Biotechnol. Prog. 1995, 11, 333-341
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Noncompetitive Immunoassays Using Bifunctional Unilamellar Vesicles or Liposomes Anup K. Singh, Peter K. Kilpatrick, and Ruben G. Carbonell* Department of Chemical Engineering, North Carolina State University, Raleigh, North Carolina 27695-7904
Small unilamellar vesicles (SWs) functionalized with an enzyme label and with specific ligands for biological molecules are useful as signal enhancement vehicles in the development of enzyme-linked immunoadsorbent assays and other biosensor applications. Bifunctional vesicles were prepared by covalently attaching horseradish peroxidase (HRP) and an antibody to the outside of the lipid bilayer of a n SUV. The reaction conditions were optimized to obtain 7-12 antibody molecules and 100-200 HRP molecules per vesicle. The enzyme retained 70-80% of its specific activity after immobilization, and the presence of immobilized proteins on the vesicle surface apparently increased the vesicle stability. To minimize the background signal and maximize the specific signal, the immunoassay protocol was optimized with respect to (1)the type and concentration of blocking agent, (2) the diluents for HRP-antibodyvesicles and sample, (3) the incubation period, and (4) the incubation temperature. The bifunctional vesicles were used in a noncompetitive immunoassay to detect d-dimer, a fibrin dimer formed a t the early stages of thrombosis. A second conjugate, HRP-antibody, was prepared, characterized, and used as a control against which to compare the assay using vesicles. The assay results using vesicles led to a detection limit for d-dimer in human plasma 9 times lower than what was achieved using the conventional enzyme-antibody conjugate assay.
Introduction Liposomes or lipid vesicles, first described by Bangham et al. (19651, are closed bilayer membrane structures that can form by suspending lipids in water. They can be prepared from a variety of lipids and lipid mixtures, with phospholipids being the most commonly used. Numerous methods exist to form liposomes, including sonication, detergent dialysis, reverse phase evaporation, and extrusion through filters (Szoka and Papahadjopoulos, 1978). In the years since their discovery, liposomes have been used as models of cell membranes (Bangham et al., 19721, while later research has centered on their use as microcarriers in drug delivery systems (Gregoriadis, 1988). Other areas in which liposomes have found applications include affinity separation of proteins (Powers et al., 1989,1990),immobilized enzyme reactors, and immunodiagnostic assays. In immunodiagnostic assays, liposomes are used as a means to obtain signal enhancement and higher sensitivities. Liposomes provide a large interior volume where one can entrap thousands of small marker molecules, and hydrophilic groups on the outside surface of liposomes offer sites for the attachment of receptor molecules like antibodies and antigens. Consequently, for a few receptors on the surface, there are thousands of marker molecules inside, as opposed to the conventional enzyme(or fluor)-receptor conjugate carrying at the most a few marker molecules per receptor molecule. In principle, this allows the liposome immunoassays to perform much better in terms of detection limit than their nonliposomal counterparts. Liposome immunoassays can be divided into two broad categories: heterogeneous and homogeneous. Most of the work done so far has concentrated on homogeneous assays, which are simpler and faster. In these immuno-
* Author to whom correspondence should be addressed.
assays, liposomes carry a ligand on the surface and marker molecules such as enzymes and fluorophores inside. The majority of these assays are based on an inhibition or a competition format, where free antigen in serum and antigens immobilized on liposomes compete to bind the antibody molecules in solution. The liposome lyses by the action of complement or cytolytic agents, or due to phase transition, upon the formation of antigenantibody complexes on the liposome surface. This leads to a release of marker molecules, which can be detected analytically. Ho and Huang (1988) provide a comprehensive list of articles published on homogeneous liposome immunoassays. The use of liposomes in solid phase or heterogeneous assays, which are more time-consuming but considerably more sensitive, has not been that widespread. A few examples are O'Connell et al. (19851, Plant et al. (1989),Locasio-Brownet al. (19901, and Durst et al. (1993). Until recently, no enzyme-linkedimmunoassays had been developed using vesicles. Direct attachment of a large marker molecule such as an enzyme results in a much larger number, up to 1000, of enzymes becoming conjugated to a vesicle compared to encapsulation of the same enzyme, which leads to the entrapment of only a few enzyme molecules. Jones et al. (1993,1994) developed a competitive ELISA to detect biotin using liposomes with biotin and HRP immobilized on the surface. These biotinylated liposomes competed with biotin in the sample to bind to antibiotin antibody immobilized on a microtiter plate. After the plate was washed to remove the nonspecifically bound liposomes, substrate was added, and the signal produced was inversely proportional to the amount of biotin in the sample. The sensitivity of vesicle immunoassays, heterogeneous and homogeneous, has been shown to be better than that of their nonvesicle counterparts in most instances. Axelsson et al. (1981) obtained results comparable to a
8756-7938/95/3011-0333$09.00/00 1995 American Chemical Society and American Institute of Chemical Engineers
