Note: Descriptions are shown in the official language in which they were submitted.
f
Jo
1~39~8~
--1--
LIPID-VESICLE-SU~.FACE ASSAY Regilt AND METHOD
. _
Background and Summary
The following publications are referred to by
corresponding number in this application:
1. Seiko, F. or., and Papahajopoulos, D., Ann. Rev.
Boyce. Bit I, 9:467 508 (1980).
2. Seiko, F ., Jr. and Papahajopoulos~ D., Pro. Nat.
clue. USA, 75:4194-4198 (1978).
lo 3. Heath, T. D., Matcher, B. A. and ~apahadjopoulos, 3.,
Biochimica et Biophysics Act, 640:66-~1 ~1981).
4. Martin, F. J., Hubbell, W. L., and Papahadjopoulos,
D., iochem sty, 20:4229-4238 ~1~81).
5. Martin, F. J. and Papahadjopoulos, D., J. Blot.
Chum., 257:286-2B8 (1982).
6. Furman, B., Klareskog, L., and Peterson, P. A ,
J tot Chum., 255:7820-7826 (1980).
7. Smith, B. A. and McConnell, H. M., Pro. Nat. Aged.
Sat. USA, 75, 2759-2763 (1978).
8. Lowry, O. H., Rosebrough, N. J., Fern, I. I,., and
Randall, R. J., J. Biol._Chem., 193:265-275 tl951).
The present invention relates to a
lipid-vesicle-surface assay reagent, and to enzyme
immunoassay methods using such a reagent.
A variety of methods for determining the
presence or concentration of biochemical analyzes is
known. The analyze to be assayed typically is one which
plays an important role in biochemical processes. Dow
molecular weight substances, such as peptize and steroid
hormones, vitamins and the like, and high molecular
weight substances such as carbohydrates and proteins are
commonly assayed analyzes.
Several important analyze assay techniques are
based on a reaction between the analyze and an
35 anti-analyte capable of binding the analyze with high
affinity and specificity. Typical analyze anti-analyte
., .
~3958~
binding pairs include an~igen-antibody,
immunoglobulin-protein A, carbohydrate-lectin,
biotin-avidin, hormone-hormone receptor protein and
complementary oligo- and polynucleotide strands. The
terms ~ligand~ and ~antiligand~ will be used herein to
designate the opposite binding members in such a binding
pair
Among the various types of assays which employ
specific binding reactions, enzyme immunoassay provide
a number of advantages in sensitivity, low cost and
simplicity. In one type of enzyme immunoassay, an
enzyme-ligand reagent is reacted in the presence of a
ligand or ligand-like analyze with a solid support
having anti-ligand binding sites carried on its surface,
lo wherein the analyze and ligand-enzyme reagent compete
for binding to the solid support. In another type of
enzyme immunoassay, a ligand analyze is capable of
binding both to anti-analyte binding sites on a solid
support, and to an anti-ligand in a anti-ligand-enzyme
reagent, to couple the reagent to the support in a
sandwich fashion. In both assay types, the amount of
analyze present is determined by separating the liquid
and solid (support) phases, and measuring the enzyme
activity associated with one or both phases. Where the
reagent and analyze compete for binding sites on the
solid support, the enzyme activity associated with the
support is inversely proportional to the concentration
of analyze present. On the other hand, a direct
relationship between the amount of analyze and the
enzyme activity associated with the solid support is
observed where the analyze functions to join the reagent
to the support by sandwiching.
Commonly, the reagent used in an enzyme
immunoassay test includes an enzyme molecule covalently
coupled to a ligand or anti-ligand molecule to form a
bimolecular pair. This type of reagent has limited
I 3 9 I
sensitivity, inasmuch as each reagent binding event is
reported by one enzyme molecule only. This limitation
has prevented general application of the enzyme
immunoassay technique to cell typing based on the
detection of selected cell surface antigens, except in
cases where antigen surface concentrations are quite
large. Another limitation is that the bimolecular
reagent must be formed from a relatively pure ligand
preparation. Otherwise, a significant portion of the
reagent (the portion composed of enzyme coupled to
non-ligand molecules) will not bind, or will bind
nonspecifically, to the solid support. As a result, a
high background attributable to unbound reagent (in the
liquid phase) and nonspecifically bound reagent (in the
solid phase) will be observed.
An enzyme immunoassay reagent composed of lipid
vehicles coated with ligand molecules and encapsulating
enzymes within the interior vehicle spaces has been
proposed in the prior art. A reagent of this type may
be relatively expensive to manufacture due to the
recognized problems of encapsulating enzymes within
liposomes efficiently. Further, many enzymes appear to
undergo loss of activity during encapsulation (reference
1). The encapsulating vehicles must be lucid before
enzyme activity associated with the vehicles can be
measured. Complement has been used for lying lipid
vehicles, but this method often lacks reproducibility
due to complement inactivation on storage. Detergent
louses has been used, but this approach may be unsuitable
for applications --e.g., cell typing-- where the cell
Cypriot to which the vehicles are bound is itself
susceptible to detergent louses.
3~5~3~
According to an aspect of the invention there is
provided a method for detecting the presence of analyze
molecules carried on biological cells, comprising: providing
a suspension of lipid vehicles having surface bound anti-
analyze molecules, at a surface concentration of at least
about 15 molecules per vehicle, and surface-bound reporter
molecules, incubating the suspension with the sample cells,
to bind the vehicles to the cell surfaces by specific
analyte/anti-analyte binding interactions, separating the
cells from unbound vehicles, and detecting the presence of
reporter associated with the cells.
