Causey L D, Dwyer D S
Department of Psychiatry, Louisiana State University Medical Center, Shreveport, USA.
Nat Biotechnol. 1996 Mar;14(3):348-51. doi: 10.1038/nbt0396-348.
Protein-protein and protein-peptide interactions that are low affinity in nature preclude the straightforward measurement of binding. To overcome this limitation, a novel method has been devised for stabilizing these weak interactions by increasing the binding avidity. These studies have focused on the binding of peptides to heat shock proteins (with a typical KD of approximately 25 to 50 microM). Multivalent ligands have been created by coupling peptides plus biotin to a neutral carrier molecule, dextran. These peptide-dextran conjugates allow for more avid binding to proteins that have been immobilized on a membrane surface. Detection of signals via enhanced chemiluminescence further increases the sensitivity of the method that has been termed Chemiluminescence of Enhanced Avidity Reactions (CLEAR). The assay is simple, reliable and consistently detects specific binding between heat shock proteins and peptide ligands. CLEAR should be generally applicable to other ligand receptor pairs where the detection of binding is limited by the low affinity of the interaction.
本质上具有低亲和力的蛋白质-蛋白质和蛋白质-肽相互作用使得直接测量结合变得困难。为克服这一限制，人们设计了一种新方法，通过增加结合亲和力来稳定这些弱相互作用。这些研究聚焦于肽与热休克蛋白的结合（典型的解离常数KD约为25至50微摩尔）。通过将肽加生物素偶联到中性载体分子葡聚糖上，制备了多价配体。这些肽-葡聚糖结合物能够更紧密地结合固定在膜表面的蛋白质。通过增强化学发光检测信号进一步提高了该方法的灵敏度，该方法被称为增强亲和力反应化学发光法（CLEAR）。该检测方法简单、可靠，能持续检测热休克蛋白与肽配体之间的特异性结合。CLEAR一般应适用于其他配体-受体对，其中结合检测受相互作用低亲和力的限制。
