Katsamba Phinikoula S, Navratilova Iva, Calderon-Cacia Maria, Fan Linsey, Thornton Kevin, Zhu Mingde, Bos Tim Vanden, Forte Carla, Friend Della, Laird-Offringa Ite, Tavares Gisele, Whatley John, Shi Ergang, Widom Angela, Lindquist Kevin C, Klakamp Scott, Drake Andrew, Bohmann David, Roell Marina, Rose Larry, Dorocke Jill, Roth Bruce, Luginbühl Béatrice, Myszka David G
Center for Biomolecular Interaction Analysis, School of Medicine, University of Utah, Salt Lake City, 84132, USA.
Anal Biochem. 2006 May 15;352(2):208-21. doi: 10.1016/j.ab.2006.01.034. Epub 2006 Feb 23.
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 1h. 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 approximately 14%. Based on the kinetic rate constants, the affinity (K(D)) of the PSA/mAb interaction was 1.1+/-0.2 nM.
为探究基于Biacore的检测方法的可靠性，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）的结合情况。为每位参与者提供了相同的试剂和详细的实验方案。将该单克隆抗体以三种不同密度固定在传感器芯片上，并采用两步分析法来确定PSA/mAb复合物的动力学和亲和力参数。首先，在2.5 - 600 nM的浓度范围内检测PSA以获取k(a)信息。其次，为准确确定这种稳定抗原/抗体复合物的k(d)，对最高PSA浓度进行重新检测，同时监测每个结合循环的解离阶段1小时。所有参与者收集的数据均可用于分析以获得相互作用的动力学参数。使用简单的1:1相互作用模型对来自三个抗体表面的结合和解离延长阶段的数据进行整体拟合。根据这22次分析计算得出的PSA/mAb相互作用的平均k(a)和k(d)分别为(4.1±0.6)×10⁴ M⁻¹ s⁻¹和(4.5±0.6)×10⁻⁵ s⁻¹。总体而言，速率常数的实验标准误差仅约为14%。根据动力学速率常数，PSA/mAb相互作用的亲和力（K(D)）为1.1±0.2 nM。
