A homogeneous, liposome-based signal amplification for assays involving enzymes.
Exploiting the fact that phosphatidylethanolamine (PE) liposome can be stabilized by a membrane lipid or protein, and that destruction of this stabilizer leads to rapid lysis of the liposome, we have designed a liposome-based signal enhancement mechanism for assays that involve enzyme as the final read-out step. Stable liposomes with entrapped glucose-6-phosphate dehydrogenase (G6PDH) were prepared with unsaturated PE stabilized with 5 mol percent of ganglioside GM1. Addition of beta-galactosidase caused rapid (3-5 min) lysis of liposomes, revealing the latent G6PDH activity, owing to the enzymatic degalactosylation of GM1. We have used Microgenic's CEDIA assay for digoxin as an example. The magnitude of signal was 25 mA/min per microgram of digoxin per liter for the unamplified assay and 1000 mA/min per microgram of digoxin per liter for the liposome-enhanced assay--i.e., a 40-fold amplification. This simple, rapid, and homogeneous signal-amplification mechanism is likely to be useful in many enzyme-dependent assays, such as ELISA, CEDIA, gene-probe assays, and immunoliposome assays.
D001769:Blood; D004077:Digoxin / Q000097:blood*; D005677:G(M1) Ganglioside / Q000378:metabolism; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism*; D006801:Humans; D007118:Immunoassay; D008081:Liposomes; D010714:Phosphatidylethanolamines; D001616:beta-Galactosidase / Q000378:metabolism
D008081:Liposomes; D010714:Phosphatidylethanolamines; D005677:G(M1) Ganglioside; D004077:Digoxin; D005954:Glucosephosphate Dehydrogenase; D001616:beta-Galactosidase