Nordstedt C, Fredholm B B
Department of Pharmacology, Karolinska Institutet, Stockholm, Sweden.
Anal Biochem. 1990 Sep;189(2):231-4. doi: 10.1016/0003-2697(90)90113-n.
A modification of a protein-binding method (B. L. Brown et al., 1971, Biochem. J. 121, 561, 562) for measurement of adenosine 3',5'-cyclic monophosphate (cAMP) in cell-culture supernatants and urine is described. With filtration over glass-fiber filters and a semiautomatic cell harvester instead of charcoal precipitation as in the original method, free [3H]cAMP tracer was rapidly and uniformly separated from that which was protein-bound. Sensitivity was increased with a high ionic strength buffer and a tritiated cAMP tracer with high specific activity. There was good agreement between cAMP values obtained with the protein-binding method and commercially available radioimmunoassays that were used as reference methods. cAMP could be determined accurately over the range 0.15-8.0 pmol/sample. More than 500 samples could be assayed in duplicate or triplicate in less than 6 h.
本文描述了一种用于测量细胞培养上清液和尿液中3',5'-环磷酸腺苷（cAMP）的蛋白质结合法（B. L. 布朗等人，1971年，《生物化学杂志》121卷，561页，562页）的改良方法。与原始方法中使用活性炭沉淀不同，该改良方法采用玻璃纤维滤膜过滤和半自动细胞收集器，可快速、均匀地将游离的[3H]cAMP示踪剂与蛋白质结合的示踪剂分离。通过使用高离子强度缓冲液和高比活性的氚标记cAMP示踪剂提高了灵敏度。用蛋白质结合法获得的cAMP值与用作参考方法的市售放射免疫测定法之间具有良好的一致性。cAMP在0.15 - 8.0 pmol/样品范围内可准确测定。在不到6小时的时间内，可以对500多个样品进行重复或三次重复测定。
