Kinetic analysis of a high-affinity antibody/antigen interaction performed by multiple Biacore users
Kinetic analysis of a high-affinity antibody/antigen interaction performed by multiple Biacore users
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DOI：
10.1016/j.ab.2006.01.034
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发表时间：
2006-05-15
影响因子：
2.9
通讯作者：
Myszka, David G.
中科院分区：
文献类型：
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作者：
Katsamba, Phinikoula S.;Navratilova, Iva;Myszka, David G.
为了探索基于Biacore的检测的可靠性，22名研究参与者测量了前列腺特异性抗原（PSA）与单克隆抗体（mAb）的结合。每个参与者都得到了相同的试剂和详细的实验方案。将mAb以三种不同的密度固定在传感器芯片上，并使用两步测定来确定PSA/mAb复合物的动力学和亲和力参数。首先，在2.5-600 nM的浓度范围内测试PSA以获得k（a）信息。其次，为了准确定义该稳定抗原/抗体复合物的k（d），重新测试最高PSA浓度，并监测每个结合循环的解离相1 h。所有参与者收集的数据可以进行分析，以获得相互作用的动力学参数。使用简单的1：1相互作用模型全局拟合来自三个抗体表面的缔合和扩展解离数据。根据22次分析计算的PSA/mAb相互作用的平均k（a）和k（d）分别为（4.1 +/- 0.6）x 10（4）M-1 s（-1）和（4.5 +/-0.6）x 10（4）M-1 s（-1）。6)x 10（-5）s（-1）。总体而言，速率常数的实验标准误差仅为14%。基于动力学速率常数，PSA/mAb相互作用的亲和力（K-D）为1.1 +/- 0.2nM。(c)2006年爱思唯尔公司All rights reserved.
To explore the reliability of Biacore-based assays, 22 study participants measured the binding of prostate-specific antigen (PSA) to a monoclonal antibody (mAb). Each participant was provided with the same reagents and a detailed experimental protocol. The mAb was immobilized on the sensor chip at three different densities and a two-step assay was used to determine the kinetic and affinity parameters of the PSA/mAb complex. First, PSA was tested over a concentration range of 2.5-600 nM to obtain k(a), information. Second, to define the k(d) of this stable antigen/antibody complex accurately, the highest PSA concentration was retested with the dissociation phase of each binding cycle monitored for 1 h. All participants collected data that could be analyzed to obtain kinetic parameters for the interaction. The association and the extended-dissociation data derived from the three antibody surfaces were globally fit using a simple 1: 1 interaction model. The average k(a) and k(d) for the PSA/mAb interaction as calculated from the 22 analyses were (4.1 +/- 0.6) x 10(4) M-1 s(-1) and (4.5 +/- 0. 6) x 10(-5) s(-1), respectively. Overall, the experimental standard errors in the rate constants were only similar to 14%. Based on the kinetic rate constants, the affinity (K-D) of the PSA/mAb interaction was 1.1 +/- 0.2nM. (c) 2006 Elsevier Inc. All rights reserved.