甲基睾酮的控温相分离荧光免疫分析

Phase Separation Fluoroimmunoassay of Methyltestosterone

本文报道一种新的甲基睾酮 ( MT)测定的免疫分析法。通过将 MT与牛血清白蛋白 ( BSA)偶联制得了 MT全抗原 MT-3 -BSA( Ag MT)。将 Ag MT经兔子免疫获得了针对MT的多克隆抗体 ( Ab MT)。与温度敏感性水凝胶偶联的抗原 ( PNIP-Ag M T)和游离半抗原 MT竞争性地与有限量的异硫氰基荧光素标记抗体 ( Ab MT-FITC)反应。根据临界析相温度以上免疫复合物沉淀的特性进行分离 ,降低背景干扰。该法对 MT测定的线性范围在 2 7.5ng/m L～ 1 .1μg/m L ,检出限为 2 7.5ng/m L。MT在人血清中的回收率比较满意。

A novel immunoassay of methyltestosterone (MT) in serum was developed. The antigen of MT (Ag MT ) was synthesized by covalently bonding MT to BSA. The antibody of Ag MT (Ab MT ) was raised from rabbit. Ag MT was conjugated to poly N isopropylacrylamide (PNIP) to form Ag MT bound thermally reversible hydrogel PNIP Ag MT . A competitive immunoassay method based on the competition of PNIP Ag MT and free MT with limited amount of fluorescein isothiocyanate (FITC) labeled Ab MT was established. The separation PNIP in the assay process was achieved by precipitation of the immunocomplex above its critical solution temperature. The amount of MT was determined by measuring the fluorescence intensity of Ab MT FITC. The linear range for MT is 27.5 ng/mL～1.1 μg/mL and the detection limit is 27.5 ng/mL. The recoveries of MT from human serum are satisfactory.