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.
Ullman
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EF
Tarnowski
T
T
Felgner
P
P
Gibbons
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I
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
D000906:Antibodies; D007202:Indicators and Reagents; D008081:Liposomes; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D013806:Theophylline; D005954:Glucosephosphate Dehydrogenase