According to a further aspect of the invention
there is provided a system for determining the presence of
a selected cellular antigen carried on the surface of a cell
comprising a soluble antibody specific against the antigen, and
a suspension of lipid vehicles having surface bound antibody
or antibody-fragment molecules which are specific against the
soluble antibody, at a surface concentration of at least about
15 molecules per vehicle, and surface-bound reporter molecules.
The reagent of the invention is composed of lipid
vehicle particles having a highly mobile, or fluid surface
array of ligand and enzyme molecules. The particles are
preferably lipid vehicles in the 0.05 to 10.0 micron diameter
size range, and include an average of at least about 15
ligand molecules and up to several thousand enzyme molecules
bound to each vehicle surface. The ligand molecules may
include one or more substantially pure ligand species, or may
include impure mixtures thereof.
The method of the invention includes reacting the
reagent with a separable support to produce
3 9
-5-
-
partitioning of the reagent between the support and
liquid reaction phases according to the concentration
of analyze present.
The invention also contemplates an assay kit
including a separable support and the reagent.
These and other objects and features of the
invention will become more fully apparent from the
following detailed description of the invention.
Detailed Description of the Invention
Preparation of Lipid Vehicles
The assay reagent of the present invention is
composed of closed lipid vehicles, each having attached
to its outer surface a laterally mobile array of enzyme
and ligand molecules. Typically, the lipid vehicles
take the form of lipid Baylor structures encapsulating
an aqueous interior region, such structures also being
referred to as liposomes. The properties and methods of
preparation of lipid vehicles have been detailed in the
literature. The reader is referred particularly to
above-numbered references 1 and 2, and references cited
therein, for a comprehensive discussion of the topic.
What will be described herein are preferred methods of
preparing liposomes used in forming the reagent of the
invention, and liposome properties which contribute to
the advantages of the invention.
Lipid vehicles are prepared from lipid mixtures
which typically include phospholipids and strolls. A
list of phospholipids used typically in liposome
preparations is given on page 471 of reference 1. One
consideration which determines the choice of lipids used
is the degree of fluid mobility and lipid packing
density which is desired in the vehicles formed. As
reported in a number of literature reports, these
characteristics can ye varied according to the lengths
and degree of saturation of the aliphatic chains in the
lipids, and the ratio of stroll to aliphatic chain
39
--6--
lipids used The significance of surface fluid mobility
in the the vehicle reaccent of the invention will be seen
below. Packing density characteristics are important to
the success of reactions used to attach Lund and
enzyme molecules covalently to the vehicle surfaces.
For example, it has been found that the inclusion of at
least about 10% mole per cent of cholesterol is
important for the success of certain protein-coupling
reactions which will be described below. The fluidity
and packing characteristics will also affect the size
and number of bowlers in the vehicles produced.
The vehicle lipid composition is also selected
to produce a requisite number of specific lipid head
groups through which the surface-bound reagent
components can be coupled to the vehicles. The head
groups, or necessary modifications thereof, may be
formed in the prepared liposomes, or in the individual
; lipids before incorporation into the liposomes.
Examples of lipids used in preferred coupling reactions
will be discussed below.
The number of and type of polar lipid groups
may also be selected to produce a desired charge
distribution on the lipid vehicles at a selected pi and
ionic strength. The charge distribution may affect the
relative reactivities of enzyme and ligand molecules in
their coupling to lipid vehicles, as will be seen in
Example III, and is an important feature in minimizing
non-specific binding of the reagent vehicles to charged
solid supports, as will be discussed.
A typical lipid composition used in preparing
lipid vehicles for the reagent of the invention
preferably includes between about 10 and 50% cholesterol
or other stroll, between about 2 and 50~ of glycolipid
or phospholipid to which the enzyme and ligand molecules
of the reagent can be individually coupled, with the
remainder lipid composed of a neutral phospholipid, such
as phosphatidylcholine, or a phospholipid mixture.
Charged lipids, such as phosphatidylserine/ phosphatidic
acid, ylycolipids and charged cholesterol derivatives
such as cholesterol hemisuccinate or cholesterol sulfate
may be included to produce a desired surface chary in
the lipid vehicles.
he lipid vehicles may be formed by one of a
variety of methods discussed particularly in reference
1. Multilamellar vehicles --that is, vehicles composed
of a series of closely packed Baylor lamely-- can be
prepared by drying a mixture of lipids in a thin film
and hydrating the lipids with an aqueous buffer. The
size and number of lamely in the formed lipid vehicles
can be controlled, within limits, by varying the
hydration time and amount of agitation used in hydrating
the lipids. Where desirable, vigorous agitation, brief
sonication or extrusion through polycarbonate membranes
can be employed to obtain smaller and more uniformly
sized multilamellar vehicles.
