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Kinetic analysis of antibodies is one of the important study for characterization of antibodies and screening of ligands. In our recent study1, we compared the antigenic profiles and binding characteristics of four HIV-1 envelope glycoprotein (Env) core immunogens using multiple monoclonal antibodies by Bio-Layer Light Interferometry (BLI). This technology enables real-time analysis of interactions on the surface of a fiber optic biosensor by accurately measuring kinetic constants such as Ka, Kd, and KD in a 96-well format.

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Kinetic Analysis of Monoclonal Antibody Binding to HIV-1 gp120-derived Hyperglycosylated Cores
对结合高糖基化HIV-1 gp120核心蛋白的单克隆抗体进行动力学分析

免疫学 > 抗体分析 > 抗体-抗原相互作用
作者: Jidnyasa Ingale
Jidnyasa IngaleAffiliation: Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, USA
Bio-protocol author page: a2626
 and Richard T Wyatt
Richard T WyattAffiliation 1: Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, USA
Affiliation 2: , IAVI Neutralizing Antibody Center at TSRI, La Jolla, USA
For correspondence: wyatt@scripps.edu
Bio-protocol author page: a2627
Vol 5, Iss 20, 10/5/2015, 1527 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1615

[Abstract] Kinetic analysis of antibodies is one of the important study for characterization of antibodies and screening of ligands. In our recent study1, we compared the antigenic profiles and binding characteristics of four HIV-1 envelope glycoprotein (Env) core immunogens using multiple monoclonal antibodies by Bio-Layer Light Interferometry (BLI). This technology enables real-time analysis of interactions on the surface of a fiber optic biosensor by accurately measuring kinetic constants such as Ka, Kd, and KD in a 96-well format.

[Abstract]

Materials and Reagents

  1. 96-well black bottom plates (Greiner Bio-One GmbH, catalog number: 655209 )
  2. Anti-human Fc capture biosensors (Pall Corporation, ForteBio®, catalog number: 18-5060 )
  3. Phosphate buffered saline (Life Technologies, GibcoTM, catalog number: 100-10023 )
  4. Monoclonal antibodies and hyperglycosylated core analytes (expressed in-house)

Equipment

  1. Octet Red system (Pall Corporation, ForteBio®, part number: 30-5048 )

Software

  1. Data Analysis 6.2 evaluation software (Forte Bio)

Procedure

Biosensors are tips coated with proprietary biocompatible matrix that can bind to Fc region of human immunoglobulin with minimal non-specific binding. In a separate black bottom plate, the biosensors were hydrated in 200 μl PBS for 10 min at room temperature prior to the experiment. The sample plate was generated according to the template shown in Figure 1. The buffer wells were filled with 200 μl PBS. Selected monoclonal antibodies were diluted at 5 μg/ml in PBS and dispensed at a volume of 200 μl into each well of the loading column as indicated in the template. The H1 well was filled with 200 μl PBS. The analyte (HIV envelope core glycoprotein is used in the example shown in Figure 1) was 2-fold serially diluted in PBS starting from 1 μM to 31.25 nM in a separate plate and transferred in a 200 μl volume in the “association column” of the template as shown (Figure 1). The sensor plate (plate with bio-sensors) and sample plate (plate with other reagents, as shown in Figure 1) were then inserted into the Octet Red machine and a basic kinetic measurement experiment was performed as follows:

  1. The sensors were dipped in buffer for equilibration.
  2. The sensors were transferred to the sensor plate and monoclonal antibodies were captured on the sensors at 1,000 rpm for 60 sec.
  3. Sensors were washed in PBS and a stable baseline was achieved at 1,000 rpm for 60 sec.
  4. The antibody-immobilized sensors were immersed in analyte-containing wells for 600 sec at 1,000 rpm to allow potential association of analyte with a given monoclonal antibody.
  5. Bio-sensors were next transferred to and immersed in wells containing a volume of 200 μl PBS at 1,000 rpm for 600 sec for the dissociation of the bound analyte.
    During each run two reference sensor were included, first where at the association step the sensors were immersed in PBS buffer as an analyte control (to be used for subtraction during analysis of the raw data) and second where no antibody was immobilized on the sensor and a 500 nM analyte was used during the association phase to assess the level of non-specific binding of analyte to the anti-human Fc capture sensors, if any such interactions were detected.
    The data was analyzed with Data Analysis 6.2 evaluation software. The data was processed using analyte control reference well for subtraction from the experimental data curves, aligning Y-axis to the baseline, and applying Savitzky-Golay as the filtering process. Next, the association and dissociation response curves were plotted using 1:1 model and global full curve fitting to compute the Ka, Kd, and KD values.