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51Cr-releaseassay for the detection of anti-rat transplantation antibody. Jones et al. (1994), in a competitive assay to detect biotin, reported that the detection limit using vesicle-biotin-HRP conjugate was 10 times lower than that using biotin-HRP. Choquette et al. (1991) demonstrated that the use of theophylline-labeled liposomes in a competitive assay provides an order of magnitude signal enhancement over theophylline derivatized with fluorescein. In the competitive-bindingassay for digoxin (O’Connellet al., 1985), signals are amplified 103-104-fold by using liposomes as compared to labeled hapten. This paper reports results on the use of liposomes to increase the sensitivity of noncompetitive (or, sandwich) enzyme-linked immunosorbent assays (ELISAs). The reason behind choosing a noncompetitiveformat was that it is inherently more sensitive than a competitive assay. In a competitive assay, the minimum detectable concentration is limited by the dissociation constant of the antibody for the analyte. In a noncompetitive assay, one can detect a single antibody-antigen binding event as long as the signal is significantly higher than the background. In a sandwich ELISA using a microtiter plate, the antibody to the antigen of interest is adsorbed to a solid surface, such as the bottom of a cuvette or a well in a microtiter plate. The surface is then blocked to eliminate nonspecific binding in subsequent steps by adsorbing a protein, such as bovine serum albumin, followed by aspiration and rinsing to remove the unbound protein. In the second step, samples containing the antigen are incubated with the solid surface and the nonspecifically bound antigen is removed by washing. In the third step, a second antibody, also specific to the antigen, which is conjugated to one enzyme molecule, is added. The amount of labeled antibody and, hence, the antigen is determined by assaying for the enzyme. In this work, liposomes bearing surface-immobilized antibody and an enzyme label were used in the third step of the assay. The advantage to using this arrangement is that in current ELISA techniques each labeled antibody carries a single enzyme molecule, but with liposomes the ratio of enzyme to antibody molecules can be increased significantly, as illustrated in Figure 1. Consequently, the use of liposomes with surface-immobilized antibody and enzyme label molecules should result in better sensitivities. The antigen chosen for this study was d-dimer, which is a fibrin dimer formed at the early stages of thrombosis. Thrombosis is a major clinical complication of atherosclerosis, and it has been established that myocardial infarction in most cases is due to the obstruction of the coronary vessels by thrombi (Liu et al., 1990). A schematic diagram of the formation of d-dimer is presented in Figure 2 (Halkier, 1991). The origin of a DD/E complex from the protofibrils is outlined by the dashed lines in Figure 2. We describe the preparation and characterization of bifunctional small unilamellar phospholipid vesicles with covalently attached antibody and enzyme label. The average vesicle diameter, the number of enzyme and antibody molecules immobilized per vesicle, and the activity of the enzyme were determined. The bifunctional vesicles were used in a noncompetitive assay to detect d-dimer. It was observed that the concentration of antibody used to coat the plate had a considerable effect on assay performance, and a concentration of 40 ,ug/mL coating antibody (8-8-G) proved to be the optimum concentration. The assay was also optimized with respect to the following parameters: blocking agent, diluents for HRP-antibody-vesicles and sample, incubation times,
Biofechnol. Prog., 1995, Vol. 11, No. 3
IS
- a\
P
+
with antibody & enzyme on the
Blocking Protein
Figure 1. Vesicles in a sandwich assay. In a conventional ELISA using antibody-enzyme conjugate, one can obtain 1-2 enzyme molecules per binding event, whereas if vesicles with immobilized enzyme and antibody are used, hundreds of enzyme molecules will be present per binding event, enhancing the signal 100-fold.
and temperatures for various incubation steps. The assay with vesicle was compared to a conventional ELISA using HRP-antibody conjugate, and the vesicles yielded a detection limit 9 times lower than that of the HRPantibody conjugate. Materials and Methods Materials. Horseradish peroxidase (HRP) type VI-A, bovine serum albumin (BSA) fraction V (RIA grade), 3,3’,5,5’-tetramethylbenzidine dihydrochloride (TMB), casein, 2,2’-azinobis( 3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), hydrogen peroxide (HZOZ), urea hydrogen peroxide tablets, dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylcholine(DSPC), cholesterol (Chol), phosphate-buffered saline (PBS), and Sepharose CL-6B were obtained from Sigma Chemical Company (St. Louis, MO). Polystyrene 96-well highbinding, flat-bottom microtiter plates were obtained from Corning Inc. (Corning, NY) and Costar Corp. (Cambridge, MA). Carnation Instant Dry Milk was bought from a local grocery store. [14C]Formaldehydewas obtained from Amersham (Arlington Heights, IL). The d-dimer antigen and the two anti-d-dimer monoclonal IgG’s, designated 5-4-C and 8-8-G, were kindly’ provided by Organon Teknika Corp. (Treyburn, NC). All other chemicals were either from Fisher Scientific or Sigma and of reagent grade or better. Methods. Vesicle Preparation. Small unilamellar vesicles were prepared with compositions of DSPC/Chol/ DMPE (40:40:20 mol %) using standard sonication procedures (Szoka and Papahadjopoulos, 1980). Mixtures of various lipids weighing 30 mg total were dissolved in chlorofordmethanol(9:l) and dried in a rotary evaporator to form a thin lipid film on the inside wall of the flask. The lipids were then hydrated in 10 mL of 50 mM citrate buffer (pH 6.0) at 70-80 “C. Unilamellar vesicles were formed by sonicating the hydrated lipid suspension at
Biotechnol. Prog., 1995, Vol. 11, No. 3
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Fibrin Dimer
180 kD
Intermediate Polymer
- , - - - - - A
Figure 2. Formation of d-dimer polymer. d-dimer is a fibrin dimer detected in the arteries of patients suffering from thrombosis, which can ultimately lead to myocardial infarction. Thrombin catalyzes the polymerization of fibrinogen to fibrin dimer, resulting in the formation of a soft clot. "he clot is stabilized and hardened by factor XIII-induced covalent bonds between adjacent D domains. The basic unit of polymer is encircled by a dashed line.
