基于温度敏感水凝胶的雌二醇荧光免疫分析

雌二醇完全抗原(Ag)与异丙基丙烯酰胺(NIPA)共聚,可得水凝胶－抗原结合物(pNIPA－Ag),pNI－PA-Ag与游离的雌二醇(E₂)竞争有限的荧光标记的单抗McAb-F,利用水凝胶温度相变的性质,分离高分子免疫复合物,并测定其荧光信号.结果表明,游离E₂在10～625ng/mL范围内呈线性关系,样品的回收率为88.9%～102.0%.

Fluoroimmunoassay of Estradiol Based on the Thermo-sensitive Hydrogel

Abstract An indirect competitive fluoroimmunoassay of estradiol was developed based on the thermo-sen-sitive hydrogel, poly-JV-isopropylacrylamide(pNIPA). Antigen was conjugated to pNIPA to form antigen bound thermally reversible hydrogel (pNIPA-Ag) after the antigen was synthesized by covalent bonding estradiol to bovine serum albumin. The anti-estradiol monoclonal antibody (McAb) was prepared by an in vivo method with McAb secreting hybridomas. Fluorescein isothiocyanate(FITC) labeled McAb(McAb-F) was prepared by bonding FITC to purified McAb. Then free estradiol reacted with limited amount of McAb-F competing with pNIPA-Ag. Separation of the immune complex, pNIPA-Ag-McAb-F, was carried out by centrifuging and precipitating at a temperature above critical solution temperature of the hydro-gel. After the above immune complex was dissolved, the fluorescence intensity was measured by a Hitachi M-850 fluorospectrometer. The calibration curve of free estradiol covered a range of 10-625 ng/mL, and the detection limit of the presented method for free estradiol was 3. 2 ng/mL. The cross-reactions and the recoveries of the immunoassay were also investigated.