Small unilamellar vehicles having diameters of
about 0.05 micron or less can be formed by sonicating a
suspension of large multilamellar vehicles either by
probe or bath sonication. Another technique for
producing small unilamellar vehicles involves the
removal of detergent from a deteryent-phospholipid
mixture by dialysis. Typical detergents include shalt
and deoxychola~e. An alternative method for the
preparation of small unilamellar vehicles that avoids
both sonication and detergents employs an ethanol
injection step in which lipids dissolved in ethanol are
rapidly injected into a buffer solution. similar
technique in which phospholipids dissolved in
ether-containing solvents has been used to produce large
unilamell~r v~sicles with a generally heterogeneous size
distribution
on one preferred method of preparing large
~395~3~
--8--
unilamellar vehicles, referred to as reverse phase
evaporation, a desired composition of lipids is
dissolved in a suitable organic solvent such as deathly
ether, isopropyl ether, or a solvent mixture such as
isopropyl ether and chloroform I n aqueous
solution is added directly to between about 3 and 6
volumes of the lipid-solvent mixture and the preparation
is sonicated for a brief period to form I homogeneous
emulsion. The organic solvent, or solvent mixture is
removed under reduced pressure, resulting in the
formation of a viscous, gel-like intermediate phase
which spontaneously forms a liposome dispersion when
residual solvent is removed by evaporation under reduced
pressure. The size of the resulting vehicles may be
varied according to the amount of cholesterol included
in the lipid mixture. The reader is referred to
references 1 or 2 for further details concerning the
reverse phase evaporation technique.
The lipid vehicles prepared may be obtained in
a defined size range by various techniques. Methods for
reducing size heterogeneity in smell unilamellar
vehicles by gel filtration and ultra centrifugation have
been described. A method of reducing the size and the
size heterogeneity of lipid vehicles by extrusion
through polycarbonate filters having selected pore sizes
is described in reference 2. The latter method is
advantageous because of its simplicity and because
essentially all of the vehicles are recovered, the
larger ones being converted to desired-sized smaller
vehicles by passage through the filter.
It can be appreciated from the foregoing that
lipid vehicles having a desired size range, morphology,
deformability, fluid mobility and surface charge and
reactivity characteristics may be prepared by proper
selection of lipid components and preparative
techniques.
I
Enzyme and Ligand Coupling
to Lipid Vehicles
This section is concerned with techniques used
to couple ligand and enzyme molecules covalently to
surface lipids in lipid veslcles. The ligand molecules
in the reagent function to bind the reagent to
anti-ligand binding sites on a separable support. ho
used herein, the term ~ligand~ refers broadly to either
species in a binding pair composed of a target molecule
having one or more specific epitopic features, and a
target-binding molecule which recognizes such features
to bind the target molecule specifically and with a high
affinity. ~Anti-ligand~ refers to the other of the two
species in the binding pair. Among the binding pairs
which are contemplated by the present invention are
antigen-antibody, immunoglobulin-protein A,
carbohydrate-lectin, biotin-avidin, hormone-hormone
receptor protein and complementary nucleated strands.
More generally, the ligand may include any fragment or
portion of a ligand molecule which is capable of
participating with the opposite member of the pair in
specific, high affinity binding. For example, in an
antibody-antigen paint the binding ligand may include
the antigen binding Phoebe or Fob' fragments. As
another example, in the protein A-immunoglobulin pair,
the target ligand may include only the Fc immunoglobulin
fragments. According to an important feature of the
present invention, relatively impure ligand mixtures
containing as little as 0.5 to 20 mole percent of
specific ligand molecules may be employed in the vehicle
reagent.
The enzyme in the reagent includes an enzyme
which can function to produce a measurable enzyme
activity in the presence of suitable substrate(s) and
necessary cofactor(s) with the enzyme covalently to the
outer surface of a lipid vehicle. Preferably the enzyme
I
--10--
can be obtained in pure or near-pure form and is
relatively staple on Starkey in solution, or is
resistant to freezing or lyophilization. For most
applications enzymes whose activity can be expressed by
an easily detectable color change will be preferred.
Representative classes of enzymes contemplated herein
include oxidoreductases, typified by luciferase, glucose
oxidize, galactose oxidize and kettles; hydrolyses,
typified by various types of phosphatases; glycoside
hydrolyses, such as beta-galactosidase; peptidases; and
leases.
One enzyme which has been used advantageously
in the reagent of the invention is beta-galactosidase
derived prom a bacterial source. Among the advantages
of this enzyme are: (1) the enzyme is available in
purified Norm with high specific activity; (2) the
enzyme contains free they'll groups that can be joined to
reactive lipids without affecting the enzyme activity;
and (3) both fluorogenic and chromogenic substrates are
available. the relatively low molecular weight of the
enzyme with respect to lipid vehicles allows for the
attachment of a relatively large number of enzyme
molecules on each vehicle, as will be seen below.
Two or more enzyme species may be attached to
the lipid vehicles in accordance with the present
invention. The plural enzymes may function
independently, or cooperatively, as where the product
generated by one enzyme is used as the substrate by
! another.
Several methods are available for coupling
biomolecules covalently to the polar head groups of
lipids. As a general consideration, it is important to
select a coupling reaction which does not significantly
reduce the enzymatic activity or ligand binding activity
of the molecules being coupled. At the same time it is
important to select a method which produces a relatively
3~3t5~;~
high coupling efficiency. In this regard, where the
enzyme and ligand components are coupled to the vehicles
in simultaneous reactions, the relative reactivities of
the two species toward the lipid sites must be taken
into account. finally, care must be exercised to avoid
reactions which would produce significant cross linking
of the vehicle lipid components to each other, or of the
individually - coupled ligand or enzyme molecules Jo one
another, since any cross linking of the reagent
components (except for the individual lipid surface
component conjugations would reduce the fluid mobility
of the surface lipids and attached molecules. Without
intending to limit the scope of the invention, two
preferred methods of coupling biomolecules, particularly
proteins, to lipid vehicles will be described herein
The first method involves Schiff-base formation
between an alluded group on the lipid or molecule to be
coupled, and a primary amino group on the other of the
two reactants. The alluded group is preferably formed
by peridot oxidation. The coupling reaction, after
removal of the oxidant, is carried out in the presence
of a reducing agent. although the non lipid molecule
being coupled may be oxidized, more commonly it is the
lipid group which is the alluded precursor since
peridot treatment inactivates many proteins. Typical
aldehyde-lipid precursors include lactosylceramide,
trihexosylceramide, galactocerebroside,
phosphatidylglycerol, phosphatidylinositol and
gangliosides.