70-80 "C for 1 h using a probe sonicator (Model W-385, Heat Systems Ultrasonics, Farmingdale, NY). Undispersed phospholipids were removed by centrifuging at 3000g on a ked-rotor table-top centrifbge (Model Centra4,International Equipment Co., Needham Heights, MA) for 20 min and filtering the solution through a 0.2 pm Acrodisc filter (Gelman Sciences, Ann Arbor, MI). Radiolabeling of Antibody. In order to determine the amount of antibody 5-4-C immobilized on the vesicle, the antibody was labeled with [14C3formaldehyde by reductive methylation (Jentoft & Dearborn, 1983). Fifty microliters of 0.1 M NaCNBH3 was added to 1 mg of antibody in 0.5 mL of PBS buffer. Ten microcuries of [14Clformaldehyde was added, and the reaction was carried out at 25 "C for 2 h at room temperature. Unreacted formaldehyde and excess reducing agent were removed by dialyzing 4-6 times against 1 L buffer (0.05 M boric acid/borax, pH 8.4) exchanges. The 14C-labeled antibody was pipeted out of the dialysis bag and stored a t -20 "C. Ten microliters of radiolabeled antibody was added to 10 mL of Cytoscint (ICN Biomedicals, Irvine, CAI in a scintillation vial, and the disintegrations per minute (DPM) were counted in a Packard 1500 liquid scintillation counter. This protocol results in a product containing (0.1-1) x lo6 DPIWmg of antibody. Immobilization o f h t i b o d y and HRP on V e h l e s . Horseradish peroxidase and the antibody were covalently linked to vesicles simultaneously using the periodate oxidation method (Nakane and Kawaoi, 1974; Heath et al., 1980). HRP (10-13 mg/mL) and 1-3 mg/mL antibody in 0.05 M citrate buffer (CB) (pH 6.0) were oxidized with 0.1 M sodium periodate for 30 min at 25 "C in the dark with gentle stirring. Excess periodate was neutralized by 0.32 M ethylene glycol for 1 h at 25 "C. Unreacted reagents were removed by desalting on a 10 cm EconoPac lODG desalting column (Bio-Rad Laboratories, Richmond, CA) equilibrated with pH 6.0 citrate buffer. The activated antibody and HRP were added to 1 mL of 3
mg lipid/mL vesicles. The reaction was carried out at 25 "C with gentle stirring, and the pH was maintained a t 8.4 for 2 h by adding 0.1 N NaOH. One hundred microliters of 20 mg/mL NaCNBH3 was added and the solution was left for 16-18 h at 4 "C. The sample was concentrated in a centrifuge using an Amicon Centriprep10 cell for 40 min at 3000 rpm. The concentrated sample was applied to a 70 x 1.5 cm gel permeation column packed with Sepharose CG6B and equilibrated with 0.05 M b o r d o r a t e buffer at pH 8.4. The flow rate was maintained at 0.5 mumin using a Masterflex peristaltic pump (Cole-Palmer Instrument Co., Chicago, IL), and 1 mL fractions were collected on a Gilson FC 204 automatic fraction collector. The fractions were analyzed by assaying for HRP activity, as will be discussed later, and measuring radioactivity. In some cases, one separation was inadequate to separate the HRP-antibody-vesicles from free HRP and antibody. The pooled fractions were then concentrated and applied to the same column to achieve a satisfactory separation. A sequential scheme was also used, where HRP was first conjugated to vesicles. The HRP-vesicles were separated from unreacted HRP by size exclusion chromatography. The size of the HRP-vesicles as determined by QLS was 780 f 60 & and approximately 110 HRP molecules were attached to each vesicle. These vesicles were later used to conjugate antibody molecules. One milligram of antibiotin antibody (ABA) in 0.5 mL of PBS was activated by periodate and reacted with 1 mL of vesicles containing 0.79 mg/mL lipid. The sample turned cloudy after the completion of the reaction, and centrifugation led to the formation of a precipitate, indicating that the vesicles cross-linkedthrough antibody molecules. The cross-linking is caused by the low concentration of antibody in the reaction mixture, as some antibody molecules bind to more than one vesicle. These HRP-ABA-vesicles bound specifically to the microtiter plate coated with biotin, but the background signal was
Biotechnol. Prog., 1995, Vol. 11, No. 3
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almost 50% of the total signal. The high nonspecific signal might be due to the denaturation of ABA caused by harsh reaction conditions (reaction at a high pH of 9.5 at room temperature for 18-20 h). The other disadvantage to this scheme is that it needs two reaction cycles and two separation steps, compared to one each in the simultaneous scheme described earlier. Characterization of Vesicles with Immobilized HRP and Antibody. HRP Specific Activity. Enzyme activity measurements were used to determine the effectiveness of separating HRP-vesicle conjugates from unreacted HRP by GPC and to determine the activity of HRP after immobilization. Activity measurements were performed in 50 mM citrate buffer, adopting the procedure outlined by Gallati (1979) using 2,2'-azinobis(3ethylbenzthiazoline-6-sulfonicacid) (ABTS) and