Xie Lei, Mark Jones R, Glass Thomas R, Navoa Ryman, Wang Yan, Grace Michael J
BiotechDev./Bioanalytical, Schering-Plough Research Institute, 1011 Morris Ave., U-13/2050, Union, NJ 07083, United States.
J Immunol Methods. 2005 Sep;304(1-2):1-14. doi: 10.1016/j.jim.2005.04.009.
Measuring a protein-ligand interaction in solution, away from the ligand's cellular environment, may not provide an affinity value applicable in vivo. Here, we present a simple, accurate and highly sensitive method for determining the antibody affinity to cell surface receptor, hIGFR, and compare this data to affinity determined for the soluble receptor. Measurements were performed on both full-length bivalent IgG and the monovalent Fab fragments to assess possible differences in apparent affinity introduced by avidity of the bivalent IgG. Affinities determined for soluble hIGFR were 4 x 10(-12) M for the bivalent IgG and monovalent Fab. Comparable affinities of 6 x 10(-12) M and 1 x 10(-11) M for the bivalent IgG and Fab, respectively, were also determined for full-length hIGFR on cell surface. The method described allows estimation of reactant concentrations (anti-IGFR antibody) relative to one known reference concentration (the concentration of soluble hIGFR in our case) allowing us to estimate the average receptor density on the cell surface. Taken together, we believe these data can provide valuable insight into antibody behavior in vivo, especially in the case of insoluble or difficult to purify transmembrane receptors.
在溶液中测量蛋白质 - 配体相互作用，而不考虑配体的细胞环境，可能无法提供适用于体内的亲和力值。在此，我们提出了一种简单、准确且高度灵敏的方法来测定抗体与细胞表面受体hIGFR的亲和力，并将此数据与针对可溶性受体测定的亲和力进行比较。对全长二价IgG和单价Fab片段都进行了测量，以评估二价IgG的亲合力所引入的表观亲和力的可能差异。针对可溶性hIGFR测定的二价IgG和单价Fab的亲和力均为4×10⁻¹² M。对于细胞表面的全长hIGFR，二价IgG和Fab的亲和力分别为6×10⁻¹² M和1×10⁻¹¹ M，二者具有可比性。所描述的方法允许相对于一个已知参考浓度（在我们的例子中为可溶性hIGFR的浓度）估算反应物浓度（抗IGFR抗体），从而使我们能够估算细胞表面的平均受体密度。综上所述，我们认为这些数据可以为体内抗体行为提供有价值的见解，特别是在不溶性或难以纯化的跨膜受体的情况下。