In practice, the vehicles are oxidized by
peridot 'or a period sufficient to produce oxidation
of a majority of the oxidizable lipid groups, and
thereafter the vehicles are separated from the peridot
by column gel filtration. Alluded groups on the
vehicle surfaces are conjugated with a primary amine,
such as a Lawson group in a protein, to form a Showoffs
{
I 1%
! -12-
base which is subsequently reduced with sodium
bordered or sodium cyanoborohydride to form a more
stable bond. Typically, for conjugation reduced with
sodium bordered, oxidized lipid vehicles at a
concentration of between about 5 and 10 micro moles of
total lipid are mixed in 1 ml with 10 to 30 milligrams
of protein at an alkaline phi The reaction is carried
out for about 2 hours at room temperature. For
conjugation reduced with sodium cyanoborohydride, the
reaction typically is carried out over longer reaction
times. The reader is referred to reference 3 for
additional details.
Using lipid vehicles prepared by reverse phase
evaporation and extruded through a 0.2 micron pore-size
polycarbonate membrane, up to about 200 micrograms of
I immunoglobulin G (Gig) per micro mole of lipid vehicle
lipid can be attached to the vehicle surfaces by the
above method. Based on a calculated number of about 1.2
x 1012 vehicles per micro mole of vehicle lipid, this
conjugation ratio corresponds to about 600 Gig molecules
per lipid vehicle. Studies conducted in the support of
the present application indicate that correspondingly
smaller molecules can be coupled to lipid vehicles in
correspondingly larger numbers. Thus up to about 1800
Fob' antibody fragments per lipid vehicle (in the 0.2
micron diameter range) can be attached. The method has
wide applicability, due to the general availability of
primary amine groups in proteins and other biomolecules
which can be reacted with oxidation-produced aldehydes
in selected lipids.
second general coupling technique is
applicable to thiol-containing molecules, involving
formation of a disulfide or thither bond between a
vehicle lipid and the molecule attached. the technique
is particularly useful for coupling Phoebe and Fob'
antibody fragments to lipid vehicles.
-13-
In the disallowed interchange reaction,
phosphatidylethanolamine is modified to provide a
pyridyldithio derivative which can react with an exposed
they'll group in a protein or other biomolecule. The
reader is referred to reference 4 for a detailed
discussion of reaction conditions used in the method.
us reported there, a coupling ratio of up to 600
micrograms of Fob' antibody ruminates per micro mole of
phospholipid can be achieved Based on calculations
lo similar to those presented above, this number
corresponds to about 6000 Fob' antibody molecules per
JO . 2 micron diameter vehicle.
The thither coupling method, which it
described in detail in reference 5, is carried out by
115 incorporating in the lipid vehicles a small proportion
of a sulhydryl-reactive phospholipid derivative, such as
No (p-maleimidophenyl) bitterly)
phosphatidylethanolamine MOPE The lipid vehicles
ware reacted with a thiol-containing protein to form an
essentially irreversible thither coupling between the
protein isle group and the MPB-PE maleimide group. It
is noted that the requisite protein they'll group may be
endogenous to the protein or may be introduced on the
protein by amino-reactive they'll groups according to
known methods. coupling ratios of up to about 350 my of
sulfhydryl containing protein per micro mole of lipid
vehicle phospholipid have been obtained.
It is also contemplated herein that enzyme or
ligand molecules can be separately and individually
attached to lipid vehicles by first coupling the
molecules covaiently to free lipids dispersed in a
detergent solution. The lipid-enzyme or lipid-ligand
couples are then incorporated into lipid vehicles,
either during vehicle formation or by diffusion into
preformed vehicles according to known techniques.
Alternatively the ligand itself may contain an
-14-
endogenous hydrophobic region --for example, a
hydrophobic stretch of amino acids-- by which the Lund
can be incorporated into the surface of a lipid
vehicle. As an example, it has been shown that human
transplantation antigens can be attached to egg lecithin
vehicles by anchoring hydrophobic peptize regions in the
antigens to the vehicles (reference 6).
t is further contemplated that immumoglobulin
or immunoglobulin fragment Lund molecules can be
attached to lipid Yesicles through pair-specific binding
to anti-immunoglobulin antibodies, or fragments thereof,
or to protein A covalently attached to the vehicles.
! The enzyme and ligand molecules may be coupled
to (or incorporated into) lipid vehicles either
sequentially, in separate coupling reactions, or in
simultaneous reactions. Sequential coupling is
indicated where different reactions are used to couple
enzyme and ligand molecules to the lipid vehicles, or
where the relative conjugation reactivities of the two
species is difficult to control. The latter problem may
arise, for example, where the reactivity of either
species varies significantly during the reaction period.
