Abstract: An indirect competitive fluoroimmunoassay of estradiol was developed based on the thermo-sen-sitive hydrogel, poly-JV-isopropylacrylamide(pNIPA).Antigen was conjugated to pNIPAto 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 FITCto purified McAb.Then free estradiol reacted with limited amount of McAb-Fcompeting 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.

Key words: Estradiol, Poly-N-isopropylacrylamide, Fluoroimmunoassay