H202 as substrates at 25 "C. Substrate (3.2 mL) (2 mM ABTS and 2.75 mM H202) was pipeted into sample and reference cuvettes. Ten microliters of enzyme solution was added to the sample and blanked against buffer, and the change in absorbance was recorded at 410 nm with a Shimadzu 160-ModelW-visible spectrophotometer.The immobilized HRP concentration was determined by measuring the absorbance of the vesicle solution at 403 nm, subtracting the contribution due to scattering by vesicles from the total absorbance, and using an extinction coefficient of 1.85 ml/(mgcm). The kinetic parameters of the enzyme were determined as described previously (Jones et al., 1993). Concentration of Phospholipids in Vesicles. The concentration of phospholipid in a vesicle sample, and ultimately the vesicle concentration, was determined by a phosphate assay using the method of Chen et al. (1956). One hundred microliters of sample (unknown and standard) containing between 0.025 and 0.25 pmol of phosphate was placed in a Pyrex test tube and heated in an aluminum block to 250 "C with 0.5 mL of 10 N H2S04 for 20 min. After cooling to room temperature, 6 drops of 30% aqueous phosphate-free H202 were added to each sample and reheated to 250 "C for 30 min. After cooling, 4 mL of deionized HzO, 0.5 mL of 2.5% aqueous ammonium molybdate, and 0.5 mL of 10%aqueous ascorbic acid were added to each sample, vortex mixed thoroughly, and heated in a boiling water bath for 7-10 min. After cooling, the absorbance was measured a t 830 nm on a Shimadzu 265 W-visible spectrophotometer. A calibration curve was obtained using standard inorganic phosphate solutions and phospholipid. Size of the Vesicles. Hydrodynamic radii of the vesicles before and after immobilization were determined using quasi-elastic light scattering (QLS). Vesicle solutions were centrifuged at 3000 rpm for 15 min to remove dust particles. Vesicle solution (1-2 mL) was pipeted into a clean borosilicate glass tube that was placed in the sample chamber of the light-scattering apparatus. Measurements were performed at a 90" scattering angle using a Coherent Innova 70-3 argon ion laser with a Brookhaven BI-2030 AT correlator and goniometer. The QLS data were analyzed using the constrained regularization method of Provencher (1982a,b), resulting in a size distribution characterized by a mean diameter and variance. Stock and Ray (1985)reported this method to be the most robust to noise present in the autocorrelation function and therefore the most reliable for analyzing experimental data. Estimation of the Number of HRP Molecules per Vesicle. The number of HRP molecules immobilized per
vesicle was estimated using the relation number of HRP - HRP concentration vesicle vesicle concentration HRP concentration = (lipid concentration
I
number of lipids vesicle
i
HRP concentration
= (lipid concentration
)N,d
1
(1)
For spherical unilamellar vesicles of radius R, with bilayer thickness t and an average area per lipid molecule A, Ntot is given by (Hutchinson et al., 1989) 4nR;
Ntot
=
A+
4n(R, - t>2
A
(2)
The vesicle diameter R , was calculated from the HRPantibody-vesicle diameter (determined by QLS) by assuming that the HRP is attached directly to the vesicle surface with no intervening spaces and that the contribution of antibody molecules to the vesicle size is negligible as the number of antibody molecules attached is small compared to the number of HRP molecules. The diffusion coefficient of HRP in pure water at 20 "C is 7.05 x cm2/s,as determined by its sedimentation velocity (Cecil and Ogston, 1951). Thus, the Stokes-Einstein relationship yields a hydrodynamic radius for HRP of 30 A. Given that the diameter of HRP is 60 A, the vesicle diameter was taken to be 120 A smaller than the mean HRP-antibody-vesicle diameter. The bilayer thickness, t, was assumed to be 40 A (Israelachvili and Mitchell, 1975; Johnson, 1973). The average area per lipid molecule was calculated by using values of 71,41, and 19 A2 for the phosphatidylcholine,phosphatidylethanolamine, and cholesterol projected head areas, respectively (Israelachvili and Mitchell, 1975). The average value of area per lipid obtained for these vesicles was 44.2 A2! lipid, Amount of Antibody on the Vesicle Surface. The antibody (5-4-C)was 14C-labeledas described previously for quantification purposes. The specific activity of antibody (DPWmg) is obtained by calculating the ratio of DPWmL to the concentration (mg/mL) of antibody. The concentration of antibody was measured spectrophotometrically at 280 nm using an extinction coefficient of 1.4 d ( m g c m ) , and DPM was measured in a liquid scintillation counter. The concentration of antibody after its immobilization on the vesicles was determined by counting the radioactivity of the vesicle solution, the underlying assumption being that specific activity of antibody is conserved. The number of antibody molecules immobilized per