In one typical protocol, ligand or a
ligand-containing mixture, is first reacted by an
I above-described coupling reaction, with vehicles to
produce a desired vehicle surface concentration of the
analyte-speciEic ligand. after the initial coupling
reaction has been completed, the vehicles are separated
from unrequited Lund molecules, then reacted with
enzyme molecules. One advantage of the present
invention is that the Tuscaloosa may be easily separated
from the unrequited solution components of the coupling
reaction(s) by centrifugation, facilitating intermediate
purification steps that may be required during reagent
preparation.
The ratio of analyze specific ligand to enzyme
:~3~582
-15-
molecules coupled to the vehicles is generally selected
to maximize the signal-to-noise ratio in an enzyme
immunoassay employing the reagent. Studies on the
kinetics and specificity of vehicle reagent binding to
various types of separable supports suggest that two
countervailing factors are important in maximizing
reagent performance. on one hand, a minimum surface
concentration of analyte-specific ligands is required to
effect stable reagent binding to a support. Binding
affinity generally increases as the surface
concentration of ligand molecules increases from an
average of about 10-15 molecules per vehicle up to about
50-100 ligand molecules per vehicle (of average diameter
of about 0.2 microns). On the other hand, as the number
of ligand molecules bound to the lipid vehicles is
increased, particularly where the ligand traction
coupled Jo the vehicle is relatively impure, the number
of sites available for enzyme attachment to the vehicles
is reduced. Ideally, as in the case where a relatively
pure ligand preparation is coupled to the lipid
vehicles, the vehicles can easily accommodate 100 or more
ligand molecules and several times that number of enzyme
molecules.
Binding studies done in support of the present
application indicate that at least two, and probably
three or more reagent ligand molecules must bind
specifically to the separable support binding sites in
order to produce stable attachment of the vehicle to the
support. The surface concentration of ligand molecules
required to promote such stable multi site vehicle
binding to a macro molecular support can be quite low, on
the order of about I molecules per vehicle. This
feature is believed to be due to in part to the highly
mobile, or fluid nature of the lipid-bound surface
molecules on the reagent vehicle surfaces. Diffusion
constants of the order of owe o 10-9 cm~Jsec for
I
-16-
phospholipid diffusion within lipid bowlers have been
measured reference I
Binding efficiency may be further enhanced
where the binding sites on the solid support are
S themsel Vow carried on lipid vehicles. Here the combined
mobility of the ligand molecules on the reagent lipid
vehicles and the binding site molecules on the separable
support lipid vehicles would facilitate multi-site
binding a low ligand and anti-liyand (binding site)
surface concentrations.
nether important advantage of the reagent
invention is the relatively high surface packing of
covalently attached molecules which is achievable in the
reagent vehicles. As noted above, it is possible to
attach up to several thousand protein molecules on a
vehicle surface, based on a protein molecular weight of
around 50,000 and a vehicle diameter size of about 0.2
microns. Thus a 0.2 micron vehicle having an average ox
about 50 Fall fragments carried on its outer surface can
carry nearly 100 times that number of an enzyme having
about a 50,000 molecular weight.
Alternatively, where the analyte-specific
ligand being coupled to the vehicles constitutes as
little as one percent of the total non-enzyme molecules
attached to the surface, each vehicle can still
accomoda~e up to several hundred or more enzyme
molecules, producing a vehicle whose enzyme to ligand
molar ratio is still substantially greater than one.
Other advantages of the instant reagent in an enzyme0 immunoassay will be considered below.
Assay Methods
The method of the invention comprises reacting
a liposome surface reagent with a separable support
carrying ~nalyte-related binding-site molecules. The
reagent binds to the support in proportion to the amount
of analyze present.
I
-17-
.
s used herein, separable support refers to any
support structure capable of being readily separated --
for example, by differential centrifugation,
precipitation or electrophoretic separation -- from
S analyze and vehicle reagent components not bound to the
support. another feature of the support is that
binding-site molecules can be attached to its surface.
The separable support may include a water-insoluble
solid support, such as one formed of glass, cellulose,
agrees, polystyrene and the like. Macro molecular
tissue homogenate structures, intact cells, and cell
membrane structures are other contemplated supports.
Also as detailed above, the support may include
surface-bound lipid vehicles to which the support
bindincJ site molecules are attached.
he analyte-related binding site molecules on
the support are selected to bind specifically to the
reagent Lund molecules, or to the analyze molecules,
or to both, depending on the type of enzyme immunoassay
I method, as considered below. The binding site molecules
may be adsorbed to the solid support, or may be
covalently attached thereto by means of a suitable
coupling reaction which may involve the use of
conventional linking agents such as glutaraldehyde.
Methods of forming solid supports having a wide variety
of attached molecules, being well known to those skilled
in the art, will not be detailed herein.
Some types of solid supports are known to have
surface irregularities, such as cavities or crevices,
which may be inaccessible to reagent liposome particles
of the type contemplated herein. The fewer binding
sites on the support available for liposome binding can
result in a proportionate reduction in assay
sensitivity. Further, the solid support may have
I surface properties which tend to promote non-specific
attachment of the liposome resent, leading to a
:~3i9~
-18-
decreased signal-to-noise ratio in the assay.
The two problems just mentioned may be reduced
or eliminated by employing a solid support in which the
binding site molecules are carried on lipid vehicles
which are themselves attached to the support. Two
representative methods by which lipid vehicles can be
attached to solid supports will now be described.
