An improved chemiluminescence-based liposome immunoassay involving apoenzyme

An improved liposome immunoassay system (LIS) combining the chemiluminescence-based LIS with an apoenzyme reactivation immunoassay system (ARIS) was developed. A low-molecular-weight co-factor, FAD (flavin adenine dinucleotide), was incorporated into liposomes instead of the high-molecular-weight enzyme GOD (glucose oxidase). FAD released from liposomes by cytolysin-hapten conjugates bound to Apo-GOD and regenerated GOD. The system allowed detection of 10 pM digoxin, the model analyte and was linear over 10 pM to 13 nM digoxin. This sensitivity was about 300 times higher than that of the homogeneous system using GOD-containing liposomes and 30 times higher than that of the heterogeneous system which we reported previously. The time required for incubation during the detection of digoxin was reduced from 20 to 3 h.