A novel minetic enzymatic fluorescence immunoassay for hepatitis B surface antigen by using a thermal phase separating polymer

摘要

Iron tetrasulfonatophthalocyanine (FeTSPc),a peroxidast mimic,was used as a labeling ragent and poly(N-isopropylacrylamide)(PNIP)as the separation support of the immune complex for the mimetic-enzymatic immunoaccay of hepatitis B surface antigen (HBsAg).PNIP was precipitated form aqueous solution when the ambient temperature was higher than its lowere critical solution temperture of 31deg C.In a sanwich immunosassay,the antigen (HBsAg)first reacted with mouse anti-human HBsAg antibody immobilized on PNIP (PNIP-antibody)and then further reacted with FeTSPc-labeled mouse anti-HBsAg antibody(antibody-FeTSPc) at room temperature in a homogeneous pormat. After changing the temerature to separate the PNIP-antibody-HBsAg-antibody-FeTSPc conjugate moiety, it was re-dissolved and determined by coupling with the fluorogenic reaction of hydrogen peroxide and p-hydroxypenylpropionic acid. The sensitivity of this method (3 ng mL~(-1))was close to that of the traditional ELISA using the same reactants. However, the assay was much faster (the assay time decreased form 100-120 to 45 min). This method was applied to determine HBsAg in human serum with satisfactory results.