In first method, glass surfaces, for instance
lass tubes or controlled-pore glass beads, are
I derivitized with glycerol, activated with
carbonyldiimidazole and converted into amino-ylass by
reaction with excess Damon Al Kane. The amino-glass is
converted into pyridyl depth glass by reaction with
N-succinimidyl 3 (2-pyridyldithio~ preappoint. The
pyridyl depth glass is then reduced with dithiothreitol
or 2-mercaptoethanol to yield a glass surface with trio
functions.
To achieve reversible attachment of liposomes,
the lipid vehicles, prepared to include
N-(3-(2-pyridyldithio)propionyl)phosphatidylethanooilmen
POPE synthesized according the method described in
reference 4 r are reacted with the trio glass at a pub
between about 7.0 and 8.5. The disulfide bond which
forms between the glass and the vehicles can be cleaved
by mild reduction, for example with dithiothreitol at
low pi.
Irreversible attachment of vehicles to a glass
support Max be achieved by reacting the trio glass with
lipid vehicles prepared to contain MPE-PE, as described
in reference 5, to form a thither linkage.
It is also contemplated that lipid vehicles may
be attached to a solid support nonequivalently through
specific, high affinity ligand/anti-ligand binding. As
one illustration, avid in molecules are attached
covalently to a solid support using conventional
methods. Lipid vehicles prepared to contain
I
-19-
biotinylated surface lipids then bind with high affinity
to the support. Binding site molecules may be attached
to, incorporated into, or formed with the lipid
vehicles, according to above described techniques.
Attachment of binding site molecules to lipid
vehicles carried on a solid support may increase the
accessibility of the liposome reagent to the binding
sites on the solid support. Another advantage inherent
in this approach is that the vehicles to which the
binding site molecules are attached may themselves be
prepared to have a selected surface charge character,
with respect to the reagent liposome particles, for
enhancing specific binding, and reducing non-specific
binding, between the liposome reagent and the solid
support. Because the binding site molecules are
themselves supported in highly mobile vesicle-surface
arrays reagent binding to the solid support may be
facilitated.
Three general types of enzyme immunoassay tests
employing a solid support in conjunction with the
reagent vehicles will now be described. In a first type
of test the reagent vehicles carry analyze or
analyte-like ligands which compete with analyze in
solution for binding to anti-analyte binding sites on
the solid support. The analyze, and the ligand attached
to the liposome reagent, may be a target-type antigen
which compete for binding to an anti-analyte attached to
the solid support, or the analyze may be a binding
protein which competes with binding proteins on the
reagent fox binding to target type binding sizes on the
solid support. In both instances, the amount of
liposome reagent binding to the solid support varies
inversely with the amount of analyze present.
The assay reaction is carried out in a suitable
reaction medium which may include a biological specimen
582
-20-
fluid, such as serum, containing the analyze. The pi of
the reaction medium is one which is compatible with
ligand/anti-ligand binding reactions, and preferably
between about S and 9. More specifically, the pi and/or
ionic strength of the reaction medium may be adjusted to
achieve a desired charge interaction between the
liposome reagent and the solid support. Generally it
can be said that the greater the charge repulsion
between the reagent and the support, the less the
reagent will bind both specifically and nonspecifically,
to the support. It is often an advantage to carry out
the binding reaction at a pi and ionic strength which
minimizes charge repulsion between the support and the
reagent, and to remove nonspecifically-bound reagent
later by a washing step, usually with a low-ionic
strength, high pi washing solution.
The reaction medium may also be adjusted to
have a specific gravity which approximates the buoyant
- density of the reagent particles. Typically, this can
be achieved in a medium having a specific gravity
between about 1.0 and 1.2. The adjustment in specific
gravity, by reducing the tendency of the vehicles to
float or sink in the medium, promotes the requisite
contact between the vehicles and the solid support.
Alternatively, it may be advantageous in some assay
methods to employ a liposome surface reagent which is
either more or less dense than the reaction medium, to
facilitate separation of the liposome reagent from the
support, or to achieve some other advantage related to
reagent partitioning.
Sensitivity in the assay requires reacting a
defined amount of solid support with a known selected
amount of the liposome reagent. With too little
liposome reagent added to the reaction mixture, the
binding sites on the solid support can accommodate a
substantial quantity of bound analyze without any
82
-21-
observed analyte-dependent displacement of the liposome
reagent from the support. With too much liposome
reagent added, excess unbound liposome reagent competes
with the Anita for binding to any displaced binding
S sites on the support. The assay background Allah tends
to be high with too much liposome reagent, due to
nonspecific binding to the solid support and excess
reagent in the liquid phase In most assays, the
optimal amount of liposome reagent is determined by
titrating a given amount of solid support with liposome
reagent to an end point which just indicates saturation
or near saturation of the support binding sites.
The method can be carried out as a single
reaction in which the solid support, a defined amount of
liposome reagent, and the analyze are coincubated for a
period sufficient to produce binding equilibrium among
the reaction components. Typical reaction times range
from about 5 minutes to several hours, at temperatures
ranging preferably from about room temperature up to
37C or somewhat higher.
Alternatively, the assay may be performed as a
two-step reaction in which the analyze is reacted first
with the solid support alone, after which the separated
solid support is reacted with the liposome reagent. The
two-step test may be advantageous where the volume of
original solution to be assayed is quite large, or where
that solution contains substrates or inhibitors of the
liposome reagent enzyme. The two-step reaction also has
the advantage that the second reaction in which the
liposome particles bind to the solid support can be
carried out in a selected reaction medium having a
desired phi ionic strength and specific gravity.
