Use of liposome encapsulation in a combined single-liquid reagent for homogeneous enzyme immunoassay.

A technique has been developed to permit mutually reactive macromolecular reagents used in immunoassays to be combined without premature reaction. A conjugate of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and theophylline has been encapsulated in 0.2-micron-diameter bi-lamellar liposomes. Suspensions of these liposomes had excellent stability. Whereas the enzyme activity of the free conjugate is rapidly inhibited by anti-theophylline antibody, a suspension of the encapsulated conjugate in a solution of the antibody and NAD+ (6.0 mmol/L) retained greater than 92% of the initial enzyme activity after standing for one year at 4 degrees C. At higher NAD+ concentrations the liposomes aggregated, and enzyme activity was inhibited by leakage of the NAD+ hydrolysis product, adenosine diphosphoryl 5-ribose (ADP-ribose), into the liposomes. Inhibition by ADP-ribose could be blocked and partly reversed by adding semicarbazide. The liposomes were efficiently lysed by Triton X-100, deoxycholate, or octyl glucoside, the kinetics and extent of lysis being affected by liposome size and correlating with the acid strength of various cholate derivatives. Addition of a serum sample and a solution of buffer, substrate, and detergent to a single reagent containing the liposomes and anti-theophylline antibody provided assay results equivalent to those obtained by conventional two-reagent EMIT homogeneous enzyme immunoassay for theophylline.