It contains: 750 mg 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), 115 mg N-hydroxysuccinimide (NHS), 10.5 ml 1.0 M ethanolamine-HCl pH 8.5 |
| HBS-EP+ Buffer 10× | GE Healthcare | BR100669 | Concentrated stock solution |
| Plastic Vials, o.d. 7 mm | GE Healthcare | BR100212 | |
| Rubber Caps, type 3 | GE Healthcare | BR100502 | |
| Plastic Vials and Caps, o.d. 11 mm | GE Healthcare | BR100214 | |
| ProteOn XPR36 Protein Interaction Array System | Bio-Rad | 1760100 | |
| ProteOn Manager Software | Bio-Rad | 1760200 | Version 3.1.0.6 |
| GLM Sensor Chip | Bio-Rad | 1765012 | |
| Amine Coupling Kit | Bio-Rad | 1762410 | It includes EDAC (EDC), sulfo-NHS, ethanolamine HCl |
| Regeneration and Conditioning Kit and Buffers | Bio-Rad | 1762210 | It includes 1 each glycine buffer (pH 1.5, 2.0, 2.5, 3.0), and NaOH, SDS, HCl, phosphoric acid, NaCl; 50 ml each solution |
| 2 L PBS/Tween/EDTA buffer | Bio-Rad | 1762730 | It includes hosphate buffered saline (PBS), pH 7.4, 0.005% Tween 20, 3 mM EDTA |
| Octet RED384 System | FortéBio | ||
| Data Analysis Software | FortéBio | Version 9.0.0.4 | |
| Dip and Read Anti-hIgG Fc Capture (AHC) Biosensors | FortéBio | 18-5060 | One tray of 96 biosensors |
| 384-Well Tilted-Bottom Plate | FortéBio | 18-5080 | |
| Biosensor Dispenser | FortéBio | 18-5016 | |
| Kinetics Buffer 10X | FortéBio | 18-1092 | 10X concentration. Contains ProClin 300. |
| IBIS MX96 SPRi System | Wasatch Microfluidics | ||
| Microfluidics Continuous Flow Microspotter (CFM) Printer | Wasatch Microfluidics | Version 2.0 | |
| SensEye COOH-G chip | Wasatch Microfluidics | 1-09-04-004 | |
| Data Analysis Software | Wasatch Microfluidics | Version 6.19.3.17 | |
| SPRint Data Analysis Software | Wasatch Microfluidics | Version 6.15.2.1 | |
| Scrubber 2 | BioLogic Software | Version 2 | |
| Pierce Recombinant Protein A/G | ThermoFisher Scientific | 21186 |
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