242
Biotechnol. Prog. 1993, 9, 242-258
Preparation and Characterization of Bifunctional Unilamellar Vesicles
?or Enhanced Immunosorbent Assays
Matthew A. Jones, Peter K. Kilpatrick, and Ruben G. Carboriell*
Department of Chemical Engineering, North Carolina State University, Raleigh, North Carolina 27695-7904
Small unilamellar phospholipid vesicles with covalently attached biotin and horseradish
peroxidase (HRP) were prepared and characterized in tetms of hydrodynamic diameter,
amount and activity of immobilized enzyme, and number of biotin molecules on the
outer vesicle surface. In addition, the specific adsorption of these bifunctional vesicles
and commercially available biotin-labeled horseradish peroxidase (B-HRP) to antibiotin antibody (ABA) coated polystyrene microtiter plate welfs was examined. At low
antibody surface densities, the signal (AAlmin) generated by the vesicles adsorbed to
the surface was approximately 100 times higher than the signal generated by B-HRP.
It was also found that the biotin-conjugated vesicles were able to compete effectively
with free biotin in solution for surface ABA sites. ThBse results indicate that this type
of vesicle may be used in competitive and sandwich-type enzyme-linked immunoassays
to improve the detection limits, increase the signal, and decrease the reaction time
necessary to detkct a given analyte concentration in solution.
Introduction
Immunodiagnostic assays rely on the specific molecular
recognition of antigens by their antibodies for the detection
of a given analyte in solution. In medical applications,
the analyte could be a viral protein, a drug, a hormone, or
an antibody generated as a result of infection (Bluestein
et al., 1990). Immunodiagnostic assays are becoming
increasingly important in other fields, including the
analysis of the levels of toxins, antibiotics, steroids,
vitamins, and amino acids in food (Allen and Smith, 1987),
as well as in the detection of pesticides, heavy metals, and
other toxic chemicals (polychlorinated biphenyls, dioxin,
phenols) in aqueous streams (Luong et al., 1988; Krull,
1990). Antibody-antigen recognition is the basis for most
of the work currently being done on the development of
biosensors for the detection of analytes at low concentration (Lowe, 1984; Turner et al., 1987). The medical
applications of immunodiagnostic kits alone resulted in
approximately 2 billion U.S.dollars in sales worldwide in
1990, and there is a significant and increasing demand for
assays that are more accurate, more reliable, easier, or
more sensitive (Bluestein et al., 1990). Most of the medical
applications require that the analyte be detected as quickly
as possible after the onset of the disease. This often
requires measurements of analytes in the nanomolar (10-9
M) range, but in many cases it would be advantageous to
M) range.
have a detection limit in the picomolar
One of the most widely used immunodiagnostic techniques is the enzyme-linked immunosorbent assay (ELISA),in which one of the reactants is adsorbed on the surface
of a solid support and the analyte is detected using an
enzyme-labeled reactant. It is clear that in ELISAs, the
larger the number of enzyme molecules conjugated to the
labeled reactant, the larger the resulting signal for a given
concentration of labeled reactant adsorbed on the solid
surface. Additional label could potentially result in either
shorter reaction times or lower detection limits for enzymelinked assays. Bates (1987)and Kricka and Thorpe (1986)
have summarized some of the approaches that have been
* Author to whom correspondence should be addressed.
8756-7938/93/3009-0242$04.00/0
used to enhance ELISA signals. These include the use of
several enzyme molecules covalently linked together with
an antibody and three biotin-conjugated enzymes bound
to three of the binding sites of avidin while the fourth site
is bound to a biotin-conjugated antibody (Bates, 1987).
However, the direct attachment of several enzyme molecules to a single antibody molecule or to a single antigen
molecule can lead to loss of specificityand binding strength
of the antigen-antibody interaction. This can come about
as a result of steric hindrance upon binding to the surface
as well as denaturation of the molecule during the coupling
procedure. It is desirable to have alternative methods to
increase the number of signal amplification labels without
sacrificing specificity or the strength of the association
constant. Recently, Janssen Biotech & Biochimica Products (Flanders, NJ) introduced poly(HRP)-streptavidin,
which is a chain of 18-20 horseradish peroxidase molecules
conjugated to streptavidin. This reagent can be used to
enhance signal generation in ELISAs by 4-20 times as
compared to the conventional assay using the standard
horseradish peroxidase-streptavidin conjugate (Janssen,
1991).
In this article, we explore the properties of bifunctional
vesicles which are conjugated with both a small ligand for
molecular recognition as well as multiple HRP molecules
for signal generation. These vesicles are a subset of the
broad category of lipid vesicles known as liposomes; being
approximately 1000 8, in diameter and having a single
lipid bilayer structure puts them in the category of small
unilamellar vesicles (Szoka and Papahadjopoulos, 1980).
Given that many enzyme molecules can be attached to the
outer vesicle surface, the bifunctional vesicles have the
potential of further increases in speed and sensitivity of
these assays. Figure 1illustrates how a bifunctional vesicle
of this type could be used in a competitive immunoassay
for a small analyte. With suitable modifications, it is also
possible to develop noncompetitive or sandwich-type
assays based on enzyme-conjugated vesicles.
In principle, any small particle could be used to attach
enzymes and ligands in an immunodiagnostic scheme such
as that shown in Figure 1. For example, latex spheres are
0 1993 American Chemical Society and American Institute of Chemical Engineers
Bbtechml. Rug., 1993, Vol. 9, No. 3
243
Vesicle with immobilized
/ enzyme (a)
and ligand ( .)
Free ligand
Figure 1. Schematic representation of bifunctional vesicles
competing with free ligand for binding sites of surface-immobilized antibody.
now available in uniform sizes with diameters less than
10oO A. On the other hand, small unilamellar vesicles
exhibit several features that make their use in this
application attractive. Vesicles can be made easily by a
variety of techniquesfrom relatively inexpensive materials
in relatively uniform sizes, As illustrated in this article,
their surface composition can be readily controlled by
mixing phospholipids of different types. The uniform,
oriented structure of the lipid bilayer in a vesicle makes
their surface properties predictable,and this allowscontrol
of the surface charge, the number of reactive groups, and
the hydrophilicity of the surface. Vesicles can also
maintain their stability either in dehydrated form or in
liquid solution for extended time periods. Latex spheres
are particularly difficult to keep in suspension without
the addition of surface active compounds whicb, in turn,
can affect surface properties. Even though other particle
materials may be suitable candidates for this application,
the unique properties of vesicles make them worthwhile
candidates for further study.
Most previous applications of vesicles to immunodiagnostics have been based on homogeneous assays which
are generallyturbidimetric or lytic. In lytic immunoassays,
vesicles prepared with antigen on the surface release
radioactiveor fluorescent marker molecules encapsulated
insidethe aqueous core of the vesicle (Ishimori et al., 1984;
Hoand Huang, 1988;Yuet al., 1987). Other homogeneous
assays use vesicles to enhance the measured turbidity
resulting from the aggregation of antigen-coated latex
beads (Martin and Kung, 1987;Cooper, 1989).
There are less than a handful of exampleswhere vesicles
have been used in solid-phase or heterogeneous immunoassays. O'Connell et al. (1985)developed a competitive
immunoassay for digoxin using anti-digoxin antibody
coated glass tubes and dipalmitoylphosphatidylethanolamine (DPPE)-digoxigenin phosphatidylcholine vesicles
with encapsulated sulforhodamine B dye. After specific
binding to the antibody-coated surface, the vesicles were
disrupted with surfactants to release the dye, which was
detected spectrophotometrically. Campbell et al. (1988),
from the same group a t Becton-Dickinson,describesimilar
vesicles adhering to antibody-coated porous membranes
for the competitive adsorption step. Other investigators
have developed assays based on the measurement of dye
fluorescence, which is inherently more sensitive than the
measurement of dye UV absorption just mentioned. Plant
et al. (1989) used avidin to bridge biotin-conjugated
proteins with biotinylated vesicles with encapsulated
5(and 6)-carboxyfluorescein in immunoassays. Recently,
Locascio-Brown et al. (1990)developed a flow injection
immunoassay system for quantifying theophylline and
anti-theophyllineusing theophylline-labeled phospholipid
vesicles with encapsulated carboxyfluorescein. Further
investigations showed that this technique resulted in an
order of magnitude signal enhancement over theophylline
derivatized with fluorescein (Choquette et al., 1992). In
these immunoassays, the label was trapped in the aqueous
core of the vesicle.
The encapsulation of dyes and other small marker
molecules inside a vesicle can be achieved readily by
suspending the phospholipid in an aqueous phase containing the label. Dye concentrations used in these
preparations are on the order of 0.01-1.0M. Small vesicles
can be prepared having relativelynarrow size distributions
with external diameters ranging from 500 to lo00 A. A
large number of small molecules can be encapsulated in
. For example,vesicles with an external diameter
of
this570 and a bilayer thickness of 40 A (Israelachviliand
L.
Mitchell, 1975)have an internal volume of 6.2 X
If a 0.1 M solution of dye is encapsulated in this space, in
principle it would lead to approximately 3700 entrapped
dye molecules per vesicle. However, as pointed out by
Wagner and Baffi (1987),the encapsulated dye often
diffuses out of the vesicle with time, and the diffusion rate
can be faster with higher concentrations of encapsulated
material. As a result, it is difficult to store vesicles with
entrapped marker for long periods of time. For this reason,
Wagner and Baffi patented the use of fluorescent lanthanide metal-chelating agent combinations which could
be attached directly to the lipids formingthe vesiclebilayer
and thus become an integral component of the membrane
structure. In some instances it may be possible to attach
a t least as many fluorescent molecules to the surface of a
vesicle as can be entrapped within the vesicle eore. In the
example above, approximately 7800fluorescent molecules
could be attached to a 570 A diameter vesicle if 25% of
the phospholipid molecules were labeled, although this
high label content could lead to vesicle insiability. One
possible advantage of utilizing vesicles with entrapped
fluorescent molecules is that the fluorescence is selfquenched uptil disruption by detergent, which allows an
extra degree of control. Thus, in the case of fluorescent
dye moleculep, encapsulation appears to be preferable to
surface labeling.
The advantageof direct attachment of marker molecules
to the vesicle surface is more apparent if one considers
enzyme molecules such as alkaline phosphatase or horseradish peroxidase. In these cases it is not possible to
achieve concentrations of 0.1 M enzyme solutions for
encapsulation. Instead, it would be typical to have enzyme
concentrations on the order of 1o-S M, resulting in only a
handful of entrapped enzymemolecules per vesicle. Heath
et al. (1'980)described a method for the covalent attachment of horseradish peroxidase to the outer surface of
vesicles by reacting phosphatidylethanolamine in the
bilayer with the carbohydrate chains of this glycoprotein,
resulting in hundreds of enzyme molecules per vesicle.