Upon completion of the assay reaction, the
solid support is separated from the liquid phase of the
reaction medium/ including the unbourld suspended
liposomes, and the support or the liquid phase, or both,
are assayed for enzyme activity. To reduce the level of
nonspecifically bound liposome reagent, the separated
support is preferably washed one or more times with a
washing solution whose pi and ionic strength act to
increase charge repulsion between the solid support and
the liposome reagent.
In a second type of enzyme immunoassay
contemplated herein, the solid support carries an
analyze or analyte--like binding molecule which competes
lo with the analyze for binding to the ligand on the
liposome reagent. The analyze, and the binding-site
molecules on the support may be either a target-type
antigen which compete for binding to a ~arget~binding
ligand on the liposome, or the analyze may be a
target-binding molecule which competes with the solid
support for binding to an antigen-like ligand on the
liposome reagent. Various considerations relating to
the phi ionic strength and specific gravity of the
reaction solution which have been discussed above are
applicable to the instant method. Lookers the
procedure used for optimizing the amount of liposome
reagent added to a given amount of solid support is
similar to that already discussed.
A third general assay type is a sandwich
technique in which the liposome reagent is bound to a
support through a multivalent analyze. The assay is
preferably performed as a two-step method in which
analyze is first reacted with the support, after which
the separated support is reacted with the liposome
reagent. The analyze may be either an antibody or an
antigen, with the solid support and liposome reagent
each carrying an opposite binding pair of the analyze.
One advantage of this method is that the amount of
liposome reagent bound to the solid support is directly
proportional to the amount of analyze present. The
method can thus be used to detect very small quantities
I
-23-
of analyze Additionally/ since the liposome reagent is
bound to the solid support through analyze sandwiching
the amount of liposome added to a given quantity of
solid support can be less than the saturating or near
saturating amounts required in the previously described
tests. In turn, the lower concentration of liposome
reagent leads to an improved siynal-to-noise ratio in
the test
The considerations relating to pi, ionic
strength and specific gravity in the reaction medium are
similar to those discussed above and will not be
described further here.
One aspect of the invention which can be
appreciated from the above is the provision of an
immunoassay kit which includes the liposome reagent of
the invention. ALSO included in the kit is a separable
support of the type described above, having
surface-attached, analyte-related binding site
molecules which may be directly bound to the support or
attached to the support surface through lipid vehicles,
as described above. The binding site molecules may be
analyze or analyte-like molecules which compete with the
analyze for binding to anti-analyte ligand molecules in
the surface reagent. Alternatively, the binding site
molecules may be an anti-analyte species, where 'the
analyze and analyte-like reagent ligand compete for
binding to the support, or where the reagent is bound to
the support in sandwich fashion through the ligand.
The assay methods just described are intended
for detecting the presence or concentration of a free
analyze in a sample solution. According to another
aspect of the invention, the reagent is used in an
enzyme immunoassay for determining the presence or
concentration of cell-specific surface antigen
analyzes. were the separable support includes a
biological cell, and more specifically, a cell membrane
.
i,, '
3S~32
-24-
whose outer surface carries the antigen. Typical
analyzes include blood type specific antigens carried on
the surface of blood cells, species and strain specific
surface antigens carried on the surface of various
animal tissues, and surface antigens characteristic of
particular cellular transformation states in various
tissues or in tissue culture. Alternatively, the
analyze may include anti-cell surface antigen antibodies
attached to the cell by incubation with the free
antibody.
In the cell-surface antigen assay, the cell
sample to be assayed is adder to defined amounts of
liposome reagent having analyte-recognition molecules.
After a suitable reaction time, under reaction
conditions which may be selected in accordance with the
considerations mentioned above, the ceils and bound
liposome reagent particles may be separated from the
unbound liposomes which remain suspended in solution by
differential centrifugation. The separated cells are
then washed to remove non-specifically bound liposomes
and the enzyme activity associated with the washed cells
is determined. Other antigen-bearing supports, such as
viral particles, spores, tissue structure, or other
suspendible particulate matter which can be separated
readily from unbound reagent and soluble components
--for example by differential centrifugation or
precipitation-- are also contemplated herein
From the foregoing it can be appreciated how
the liposome reagent of the invention contributes to the
improved signal-to-noise ratio achievable in various
types of enzyme immunoassay tests. A high signal level
is achieved by virtue of the large number of enzyme
molecules which report each binding event in the
assay AS seen, up to several thousand enzyme molecules
can be attached to a vehicle which binds to a support
through a small number of binding sites on the support.
-25-
The fact that the enzyme whose activity is being
measured is bound to a liposome surface may further
enhance the signal level. Several enzymes are known to
have increased activity in an immobilized state, a
phenomenon thought to be related to favorable surface
reaction kinetics. Immobilized enzymes are often less
susceptible to inactivation as well.
The noise level in the assay methods described
is reduced by limiting non-specific binding of the
liposome reagent to a separable support. This may be
accomplished, according to the invention, by reacting
the liposome reagent with a solid support under pi and
ionic strength conditions which favor specific binding
between the two, followed by exposing the separated
lo support to a washing medium which removes
non-specifically bound liposomes through a charge
repulsion effect.
