Fowler C J, Martinsson T, Brännström G
Department of Biochemical Pharmacology, Astra Pain Control AB, Södertälje, Sweden.
Methods Find Exp Clin Pharmacol. 1993 Jul-Aug;15(6):337-43.
Following incubation at 4 degrees C, [3H]-[Sar9,Met(O2)11]-substance P bound to tissue culture plates in a manner displaceable by substance P (SP). The IC50 for SP was about 100 nM. Although the total binding to the wells could be reduced by increasing the assay bovine serum albumin concentration, by the presence of divalent ions and by gelatin-precoating of the wells, the remaining binding could be inhibited by about 70% by SP, and would thus appear as "specific" binding in assays. The non-peptide antagonist (+/-)CP-96345 also inhibited the binding to the wells at high concentrations (IC50 about 2 mcM), whereas physalaemin did not. Physalaemin is thus a better agent to define non-specific binding. The consequences of the "specific" binding to the culture wells observed with SP to define non-specific binding are demonstrated in two cell systems: in cultured human umbilical vein endothelial cells, an apparent receptor density of about 100,000/cell is shown to be due mainly to binding to the wells, rather than the cells. In UC11MG cells, which express 40,000-60,000 NK-1 recognition sites/cell, the use of SP to define binding gives artifactually high KD and Bmax values, and an artifactually low potency (and Hill slope) of (+/-)CP-96345 when compared with data obtained using physalaemin to define the non-specific binding.
在4℃孵育后，[3H]-[Sar9,Met(O2)11]-P物质以可被P物质（SP）置换的方式结合到组织培养板上。SP的半数抑制浓度（IC50）约为100 nM。尽管通过增加测定体系中的牛血清白蛋白浓度、存在二价离子以及对培养板进行明胶预包被可减少与孔的总结合，但剩余的结合仍可被SP抑制约70%，因此在测定中会表现为“特异性”结合。非肽拮抗剂(+/-)CP - 96345在高浓度时（IC50约为2 μM）也抑制与孔的结合，而雨蛙肽则无此作用。因此，雨蛙肽是界定非特异性结合的更好试剂。用SP观察到的与培养孔的“特异性”结合对界定非特异性结合的影响在两种细胞系统中得到了证实：在培养的人脐静脉内皮细胞中，约100,000个/细胞的表观受体密度主要是由于与孔而非细胞的结合所致。在表达40,000 - 60,000个NK - 1识别位点/细胞的UC11MG细胞中，与使用雨蛙肽界定非特异性结合所获得的数据相比，用SP界定结合会人为地给出高解离常数（KD）和最大结合容量（Bmax）值，以及(+/-)CP - 96345人为地给出低效价（和希尔斜率）。