This development offers the opportunity for the enhancement of ELISA signals and sensitivity using ligand- and
enzyme-conjugated vesicles.
To the best of our knowledge, no ELISAs have been
developed using vesicles with surface-immobilized en-
"1
Biotechnol. Prog., 1993,Vol. 9,No. 3
244
zymes. There are a number of issues that must be resolved
to establish the feasibility of such an approach. The
enzyme immobilized to the vesicle surface must be shown
to maintain high specific activity and must be bound in
sufficiently large quantities to expect a significant enhancement in the signal in the course of the immunoassay.
In addition, it must be demonstrated that the binding of
ligand- and enzyme-conjugatedvesicles to antibody-coated
surfaces is specific; there should be little vesicle break-up
or nonspecific adsorption to the plate. Finally, the strength
of adsorption of ligand-conjugated vesicles to antibodycoated surfaces must be compared to the strength of
adsorption of free ligand in bulk solution. This is
particularly important in the design of competitive assays,
in which the vesicles must be able to compete with free
ligand in bulk solution. The strength of adsorption of the
vesicles to the surface is likely to be a function of the
antibody surface density on the microtiter plate and the
ligand concentration on the vesicle surface. At low
antibody surface densities, the signals generated from
ligand- and enzyme-conjugated vesicles should be significantly higher than those obtained from traditional
enzyme-conjugated ligands. This would tend to make the
use of vesiclesparticularly advantageous in noncompetitive
immunoassays at low surface concentrations of adsorbed
analyte on the plate.
This article addresses all of the issues noted above. It
describes the preparation and characterization of bifunctional small unilamellar phospholipid vesicles with covalently attached antigen (biotin) and enzyme label
(horseradish peroxidase), with an aim toward application
to solid-phase immunoassays. The average vesicle diameter, the number of enzyme molecules immobilized per
vesicle, and the activity and kinetic parameters for the
immobilized enzyme were determined. In addition, the
specificity and strength of vesicle binding to microtiter
plates coated with a polyclonal anti-biotin antibody (ABA)
were determined. The magnitude of the association
constant was calculated over a range of antibody loadings.
As will be shown in the following sections, a large number
of enzyme molecules (>loo) can be attached to the vesicle
surface,while at the same time maintaining good specificity
and binding strength to the ABA-coated plates. At low
ABA surface coverages the signals generated by the
bifunctional horseradish peroxidase and biotin-conjugated
vesicles (HBVs)were found to be approximately 100times
greater than those generated by commercially available
biotinylated horseradish peroxidase (B-HRP). A t low
antibody loadings, it was shown that the HBVs are able
to compete effectively with free biotin in solution, allowing
for the possibility of designing vesicle-mediated competitive immunosorbent assays for signal enhancement and
improved detection limits.
Materials and Methods
Materials. Horseradish peroxidase (HRP) type VI-A,
biotin-conjugated horseradish peroxidase (B-HRP) type
VI-A, goat affinity-purified polyclonal anti-biotin antibody
(ABA), bovine serum albumin (BSA), 2,2'-azinobis(3ethylbenzthiazoline-6-sulfonicacid) (ABTS), hydrogen
peroxide (HzOz), biotin, dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylcholine (DSPC),
cholesterol (Chol),phosphate-buffered saline (PBS), and
Sepharose CL 6B-200 were obtained from Sigma Chemical
Company (St. Louis, MO). Polystyrene 96-well roundbottom microtiter plates were obtained from Corning, Inc.
(Corning, NY). Biotinylimidohexanoic acid succinimido
ester, or long-chain biotin (biotin-LC-NHS),was obtained
from Pierce Chemical Company (Rockford, IL). [14C]Formaldehyde was obtained from Amersham (Arlington
Heights, IL). All other chemicals were from Fisher
Scientific and of reagent grade or better.
Methods. Synthesis of DMPE-LC-biotin. Biotinylated phospholipid was synthesized by coupling DMPE
with biotin-LC-NHS using the method of Bayer and
Wilcheck (1984). DMPE (30 mg) and biotin-LC-NHS (25
mg) were dissolved in 2 mL of chloroformimethanol(2:l)
by vortexing. After the addition of 20 pL of triethylamine,
the mixture was allowed to react a t room temperature for
2 h. DMPE-LC-biotin was purified by preparative thinlayer chromatography on silica gel plates (Analtech Inc.,
Newark, DE) using chloroformimethanoliwater (70:26:4
vol % ) as the mobile phase. The product was identified
using a cinnamaldehyde spray to detect biotin (McCormick
and Roth, 1970) and a molybdenum spray to detect
phospholipid (Dittmer and Lester, 1964). The silica gel
containing DMPE-LC-biotin was removed from the plate
and extracted with chloroformimethanol(2:l viv). After
removal of the solvent on a rotary evaporator at 40 "C, the
product was stored a t -20 "C until use. Product purity
was assessed by high-performance thin-layer chromatography on silica gel plates (Whatman, Clifton, NJ) using
chloroformimethanoliwater (75:23:2vol 96 ) as the mobile
phase. The plate was treated with the cinnamaldehyde
and molybdenum sprays as above to detect biotin and
phospholipid. The treatment with molybdenum spray
resulted in two spots, and the cinnamaldehyde spray
yielded a single spot, indicating that the impurity was
unreacted phospholipid. The percent of unreacted phospholipid was estimated by scanning the lane treated with
the molybdenum spray using a GS 300 scanning densitometer (Hoefer Scientific Instruments, San Francisco, CA)
in the reflectance mode and then cutting out and weighing
the peaks resulting from the scan.
VesiclePreparation. Small unilamellar vesicles were
prepared with compositions of DSPCICholIDMPE (40:
40:20 mol % ) and DSPCiCholiDMPEIDMPE-LC-biotin
(40:37.5:20:2.5 mol 7%) using standard sonication procedures (Szoka and Papahadjopoulos, 1980). The vesicles
containing DMPE-LC-biotin were used in the preparation
of horseradish peroxidase-biotinylated vesicles (HBVs),
while vesicles containing no biotinylated phospholipid were
used to prepare horseradish peroxidase-conjugated vesicles
(HVs) to determine the effects of biotin on enzyme
immobilization. Two vesicle preparations, one at each of
the compositions above, were used in the investigation.
Briefly, 60 mg of lipid was dissolved in chloroform/
methanol (2: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 20 mL of 50 mM citrate buffer (pH
6.0) at 70-80 "C. Unilamellar vesicles were formed by
sonicating (Model W-385, Heat Systems Ultrasonics,
Farmingdale, NY) the hydrated lipid suspension a t 70-80
"C for 1 h. Undispersed phospholipid was removed by
centrifuging at 3000g on a fixed-rotor table-top centrifuge
(International Equipment Co., Needham Heights, MA)
for 20 min and filtering the solution through a 0.2-km
Acrodisc filter (Gelman Sciences, Ann Arbor, MI). The
vesicle sizes were determined by quasi-elastic light scattering as will be described later. Vesicles were stored at
4 "C.
HRP Immobilization and Isolation. Horseradish
peroxidase was covalently linked to vesicles using the
periodate method (Nakane and Kawaoi, 1974; Heath et
al., 1980) with modifications as shown in Figure 2. Three
batches of HBVs were prepared by immobilizing HRP to
Bbtechml. Rug., 1993, Vol. 9, No. 3
10;-
245
10;
CH2-Oli
pH 6 . 30 min
C11=0
Room Temp.
I
I
\
\
Figure 2. Chemistry of attaching HRP to vesicle surface:
glycoprotein immobilization method (Heath et al., 1980).
vesicles containing 2.5 mol % biotinylated phospholipid,
while one batch of HVs was prepared by immobilizing
HRP to vesicles without biotin. Peroxidase (50mg) in 5
mL of 50 mM citrate buffer (pH 6.0) was combined with
5 mL of 0.06 M sodium periodate and allowed to react in
the dark for 30 min at 25 "C, during which the periodate
oxidizes the hydroxyl groups of the enzyme carbohydrate
chains to aldehyde groups. Ethylene glycol (5mL of 0.32
M) was then added to the mixture which was kept in the
dark for 1h to neutralize excess periodate a t 25 "C. After
the activated HRP was isolated on a 10-cm Econo-Pac
lODG desalting column (Bio-Rad Laboratories, Richmond,
CA), 5 mL of 3 mg/mL vesicles were added to the HRP,
the pH was adjusted to 9.5 with 1N NaOH, and the mixture
was allowed to react with gentle stirring for 2 h at 25 "C.
Under these conditions,the aldehyde groups on the protein
reacted with the surface amines on the vesicles to form an
unstable imine intermediate. The imine was then reduced
to a stable secondary amine linkage by adding 2-3 mg of
sodium borohydride and incubating overnight a t 25 "C.
The pH was then adjusted to 6.0 with 1N HCl, and the
reaction mixture was concentrated to 2 mL using an
Amicon Centriprep- 10 concentrator. Vesicles were separated from unreacted enzyme by gel permeation chromatography (GPC). The concentrated reaction mixture
was applied to a 1.5 X 70 cm column packed with Sepharose
CL-6B (exclusion limit MW 4 X lo6) in 50 mM CB (pH
6.0) at a flow rate of 0.5mL/min, and 1-mL fractions were
collected. The gel excluded the vesicles and allowed HRP
to penetrate, so that vesicles eluted in the column void
volume, while the unreacted HRP was retarded in the
column. An enzyme assay described in the following
section was used to estimate the amount of enzyme present
in each fraction and to gauge the effectiveness of the
separation of vesicles from free enzyme.
In addition to the covalent attachment of HRP to
vesicles, a control experimentwas performed to determine
the extent of noncovalent binding of HRP to vesicles during
the immobilization procedure. Biotinylated vesicles and
HRP that had not been activated by periodate were
combined in the same proportions as above, incubated a t
pH 9.5 for 2 h, and treated with sodium borohydride
overnight a t 25 "C. The GPC column above was used to
separate vesicles from the free enzyme. The amount of
HRP noncovalently associated with the vesicles was
estimated by enzyme activity as described in the next
section. The immobilization procedure used by Heath et
al. (1980)included the additional step of reacting the lysine
residues on HRP with fluorodinitrobenzene (FDNB) to
change the isoelectric point from approximately 7.7 to
6.5. The reasons for inclusion of this preliminary step
were to minimize the possibility of noncovalent association
of protein with the vesicles by shifting its p l far from the
pH value of 9.5 used in the reaction and to minimize crosslinking between enzyme amines and carbohydrate aldehydes. According to Heath et al. (1980), the FDNB
blocking step resulted in an irreversible loss of 33 % of the
original HRP activity. In order to minimize activity loss,
the FDNB blocking step was omitted in this work. Also,
the HRP was reacted with vesicles immediately after
activation to minimize the homopolymerization of HRP,
which could occur upon storage.