vesicle is given by the ratio of antibody concentration to vesicle concentration, which was later determined by a phosphate assay as described earlier. Preparation of Enzyme-Antibody Coqjugate. Six milligrams of HRP was dissolved in 0.5 mL of CB (pH 6.01, and 0.182 mL of 0.3 M sodium periodate was added to it. The reaction was carried out for 0.5 h in the dark with gentle stirring. The oxidation of HRP was terminated by adding 0.32 M ethylene glycol. The excess reagents were separated from HRP on a desalting column. To the activated HRP was added 2 mg of IgG 5-34!, and the pH was raised to 8.4. The reaction was carried out at room temperature for 4 h, followed by reduction with 3 mg of sodium cyanoborohydride at 4 "C overnight. The HRP-IgG conjugate was separated from
Biotechnol. Prog,, 1995,Vol. 11, No. 3 excess HRP on a 100 cm Bio-gel P-300 column equilibrated with 50 mM PBS at pH 7.4. Protocol for Microtiter Plate Preparation. The inner 60 wells of a 96-well Costar microtiter plate were coated with monoclonal antibody 8-8-G. The outer wells exhibited high well-to-well variation, possibly due to uneven temperature distribution. Monoclonal 8-8-G was dissolved in 50 mM carbonatehicarbonate buffer (pH 9.6) to obtain a concentration of 10-40 pg/mL. One hundred and fifty microliters of coating solution was dispensed in each well. f i r the plate was covered with a plate sealer, it was incubated at 4 "C on a plate-shaker at 500 rpm for 21 h. The coating solution was aspirated and the plate was washed twice with PBS on a manually operated plate-washer. The wells were then blocked with a blocking protein to saturate the unbound sites on the polystyrene surface. This was done by incubating the wells with 300 +well of either 1 wt % BSA or milk protein, dissolved in PBS, for 1 h at room temperature. The wells were aspirated dry and then washed three times with PBS. Protocol for the Sandwich ELISA with Vesicles. The stock solution of antigen, d-dimer, was serially diluted in 1 wt % BSA in PBS (pH 7.4). One hundred microliters of either diluted sample or control was applied to the coated wells. The plate was covered with a plate sealer and incubated at 37 "C for 1 h in a mechanical convection incubator. After the plate was washed four times with PBS, 100 pLJwell of HRP-antibody-vesicles was added and the plate was incubated at 37 "C for 1h. The wells were washed six times with PBS to remove the unbound and nonspecifically bound vesicles. Substrate TMB urea hydrogen peroxide (100 pLJwell) was added, and the plate was incubated at room temperature for 30 min. The reaction was then stopped with 2 N sulfuric acid, and the absorbance at 450 nm was read in a Biotek EL 340 plate-reader. Protocol for the Sandwich EUSA with HRPAntibody-Coqjugate. The ELISA was performed in exactly the same way as described in the previous section for vesicles, except with a modification in the washing steps. A nonionic detergent, Tween-20, was added to PBS buffer (pH 7.4) to make a 0.05 wt % solution. The wells were washed four times with this solution after each of the three steps: blocking, incubation with ddimer, and incubation with the conjugate. Ti#% Assay. The signals in ELISA were evaluated by measuring the HRP activity using TMB as substrate. The buffer used was 0.05 M phosphatekitrate (pH 5.0) containing 0.014% HzOz. One milligram of TMB was dissolved in 10 mL of buffer solution, and 100 pL was applied to each microtiter well. Reaction of HRP with TMB produces a soluble product blue in color. The reaction was stopped by adding 100 pL of 2 N HzSO4 as the color changed from blue to yellow, and the absorbance was read at 450 nm. Nonspecific Binding Experiments. To decrease the nonspecific signal, the following parameters were varied: blocking protein, coating buffer, wash buffer, type of plate, diluent, and incubation time. The optimum choice of these parameters is dependent on the antigenantibody system being considered, and hence these studies have to be repeated for every new assay. Various concentrations of milk protein, casein, BSA, and gelatin were considered as potential blocking proteins. As the diluent, milk protein, BSA, casein, and gelatin were tested. The different polystyrene plates tested were Costar high-binding, Corning high-binding easy-wash, and Corning round-bottomedwell plates. Different dura-
+
337
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3a
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a30E
F h
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f3 2
v
0
0
20
40
60
80
100
120
Elution Volume (ml) Figure 3. Elution profile of HRP-goat IgG-vesicle conjugates from unreacted HRP and goat IgG on a 1.5 x 70 cm Sepharose CL-6B column after the periodate coupling reaction. The fractions were analyzed by measuring disintegrations per minute (DPM) ( 0 )and enzyme activity (0).
tion times were tested for incubating the wells with sample and with HRP-antibody-vesicles.