    Figure 1. Schematic diagram of sample plate and representative data of a kinetic experiment by bio-layer interferometry (BLI). A. Sample plate setup for measuring the kinetics of binding interaction between a monoclnal antbody and its ligand. REF 1 well is filled with PBS and used for reference subtraction. REF 2 well is a control experiment to check the non-specific binding of an analyte to the Fc capture sensors. B. The binding kinetics of a broadly neutralizing HIV 1 monoclonal antibody to a stable core envelope protein at various concentrations of the analyte. The black lines represent the theoretical Langmuir fits generated by 1:1 binding kinetics. The colored lines are the experimental curves for association and dissociation of the binding event.

Acknowledgements

This protocol was previously used in Ingale et al. (2014).

References

  1. Ingale, J., Tran, K., Kong, L., Dey, B., McKee, K., Schief, W., Kwong, P. D., Mascola, J. R. and Wyatt, R. T. (2014). Hyperglycosylated stable core immunogens designed to present the CD4 binding site are preferentially recognized by broadly neutralizing antibodies. J Virol 88(24): 14002-14016.

材料和试剂

  1. 96孔黑色底板(Greiner Bio-One GmbH,目录号:655209)
  2. 抗人Fc捕获生物传感器(Pall Corporation,ForteBio ,目录号:18-5060)
  3. 磷酸盐缓冲盐水(Life Technologies,Gibco TM ,目录号:100-10023)
  4. 单克隆抗体和高糖基化核心分析物(内部表达)

设备

  1. Octet Red系统(Pall Corporation,ForteBio ,部件号:30-5048)

软件

  1. 数据分析6.2评估软件(Forte Bio)

程序

生物传感器是用专有的生物相容性基质包被的末端,其可以以最小的非特异性结合结合人免疫球蛋白的Fc区。在单独的黑色底板中,在实验前将生物传感器在室温下在200μlPBS中水合10分钟。根据图1所示的模板产生样品板。用200μlPBS填充缓冲液孔。将选择的单克隆抗体在PBS中以5μg/ml稀释,并以200μl的体积分配到如模板所示的加载柱的每个孔中。用200μlPBS填充H1孔。在单独的平板中,分析物(HIV包膜核心糖蛋白用于图1所示的实例中)在PBS中以1μM至31.25nM开始连续稀释2倍,并在200μl体积中转移到模板如图所示(图1)。然后将传感器板(具有生物传感器的板)和样品板(具有其它试剂的板,如图1所示)插入Octet Red机器中,并如下进行基本动力学测量实验:

  1. 将传感器浸入缓冲液中用于平衡。
  2. 将传感器转移至传感器板,并在1,000rpm下在传感器上捕获单克隆抗体60秒。
  3. 在PBS中洗涤传感器,在1,000rpm下获得稳定的基线60秒。
  4. 将抗体固定的传感器浸没在含分析物的孔中,在1,000rpm下600秒,以允许分析物与给定的单克隆抗体的潜在缔合。
  5. 然后将生物传感器转移至含有体积为200μlPBS的孔中,并以1,000rpm浸渍于600秒以解离结合的分析物。
    在每次运行期间,包括两个参考传感器,首先,在关联步骤,将传感器浸没在作为分析物对照的PBS缓冲液中(用于在原始数据分析期间进行减法),其次,其中没有抗体固定在传感器上,在结合期期间使用500nM分析物以评估分析物与抗人Fc捕获传感器的非特异性结合的水平(如果检测到任何这样的相互作用)。
    使用Data Analysis 6.2评估软件分析数据。使用分析物对照参考孔处理数据以从实验数据曲线中减去,将Y轴与基线对准,并应用Savitzky-Golay作为过滤过程。接下来,使用1:1模型和全局全曲线拟合来绘制缔合和解离响应曲线,以计算K sub,K d和K D,/sub>值。


    图1.样品板和通过生物层干涉测量(BLI)的动力学实验的代表性数据的示意图。 A。用于测量单核体抗体与其配体之间的结合相互作用的动力学的样品板设置。 REF 1孔用PBS填充并用于参考减除。 REF 2孔是检查分析物与Fc捕获传感器的非特异性结合的对照实验。 B.在各种分析物浓度下广泛中和性HIV 1单克隆抗体与稳定的核心包膜蛋白的结合动力学。黑线代表通过1:1结合动力学产生的理论Langmuir拟合。彩色线是结合事件的缔合和解离的实验曲线

致谢

此协议以前用于Ingale等人(2014)。

参考文献

  1. Ingale,J.,Tran,K.,Kong,L.,Dey,B.,McKee,K.,Schief,W.,Kwong,P.D.,Mascola,J.R.and Wyatt,R.T。 设计用于呈递CD4结合位点的高糖基化稳定核心免疫原优先被广泛中和抗体识别。 a> J Virol 88(24):14002-14016。
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How to cite this protocol: Ingale, J. and Wyatt, R. T. (2015). Kinetic Analysis of Monoclonal Antibody Binding to HIV-1 gp120-derived Hyperglycosylated Cores. Bio-protocol 5(20): e1615. DOI: 10.21769/BioProtoc.1615; Full Text



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