Characterization of Vesicles with Immobilized
HRP. Enzyme activity measurementswere used to gauge
the effectiveness of separating HRP-vesicle conjugates
from unreacted HRP by GPC, as well as to determine
kinetic parameters for native HRP, B-HRP, and the
enzyme immobilized to biotinylated vesicles. Activity
measurements were performed in 50 mM citrate buffer
adopting the procedure outlined by Gallati (1979) and
using ABTS and H202 as substrates a t 25 "C. The HRP
activity in the GPC fractions was measured at pH 4.0 with
2 mM ABTS and 2.75 mM H202. First, 3.2 mL of substrate
was pipeted into sample and reference cuvettes. Next, 10
pL of enzyme solution was added to the sample with
agitation, zeroed against buffer, and the change in absorbance recorded at 410 nm with a Shimadzu 160-Model
UV-visible spectrophotometer. Enzyme solutions from
fractions containing high enzyme concentrations were
diluted between 10and 100times to obtain AA/min values,
and the reported activity of the fraction was based on the
undiluted fraction. After 8-10 fractions containing the
highest concentration of HRP-vesicle conjugates on the
basis of the enzyme assay were pooled, immobilized enzyme
concentrations were determined from the absorbance of
the solutions at 403 nm. A t this wavelength, there are two
contributions to the total absorbance for solutions containing HRP immobilized to vesicles. One is from absorbancedue to the enzyme and the other is from turbidity
due to light scattering by the vesicles. The absorbance
due to the scattering was subtracted from the total
absorbancewhen the immobilized enzyme concentrations
were determined. The HRP concentration was then
calculated using an extinction coefficient st 403 nm of
1.85 mL/mgcm, which was determined by measuring the
absorbance of HRP solutions between 0.02 and 0.10 mg/
mL on a Shimadzu 265-Model UV-visible spectrophotometer.
Activity measurementsto characterize the enzymewere
performed with HBVs, B-HRP, and native HRP. In the
characterization experiments, a fixed hydrogen peroxide
concentration (2.75mM) and various ABTS concentrations
(0.0156-2.0 mM) were used. Kinetic parameters were
determined using the Michaelis-Menten equation, given
Biotechnol. Prog., 1993,Vol. 9,
246
by
V=
m
' a,
[I'
K,+ [Cl
(1)
where Vis the reaction rate, [C] is the ABTS concentration,
V, is the maximum reaction rate, and K , is the Michaelis
constant. The kinetic parameters K , and V,, were
determined from a plot of reaction rate versus substrate
concentration using a nonlinear least-squares fit of the
experimental data. The maximum reaction rate V,,, can
also be written as
V,, = k,[E,l
(2)
where k , is the enzyme specific activity and [E,] is the
enzyme concentration. The enzyme specific activity was
calculated by dividing V,, by the enzyme concentration
used in the assay. Kinetic parameters for native HRP,
batch 3 HBVs, and B-HRP were determined at pH values
ranging from 3.5 to 5.0. In addition, the kinetic parameters
at pH 4.0 for batch 2 HBVs were measured immediately
after preparation and at pH 4.0 and 5.0 after storage at
4 "C for 50 days.
The concentration of phospholipid in a vesicle sample
was determined by phosphate assay using the method of
Chen et al. (1956). This technique relies on the digestion
of phospholipid to inorganic phosphate and COZby HzSO4 and H202. The phosphate then reacts with molybdate
and ascorbic acid to form a blue phosphomolybdate
complex that can be detected spectrophotometrically.
Briefly, 100 pL of solution (unknown or standard) containing between 0.025 and 0.25 pmol of phosphorus was
placed in a 30-mL (18 mm outer diameter) Pyrex test tube
and heated in an aluminum block to 250 "C with 0.5 mL
of 10 N HzS04 for 20 min. After the test tube cooled to
room temperature, six drops of 30% aqueous Hz02 were
added to each sample and the samples were reheated to
250 "C for 30 min. After cooling, 3.9 mL of deionized
HzO, 0.5 mL of 2.5 % aqueous ammonium molybdate and
0.5 mL of 103' % aqueous ascorbic acid were added to each
sample, and the sample tubes were vortexed thoroughly
and heated in a boiling water bath for 7-10 min. After
cooling, the absorbance was measured at 830 nm on a
Shimadzu 265-Model UV-visible spectrophotometer. A
calibration curve was obtained using standard inorganic
phosphate solutions and phospholipid. The molar concentration of total lipid was calculated by dividing the
phospholipid concentration determined using the phosphate assay by the mole fraction of phospholipid in the
vesicle preparation to account for the presence of cholesterol. The HRP to lipid ratio (pgipmoL)was calculated
by dividing the immobilized HRP concentration (mg/mL)
by the lipid concentration (mmolimL).
Hydrodynamic radii of the vesicles before and after
immobilization were determined using quasi-elastic light
scattering (QLS). In this method, the time-dependent
fluctuations of scattered light intensity are measured to
determine the particle diffusion coefficient in dilute
solution (Ford, 1985). For noninteracting Brownian
particles, the average hydrodynamic radius, Rh, can be
calculated from the Stokes-Einstein relationship (Pecora,
1964)
kT
R, = (3)
6rDp
where k is Boltzmann's constant, T is the absolute
temperature, D is the average diffusion coefficient, and p
is the solvent viscosity. Vesicle solutions were prepared
for QLS measurements by centrifuging 2 mL of diluted
No. 3
sample at 500g for 15 min to force residual dust to the
bottom of the borosilicate scattering cell. Measurements
were performed at a 90" scattering angle using a Coherent
Innova 70-3 argon ion laser with a Brookhaven BI-2030
A T 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 standard deviation.
Estimation of the Number of HRP Molecules and
Outer Biotin Molecules p e r Vesicle. The number of
HRP molecules immobilized per vesicle was estimated
from two experimental quantities: the mean HRP-vesicle
diameter and the HRP to lipid ratio in pg/pmol. First,
the vesicle diameter was calculated from the HRP-vesicle
diameter, assuming that the HRP is attached directly to
the vesicle surface with no intervening spaces. The
diffusion coefficient of HRP in pure water at 20 "C is 7.05
x
cm2/sas determined by 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-vesicle
diameter. Next, the total number of lipid molecules per
vesicle, Ntot,was estimated. For spherical unilamellar
vesicles of radius R, with bilayer thickness t and an average
area per lipid molecule A , Ntotis given by (Hutchinson et
al., 1989a)
Ntot
=
4rRV2 4r(R, - t ) 2
A
+ A
(4)
The bilayer thickness was assumed to be 40A (Israelachvili
and Mitchell, 1975;Johnson, 1973). The average area per
lipid molecule was calculated using values of 71, 41, and
19A2for phosphatidylcholine, phosphatidylethanolamine,
and cholesterol, respectively (Israelachvili and Mitchell,
1975), weighted by the appropriate mole fraction of each
component. The A value obtained for the biotinylated
vesicles was 44.8 A2/lipid,while the value obtained for the
nonbiotinylated vesicles was 44.2 A2/lipid. Finally, the
number of HRP molecules per vesicle was calculated by
multiplying the HRP to lipid ratio in pg/pmol by Ntotand
dividing by 40 000, the molecular weight for HRP (Keilin
and Hartree, 1951; Maehly, 1955).
The theoretical maximum number of HRP molecules
per vesicle of a given size can be calculated by dividing the
available packing area per vesicle by the cross-sectional
area per HRP molecule (2800 A2). The radius of the
packing area is equal to the vesicle radius plus the protein
radius. The maximum available area per vesicle for HRP
immobilization can then be calculated by multiplying the
available packing area by 0.91, the packing factor for
hexagonal closed-packed spheres adsorbing to the vesicle
surface. The number of lipid molecules on the outer vesicle
surface, Neuter, given by
4rR:
Nouter
=A
(5)
was multiplied by the mole fraction of DMPE-LC-biotin
(0.025) to obtain the number of biotin molecules on the
external vesicle surface (Nbiotin). The biotin surface density
on the vesicle was calculated by dividing Nbiotin by the
surface area per vesicle to give 5.58 X
molecules/82
(1792 A2imolecule).
Total ABA per Unit Area on Polystyrene Wells.
Measurements of the binding of HBVs and B-HRP to
ABA adsorbed to polystyrene wells, as described in the
following section, were performed at various antibody
surface loadings. The total antibody adsorbed to the
Biotechnol. Prog.. 1993, Vol. 9, No. 3
polystyrene well surface was determined using radiolabeled
antibody. Polystyrene 96-well round-bottom microtiter
plates were obtained from Corning. Wells from the plates
were cut into sets of two to facilitate radiocounting.
Radiolabeled ABA was prepared by reductive methylation
(Jentoft and Dearborn, 1983). Briefly, 50 pL of 6.5 mg/
mL NaCNBH4 was added to 2 mg of antibody in 1mL of
phosphate-buffered saline (PBS). The PBS (Sigma) was
120 mM NaCl and 2.7 mM KC1 in 10 mM NaP04,pH 7.4.
Next, 40 pL of 0.25 pCi/pL [l4C1formaldehydewas added
and allowed to react for 2 h at 25 "C. Unreacted [l4C1formaldehyde was removed by dialysis with four 1-L
changes of PBS over a period of 2 days. The concentration
of antibody was determined using a mass-based extinction
coefficient of 1.4 L/gcm at 280 nm (Sigma).
The specific radioactivity of the labeled antibody was
determined by placing a sample of known concentration
of 20 mL of Scintiverse I1 scintillation cocktail (Fisher
Scientific) and counting on a Packard 1500 liquid scintillation counter. At each ABA concentration tested, eight
wells were incubated with 200 pL of ABA solution per well
at antibody concentrations ranging from 1to 40 pglmL in
PBS for 2 h at 25 O C . After they were rinsed three times
with 300 p L of PBS, the wells were immersed in 20 mL
of Scintiverse I1 scintillation cocktail and counted as above
to determine the total amount of ABA adsorbed per well.
The available surface area per well was calculated from
the manufacturer-specified area (which is based solely on
well geometry) and the additional area resulting from
capillary forces. The difference in height between two
wells, one containing PBS only and one containing 1 wt
5% BSA in PBS, was found to be 1 mm and was used to
estimate the additional area due to the meniscus. The
total available area was thus taken to be 1.63 cm2. The
area per ABA molecule was calculated from the surface
density assuming a molecular weight of 160 000 (Goding,
1986).
