Petrossian A
XOMA Corporation, Santa Monica, CA 90404.
Cell Biophys. 1993 Aug-Dec;23(1-3):111-37. doi: 10.1007/BF02796509.
We have obtained equilibrium and rate constants for the interaction of monoclonal IgG and its monovalent Fab fragment with a hapten (fluorescein) attached to the surface of a liposome. Binding was detected at nanomolar hapten concentrations by the quenching of the hapten's fluorescence on antibody binding. The binding parameters were computed from nonlinear least squares fits, using mass-action models. Crypticity of the hapten was observed and interpreted as an equilibrium between two states, extended and sequestered, the latter representing haptens associated with the membrane surface. Depending on the lipid composition of the liposomes, the fraction of sequestered hapten ranged from 0.25 to 0.975; transitions between the two states took place on the time scale of minutes. Fab interactions with extended hapten on the membrane were similar to interactions with water-soluble hapten. The ability of IgG to bind bivalently to membrane gave it an avidity two to six times the affinity for purely monovalent binding. However, the equilibrium constant for the monovalent-bivalent binding equilibrium was effectively four to five orders of magnitude less than that for the initial binding step. This probably reflects steric penalties for the simultaneous binding of two haptens on a membrane.
我们已获得单克隆IgG及其单价Fab片段与附着在脂质体表面的半抗原（荧光素）相互作用的平衡常数和速率常数。通过抗体结合时半抗原荧光的淬灭，在纳摩尔浓度的半抗原下检测到结合。使用质量作用模型，通过非线性最小二乘法拟合计算结合参数。观察到半抗原的隐蔽性，并将其解释为两种状态（伸展态和隔离态）之间的平衡，后者代表与膜表面相关的半抗原。根据脂质体的脂质组成，隔离态半抗原的比例范围为0.25至0.975；两种状态之间的转变发生在分钟时间尺度上。Fab与膜上伸展态半抗原的相互作用类似于与水溶性半抗原的相互作用。IgG二价结合膜的能力使其亲和力比纯单价结合的亲和力高两到六倍。然而，单价-二价结合平衡的平衡常数实际上比初始结合步骤的平衡常数小四到五个数量级。这可能反映了两个半抗原在膜上同时结合的空间位阻。
