Real-time Characterization of Antibody Binding to Receptors on Living Immune Cells.
Real-time Characterization of Antibody Binding to Receptors on Living Immune Cells.
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DOI：
10.3389/fimmu.2017.00455
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发表时间：
2017
影响因子：
7.3
通讯作者：
Buijs J
中科院分区：
文献类型：
--
作者：
Bondza S;Foy E;Brooks J;Andersson K;Robinson J;Richalet P;Buijs J
了解免疫细胞的分子相互作用对于开发治疗癌症和自身免疫性疾病的药物至关重要。当描述分子相互作用时，使用相关的活模型系统是很重要的，因为受体寡聚化和聚类等过程会影响结合模式。我们开发了一种方案，使配体结合到活悬浮细胞上的受体的时间分辨分析。利用生物分子锚定分子将不同的悬浮细胞系和弱粘附细胞拴在培养皿上，用配体示踪剂分析抗体结合情况。本报告中描述的方案和试验用于表征涉及8个细胞系的相互作用。实验成功地在三个不同的实验室进行，证明了协议的鲁棒性。对于不同的抗体，分别与Daudi和Ramos b细胞上的CD20、Jurkat细胞上的t细胞共受体CD3和转染HEK293细胞上的Fcγ受体CD32结合获得了亲和性和动力学速率常数。分析利妥昔单抗与b细胞的结合结果表明，其亲和力为0.7-0.9 nM，这与之前报道的活b细胞的亲和力相似。然而，我们观察到利妥昔单抗与b细胞相互作用的异质性行为，据我们所知，这在以前没有被描述过。对复杂相互作用的理解将有助于实时表征活免疫细胞上的结合过程。这为拓宽结合动力学与生物功能的关系提供了机会。
Understanding molecular interactions on immune cells is crucial for drug development to treat cancer and autoimmune diseases. When characterizing molecular interactions, the use of a relevant living model system is important, as processes such as receptor oligomerization and clustering can influence binding patterns. We developed a protocol to enable time-resolved analysis of ligand binding to receptors on living suspension cells. Different suspension cell lines and weakly adhering cells were tethered to Petri dishes with the help of a biomolecular anchor molecule, and antibody binding was analyzed using LigandTracer. The protocol and assay described in this report were used to characterize interactions involving eight cell lines. Experiments were successfully conducted in three different laboratories, demonstrating the robustness of the protocol. For various antibodies, affinities and kinetic rate constants were obtained for binding to CD20 on both Daudi and Ramos B-cells, the T-cell co-receptor CD3 on Jurkat cells, and the Fcγ receptor CD32 on transfected HEK293 cells, respectively. Analyzing the binding of Rituximab to B-cells resulted in an affinity of 0.7–0.9 nM, which is similar to values reported previously for living B-cells. However, we observed a heterogeneous behavior for Rituximab interacting with B-cells, which to our knowledge has not been described previously. The understanding of complex interactions will be facilitated with the possibility to characterize binding processes in real-time on living immune cells. This provides the chance to broaden the understanding of how binding kinetics relate to biological function.