HBV and B-HRP Adsorption to ABA-Coated P S
Wells. Polystyrene 96-well round-bottom microtiter
plates were coated with antibody as in the previous section
using 200-pL aliquots of antibody solutions ranging from
1 to 45 pg/mL. Wells were rinsed with a Biotek EL401
platewasher by aspirating the well contents, filling the
wells with 300 pL of PBS wash buffer, and aspirating the
well contents. Wells were rinsed two times using this
procedure. Next, the wells were blocked by incubating
200 pL of 1wt 96 BSA in PBS for 1h at room temperature,
followed by rinsing three times as above. Additional wells
that were blocked only with BSA were used to determine
the extent of nonspecific binding and were treated as
background wells. Aliquots (100 pL) of HBVs or B-HRP
were added to the wells at concentrations ranging from
to 10-13M and allowed to bind. Preliminary investigations indicated that binding was complete after
incubation times of 2 h for vesicles and 1 h for B-HRP,
and these equilibration times were therefore used.
In early measurements it was found that the signal
generated by B-HRP adsorbed to ABA was very sensitive
to the rinsing technique and that the B-HRP was being
rinsed from the wells when aspiration was used as above.
As a result, the wells containing B-HRP were rinsed twice
with 100-pL aliquots of buffer by hand decanting. The
signals obtained with HBVs were not very sensitive to
washing with PBS, and these were rinsed four times with
aspiration as above.
Next, 100 pL of substrate solution containing ABTS
and H202 was added to each well. The substrate concentrations were chosen such that the ABTS concentration
247
2oo
0
;
i
0
20
40
60
80
100
Fraction Number (lml)
120
Figure 3. Elution profile of HRP conjugates from unreacted
HRP on a 1.5 X 70 cm Sepharose CL-6B column after the
periodate coupling reaction. Enzyme activity was measured with
2.0 mM ABTS and 2.75 mM HrOe in 50 mM citrate buffer (pH
4.0) a t 25 "C.
was higher than the Michaelis parameter (K,)determined
in the kinetic assays described earlier, but that substrate
inhibition would be minimized. For HBVs, 2 mM ABTS
with 2.75 mM H202 in 50 mM citrate buffer (pH 5.0) was
used, while for B-HRP 1.0 mM ABTS with 2.75 mM H202
in 50 mM citrate buffer (pH 4.0) was used. The pH of the
substrate solution used for HBVs was chosen to be 5.0
rather than 4.0 because the reaction rate was so fast at
high HBV concentrations when using pH 4.0 buffer that
accurate AA/min values could not be obtained. The AAI
min values were measured on a Biotek EL 340 platereader
at 410 nm at room temperature. The specific signals were
calculated by subtracting the signal obtained in the BSAcoated wells from the signal obtained in the ABA-coated
wells. It was assumed in subsequent calculations that the
specific signal measured in the wells was directly proportional to the number of vesicles (or B-HRP molecules)
adsorbed to the antibody-coated well surface.
The adsorption data for B-HRP and HBVs were fit to
the Langmuir equation, and the maximum surface coverage
of ligand was calculated from the maximum signal
measured in the wells as described in the Results and
Discussion section. In addition, the HBV adsorption data
was fit to an equation that considers the surface exclusion
effects of large ligands adsorbing to the antibody-coated
surface. The adsorption of batch 3 HBVs was performed
immediately after preparation, while the adsorption of
batch 2 HBVs was performed only after storing the vesicles
at 4 OC for 50 days.
Comparison of HBV and B-HRP Signals. The
specific signals from batch 3 HBVs were compared to the
specific signal from B-HRP at ABA surface densities
ranging from 104 to 208 ng/cm2. The HBV concentration
in the wells was 5.0 X
M while the B-HRP concenM. These concentrations were
tration was 2.7 X
chosen to be approximately equal to the inverse of the
association constant. The methods reported in the previous section were used to obtain AAimin values for
adsorbed B-HRP and HBVs, except the pH of the
substrate solution for the vesicles was changed from 5.0
to 4.0. This was done in order to use the optimum substrate
conditions for immobilized HRP.
Competitive Assays Using HBVs and B-HRP.
Corning microtiter plates were prepared for the assays by
adsorbing ABA and blocking with BSAas described earlier.
Batch 3 HBVs and B-HRP were used in competitive assays
for biotin at ABA surface densities of 112 ng/cm2 (bulk
[ABAI 2.5 pg/mL) for HBVs and 138ng/cm2 (bulk [ABAI
4.0 pg/mL) for B-HRP. Aliquots (100 pL) of 1.6 X
M HBVs and biotin at concentrations ranging from 10-14
248
Biotechnol. Prog., 1993,Vol. 9, No. 3
Table I. Results of Immobilizing HRP to Vesicles
batch
HBV 1
HBV 2
HBV 3
Hv
HRP:lipid
(icglrmol)
65
69
-0
I1
a3
diameter (A)
1040 f 240
1010 rt 140
870 f 106
780 i 60
N, ,
(XI0
N
)
1.09
1.02
0.71
0.55
to
M were added to the wells which were then
incubated for 2 h at room temperature. For B-HRP,
aliquots with 2.2 x
M B-HRP and biotin over the
same concentration range above were added to the wells
which were incubated for 1h at room temperature. After
the HBV wells were rinsed four times and the B-HRP
wells rinsed two times, as described earlier, 100 pL of
substrate solution in 50 mM citrate buffer (pH 4.0) was
added. For HBVs, the substrate contained 2 mM ABTS
and 2.75 mM H202, while for B-HRP the substrate
contained 1mM ABTS and 2.75 mM H202, which are the
optimum substrate conditions discussed earlier. A t each
biotin concentration, the signals ( AAimin) from four ABAcoated wells and from four BSA-blocked wells were
measured with the Biotek EL 340 platereader and averaged.
Results and Discussion
Isolation and Characterization of Vesicles with
Immobilized HRP. Conjugate Dimension and Structure. The separation of HRP-conjugated vesicles from
unreacted HRP by size exclusion chromatography with
Sepharose CL-6B for a typical immobilization is shown in
Figure 3. The enzyme activity in the 1-mL fractions was
measured using 2 mM ABTS and 2.75 mM HzOz in 50 mM
citrate buffer at pH 4.0. The vesicles are essentially
excluded from the column packing and elute in the void
volume (37 mL), while the unreacted enzyme elutes a t 87
mL. Resolution (defined as the volume difference between
two neighboring peak maxima divided by the mean of the
peaks' widths) of at least 1.0 is required for adequate
separation (Scopes, 1982). The resolution achieved for
the separation depicted in Figure 3 was approximately 2,
indicating base-line separation.
Table I summarizesthe results of immobilizationof HRP
to biotinylated vesicles (batches 1-3 HBVs) and to
nonbiotinylated vesicles (HVs)in terms of the micrograms
of HRP immobilized per micromole of total lipid, average
hydrodynamic diameter, total number of lipid molecules
per vesicle, number of lipid molecules in the outer bilayer
per vesicle, number of HRP molecules immobilized per
vesicle, and the number of biotin molecules on the outer
vesicle surface. Table I shows that between 65 and 77 pg
of HRP was coupled per pmol of lipid to biotinylated
vesicles, while 83 pg of HRP was coupled per pmol of lipid
to nonbiotinylated vesicles. The biotinylated phospholipid
in the vesicle had little effect on the amount of enzyme
immobilized. These HRP to lipid ratios are approximately
70 % lower than the value of 250 reported by Heath et al.
(1980). The reason for the discrepancy could be due to
the differences in vesicle composition used in the investigations. Those used by Heath et al. (1980) contained 33
mol 3' % phosphatidylethanolamine, while the vesicles in
this work contained 20 mol % DMPE. It might be possible
to increase the amount of HRP immobilized to the vesicle
surface by stirring during the enzyme activation step of
the immobilization, by changing the vesicle composition
in favor of phosphatidylethanolamine, or by changing the
reaction chemistry. These possibilities, however, were not
, , , I ,
(X10
5.94
5.56
3.9
3.1
1)
HRP;
vesicle
theoretical
maximum
HRPjvesicle
177
176
136
114
1067
1003
729
576
biotin;
vesicle
1500
1400
900
investigated. The DMPE-LC-biotin used to prepare the
vesicles contained approximately 20 % DMPE by weight
using scanning densitometry, which was taken into account
when the vesicles were prepared.
It was found from QLS measurements that the HBVs
had mean diameters ranging from 870 to 1040A, while the
HVs had a mean diameter of 780 8, (see Table I). Prior
to protein attachment the biotinylated vesicles exhibited
a mean diameter of 650 f 90 A, while nonbiotinylated
vesicles had a mean diameter of 600 f 85 A. The diameter
after conjugation is expected to be at least 120 A larger
if the vesicle surface is coated with HRP, which has a
diameter of 60 A. Thus, the expected HBV diameter is
770 A, and the expected HV diameter is 720A. The average
HBV diameters are 13-35 % larger than the expected HBV
diameter, while the HV diameter is 8% larger than the
expected HV diameter. The additional growth may be
due to slight vesicle fusion during the immobilization
process or vesicles cross-linking together through HRP
molecules. The likelihood of this occurring was kept small
by using a large HRP to vesicle ratio (-8500 mol of HRPi
mol of lipid) during reaction. The extent of noncovalent
association of HRP with biotinylated vesicles during the
immobilization procedure was estimated by bringing
vesicles into contact with HRP that had not been activated
by periodate, as described earlier in the Materials and
Methods section. The amount of HRP noncovalently
associated with vesicles was found to be less than 1%of
the amount immobilized covalently, indicating little
nonspecific adsorption.
Table I also lists the total number of lipids per vesicle
(Ntot,from eq 4) and the number of lipids in the outer
from eq 5) for each of the immovesicle bilayer (NoUte,,
bilizations. The Ntotvalues were used to estimate the
number of HRP molecules per vesicle, while Neuter values
were used to estimate the number of biotin molecules on
the outer vesicle surface. Values of Ntotfor HBVs ranged
from 1.09 X lo5 lipid molecules per vesicle for batch 1
HBVs to 7.09 X lo4 lipid molecules per vesicle for batch
3 HBVs. The Ntotvalue for HVs was 5.49 X lo4 lipid
molecules per vesicle. Values of NoUte, for HBVs ranged
from 5.94 X lo4 lipid molecules per vesicle for batch 1
HBVs to 3.94 X lo4 lipid molecules per vesicle for batch
3 HBVs. The N,,, value for HVs was 3.10 X lo4 lipid
molecules per vesicle. For HBVs, it was found that
between 177 and 136 HRP molecules could be immobilized
per vesicle, while 114 HRP molecules were immobilized
per vesicle with HVs, as seen in Table I. The theoretical
maximum number of HRP molecules per vesicle for each
vesicle size is also listed in Table I. The experimentally
determined HRPivesicle ratios represent between 17 and
20% of the theoretical maximum number of HRP molecules that can be attached to the vesicles under closepacked conditions. Clearly, conjugates with a large amount
of enzyme label immobilized to the vesicle surface that
still maintain sufficient specificity for affinity binding
would be the most useful in immunoassays.
