O'Shannessy D J, Brigham-Burke M, Soneson K K, Hensley P, Brooks I
Department of Protein Biochemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406.
Anal Biochem. 1993 Aug 1;212(2):457-68. doi: 10.1006/abio.1993.1355.
Surface plasmon resonance (SPR) is a label-free, real time, optical detection method which has recently been commercialized as the BIAcore (Pharmacia). The technique relies on the immobilization of one of the interactants, the ligand, onto a dextran-coated gold surface. The second interactant, the ligate, is then injected across the surface and the interaction of the soluble ligate with the immobilized ligand is observed continuously and directly. The process of dissociation of bound ligate may also be observed directly after the sample plug has traversed the layer. Thus, the data generated contain information on the kinetic rate and equilibrium binding constants for the interaction under investigation. Historically, data from this instrument have been analyzed in terms of linear transformations of the primary data and requires that data from several ligate concentrations be analyzed to determine a single value for the association and dissociation rate constants. Here we discuss the analysis of untransformed BIAcore data by nonlinear least squares methods. The primary data are analyzed according to the integrated rate equations which describe the kinetics of the interaction of soluble ligate with immobilized ligand and the dissociation of the formed complex from the surface, respectively. Such analyses allow the direct determination of the association and dissociation rate constants for each binding experiment and, further, allow the analysis of data over a wider concentration range with lower associated errors compared to previously described methods. Through the use of modeling these interactions, we also demonstrate the limitations in determining the dissociation rate constant from the association phase of the interaction, thereby requiring that the dissociation process be analyzed. Indeed, the dissociation phase should be analyzed first to yield a relatively precise and unambiguous value of the dissociation rate constant, kd, which can then be used to constrain the analysis of the association phase to yield a better estimate of the association rate constant, k(a). We further demonstrate that, at least for the interaction investigated, the apparent rate and equilibrium binding constants determined using SPR are concentration independent and can be determined with good reproducibility.
表面等离子体共振（SPR）是一种无标记的实时光学检测方法，最近已作为BIAcore（Pharmacia）实现商业化。该技术依赖于将其中一种相互作用分子，即配体，固定在葡聚糖包被的金表面上。然后将第二种相互作用分子，即被连接物，注入穿过该表面，并连续且直接地观察可溶性被连接物与固定化配体之间的相互作用。在样品塞穿过该层之后，也可以直接观察结合的被连接物的解离过程。因此，所产生的数据包含有关所研究相互作用的动力学速率和平衡结合常数的信息。从历史上看，该仪器的数据一直是根据原始数据的线性变换进行分析的，并且需要分析来自几种被连接物浓度的数据，以确定缔合和解离速率常数的单个值。在这里，我们讨论通过非线性最小二乘法对未变换的BIAcore数据进行分析。根据积分速率方程对原始数据进行分析，这些方程分别描述了可溶性被连接物与固定化配体相互作用的动力学以及形成的复合物从表面解离的过程。这样的分析允许直接确定每个结合实验的缔合和解离速率常数，并且进一步允许在比先前描述的方法更宽的浓度范围内分析数据，且具有更低的相关误差。通过对这些相互作用进行建模，我们还证明了从相互作用的缔合阶段确定解离速率常数的局限性，因此需要对解离过程进行分析。实际上，应该首先分析解离阶段，以得出解离速率常数kd的相对精确和明确的值，然后可以使用该值来约束缔合阶段的分析，以更好地估计缔合速率常数k(a)。我们进一步证明，至少对于所研究的相互作用，使用SPR确定的表观速率和平衡结合常数与浓度无关，并且可以以良好的重现性进行确定。
