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 com-bined without premature reaction. A conjugate of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and theophylline has been encapsulated in O.2-m-diameter bi-lamellar lipo-somes. Suspensions of these liposomes had excellent stabil-ity. 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>92 % of the initial enzyme activity after standing for one year at 4#{176}C.At higher NAD concentrationsthe liposomesaggregated, and enzyme activ-ity was inhibited by leakage of the NAD hydrolysis product, adenosine diphosphoryl 5-ribose (ADP-ribose), into the lipo-somes. Inhibition by ADP-nbose 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 anti-body provided assay results equivalent to those obtained by conventional two-reagent EMIT#{174}homogeneous enzyme im-munoassay for theophylline. AdditIonal Keyphrases: theophylline. glucose-6-phosphafede