As shown in Table I, the number of biotin molecules on
the outer vesicle surface with batch 1 HBVs was 1500,
while with batch 2 HBVs it was 1400 and 990 with batch
Biotechnol. Prog., 1993, Vol. 9, No. 3
3.0
3.5
4.0
249
4.5
5.0
5.5
PH
3.0
3.5
4.5
4.0
PH
5.0
I
5.5
0.0
0.5
1.0
1.5
[ABTS] (mM)
2.0
2.5
0.0
0.5
1.0
1.5
[ABTS] (mM)
2.0
2.5
0.3 0.2 0.1 1
t
Figure 4. Enzyme activity as a function of pH at constant ABTS
concentration values ranging from 0.0156 to 2.0 mM with 2.75
mM H 2 0 2in 50 mM citrate buffer a t 25 "C: (a) native HRP, [E,,]
= 7.23 X 10 '> g/L; (b) batch 3 HBVs, [E,,] = 4.46 X
g/L; (c)
B-HRP [E,,] = 3.69 X 10 g/L. ABTS concentrations: 2 ( 0 ) ;1
(0);0.5 (A);0.25 (A);0.125 (+); 0.0625 (X); 0.0132 (0);
0.0156
mM (w).
3 HBVs. The surface density of biotin on the HBV surface
was 5.58 X lo1*molecules/cm2and will be compared to the
ABA surface density in the wells in the next section. Since
HVs do not contain biotinylated phospholipid, no value
is reported for the number of outer biotin molecules per
vesicle.
Kinetics of Immobilized HRP. The HBVs were also
characterized in terms of the immobilized enzyme activity
and compared to native HRP and B-HRP. Enzyme
activity values for native HRP, batch 3 HBVs, and B-HRP
at pH values ranging from 3.5 to 5.0 at 2.75 mM H2Oz with
ABTS concentrations ranging from 0.0156 to 2.0 mM in
50 mM citrate buffer are shown in Figure 4. The enzyme
concentrations used in the assays were 1.8 X 10-loM with
native HRP, 1.1X 10-loM with HBVs, and 9.2 X
M
with B-HRP. The HBV concentration used in the enzyme
assays corresponds to 8.1 X 10-13 M at the enzyme
concentration above. It was found that the pH optimum
was between 4.0 and 4.2 using 1-2 mM ABTS and 2.75
mM Hz02 for all three types of HRP tested. The results
agree with the reported activity optimum for native HRP,
which occurs at pH 4.2 using 2 mM ABTS and 2.75 mM
H202(Gallati, 1979). In addition, the trends in the pH
profile agree with results obtained by Gallati (1979), who
Figure 5. Enzyme activity as a function of ABTS concentration
with 2.75 mM HrOL in 50 mM citrate buffer (pH 4.0) a t 25 "C:
(a) native HRP, [E,,] = 7.23 X
g/L; (b) batch 3 HBVs, [E,,]
= 4.46 X
g/L; (c) B-HRP, [E,,] = 3.69 X D f i g / L . The activity
data from Figure 4 was replotted to determine kinetic parameters.
reported that with higher ABTS concentrations the optima
are shifted toward higher pH values.
Kinetic parameters ( K , and V,,,) were obtained by
replotting the activity data in Figure 4 as a function of
ABTS concentration and using nonlinear least-squares
regression analysis to fit the data with the MichaelisMenten equation. Representative examples for native
HRP, HBVs, and B-HRP at pH 4.0 are shown in Figure
5. Slight deviation from the Michaelis-Menten model
occurs above 1mM ABTS for both native and immobilized
HRP, while for B-HRP the deviation occurs above 0.5
mM ABTS. The decrease in activity is probably due to
substrate inhibition at high substrate concentrations
(Childs andBardsley, 1975). It can be seen that the activity
values are essentially invariant between 0.5 and 2 mM
ABTS, indicating that substrate concentrations in this
range should be used in an ELISA. Michaelis parameters
(K,) for native and immobilized HRP are shown in Figure
6 at pH values ranging from 3.5 to 5.0. It was found that
K , increases monotonically with increasing pH from a
value of 0.085 mM at pH 3.5 to a value of 0.304 mM at pH
5.0. The Michaelis parameter, which is generally associated with the enzyme affinity for substrate, was not
significantly changed by immobilization, indicating that
the enzyme affinity for ABTS and H202 was not affected.
Biotechnol. Prog., 1993, Vol. 9,No. 3
250
250
, ,
,
I "
x
*
.a
C
B
200
150
100
50
8
E
p
I
i
01'
0
'
1
10
20
30
40
Bulk [ABA] (pg/ml)
50
60
Figure 7. ABA adsorbed to Corning round-bottom polystyrene
microplate wells after 2 h a t room temperature versus the bulk
antibody concentration in the wells.
These K, values are slightly lower than the values reported
by Gallati (1979), who reported a constant value of
approximately 0.36 mM at pH values ranging from 3.5 to
4.2, with an increase to approximately 0.75 mM at pH 5.0.
The discrepancy could be due to the use of different buffer
systems, which could affect the enzyme activity (Scopes,
1982). Gallati (1979) used a buffer of 50 mM NaH2P04
with 100 mM NaC1, while 50 mM citrate buffer was used
in this investigation.
Specific activity values (k,) for both free and immobilized HRP are compared in Table I1 at pH values ranging
from 3.5 to 5.0. When compared to native HRP, the
percent specific activity of the immobilized enzyme ranges
from 65 7% at pH 3.5 to 84 % at pH 5.0. A slight decrease
in activity is expected upon immobilizing enzymes to a
support (Klibanov, 1979). The percent specific activity
values for immobilized HRP in Table I1 are substantially
higher than the value of 44% reported by Heath et al.
(1980). A likely cause for activity loss in their report was
the initial FDNB blocking step, which was omitted in this
work. It should be noted that, even though Nakane and
Kawaoi (1974)report no irreversible loss of enzyme activity
using the FDNB treatment, Heath et al. (1980)consistently
observed HRP inactivation after immobilization and
attributed 33 7% activity loss to the FDNB treatment. The
FDNB blocking step is probably not necessary, since the
amount of HRP noncovalently associated with vesicles
was found to be small, and little if any cross-linking was
observed when this step was omitted. The specific activity
values for batch 2 HBVs were measured immediately after
enzyme conjugation and then after storage at 4 O C for 50
days at pH 4.0 only. The initial K, value was 0.240 mM
and the initial k, value was 67 800 (AA/min)/(mg/mL).
These values agree with the values determined for batch
3 HBVs. After storage at 4 O C for 50 days, the K, value
was 0.254 mM and the k, value was 53 000 (AAlmin) (mg/
mL). The Michaelis constant did not change significantly,
while the specific activity decreased by 22%. This
indicates that HRP immobilized to vesicles retains approximately 80% of the initial enzyme specific activity
after 50 days when stored at 4 "C.
Adsorption of ABA to Polystyrene Wells. The
surface concentration of radiolabeled ABA adsorbed to
polystyrene microplate wells is shown as a function of the
bulk antibody concentration used in the incubating
solution in Figure 7. It should be noted that the antibody
adsorption data depicted in Figure 7 might not represent
a true adsorption isotherm since the adsorption kinetics
were not examined. It was found that bulk ABA concentrations between 1and 20 pg/mL resulted in antibody
surface concentrations ( r A B A ) ranging from 60 to 173 ng/
cm2. Higher bulk ABA concentrations did not result in
increased adsorption of antibody. The maximum surface
Table 11. Specific Activity (kp)Values for Native HRP
and Batch 3 HBVs
3.5
3.8
4.0
4.2
4.5
5.0
0.60
0.94
0.93
0.87
0.70
0.32
0.39
0.64
0.66
0.65
0.53
0.27
65
68
71
15
76
84
concentration (rABA,max) of ABA adsorbed was found to
be 208 f 26 ng/cm2at 40 kg/mL bulk ABA concentration.
These results are in agreement with those of Cantarero et
al. (1980),who found that incubation of polystyrene tubes
with bulk bovine IgG concentrations above approximately
10 pg/mL resulted in a surface density between 246 and
277 ng/cm2. The plateau in Figure 6 represents an area
per antibody molecule of 1.28 X lo4Az or a surface density
of 7.83 X lo1' molecules/cm2. The biotin surface density
on the vesicle (5.58 X 10l2molecules/cm2)is about 7 times
larger than the antibody surface density, so that as avesicle
contacts the well surface there is ample opportunity for
specific binding to occur. When vesicles bind to antibodycoated surfaces, many antibody molecules are covered
because of the large difference in size between antibody
and vesicle. For example, a vesicle with a lOOOA diameter
has a cross-sectional area of 7.85 X lo5A2. When adsorbed
to a surface containing 208 ng/cm2 antibody, it follows
that the vesicle would cover approximately 60 antibody
molecules. The implications of the so-called "large ligand
effect" with respect to ligand binding are discussed in the
following sections.
Modeling of HBV and B-HRP Adsorption Data.
The Langmuir model (Langmuir, 1918) was used to
describe the adsorption of ligands (B-HRP or HBVs) to
the antibody-coated surface:
In this equation, r is the ligand surface density (number/
is the ligand surface density at high ligand
cm2), rmax
concentration, K is the equilibrium association constant,
and [Ll is the bulk concentration of adsorbing species.
The association constant is of interest since it quantifies
the strength of binding between antibody and ligand at
a fixed antibody surface density. A ligand concentration
of l / K results in binding one-half of the available surface
binding sites, and K increases as the strength of binding
increases. The ligand surface density was calculated from
Biotechnol. Prog., 1993, Vol. 9, No. 3
25 1
(7)
where [E,], is the effective enzyme concentration from eq
8, u is the volume of the substrate solution in the wells
(100 pL), N , is Avogadro's number, n is the number of
HRP molecules per ligand (e.g., 136 for batch 3 HBVs and
1 for B-HRP), M is the HRP molecular weight (40 000),
and A , is the area of the well exposed to the solution. The
available area at 100 ,uL of well volume, calculated from
the manufacturer-specified area and the additional area
resulting from capillary forces as described in the Materials
and Methods section on total ABA per unit area on
polystyrene wells, was taken to be 1.05 cm2.
The effective enzyme concentration in the wells was
calculated from the specificsignal (S,in AAlmin) measured
in the wells, assuming that the kinetic parameters ( K ,
and k,) reported earlier for the bulk enzyme are the same
when the ligand is adsorbed to the antibody-coated surface.
It must be noted that this assumption may not be strictly
correct, since diffusion limitations of substrate to the
enzyme at the surface could result in underestimating the
HBV (or B-HRP) surface density. Using the MichaelisMenten equation, the effective enzyme concentration in
the wells may be calculated as
where K,, k,, and [Cl were defined earlier and F is a
factor that accounts for the difference in the optical path
lengths of the instruments used in the experiments. That
is, in enzyme activity measurements to determine kinetic
parameters performed on the Shimadzu 160 spectrophotometer the path length was 1 cm, while in experiments
performed on the Biotek 340 platereader the path length
at a volume of 100 p L is 0.4 cm according to the plate
manufacturer (Corning). This was confirmed by comparing the absorbance of an ABTS solution on the
spectrophotometer with the absorbance of the solution on
the platereader with 100 ,uL in the wells. Thus, F was
taken to be 0.4.
