Use of Liposome Encapsulation in a Combined Single ... - CiteSeerX

CLIN. CHEM. 33/9,

1579-1584

(1987)

Use of Liposome Encapsulation in a Combined Single-Liquid Reagent for Homogeneous Enzyme Immunoassay Edwin F. UIIman,Thomas Tarnowekl, PhilIp Feigner, and Ian Gibbons1 A technique has been developed to permit mutually reactive

macromolecular reagents used in immunoassays to be combined without premature reaction. A conjugate of glucose-6phosphate 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 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 >92% of the initial enzyme activity after standing for one year at 4#{176}C. At higher NAD

concentrationsthe liposomesaggregated, and enzyme activity was inhibited by leakage of the NAD hydrolysis product, adenosine diphosphoryl 5-ribose (ADP-ribose), into the liposomes. 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 antibody provided assay results equivalent to those obtained by

conventional two-reagent EMIT#{174} homogeneous enzyme immunoassay for theophylline. AdditIonal Keyphrases: theophylline hydrogenase

EMIT

compared

.

. glucose-6-phosphafedeADP-ribose

Homogeneous immunoassays for drugs usually require two reagents: an antibody and a conjugate of the drug with a label such as an enzyme (1) or fluorophore (2). Normally, these reagents are not combined before the assay because the immune complex formed between the antibody and the labeled drug equilibrates only slowly with the free drug in the sample. Nevertheless, minimizing the required number of reagents by combining these reagents would offer greater simplicity and increase throughput and assay precision. One approach to combining incompatible reagents is to

blend the components in dry form. No reaction occurs until sample and diluent are added. This method is used commercially in EMIT-qst, a homogeneous enzyme immunoassay for monitoring concentrations of therapeutic drugs (3)#{149}2 However, the convenience of this method is in part offset by the need to shake the reagents vigorously after addition of the sample to ensure complete dissolution of the solids. Here we describe a homogeneous enzyme inununoassay in which the reactive components are combined in a single Syva Co., 900 Arastradero Road, P.O. Box 10058 Palo Alto, CA 94303. ‘Present address: Biotrack, 430 Oakmead Parkway, Sunnyvale, CA 94086. 2E and EMIT-qst are registered trademarks of Syva Co. ReceivedApril 9, 1987; accepted June 4, 1987.

liquid reagent but are prevented from reacting because one of the regions is encapsulated in liposomes. The method is, in principle, applicable to any immunoassay involving mutually reactive antigen and antibody reagents. Materials and Methods Materials Chemicals. We used tert-butanol (Schwartz/Mann, Spring Valley, NY 10977); sodium azide (Eastman Kodak, Rochester, NY 14650); G6P, monosodium salt (Calzyme, San Luis Obispo, CA 93403); NAD (US Biochemicals, Cleveland, OH 44122); G6PDH (EC 1.1.1.49, from Leuconostoc mesenteroides; US Biochemicals, or Cooper Biomedical, Malvern, PA 19355); BSA (Miles, Napierville, IL 60566); RSA (PelFreeze, Rodgers, AR 72756); and synthetic POPC and POPG (Avanti Polar Lipids, Birmingham, AL 35216). All other chemicals were from Sigma Chemical Co., St. Louis, MO 63178. Cholesterol was recrystallized three times from ethanol. The buffer contained 55 mmol of Tris and 5 mmol of NaN3 per liter, pH 8.0, unless otherwise stated. G6PDH-drug conjugates. G6PDH was covalently coupled to theophylline as described by Singh et al. (4). Antibodies. .-Globulin fractions of sheep and rabbit antibodies were provided by Syva Co. The theophylline immunogen was prepared as previously described (4). Murine monoclonal antibody to theophylline was prepared by Celitech, Slough, SL1 4DY, U.K., from a cell line (6C2) produced by Syva Co., and was purified by using the Affi-gel#{174} Protein A system of Bio-Rad, Richmond, CA 94804. Procedures Protein concentrations. The concentrations of G6PDH stock solutions were determined spectrophotometrically, with #{128} = 11.5 at 280 nm (5). Concentrations of enzyme conjugates were based on the specific activity of the conjugates, which was estimated from the loss of activity of the native enzyme during conjugation. IgG concentrations were measured spectrophotometrically, with #{128}f 14 at 280 nm (6). Other protein concentrations were estimated from spectrophotometrically or gravimetrically known amounts, assuming no mechanical losses. Liposome composition. The weight percentages of lipids in the liposomes were calculated by assuming no mechanical loss or fractionation during liposome formation. G6PDH assay. Enzyme (-1 pmol, -110 ng) was added to 1 mL of pH 8 Tris buffer containing 6.6 imol of G6P, 4 tmol of NAD, and 1 mg of BSA. The absorbance at 340 nm was measured 10 and 40s after aspiration into a Gilford Stasar

Nonstandard abbreviations: ADP-ribose, adenosine diphosphoryl 5-ribose; G6P, glucose 6-phosphate;G6PDH, glucose.6phosphatedehydrogenase; POPC, i.-a-palmitoyloleoylphosphatidylcholine;POPG, z-a-palmitoyloleoylphosphatidylglycerol; BSA, bovine serum albumin; RSA, rabbit serum albumin. CLINICALCHEMISTRY, Vol. 33, No. 9, 1987