Results and Discussion The small unilamellar vesicles made with DSPC, DMPE, and cholesterol by sonication were very stable with time. They were stored at 4 "C, and no significant change in size was observed for over 6 months. The average diameter of these vesicles was 600 f 50 A for various batches. A simultaneous immobilization of both HRP and antid-dimer antibody (5-44) was carried out using mild reaction conditions. Simultaneous reaction solves two problems: (1)only one purification step and one reaction cycle are required; (2) the possibility of cross-linking is minimized as the amount of total protein is large. Milder reaction conditions were achieved by the use of sodium cyanoborohydride, which is a milder reductant than sodium borohydrate, at pH 8.4 and a temperature of 4 "C. Polyclonal goat IgG and HRP were conjugated to the vesicles to study the effect of various parameters on the coupling and purification procedures. The chromatogram from a typical separation on a GPC column is shown in Figure 3. The vesicles eluted at around 35 mL and the unreacted HRP and goat IgG eluted at approximately 80 mL. The vesicle diameter remained almost constant at around 1150 A for the different amounts of IgG immobilized. The number of IgG molecules per vesicle increased from around 3 to 15 as the IgG bulk concentration was increased from 1to 6 mg/mL, and the increase was almost linear with respect to the IgG concentration (Figure 4). On the other hand, the number of HRP/ vesicle decreased from 100 to 60. Although the efficiency of immobilization is dependent on the antibody used, Figure 4 can be used as a guide to determine the bulk concentration of IgG needed to immobilize a certain number of antibodies on the vesicle. As long as there is at least one active antibody per vesicle, vesicles can be used in an assay, and Figure 4 predicts that a bulk concentration of 1-2 mg/mL should be sufficient. The specific activity of immobilized enzyme varied between 5.5 x lo4 and 7.4 x lo4 (AA*mL)/(min-mg).The specific activity of free enzyme was 9.34 x lo4 (AA*mL)/(min.mg) at the same conditions. Hence, the HRP retained 5979% of its activity aRer immobilization. The specific activity of immobilized HRP is almost independent of the number of IgG's per vesicle (Figure 5))implying that the larger IgG molecules do not cause any steric hindrance of the substrate-enzyme interaction.
Biofechnol. frog., 1995, Vol. 11, No. 3
338 16 14
12
10 8
i" t 0
1
2
3
4
mg/ml IgG
5
6
7
Figure 4. Effect of the bulk concentration of IgG on the number of IgG and HRP per vesicle.
lo^"""'"""'""'""'""'""^
*
b
t
0 0.01
0.1
[D-dimer]
1
10
, (pg/ml)
Figure 6. Effect of the concentration of coating antibody (88-G) on ELISA with vesicles. The [8-8-G] used to coat the plate was varied from 2 to 100 pglmL.
1 0
1
2
3
4
5
6
7
mg/ml IgG Figure 5. Effect of the bulk concentration of IgG on immobilized HRP activity.
One problem associated with the use of vesicles in immunoassays is the possible nonspecific binding of vesicles to the proteins. This could be due to HRP and/ or antibody on the surface or due to the lipids in the bilayer. Nonspecific control binding experiments done with HRP-conjugated vesicles and free HRP indicated that neither HRP nor lipids on the vesicle surface bind nonspecificallyto the proteins on the plate surface. BSA (1 wt %, RIA grade) worked the best both as diluent and as blocking protein. Carbonatehicarbonate (50 mhf) at pH 9.6 was determined to be a good choice as coating buffer. Polystyrene plates by different manufacturers were used in the assays, and both Costar high-binding plates and Corning high-binding easy-wash plates performed well. The Costar plates were used as they yielded smaller well-to-well variations in measured enzyme activity. For incubations with sample and the vesicles, 1 h at 37 "C proved to be the optimum choice. The vesicles undergo lysis in the presence of detergents, so that high concentrations of detergents could not be used in the vesicle diluent or wash buffer. The maximum concentration of a nonionic detergent such as ClzEs that M, but at such will not destabilize the vesicle is 5 x M), detergents are low concentrations (cmc = 1 x not effective as the washing medium. Another variable investigated was the concentration of antibody used to coat the plate. Different concentrations ranging from 1 to 100 pglmL capture IgG (8-8-G) were used for coating the plate, and the results are summarized in Figure 6. The signal increased as the concentration of 8-8-G was increased from 1 to 40 pgl mL, attained a maximum at 40 pglmL, and then dropped slightly for 100 &mL 8-8-G. The lower signal at 100 pgImL can be attributed to the interference by other serum proteins present in the ascites fluid containing the
antibody. At higher concentrations, these nonspecific proteins are adsorbed preferentially to the solid surface, reducing the surface concentration of specific antibody. On the basis of this result, 40 pglmL was considered to be the optimum concentration of 8-8-G to coat the plate. For the d-dimer ELISA, two different conjugates were made: vesicles with 5-44 (anti-d-dimer monoclonal IgG) and HRP on the surface and (5-4-C)-HRP conjugate. The latter worked as a control against which the vesicle ELISA was judged. The chromatogram in Figure 7 shows the separation of HRP-(5-4-C) conjugate from free HRP by gel permeation chromatography. HRP retained almost 70%of its activity, and 2-3 HRP molecules were conjugated to each IgG molecule. The efficiency of conjugation was excellent, and 22%of HRP and greater than 90% of IgG were conjugated. Five batches of HRP-(5-4-C)-vesicles were made to be used in d-dimer ELISA. Table 1 summarizes the reaction conditions and results of the five immobilizations. The diameter of the vesicles varied from 1000 to 1200 A over the different runs. The number of HRP per vesicle dropped from 204 to 130 as the ratio of IgG concentration to HRP concentration in the reaction mixture was increased from 0.0877 to 0.329, but the number of IgG molecules increased from 2 to 12 per vesicle. All five batches were used in the ELISA. Batch 1, for which vesicles had only two IgG molecules per vesicle, did not work, implying that the antibody molecules either were not active or were not oriented properly. With batch 2, a higher IgG bulk concentration was used, leading to 8 IgG molecules immobilized per vesicle. This worked better than the first batch, but the signals were still lower than that obtained in the assay with HRP-antibody conjugate. With batch 3, the bulk concentration was raised even higher, which led to the conjugation of 12-13 IgG molecules per vesicle, but the number of HRP molecules dropped to 130 per vesicle. This batch led to the best assay performance. The enzyme activity was approximately 1 x IO5 AA-mgl min-mL for the three batches. Batches 4 and 5 were made with about the same IgG to HRP ratio in the reaction mixture as batch 3. The IgG used for the last two batches was not radiolabeled for two reasons: (1)use of the lysines in IgG for radiolabeling may reduce the activity of antibody; (2) the handling and disposal of
339
Biotechnol. Prog,, 1995, Vol. 11, No. 3
35
Free H R P y
A
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20
-
1 0.8 M
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15 ..
