Ullman E F, Schwarzberg M, Rubenstein K E
J Biol Chem. 1976 Jul 25;251(14):4172-8.
A general immunochemical method for the assay of haptens and proteins has been devised and applied to morphine, a morphine-albumin conjugate, and human immunoglobulin G. A fluorescein-labeled antigen and a quencher-labeled antibody are employed. By use of fluorescein and rhodamine as the fluorescer and quencher, respectively, dipole-dipole-coupled excitation energy transfer can occur within the antigen-antibody complex. The resulting quenching of fluorescence can be inhibited by competitive binding with unlabeled antigen, Alternatively, separate antibody samples can be labeled with fluorescein and rhodamine, respectively. Unlabeled antigen causes aggregation of the separately labeled components with resultant quenching. Using the latter method, experiments suggest that up to about 20 anti-morphine antibody binding sites will associate with morphine-albumin conjugates. When an excess of the conjugate is present the antibodies appear to assemble in clumps on the protein surface. Mathematical analysis of the quenching of fluorescein-labeled morphine by rhodamine-labeled anti-morphine gives an approximate fit to the quenching data, but the calculations are very dependent on the assumptions used.
已设计出一种用于测定半抗原和蛋白质的通用免疫化学方法，并将其应用于吗啡、吗啡 - 白蛋白偶联物和人免疫球蛋白G。使用了一种荧光素标记的抗原和一种猝灭剂标记的抗体。分别使用荧光素和罗丹明作为荧光剂和猝灭剂，抗原 - 抗体复合物内可发生偶极 - 偶极耦合激发能量转移。与未标记抗原的竞争性结合可抑制由此产生的荧光猝灭。另外，可分别用荧光素和罗丹明标记不同的抗体样品。未标记抗原会导致分别标记的组分聚集并产生猝灭。使用后一种方法，实验表明多达约20个抗吗啡抗体结合位点将与吗啡 - 白蛋白偶联物结合。当存在过量的偶联物时，抗体似乎会在蛋白质表面聚集成团。用罗丹明标记的抗吗啡对荧光素标记的吗啡的猝灭进行数学分析，与猝灭数据大致相符，但计算结果非常依赖于所使用的假设。
