Sidki A M, Landon J, Rowell F
Clin Chem. 1984 Aug;30(8):1348-52.
Raising antibodies to a hapten (drug or steroid) requires that it be coupled to a carrier protein through a bridge such that the hapten has maximum exposure with minimum changes in its configuration. In raising antisera in six sheep against carbamazepine (CBZ) coupled to bovine albumin, we found that some subpopulations of the antibodies recognized the bridge linking the drug to the carrier protein. To study the influence of the bridge on a fluoroimmunoassay for CBZ, we prepared four tracers by linking the carbamyl nitrogen of CBZ to fluorescein via four alkyl bridges of different lengths and structures. We calculated various binding parameters--including antibody affinity, binding capacity, and heterogeneity index--for each tracer and chose for the final fluoroimmunoassay the tracer that gave the best displacement with CBZ. We then optimized and validated the assay for direct measurement of CBZ in serum or plasma. The antibodies are coupled to magnetizable particles, which greatly facilitates separation and ensures removal of endogenous interferents.
产生针对半抗原（药物或类固醇）的抗体需要将其通过一个桥连接到载体蛋白上，以使半抗原在其构型变化最小的情况下得到最大程度的暴露。在对六只绵羊进行抗血清制备以对抗与牛血清白蛋白偶联的卡马西平（CBZ）时，我们发现某些抗体亚群识别连接药物与载体蛋白的桥。为了研究桥对CBZ荧光免疫测定的影响，我们通过将CBZ的氨甲酰氮经由四种不同长度和结构的烷基桥连接到荧光素上，制备了四种示踪剂。我们计算了每种示踪剂的各种结合参数，包括抗体亲和力、结合容量和异质性指数，并为最终的荧光免疫测定选择了与CBZ产生最佳置换效果的示踪剂。然后我们对该测定法进行了优化和验证，以直接测量血清或血浆中的CBZ。抗体与可磁化颗粒偶联，这极大地促进了分离并确保去除内源性干扰物。