0.4
5
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l50 I
oL-,,,--,3 0
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30
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40
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I
80
Elution Volume (ml) Figure 7. Separation of the HRP-5-44 conjugate from unreacted HRP and 5-4-C on a 100 x 1.5 cm Bio-gel P-300 gel permeation column. The column was equilibrated with 50 mM PBS a t pH 7.4, and the flow rate was 10 mUh. The enzyme activity was measured with 2.0 mM ABTS and 2.75 mM HzOz in 50 mM citrate buffer (pH 4.0) a t 25 "C.
Table 1. Reaction Conditions and Results of Immobilization of H R P and 5 - 4 4 on the Vesicles for Five Batches batch of HRP-(5-4-C)-vesicles 1 2 3 4 5 reaction conditions mg of IgG mg of HRP mL ofvesicle (3 mg/mLlipid) results vesicle size (A) HRPkesicle IgGhesicle enzyme activity ( x 104 AA*mg/min.mL) a
1
1
1.02 4.1 3.1 13 1 1
1110 204 2 12.6
1050 190 8 9.7
1200 130 1 2 12.1
1.14 2.5 13 13
1090 103 a 7.0
3 10 1 1150 120 a
10.4
IgG not radiolabeled.
radioactive vesicles were quite cumbersome. Also, at this point we had sufficient information to predict the approximate number of IgG molecules immobilized by knowing the initial reaction conditions. Both batches 4 and 5 performed better than the HRP-antibody conjugate. Still higher concentrations of IgG in the reaction mixture were not attempted for fear that the number of HRPs immobilized per vesicle might become too low. Figure 8 represents ELISAs performed with three batches (batches 3-5) of HRP-antibody-vesicles, all having approximately 10-12 antibody molecules on the surface, and HRP-antibody conjugate. AU four curves appear to be sigmoidal in nature, as is generally the case with sandwich ELISAs. A Langmuir equation of the form y = Xi/(1 I&) was used to fit the data, employing a nonlinear least-squares method. For comparison and quantitation purposes, we define the term least detectable dose (LDD)as the antigen concentration that results in a normalized signal of 2u higher than the blank (response for zero antigen concentration), where u is the standard deviation of blank. The average value of u for these assays was 0.035 absorbance unit, and hence LDD would be the d-dimer concentration corresponding to NA,, = 0.07. The hwest LDDs obtained with HRPantibody-vesicles were 2.4 ng/mL with batch 3 and 21.5 ng/mL with HRP-antibody conjugate. Hence, the ELISA with vesicles yielded a 9-fold improvement over that with conjugate. Batches 4 and 5 of HRP-aqtibody-vesicles resulted in LDDs of 7.5 and 4.7 ng/mL, respectively, indicating that they performed 3-4-fold better than the HRP-antibody conjugate. The detection limits with
+
0
0.001
0.01
0.1
1
10
pg/ml d-dimer
Figure 8. d-dimer ELISA using various batches of vesicles: batch 4 (e),batch 5 (O),and HRP-5-44 conjugate batch 3 (O), (0).Least detectable dose (LDD) is defined as the x-intercept of the least-squares fit to the linear region of the signal versus the d-dimer concentration plot. LDD for different batches of HRP-Ab-vesicle are 4.1 (batch 5), 4.5 (batch 41, and 1.6 ngl mL (batch 31, and LDD for HRP-Ab conjugate is 18 ng/mL. batches 4 and 5 were lower than that of batch 3, and possible reasons might be the slightly lower number of immobilized enzymes and a drop in the enzymatic activity compared to batch 3 (Table 1). One problem with the use of vesicles is the high background resulting from the nonspecific binding of vesicles to the proteins on the plate surface. The possible sources of nonspecific binding are HRP or/and IgG molecules on the vesicle surface, the vesicle itself, or the IgG molecules on the plate. To ascertain the exact source, experimentswere done with various formulations, and the results are presented in Table 2. Percent background is defined as [(backgroundsignal)/(maximum total signal)llOO. The percent background with HRPvesicles is only 2-5%, leading to the conclusion that neither HRP molecules nor vesicles bind nonspecifically. Both HRP-antibody-vesicles and HRP-antibody conjugate give a high background, implying that the IgG 5-44 in the conjugate and HRP-antibody-vesicles is the culprit. In the case of the conjugate, the nonspecifically bound molecules can be removed by washing with a M C12E6 or 0.05 wt detergent solution such as 5 x % Tween-20 in PBS. The maximum allowable C E E ~ M (=0.008 wt concentration in wash buffer is 5 x % Tween-20) with vesicles, which is much below the cmc, and it does not lead to any significant reduction in the background.
