Hsieh H V, Poglitsch C L, Thompson N L
Department of Chemistry, University of North Carolina, Chapel Hill 27599-3290.
Biochemistry. 1992 Nov 24;31(46):11562-6. doi: 10.1021/bi00161a039.
Total internal reflection fluorescence microscopy (TIRFM) has been used to directly measure the weak dissociation constants of IgG with a mouse IgG receptor (moFc gamma RII) that has been purified and reconstituted into substrate-supported planar membranes. Dissociation constants were measured for three different mouse monoclonal anti-dinitrophenyl (DNP) IgG1 antibodies and for polyclonal mouse IgG, in the absence and presence of saturating amounts of hapten (DNP-glycine). The dissociation constant for polyclonal mouse IgG was 3 microM, which agrees well with previous results. The dissociation constants for the three monoclonal antibodies with moFc gamma RII ranged from 2 microM to 3 microM and were not statistically different, suggesting that changes in moFc gamma RII dissociation constants which may exist within the IgG1 subclass are less than the error of the TIRFM measurements (approximately 20%). The measured IgG1-moFc gamma RII dissociation constants were not different for individual monoclonal antibodies in the absence or presence of saturating concentrations of DNP-glycine, directly showing that possible allosteric changes which might occur upon hapten binding and affect the equilibrium characteristics of Fc receptor binding are small. This work demonstrates a new approach for quantitatively examining the effects of solution components on weak receptor-ligand interactions.
全内反射荧光显微镜（TIRFM）已被用于直接测量IgG与已纯化并重构到底物支持的平面膜中的小鼠IgG受体（moFcγRII）的弱解离常数。在不存在和存在饱和量半抗原（DNP-甘氨酸）的情况下，测量了三种不同的小鼠单克隆抗二硝基苯基（DNP）IgG1抗体和多克隆小鼠IgG的解离常数。多克隆小鼠IgG的解离常数为3 microM，与先前的结果非常吻合。三种单克隆抗体与moFcγRII的解离常数范围为2 microM至3 microM，且无统计学差异，这表明IgG1亚类中可能存在的moFcγRII解离常数变化小于TIRFM测量误差（约20%）。在不存在或存在饱和浓度的DNP-甘氨酸时，单个单克隆抗体的测量IgG1-moFcγRII解离常数没有差异，直接表明半抗原结合时可能发生的变构变化并影响Fc受体结合的平衡特性很小。这项工作展示了一种定量研究溶液成分对弱受体-配体相互作用影响的新方法。
