Larsson A, Axelsson B
Department of Immunology, University of Stockholm, Sweden.
J Immunol Methods. 1991 Mar 21;137(2):253-9. doi: 10.1016/0022-1759(91)90031-a.
Homologous displacement experiments are described in which the binding of labelled ligand (tracer) was inhibited with the same ligand unlabelled (inhibitor). Data were evaluated with a direct curve-fitting method. The equation used employs the concentration of unlabelled ligand as the independent variable and the fraction of tracer bound (p) as the dependent variable. The other entities of the equation are the dissociation constant (Kd), the maximal fraction of tracer bound (Po) and the tracer concentration (T). Different options exist regarding the insertion of Po and T as parameters or constants in the curve-fitting. When applied to experimental data obtained from the binding of dinitrophenyl-hapten by a monoclonal antibody, the least interexperimental variation in affinity was obtained when both Kd and Po were estimated as parameters, and T was inserted as a constant. This calculation procedure achieves more reproducible results than those obtained with the Scatchard plot and the Langmuir curve, even after exclusion of the most scattered data points.
本文描述了同源置换实验，其中标记配体（示踪剂）的结合被相同的未标记配体（抑制剂）抑制。数据采用直接曲线拟合方法进行评估。所使用的方程以未标记配体的浓度作为自变量，结合的示踪剂分数（p）作为因变量。方程的其他参数是解离常数（Kd）、示踪剂结合的最大分数（Po）和示踪剂浓度（T）。关于在曲线拟合中插入Po和T作为参数或常数存在不同的选择。当应用于从单克隆抗体与二硝基苯基半抗原结合获得的实验数据时，将Kd和Po都估计为参数并将T作为常数插入时，亲和力的实验间变化最小。即使排除了最分散的数据点，该计算程序也比用Scatchard图和Langmuir曲线获得的结果更具可重复性。