Conclusions The liposome immunoassay described here combines the higher sensitivity of a noncompetitive ELISA, which is not limited by the association constant of antibody to antigen, and a large signal amplification achieved by linking hundreds of enzyme molecules to a liposome. Two conjugates were prepared to perform ELISA for the detection of d-dimer in a blood sample. The first was vesicle with HRP and anti-d-dimer immobilized on the surface and the second was HRP-anti-d-dimer conjugate; the latter functioned as a control against which the assay with the vesicle was judged. The vesicles performed an order of magnitude better than the conventional enzymeantibody conjugate, and there is room for further improvement. Theoretically, the vesicles should perform 100-200 times better than the HRP-antibody conjugate as the number of enzyme molecules per vesicle is 100200 compared to 1-2 per HRP-antibody conjugate. On
340 Table 2. Nonspecific or Background Signals for Various Conjugatesa % background with Hv 2-5% HAV 14-40% HAb 10-33% HA‘ 5-14% ratio of % background HAVIHAb 1.24f 0.11 HAVIHAd 1.92f 0.04 a Abbreviations: Hv, HRP-conjugated vesicle; HAV, HRP- and antibody-conjugatedvesicle; HA, HRP-antibody conjugate. Wells washed with PBS. Wells washed with 5 x M C1&. Wells incubated with HAV washed with PBS and those incubated with HA washed with 5 x M C1&.
the other hand, vesicles are much larger (1100 A) than the IgG molecule (100 A),and hence the binding of vesicle to one IgG on the plate will make the neighboring 4-5 IgG molecules sterically unavailable for binding to other vesicles. This does not happen in the case of the smaller HRP-antibody conjugate (150 A). If we combine these two effects, an order of magnitude improvement obtained by using vesicles appears reasonably close to the theoretical maximum enhancement possible. Vesicles have been used in immunoassays before, predominantly in the homogeneous assay format involving the lysis of vesicles by a complement, resulting in the leakage of encapsulated marker molecules to produce a signal. In a majority of the cases, the markers are low molecular weight fluorophores that can be encapsulated in very large numbers, giving rise to a large signal amplification for a single antibody-antigen complexation event. The major drawbacks associated with these assays are the low shelf-life of the complement-the guinea pig complement is inactivated upon storage-and the nonspecific leakage of fluors upon complement addition. In some cases leakage of fluors from vesicles with time has been reported too. Leakage can be reduced by encapsulating large molecular weight markers, such as enzymes, but then only a few can be entrapped in a vesicle, lowering the amplification in signal. We have developed an assay that does not require a complement, and a very large number of enzyme molecules are covalently bound to the s d a c e , eliminating the problem of leakage. One obstacle encountered with the use of vesicles in ELISA was high background. The IgG linked to the vesicle is the possible source, and one alternative to solve the problem would be to use Fab fragments instead of the whole IgG molecule. The Fc region of an IgG molecule is primarily hydrophobic and can bind nonspecifically to hydrophobic domains in other proteins. Another disadvantage of liposome immunoassay is the difficulty in preparing uniform batches of sensitized liposomes. This was alleviated by using small unilamellar vesicles that were prepared reproducibly by sonication and then carrying out experiments to precisely relate the bulk concentration of HRP and antibody used to the number of HRP and antibody molecules immobilized on a vesicle. The method used for immobilizing proteins on liposomes was periodate oxidation, as it offers the advantage of site-selective antibody labeling. The carbohydrate residues used for conjugation are localized solely in the hinge region of the Fc fragment of the antibody molecule. This has two desirable outcomes: (1)Since the antibody is bound to the liposome through the Fc region, the antibody acquires a favorable orientation where the Fab fragments are free to bind to the antigen molecules. (2) The amines in the antibody molecules are not modified, minimizing the probability of the loss of antibody activity as these amines could participitate in antigen recognition.
Biotechnol. Prog., 1995,Vol. 11, No. 3
The yield of bound protein was relatively low (-10-15%), in agreement with other published results (Torchilin and Klibanov, 1981). Alternative chemistries utilizing heterobifunctional linking agents resulting in higher yields are available, but they do not have the advantage of antibody orientation. HRP is a relatively large molecule with a molecular weight of 40 000, and not more than 150-250 molecules can be attached to a 60 nm vesicle. Fluorophores, on the other hand, are relatively small molecules with molecular weights in the range of 500-1000 and, consequently, a very large number of them can be immobilized on a vesicle. For example, if we link a fluorophore to DMPE and use it in conjunction with cholesterol and DSPC to form a vesicle, and if the mole percentage of DMPE in the vesicle is 20%,then we can get approximately 3000 fluor molecules on one vesicle. A vesicle containing 30 mol % DMPE will have 4500 fluor molecules. The mole percent of phosphatidylethanolamine was 20% in the vesicles used in the assays until now. Heath et al. (1980) prepared vesicles containing 33 mol % PE, but they did not report any information on vesicle stability. Vesicles containing 30 mol % PE were prepared and appear to be stable on the basis of size monitoring over 1 month, but stability has to be tested over longer period of time before they can be used for immobilization. Experiments to test this approach are currently under investigation.
Acknowledgment The authors gratefully acknowledge AKZO Corporate Research America, Inc., and the North Carolina Biotechnology Center for the financial support of this work. The authors also thank Organon Teknika (Durham, NC) for kindly providing the monoclonal antibodies and the positive sera for d-dimer.
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