A competitive indirect fluoroimmmoassay of free estradiol (4 ) was established b a d on the thennal sensitivity of hydrogel-pdy-N-isopropyIacrylamide. Free estradid was covaplete antigen (&-=A) , which was in turn labeled by fluorescein isothiocyanate ( m c ) as the fluorescence probe. The anvo method, and coupled with N-isopmpyiacrylamide ("A) to make an immune copolymer, pdy-N-isopmpylacyhnidemonodonal antibody ( pWA-McAb) , for the determination offree &. The i mm-y method was based on the competitive b i of free & and fluoresceinateti antigen (&-BSA-FITC) with limited amount of pNIPA-McAb. When the immundogical reaction was over, precipitation and centrifqal procedures were carried out to sepatate pNJPA-MCAtt&-BSA-FlTC from other constituents in solution. The precipitate pNIPA-MCAb&-BSA-F'ITC was dissolved in sdution and then the fluorescence intensity was mesnued. The calibration cvrvecoveredarangeof78--500ng/mLforfree&. Therecoveries were 91.2-107.2% . lently bound to bovine ~e ~m albumin ( BSA) to form cornti-& m~noclonal a n t i y (McAb) w a ~ p~~pared by in vi-tiality in fm & detection for a high sensitivity of fluorescence, especially when it is coupled with temperature responsible hydrogel-poly-N-isopropylacrylamide ( pN- IPA) .
pNPA is a functional polymer exhibiting unusual thermally reversible soluble-insoluble behavior at its lowest critical solution temperature (LCST: near 32%) in aqueous solution. * pNIPA is water soluble below the LC-ST and becomes insoluble above the LCST. This ther- mal-sensitive polymer and its copolymer with other monomers have become the basis of bioreactors , bioseparations ,4 controlled drug releases,' enzyme immobilizations.6 Proteins have been conjugated to pNPA for potential use in diagnostic testing since the precipitation immunoassay was first presented.' This timesaving im- munoassay combines the advantages of homogeneous immunoassay and heterogeneous immunoassay. Its sensitivity is equivalent to other nonisotopic heterogeneous immunoassay.