HiBiT-qIP, HiBiT-based quantitative immunoprecipitation, facilitates the determination of antibody affinity under immunoprecipitation conditions
HiBiT-qIP, HiBiT-based quantitative immunoprecipitation, facilitates the determination of antibody affinity under immunoprecipitation conditions
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HiBiT-qIP，基于 HiBiT 的定量免疫沉淀，有助于在免疫沉淀条件下测定抗体亲和力
DOI：
10.1038/s41598-019-43319-y
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发表时间：
2019
影响因子：
4.6
通讯作者：
Kamachi Yusuke
中科院分区：
文献类型：
--
作者：
Ranawakage Deshani C.;Takada Takuya;Kamachi Yusuke
抗体与其抗原的亲和力是抗体评价的重要参数。抗体-抗原亲和力的评估对于基于抗体的分析是至关重要的，特别是免疫沉淀(IP)，因为它严格依赖于抗体的性能。然而，在IP条件下，抗体亲和力或其定量决定因素-解离常数(Kd)的测定是困难的。在本研究中，我们使用基于纳米Luc的HiBiT系统建立了基于HiBiT的定量免疫沉淀(HiBiT-QIP)方法，用于检测溶液中Kdof抗原-抗体的相互作用。HiBiT-QIP方法以一种简单而定量的方式测量标记了HiBiT的免疫沉淀蛋白质的数量。我们用这种方法测量了表位标签-抗体相互作用的Kd值。将FLAG、HA、V5、PA和TY1的单体、二聚体或三聚体表位标签与谷胱甘肽S转移酶(GST)和HiBiT多肽融合，并在IP缓冲液中与同源单抗混合，计算表观Kd值。这项HiBiT-QIP试验表明，所检测的抗体克隆的Kd值有相当大的差异。此外，多聚体形式的表位标签的使用揭示了表观亲和力的拷贝数依赖的增加。
The affinity of an antibody for its antigen serves as a critical parameter for antibody evaluation. The evaluation of antibody-antigen affinity is essential for a successful antibody-based assay, particularly immunoprecipitation (IP), due to its strict dependency on antibody performance. However, the determination of antibody affinity or its quantitative determinant, the dissociation constant (Kd), under IP conditions is difficult. In the current study, we used a NanoLuc-based HiBiT system to establish a HiBiT-based quantitative immunoprecipitation (HiBiT-qIP) assay for determining the Kdof antigen-antibody interactions in solution. The HiBiT-qIP method measures the amount of immunoprecipitated proteins tagged with HiBiT in a simple yet quantitative manner. We used this method to measure the Kdvalues of epitope tag-antibody interactions. To accomplish this, FLAG, HA, V5, PA and Ty1 epitope tags in their monomeric, dimeric or trimeric form were fused with glutathione S-transferase (GST) and the HiBiT peptide, and these tagged GST proteins were mixed with cognate monoclonal antibodies in IP buffer for the assessment of the apparent Kdvalues. This HiBiT-qIP assay showed a considerable variation in the Kdvalues among the examined antibody clones. Additionally, the use of epitope tags in multimeric form revealed a copy number-dependent increase in the apparent affinity.