A homogeneous, lipsome-based signal amplification method for assays involving enzymes

摘要

The present invention is directed to a procedure for enhancing the enzyme-dependent readout of diagnostic assays using specially prepared liposomes.;Microgenic's CEDIA assay for digoxin was employed as an illustrative example of the present invention. The magnitude of signal was only 25 mA/min/ng digoxin/ml for the unamplified assay, while the signal magnitude for the amplified (i.e., liposome-enhanced) assay was 1,000 mA/min/ng digoxin/mL, i.e., a 40-fold amplification.;Stable liposomes were prepared with unsaturated PE stabilized with 5 mole percent of ganglioside GM₁. Glucose-6-phosphate dehydrogenase (G6PHD) was entrapped in these unilamellar liposomes in high concentrations. Addition of beta-galactosidase (B-gal) caused rapid (3-5 min.) lysis of liposomes revealing the latent G6PDH activity, due to the enzymatic degalactosylation of MG₁.;This simple, rapid and homogenous signal amplification procedure will be useful in many enzyme dependent assays, such as ELISA, CEDIA, Gene-probe assays and Immunoliposome assays.