The adsorption data for B-HRP and HBVs were fit to
eq 6 using weighted nonlinear least-squares fitting to
determine K. The experimental maximum ligand surface
density (I'max,exp)
was calculated from the signal measured
at high ligand concentration (S,,x,ex,) using eq 7. In
addition, the maximum theoretical ligand surface density
(rmax,theo) was calculated and compared to the experimental
value. For B-HRP, the maximum surface coverage was
estimated from the antibody surface density. Since
antibodies have two specific binding sites per molecule,
and assuming that the adsorbed antibody is fully active,
the maximum B-HRP surface density was estimated as 2
times the ABA surface density. Vesicles are large compared to the adsorbed antibody, and multiple binding can
occur. For example, at an ABA surface density of 104
ng/cm2, batch 3 HBVs cover approximately 26 antibody
molecules. Assuming that vesicles adsorb as disks in a
close-packed configuration to the surface, the maximum
vesicle surface density was calculated as the reciprocal of
the vesicle cross-sectional area multiplied by a packing
factor of 0.91. The experimental percent coverages were
calculated by dividing the I'max,exp
by the corresponding
rmax,theo and multiplying by 100.
The large ligand effect probably does not play a
significant role in B-HRP binding to adsorbed ABA, since
the cross-sectional area of an HRP molecule (2830 A2) is
over 4 times smaller than the cross-sectional surface area
of an ABA molecule adsorbed to the plate at 208 ng/cm2
(1.28 X lo4 A2). On the other hand, the adsorption of
vesicles to an antibody-coated surface is likely to have two
unique features. The first is that, because of the large size
of the vesicles relative to the distances separating antibodies on the surface, multiple-point attachment of
antigens on the vesicle to the antibody are likely at high
antibody loadings on the plate and high antigen loadings
on the vesicle surface. The second is that, since the vesicles
are spherical and large, the adsorption of a finite number
of vesicles per unit area can significantly reduce the
probability that other vesicles can find additional area
with dimensions large enough to accommodate their size.
This phenomenon has been called the large ligand effect,
and some statistical mechanical theories have been developed to estimate its importance (Schaaf and Talbot,
1989; Stankowski, 1983; Andrews, 1976). An equation
describing the fractional surface coverage of vesicles, 6, as
a function of the bulk ligand concentration, [L], is (Schaaf
and Talbot, 1989; Andrews, 1976)
6 = K,,LIP(O)
(9)
where Keffis the effective association constant and P(6)
is a term describing the probability of finding an area
large enough to accommodate a finite sphere. Schaaf and
Talbot (1989) obtained an expression for the probability
term as a function of the fractional surface coverage of the
form
8
The fractional coverage for vesicles, 6, is equal to the
ligand surface density (r)multiplied by the area per vesicle
(a). The Keffand a values were determined by weighted
nonlinear least-squares from experimental plots of r versus
[Ll using eq 9. The theoretical area per vesicle (atheo) was
calculated from the vesicle cross-sectional area and compared to the area per vesicle from the large ligand model.
The percent coverage from the large ligand model was
calculated as (atheo/a)100 and compared to the experimental percent surface coverage, which was described
earlier.
B-HRP Adsorption to ABA-Coated Wells. The
adsorption of B-HRP to ABA-coated polystyrene wells
was examined at r A B A values ranging from 100 to 208 ng/
cm2and with B-HRP concentrations ranging from 91 pM
to 45 nM. Figure 8 shows the adsorption of B-HRP to
ABA at 208 ng/cm2. The signal generated (AAlmin) by
B-HRP in ABA-coated wells is compared to the signal
generated by B-HRP in BSA-coated wells in Figure 8a.
The latter signal can be considered to be a nonspecific or
background signal. The background signal is less than
1% of the total signal generated from the ABA-coated
wells throughout the range of B-HRP concentrations used.
At B-HRP concentrations above 10 nM, the signal
decreases. This could be due to enzyme inhibition
resulting from surface crowding effects (Pesce et al., 1981).
As the surface concentration of labeled enzyme increases,
the distance separating the enzyme molecules decreases.
When the enzyme molecules become closely packed on
the surface, the activity may decrease due to steric
hindrance. The specific signal (Le., total signal with the
background subtracted), shown in Figure 8b, levels out
near a B-HRP concentration of 10 nM, giving a maximum
signal (Smax)
of 0.79 AA/min. Using this S,, in eq 7 results
in a maximum B-HRP surface density value (I'max)of 3.37
X 1O1O B-HRP/cm2. The maximum theoretical B-HRP
surface density at this antibody concentration is 1.56 X
Biorecbnol. Prog., 1993,Vol. 9,No. 3
252
h
.5
5
-
$,
.-6
0.1 -
a
a '
0
0
0
0
0
0.01 -
0
0
0.001 -
0
0
1
104
0
0
l o
0.001
loLo
1o
-~
1o 8
1
0
O
.I
10LO
1OI2
10'
1o-8
[B-HRP] (M)
11
I
a
1
a
a
a
a
10l0
1o
'~
10'
108
[B-HRP] (M)
h
10"
e
1
1
l
.r.
I
l o L oI
I
A
_.....
$2
3
1o
io9
10LO
-~
lo-*
[B-HRP] (M)
1O g
W V I (MI
Figure 8. Adsorption of B-HRP to microplate wells coated with
208 ng/cm' ABA as a function of the B-HRP concentration: (a)
enzyme activity measured in wells coated with ABA ( 0 )and
BSA (0);
(b) specific signal obtained by subtracting the BSA
signal from the ABA signal; (c) B-HRP surface density calculated
from eq 7 ( 0 )and with the Langmuir equation, K = 5.0 X IO*
' (-).
M
Table 111. Summary of Binding Parameters for B-HRP
Adsorbing to ABA-Coated Wells
total ABA
exptl
surface
r,,,,,
r,,,,,, ,,,
percent
(ligand/
density
s,,, (ligand/
(ngicm ) (PAimin) cm- X 10 I") cm- X 10 I - ) coverage
\
208
185
179
169
162
144
134
100
(I
0.791
0.766
0.856
1.67
1.80
0.94
0.20
0.02
3.37
3.27
3.65
7.12
7.68
4.01
0.85
0.085
1.56
1.39
1.35
1.27
1.22
1.09
1.01
0.76
2.2
2.4
2.7
5.6
6.3
3.7
0.84
0.11
0.50
0.48
0.44
0.27
0.34
0.45
nd
nd
K was determined from the Langmuir model.
lo1*B-HRP/cm2, indicating that B-HRP occupies only
2.2% of the total antibody binding sites.
One possible explanation for the low apparent surface
coverage by B-HRP is that the enzyme kinetics for B-HRP
bound to ABA might not be the same as for bulk enzyme,
Figure 9. Adsorption of batch 3 HBVs to microplate wells coated
with 208 ng/cm2 ABA as a function of the HBV concentration:
(a) enzyme activity measured in wells coated with ABA ( 0 )and
BSA (0);
(b) specific signal obtained by subtracting the BSA
signal from the ABA signal; (c) HBV surface density calculated
from eq 7 (a),with the Langmuir equation, K = 2.1 X IOy M-I
(-), and with the large ligand model, Kerf= 9.6 X loHM-I (- - -).
as described earlier. For example, homogeneous assays
for antigen (e.g., theophylline, phenytoin, and phenobarbital) have been developed in which binding between
antibody and enzyme-labeled antigen results in enzyme
inhibition (Pesce et al., 1981). In these assays, the antibody
reacts with the antigen-enzyme conjugate and inhibits
the enzyme activity when no free antigen is present. In
the presence of free antigen, the antibody is displaced and
the enzyme activity approaches its uninhibited value. Also,
the radiolabeling technique used to determine the ABA
surface density only detects total antibody, not active
antibody. If some of the antibody becomes inactive
through adsorption to the polystyrene well surface, the
maximum theoretical B-HRP density would be overestimated. Plant et al. (1991) examined the adsorption of
anti-theophylline antibody to polystyrene in terms of the
total antibody adsorbed as well as the active antibody and
found that only 10% of the adsorbed antibody was active.
The activity of the adsorbed ABA could be determined
Blotechnol. hog., 1993, Vol. 9, No. 3
253
10
m
1
I
#
.
.9
a "
a m
'
0.001
lo'*
0
0
I
10"O
WBVI (M)
1o-8
108
[HBVI (MI
Figure 10. Adsorption of batch 3 HBVs to microplate wells
coated with 104 ng/cm2 ABA as a function of the HBF
concentration: (a) enzyme activity measured in wells coated with
ABA ( 0 )and BSA (0);
(b) specific signalobtained by subtracting
the BSA signal from the ABA signal; (c) HBV surface density
calculated from eq 7 (a),with the Langmuir equation, K = 6.0
X 109 M-I (-), and with the large ligand model, Keff= 2.5 X 109
M-' (- - -).
using radiolabeled biotin, although these measurements
were not performed.
Another reason for low surface coverage could be the
dissociation of B-HRP from ABA during rinsing. In
preliminary work, it was found that the signal generated
by B-HRP adsorbed to ABA was very sensitive to the
rinsing technique, and B-HRP eluted from the wells with
repeated washing steps. While the practical minimum
washing volume (300pL) was used to rinse nonspecifically
bound B-HRP from the wells, the possibility remains that
some specifically bound B-HRP could have been removed
during the washing step. The B-HRP surface density as
a function of the bulk B-HRP concentration is shown in
Figure 8c. The Langmuir equation was used to fit the
adsorption data in Figure 8c, giving an association constant
of 5.0 X los M-l.
Table I11 lists the values for S m a x , I"ax,exp, rmax,theo,
percent coverage,and the association constants for B-HRP
at r A B A values ranging from 208 to 100 ng/cm2. At an
ABA surface density of 208 nglcm2,the S m a x value is 0.79
AAlmin,increases to 1.8 AAlmin at an ABA surface density
of 162 ng/cm2, and decreases to 0.02 AAlmin at an ABA
surface density of 100 ng/cm2. This trend indicates that
there is an optimum surface density near an ABA surface
density of 165 ng/cm2, where the maximum signal is
approximately 1.8 AAlmin. Others have reported similar
optima. For example, Engvall and Perlman (1972) observed an optimum in an assay for anti-human serum
albumin with different surface concentrations of human
serum albumin. The percent fractional surface coverage
values in Table I11show the same trend as the S,, values,
increasing from 2.2% at an ABA surface density of 208
ng/cm2 to a value of 6.3% at an ABA surface density of
162ng/cm2and then decreasing to 3.7 % at an ABA surface
density of 144 ng/cm2. The K values listed in Table 111
do not change much as the ABA surface density changes,
indicating that the association constant is not sensitive to
the antibody surface density between 208 and 144 ng/cm2.