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III spectrophotometer flow cell adjusted to 30#{176}C. The assay response (M) varied linearly with respect to time and enzyme concentration up to an absorbance of 1.0. Liposome preparation. Glassware was sequentially rinsed with water and methanol and then dried. A chloroform solution of the lipids (POPG, POPC, and cholesterol) was evaporated without heating in a glass vial under a gentle stream of N2, the last traces of solvent being removed under reduced pressure. The lipids were dissolved (final concentration, 30 gIL) in tert-butanol at 60 #{176}C with gentle vortexmixing. This solution was lyophilized to give a bulky powder that was kept dry and cold until used. A solution of the antibody, G6PDH, or 6GPDH-theophylline conjugate to be encapsulated was dialyzed against pH 7-8 Tris buffer, and the concentration was adjusted to 5-16 g/L. Large liposomes were prepared by vortex-mixing 0.1-1.5 mL of the protein solution for 1 mm with the lipid mixture (about 10% by weight). This solution was further diluted threefold with buffer and again vortex-mixed to provide a homogeneous, thick, milky suspension with no unsuspended lumps. After an additional sevenfold dilution with buffer, the liposomes were separated by centrifligation (20 mm, 15 000 rpm, 4#{176}C) and washed twice by resuspending with vigorous shaking followed by centrifugation. Suspensions of liposomes to be freeze-thawed (0.5-1.0 mL) were immersed in liquid nitrogen until frozen and then thawed at room temperature. The liposomes were then separated by centrifugation and washed. Small liposomes were prepared from a lyophilized lipid mixture consisting of 6mg of POPG, 114 mg of POPC, and 29.9 mg of cholesterol (4/76/20 by wt). This mixture was combined with 1 mL of a solution of the protein to be encapsulated, in pH 7 Tris buffer. After vortex-mixing these materials for 1 mm, we extruded (The Extruder’TM;Lipex Biomembranes Inc., Vancouver, B.C., Canada) the resulting milky suspension through a 0.2-.Lm (pore size) filter (Nucleopore, Pleasanton, CA 94566) at 3450-5175 kPa. The extrusion was repeated four times with intervening freezethaw steps. The small liposomes were purified by gel ifitration over a 43 x 1.5 cm column of Sephacryl S-bOO (Pharmacia, Piscataway, NJ 08854), eluting with pH 7 Tris buffer at 30 mL/h. The fractions were monitored for enzyme activity in the presence and absence of Triton X-100, 5 gIL. The liposomes were eluted as an asymmetric peak with a long trailing edge preceding a peak of unencapsulated protein. The fractions that were free of unencapsulated protein were pooled. Liposome characterization. Freshly prepared liposomes encapsulating enzyme had little enzyme activity in the absence of detergents. Activity measured in the presence of Triton X-100 was independent of detergent concentrations in the range of 0.3-5.0 gIL. Because >94% of the initial enzyme activity present before encapsulation was consistently recovered from all preparations, we used the enzyme activity released by addition of Triton X-100 to estimate the total amount of encapsulated enzyme. Leakage of enzyme from the liposomes was determined by monitoring the enzyme activity over time in the absence of detergent. The amount of anti-theophylline antibody encapsulated in liposomes was determined by measuring the enzyme activity of a suspension of the liposomes in a solution of the G6PDH-theophylline conjugate, both in the presence and the absence of Triton X-100. The resulting increase in inhibition of enzyme activity produced by the release of antibody and its binding to the enzyme conjugate was 1580 CLINICAL CHEMISTRY, Vol. 33, No. 9, 1987

compared with the inhibition produced by known concentrations of unencapsulated antibody. An estimate of the average size and polydispersity of the liposomes was carried out by dynamic light scattering measurements (7, 8) of liposome suspensions in Tris buffer containing 50 g of lipid per liter. For this we used a Nicomp laser light-scattering spectrometer (Model HN5-90) with a computing autocorrelator (Model TC-100; Nicomp Instruments, Santa Barbara, CA 93110).

Results Liposome Preparation The “large” liposomes in this study were relatively unstable; they were prepared by vortex-mixing a lyophilized mixture of lipids with a solution of the protein to be encapsulated and purifring the liposomes by centrifugation and washing. To maximize liposome stability, we used pure synthetic phospholipids that were free of auto-oxidizable polyunsaturated fatty acids. A study of the effect of different ratios of POPC, POPG, and cholesterol on the rate of leakage of encapsulated proteins showed that lowering the fraction of POPG or increasing the fraction of cholesterol decreased the rate of leakage of encapsulated G6PDH. When cholesterol constituted about one-third of the mass of the total lipids, leakage was suppressed even in liposomes with high POPG content (Figure 1). Large liposomes with encapsulated G6PDH-theophylline conjugate were estimated by dynamic light scattering to average about 3 im in diameter and to have a broad size distribution (range of variation >110%). They remained fully suspended in Tris buffer for up to 1 h, as demonstrated by replicate sampling of an unstirred suspension (CV