β-七叶皂苷钠血浆药物浓度ELISA测定方法的研究

Study on ELISA method for determination of sodiumβ-aescinate concentration in plasma

目的:建立测定β-七叶皂苷钠血浆浓度的酶联免疫吸附分析(ELISA,enzyme-linked immunosorbent assay)检测方法。方法:将半抗原β-七叶皂苷钠分别与载体蛋白牛血清白蛋白(BSA)或钥孔嘁血蓝蛋白(KLH)相连制备全抗原。将所得的免疫原免疫家兔,制备抗β-七叶皂苷钠的抗体,用免疫亲和柱纯化抗体,用辣根过氧化氢酶(POase)标记的抗原。在此基础上,建立测定β-七叶皂苷钠血浆药浓的竞争ELISA法。在波长为450nm处用酶标仪测定已标记的抗原抗体复合物的量。结果:血浆样品在0.002～0.128μg·mL^(-1)的浓度范围内相关性较好;该方法的回收率为(97.70±1.58)%;精密度试验的日内、日间RSD均小于10%。结论:该方法具有简便、快速、灵敏、准确等特点,测定条件下不受血浆中其他杂质的影响,适用于β-七叶皂苷钠血浆浓度的测定及其动物体内药代动力学研究。

Objective：To establish the ELISA（ enzyme -linked immunosorbent assay）method for detecting plasma concentration of sodium β-aescinate. Methods： Sodium β-aescinate coupled with bovine serum albumin（BSA）or with KLH to synthesize an artificial antigen. Then immunize the rabbits with the BSA - sodium β-aescinate or with the KLH - sodium β-aescinate. The antibodies were purified by the method of immunoaffinity chromatography. The antigen was labelled with the enzyme-horseradish peroxidase （POase）. Results： Under these conditions, the assay is quantitative between 0. 002 -0. 128 μg ·mL^-1 for sodium β-aescinate in plasma. The wavelength of determination was 450 nm. The recovery of this method was （ 97.70 ±1.58 ） % ; the within - day and between - day RSDs were all below 10%. Conclusion：The method is shown to highly specific, sensitive and rapid ,which can be used to determine the plasma concentration of sodium β-aescinate and its pharmacokinetics in animals.