As the antibody surface density decreases below 140 ngl
cm2,the specific signal drops rapidly and is too scattered
to determine a value of the association constant.
HBV Adsorption to ABA-Coated Wells. The adsorption of HBVs to ABA-coated polystyrene wells was
examined at ABA surface densities ranging from 71 to 208
ngIcm2 with HBV concentrations ranging from 1 pM to
6 nM. Figure 9 shows the adsorption of batch 3 HBVs to
ABA at 208 ng/cm2. The signal generated (AAlmin) by
HBVs in ABA-coated wells is compared to the signal
generated by HBVs in BSA-coated wells in Figure 9a.
When compared to the total signal generated from the
ABA-coated wells, the background signal varies from 5%
at 1 pM HBV concentration to 25% at 6 nM HBV
concentration. The percent background signals with
HBVs are about 10 times larger than the percent background signals with B-HRP. The higher background
signals can probably be attributed to nonspecific adsorption by vesicles. For example, hydrophobic interaction
could occur between the bilayer membrane and the exposed
hydrophobic residues of adsorbed protein or unblocked
polystyrene surface. The specific signal (i.e., with the
background subtracted), shown in Figure 9b, levels out at
value of 2.8
an HBV concentration of 3 nM, giving an SmaX
AAlmin. For this SmaX
value, the maximum HBV surface
density is 3.26 X lo9 (HBV/cm2). The maximum theoretical HBV surface density for batch 3 vesicles is 1.53 X
1O1O (HBVIcm2),indicating that the HBVs cover 21% of
the available surface area.
Hutchinson et al. (1989b) investigated the adsorption
of wheat germ agglutinin-conjugated vesicles to glycophorin A-coated surfaces and reported 78% coverage when
small unilamellar vesicles (diameter, 600-1200 A) adsorbed
to the surface and 10% coverage with vesicles prepared
by reverse evaporation (diameter, 2000 A). Vesicle flattening was suggested as a possible explanation for the low
surface coverage by the reverse evaporation vesicles. They
alsoreported that, for vesicleswith broad size distributions,
larger vesicles were preferentially adsorbed to the surface,
which could decrease the percent coverages. These
conflicting results indicate that ambiguity exists as to the
extent of coverage by vesicles adsorbing to surfaces.
Low surface coverage by vesicles could also be attributed
to surface exclusion effects (Schaaf and Talbot, 1989), in
which surface saturation by large disk-shaped ligands
occurs at surface coverages of between 45 and 60%. Figure
9c shows the HBV surface density data fits with the
Langmuir model and the large ligand model. The two
models are nearly indistinguishable and give association
254
Biotechnol. Prog., 1993,Vol. 9, No. 3
Table IV. Summary of Binding Parameters for HBVs Adsorbing to ABA-Coated Wells
total ABA
surface
Smax
rmex.exp
exptl percent
Langmuir large ligand
surface coverage Ka (nM-') Kerfb(nM-I)
batch density (ng/cm2) (AAlmin) (ligand/cm2X
3.26
21
2.1
0.96
3
208
2.8
1.0
1.16
7.6
6.0
2.5
3
100
2
208
2.1
2.18
18
7.2
4.2
2
164
1.8
1.86
16
6.2
3.5
1.35
12
8.3
5.1
2
138
1.3
4.4
10
5.1
0.48
0.497
2
104
0.016
0.14
nd
nd
2
71
0.015
0
a
(cmZ/ligand X 1010)
1.02
2.95
2.29
2.62
3.82
8.64
nd
a/atheo
1.6
4.5
2.6
3.0
4.3
9.8
nd
K was determined from the Langmuir model. * Keffwas determined from the large ligand model.
constants of 2.1 X lo9M-l for the Langmuir model and 9.6
X 108 M-l for the large ligand model. The Langmuir
association constant is about an order of magnitude larger
than the K value for B-HRP, indicating stronger binding
by the vesicles. The higher K value is probably a result
of multipoint attachment by vesicles. Locasio-Brown et
al. (1990) reported similar results for theophylline and
theophylline-derivatized vesicles adsorbing to an antitheophylline-coated surface. The association constant for
theophylline was 2.1 X lo7M-l, while for vesicles prepared
with 5 mol % theophylline-derivatized phospholipid the
association constant was 8 X 1O1O M-l. The area per ligand
( a ) from the large ligand model is 1.02 X 10-locm2/HBV,
which is 1.6 times larger than the area per ligand assuming
a close-packed monolayer of vesicles (6.53 X 10-l1 cm2/
HBV). This a value corresponds to an HBV surface
density of 9.8 X lo9HBV/cm2and a surface coverage which
is 64 % of the maximum possible coverage. This surface
coverage is three times the surface coverage determined
from the kinetic analysis discussed above. The difference
between the experimentally determined surface coverage
and the surface coverage from the large ligand model could
be attributed to occlusion of enzyme molecules on the
underside of vesicles adsorbing to the surface. If vesicles
flatten on the surface after adsorption, as many as onehalf of the enzyme molecules on the vesicle surface could
be buried and unable to react with substrate.
HBV adsorption to wells coated with 104 ng/cm2 ABA
is shown in Figure 10. The signal from ABA-coated wells
in Figure 10a is lower than the signal from ABA-coated
wells at 208 ng/cm2(Figure 9a), and the background signal
increased slightly. As a result, the percent background
signal is larger when HBVs adsorb to 104ng/cm2and ranges
from 14% at 1 pM HBV concentration to 50% at 3 nM
HBV concentration. Figure 10b shows that the specific
signal levels out at a vesicle concentration of 1.6 nM at an
Sma,value of 1.0 W m i n . For this Sma, value, the
maximum HBV surface density is 1.16 X lo9 HBV/cm2.
The maximum theoretical HBV surface density for batch
3 vesicles is 1.53X 10l0HBV/cm2,indicating that the HBVs
cover 7.6% of the available surface area. This percent
coverageis almost 3 times lower than when the ABA surface
density is 208 ng/cm2. Figure 1Oc shows the HBV surface
density with the Langmuir model and the large ligand
model. As before, the two models are nearly indistinguishable, giving association constants of 6.0 X lo9M-' for
the Langmuir model and 2.5 X 109M-' for the large ligand
model. The area per ligand ( a )from the large ligand model
for this case is 2.95 X 10-locm2/HBV,which is over 4 times
larger than the theoretical value of 6.53 X lo-" cm2/HBV.
This value of a indicates a surface coverage which is 22 %
of the maximum and about twice the surface coverage
determined from the kinetic analysis (7.6%). The low
surface coverage by HBVs and the large area per vesicle
at an antibody surface density of 104 ng/cm2are probably
due to the low ABA surface density, and not size exclusion
a
a
0
1000
10
20
30
40
Bulk [ABA] (pdml)
50
I
I
1
Bulk [ABA]
10 (pgiml)
I
100
Figure 11. Comparison of batch 3 HBV signal with B-HRP
signal as a function of the bulk ABA concentration used to coat
the wells. The HBV concentration was 5.0 X 10-10 M and the
M. (a) Specific signal for
B-HRP concentration was 2.7 X
HBVs ( 0 )and B-HRP (0).
(b) Ratio of the specific signals.
effects. As the antibody surface density decreases, the
distance between adsorbed vesicles increases. Another
reason for the low percent coverage by vesicles may be
lower ABA activity on the well surface at low antibody
loadings. When antibody adsorbs to the well surface at
low ABA concentrations, molecules occupy more space on
the polystyrene support and could tend to denature more
readily than when adsorbed to the surface at high ABA
concentrations.
Table IV summarizes the values for S,
percent
coverages, K, Keff,and a for batch 3 and batch 2 vesicles
at ABA surface density values ranging from 208 to 104
ng/cm2. When the ABA surface density decreased, the
Sm,, rm,,exp,
and percent surface coverage decreased. For
batch 2 HBVs the S, value (2.1 AAlmin) is 25% lower
than the Sm, for batch 3 HBVs (2.8 AAlmin) adsorbing
to 208 ng/cm2ABA. As noted earlier, the specific activity
of batch 2 HBVs decreased by 22% after 50 days at 4 "C,
when the adsorption data was collected. Thus, the lower
signals measured for batch 2 HBVs are probably due to
less active enzyme. The rmar,exp
values for batch 2 HBVs
listed in Table IV are divided by the theoretical maximum
HBV surface density (rmax,theo) of 1.14 X 1O'O HBV/cm2
to compute the percent coverages. The percent coverage
256
Blotechnol. h ~1993,
. , Vol. 9, No. 3
I' """1 ' """1
1.2
' ''"'7
1
"
t
0.4
€
T
0
s
0.6
i
t t t
1
i
1
1::
,,,,,, ,,,,,*,
~
1
0
10-15
10-13
10-l~
10.~
10'~
lo5
[biotin] (M)
Figure 12. Competitive assay results (a) normalized specific
signal (S/S,) of 1.6 X 10-lO M batch 3 HBVs in competition with
M at an ABA
biotin in concentrations ranging from 10-14to
surface density of 112 ng/cm2;(b) normalized specific signal (S/
M B-HRP in competition with biotin in
SJ of 2.2 X
concentrations ranging from 10-14to
M a t an ABA surface
density of 138 ng/cm2.
values decrease from 18% at 208 ng/cm2 to 4.4% a t 104
ng/cm2. These values are comparable to the percent
coverages for batch 3 HBVs at similar ABA loadings.
The association constants for HBVs at ABA surface
densities between 208 and 104ng/cm2as determined from
the Langmuir model and the large ligand model are
summarized in Table IV. The association constants do
not appear to be very sensitive to the antibody loading.
The differences between the K values reported in Table
IV for the two vesicle preparations are not considered
significant. The association constants for batch 2 HBVs
were slightly larger than the association constants for batch
3 HBVs at a given ABA surface density, possibly indicating
that larger vesicles bind more strongly to the surface. The
association constants determined for vesicles are approximately an order of magnitude larger than the B-HRP
association constant, indicating that vesicles bind more
strongly to the antibody-coated surface. The high association constants exhibited by HBVs are probably due to
multiple ligand binding. By virtue of their large size,
vesicles cover many antibody binding sites and are able
to bind through multipoint attachment to the surface.
When the ABA surface density was 71 ng/cm2, the total
and background signals were nearly equal, making it
difficult to determine the association constant. The area
per ligand values (a)from the large ligand model are shown
in Table IV and are compared to the theoretical area per
ligand (l/rm=,theo). The area per ligand increases as the
ABA surface density decreases,which agrees with the trend
shown by the rmax,exp
values. The experimentally determined percent coverages are approximately one-half of
the percent coverages determined from the large ligand
model. As discussed earlier, this discrepancy could be
explained by occlusion of enzyme molecules upon vesicle
adsorption.
