A novel mimetic enzymatic fluorescence immunoassay for hepatitis B surface antigen by using a thermal phase separating polymer.
Iron tetrasulfonatophthalocyanine (FeTSPc), a peroxidase mimic, was used as a labeling reagent and poly(N-isopropylacrylamide) (PNIP) as the separation support of the immune complex for the mimetic-enzymatic immunoassay of hepatitis B surface antigen (HBsAg). PNIP was precipitated from aqueous solution when the ambient temperature was higher than its lower critical solution temperature of 31 degrees C. In a sandwich immunoassay, 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 format. After changing the temperature 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-hydroxyphenylpropionic 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 from 100-120 to 45 min). This method was applied to determine HBsAg in human serum with satisfactory results.
Zhu
Q Z
QZ
Key Laboratory of Analytical Sciences of MOE and Department of Chemistry, Xiamen University, Xiamen 361005, China.
Yang
H H
HH
Li
D H
DH
Chen
Q Y
QY
Xu
J G
JG
D006514:Hepatitis B Surface Antigens / Q000097:blood*; D006801:Humans; D007124:Immunoenzyme Techniques; D013050:Spectrometry, Fluorescence
D006514:Hepatitis B Surface Antigens