title: Use of liposome encapsulation in a combined single-liquid reagent for homogeneous enzyme immunoassay.
abstract: 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.
authors.forename: E F
authors.initials: EF
authors.lastname: Ullman
authors.forename: T
authors.initials: T
authors.lastname: Tarnowski
authors.forename: P
authors.initials: P
authors.lastname: Felgner
authors.forename: I
authors.initials: I
authors.lastname: Gibbons
mesh_terms: D000246:Adenosine Diphosphate Ribose; D000906:Antibodies; D000937:Antigen-Antibody Reactions; D005954:Glucosephosphate Dehydrogenase; D006801:Humans; D007124:Immunoenzyme Techniques; D007202:Indicators and Reagents; D008081:Liposomes; D009243:NAD; D011786:Quality Control; D013806:Theophylline / Q000032:analysis* / Q000276:immunology
chemical_list: D000906:Antibodies; D007202:Indicators and Reagents; D008081:Liposomes; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D013806:Theophylline; D005954:Glucosephosphate Dehydrogenase