Comparison of HBV and B-HRP Signals. The
specific signals generated by HBVs and B-HRP at ABA
surface densities ranging from 104 to 208 ng/cm2 are
compared in Figure l l a . The HBV concentration was 5.0
X 10-lO M, and the B-HRP concentration was 2.7 X 10-9
M. These concentrations were chosen to be approximately
1/K of the conjugated species, as determined from the
Langmuir model, to give a representative view of the signal
generation. The ABTS and H202 concentrations used for
the HBVs were the same as in the adsorption experiments
(2.0 and 2.75 mM, respectively), but the pH was changed
from 5.0 to the optimum 4.0. The signal ratio (HBV/BHRP) in Figure l l b is replotted from the data in Figure
l l a . Above an ABA surface density of approximately 175
ng/cm2,the ratio is constant at approximately 5. At this
antibody surface density, vesicles bound to the surface
cover many ABA molecules, and the signal ratio is low
since B-HRP can bind to the ABA individually. As the
ABA surface density decreases below 175 to 104 ng/cm2,
the signal ratio increases to a limiting value of about 200.
A ratio of at least 100 was expected, since there are
approximately 100 HRP molecules per vesicle. The
additional increase in signal at low ABA surface coverage
results from relatively low B-HRP signals as the ABA
surface density decreases below 140 ng/cm2. Figure 11
shows that vesicles having multiple enzyme labels could
give signal enhancements of at least 100 times compared
to singly labeled antigen. The highest ratio is achieved at
a low surface concentration of binding sites because vesicles
bind more strongly to the surface than B-HRP, even at
low antibody surface densities, and vesicles with multiple
enzymes generate a higher signal than a single enzyme
molecule bound to the surface.
Competitive Assay Results. The specific signal (S)
from the adsorbed enzyme label (HBVs or B-HRP) was
calculated by subtracting the signal obtained in BSAfrom the signal in ABA-coated wells
coated wells (SBSA)
(SABA).
The results of the competitive assays for biotin
at concentrations from 10-14to 10-1M are shown in Figure
12, in which the specific signal (S) has been normalized
by the average maximum specific signal (So)and plotted
versus the biotin concentration. The average maximum
specific signal was calculated from the specific signals at
low biotin concentration. The So value for HBVs was 1.4
AAlmin, while for B-HRP it was 0.3 AAlmin. For HBVs,
the response (S/So)was constant between 10-14and lo-"
M biotin and then decreased monotonically to 0.3 &I/
min at 10-1 M biotin. For B-HRP, the response was
M biotin and then
constant between
and
decreased sigmoidally to 0.001 AAlmin at lo4 M biotin.
Although the vesicles did not exhibit the sigmoidal shape
characteristic of competitive assays (Campfield, 1983),
probably due to multiple binding of HBVs with the ABAcoated surface, they clearly competed with free biotin.
The ABA surface density used for vesicles was 112 ng/cm2
(bulk [ABAI 2.5 pg/mL), while the ABA surface density
for B-HRP was 138ng/cm2 (bulk [ABAI 4.0 pg/mL). The
error bars in Figure 12 are the standard deviations
calculated from the four replicates at each biotin concentration (Bevington, 1969).
The least detectable dose (LDD) and the slope of the
linear region are two criteria used to assess the performance
of competitive immunoassays (Campfield, 1983). The
LDD is the lowest antigen concentration that results in a
Biotechnol. Prog., 1993,Vol. 9, No. 3
256
displacement of the response equal to twice the standard
deviation (CT) of the SIS, value (SIS, - 2a). A lower LDD
indicates that a lower antigen concentration can be
detected. The slope of the curve in the linear region is an
indication of the sensitivity of the assay (Aresponsel
Aantigen concentration). A large negative slope is desired.
The LDD for HBVs was 1.6 X 10-9 M, while for B-HRP
it was 3.6 X 10-9 M. This indicates that the detection
limit when using HBVs is at least as low as when using
B-HRP and could be improved under optimum assay
conditions. The slope in the linear region for HBVs was
-0.011 M-l, while for B-HRP it was-0.027 M-l. Multipoint
binding of vesicles to the ABA-coatedsurface would require
a higher biotin concentration to displace the vesicles and
therefore increase the slope. One way to overcome this
effect is to lower the amount of DMPE-LC-biotin in the
vesicles. Another way to minimize multiple binding is to
perform the assay at a lower antibody surface density.
The most important result in Figure 12 is that these
bifunctional vesicles can compete effectively with small
ligands for surface antibody binding sites. Work is
currently underway to optimize the assay conditions.
Conclusions
Bifunctional vesicles having both an enzyme label and
a small model antigen were prepared by conjugating HRP
to small unilamellar vesicles prepared with 2.5 mol %
biotinylated phospholipid. After conjugation, the enzyme
specific activity decreased between 35% and 15% when
compared to native enzyme, but the pH optimum at 4.0
with 1.0-2.0 mM ABTS and 2.75 mM H202 was unchanged
from native HRP. The HBVs had on the order of 1000
biotin molecules and 100 HRP molecules per vesicle. The
association constants from the Langmuir model for HBVs
adsorbing to ABA-coated microplate wells are about 10
times larger than the association constant for B-HRP,
indicating that vesicles bind more strongly than B-HRP,
probably due to multipoint attachment. The background
signals for the HBVs are about 10 times higher than the
background signals for B-HRP, most likely due to nonspecific adsorption through the hydrophobic interaction
of vesicles with the protein-coated surface.
Although not investigated in this work, blocking agents
other than BSA (milk proteins, detergents, serum proteins)
can be tested to minimize nonspecific adsorption. The
maximum percent surface coverages by HBVs were 21 7%
at an ABA surface density of 208 ng/cm2, and they
decreased to 4-8% at an ABA surface density of 104 ngl
cm2. These estimates were obtained by assuming that the
kinetic parameters determined for the immobilized enzyme
in bulk solution were the same as when the vesicles were
adsorbed to the well surface. The actual surface coverages
by vesicles might be higher than these estimates suggest,
since enzyme molecules on the side of the vesicle adsorbed
to the surface could be occluded or because of diffusion
limitations of substrate to the enzyme. Work is currently
underway to determine the vesicle surface coverage with
more certainty.
The large ligand model fit the HBV adsorption to ABA
at 208 and 104 ng/cm2. The adsorption of HBVs and
B-HRP to ABA-coatedsurfaces indicated that at low ABA
surface density the signal from HBVs was about 100 times
higher than the signal from B-HRP. This suggests that
vesicles could be useful in both sandwich-type ELISAs
and competitive ELISAs. In sandwich assays low bulk
concentrations of analyte would result in a low density of
surface binding sites. Since the association constant for
vesicles is larger than for singly labeled antigen, a lower
concentration of vesicles can be used in a competitive
ELISA, and this could result in improved detection limits.
Competitive assays for biotin were performed using HBVs
or B-HRP as enzyme-labeled antigen. It was found that
the HBVs competed effectively with biotin for ABA surface
binding sites. Even though the LDD and sensitivity were
comparable to the results obtained using B-HRP, we
believe there is considerable room for improvement in these
results by optimizing the assay conditions.
Additional work is required to explore and optimize the
performance of HBVs in immunosorbent assays. While
numerous immunoassays have been developed that utilize
vesicles with encapsulated label molecules, to the best of
our knowledge no ELISAs have been developed using
vesicles with surface-immobilized enzymes. Multiple
binding between HBVs and the ABA-coated surface must
be minimized in order for vesicles to compete effectively
with free antigen. This can be controlled by performing
the assay at low antibody surface densities and at low
antigen levels in the vesicle bilayer. Also, lower background signals would allowlower antibody surface densities
to be used. This could be achieved by blocking the wells
with a more suitable agent (i.e., milk proteins, detergents,
serum proteins). These aspects of assay optimization will
be described in a later publication.
Notation
a
atheo
A
ABA
ABTS
Aw
B-HRP
biotin-LCNHS
[CI
Chol
D
DMPE
DMPE-LCbiotin
DSPC
[E,]
[E01w
ELISA
F
FDNB
HzO2
HBV
HRP
HV
K
k
Keff
K,
k,
well surface area occupied per vesicle from the
large ligand model
theoretical well surface area occupied per
vesicle
average area per lipid molecule in vesicle
bilayer
anti-biotin antibody
2,2’-azinobis(3-ethylbenzthiazoline-6-sulfonic acid)
area of well exposed to 100 p L of solution
biotinylated horseradish peroxidase
biotinylimidohexanoicacid succinimidoester
substrate (ABTS) concentration
cholesterol
average diffusion coefficient
dimyristoylphosphatidylethanolamine
dimyristoylphosphatidylethanolamine coupled to biotin
distearoylphosphatidylcholine
enzymeconcentrationin activitymeasurement
effective enzyme concentration adsorbed to
wells
enzyme-linked immunosorbent assay
ratio of path length in the spectrophotometer
to the path length in the platereader
fluorodinitrobenzene
hydrogen peroxide
horseradish peroxidase conjugated to biotinylated vesicles
horseradish peroxidase
horseradish peroxidase conjugated to vesicles
association constant for HBVs or B-HRP
adsorbing to ABA-coated wells from the
Langmuir equation
Boltzmann’s constant
effective association constant from the large
ligand model
Michaelis constant
enzyme specific activity
Biotechnol. Prog., 1993, Vol. 9,No. 3
[Ll
LDD
M
n
N*
Nbiotin
Smax,exp
so
T
t
V
Vmax
bulk concentration of HBVs or B-HRP in
adsorption experiments
least detectable dose
H R P molecular weight
number of H R P molecules per HBV or B-HRP
Avogadro's number
number of biotin molecules in outer leaflet of
vesicle bilayer
number of lipid molecules in outer leaflet of
vesicle bilayer
total number of lipid molecules per vesicle
phosphate-buffered saline
quasi-elastic light scattering
hydrodynamic radius
vesicle radius
specific signal ( S ~ -A
signal measured in ABA-coated wells
signal measured in BSA-coated wells
experimentally determined maximum specific
signal a t high HBV or B-HRP concentration
specific signal for HBVs or B-HRP measured
in competitive assays a t low biotin concentrations
absolute temperature
bilayer thickness
enzyme reaction rate
maximum enzyme reaction rate
Greek Letters
r
FABA
rABA,max
rmax,exp
rmax,theo
e
P
surface density of HBVs or B-HRP
antibody surface density in wells
antibody surface density in wells a t high
antibody concentration
experimentally determined surface density of
HBVs or B-HRP a t high concentration
theoretical maximum surface density of HBVs
or B-HRP
fractional surface coverage of vesicles
solvent viscosity
Acknowledgment
T h e authors gratefully acknowledge AKZO Corporate
Research America, Inc., for t h e financial support of this
work.
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Accepted January 21, 1993.