The improved signal-to-noise ratio is observed
where the ligand molecules in the reagent represent only
a small fraction of the total non-enzyme molecules
carried on the reagent vehicles, and where the liposome
reagent carries two or more distinct types of ligand
molecules or more than one type of enzyme.
The following examples describe particularS embodiments of making and using the invention.
Example I
Lipid Vehicle Preparation
The following procedure was used to produce a
suspension of lipid vehicles containing the
sulfhydryl-reactive phospholipid derivative MPB-PE.
The synthesis of MPB-PE was performed substantially as
described in reference S. Briefly, transesterified egg
Pi was reacted with freshly distilled triethylamine and
succinimidyl 4-(p-maleimidophenyl) bitterroot in an hydrous
methanol under an argon atmosphere at room temperature
for two hours The POPE formed was purified by
3~35~
-26-
silicic acid chromatography.
Large, unilamellar vehicles were prepared by a
reverse phase evaporation method described generally in
references 1 and 2. Cholesterol (10 micro mole),
phosphatidylcholine (9.5 micro mole), MPB~PE (.5
micro mole) and a trace amount of initiated
dipalmitoylphosphatidylcholine were dissolved in 1 ml of
deathly ether. A buffer, at pi I containing 20 my
citric acid, 35 my disodium phosphate, 108 my sodium
chloride and 1 my ETA was added (300 micro liter) r and
the two phases emulsified by sonication for one minute
at 25C in a bath sonicator. Ether was removed under
reduced pressure at room temperature and the resulting
dispersion was extruded successively through 0.4 micron
and 0.2 micron Unhip polycarbonate membranes (Byrd
Laboratories, Richmond, CA).
The lipid vehicle preparation was examined by
electron microscopy. Most of the vehicles were in the
0.2 micron diameter size range and had one or a few
Baylor lamely. Based on the microscopic examination
of the vehicles, and the known lipid concentration
thereof, a vehicle concentration of about 1.2 x 1012
vehicles per micro mole of lipid was calculated.
Example II
_ uplin~ of Fob' Fragments to Vehicles
This example examines optimal conditions for
coupling antibody Fob' fragments to lipid vehicles
formed in accordance with Example It
Rabbit anti-human immunoglobulin G (Gig)
antibodies were isolated and purified according to
conventional methods. Libya divers were prepared by
pepsin digestion of the purified antibodies. The diver
fragments were reduced with dithiothreitol at a pi of
about 4.8 to produce Fob' monomer fragments. The pi of
the reduction reaction is important in that when tune
reaction is performed significantly above pi 4.8~ (i.e.
' i
i r 3 9 5 I
-27-
pi OWE) over reduction may occur which leads to
inactivation of the antibody fragments; while
under reduction, which may occur which at a lower pi
(i.e., pi o'er is characterized by relatively poor
coupling efficiency to the vehicles.
Freshly prepared vehicles at a concentration of
about l micro mole of phospholipid per ml were reacted
with freshly prepared Fob' fragments at a concentration
selected between 0.5-4.0 my per ml. The reaction was
lo carried out in a pi 6.5 buffer under a stream of argon
for up to 12 hours at zoom temperature. The vehicles
were separated from unconjugated antibody fragments by
differential centrifugation. The amount of protein
conjugated to the vehicles varied according to the
initial concentration of antibody fragments in the
reaction. At an initial protein concentration of 4
mg/ml, approximately 500 micrograms of Fob' per
micro mole lipid were coupled to the vehicles in eight
hours. For vehicles in the 0.2 micron diameter size,
this corresponds to about 5,000 molecules of Fall
monomer fragments per vehicle. This number is somewhat
higher than that reported in the literature and may be
due it part to the reducing conditions used to reduce
Phoebe divers to Fob' monomers.
To test the specificity of binding of the
Fab'-liposome reagent, ho human red blood cells were
sensitized with human anti-D Gig and incubated with the
anti-IgG carrying vehicles. Vehicle concentrations
between 10 and 125 nanomoles of phospholipid per ml were
mixed with an equal volume of a 2 percent suspension of
the sensitized erythrocytes. The erythrocytes were
incubated with the reagent for two hours at room
temperature, after which they were separated and washed
by low speed centrifugation in a clinical centrifuge.
Radioactivity associated with the cell-bound vehicles
increased quantitatively with increasing amounts of the
liposom~e added, up to a saturation point corresponding
to about 5,000 liposome vehicles per red blood cell.
Binding of liposomes to human erythrocytes not coated
with human Gig was less than about 5 percent of that of
antibody-specific liposome binding.
Example III
Coupling ens and Beta-Galactosidase
to Lipid Vehicles
This example demonstrates the effect of lipid
vehicle surface charge on the relative amounts of
coupling of Fob' fragments and beta-galactosidase to
lipid vehicles.
ibid vehicles were prepared according to the
procedure described above, except that the vehicles were
prepared to contain either 10 percent or 20 percent
phosphatidylglycerol and proportionately less
phosphatidylcholine.
Purified rabbit anti-human Gig antibodies were
pepsin-digested and reduced with dithiothreitol for
twenty minutes at pi 4.8, according to the method
above. Beta-galactosidase (847 IU/mg), was obtained
from Boehringer-Mannheim.
Enzyme and freshly reduced loosened preparations
were reacted with one of the two lipid vehicle
preparations under reaction conditions similar to those
described in Example II. Specifically, 1 my per ml Fob'
and 0.25 my per ml beta-galactosidase were reacted with
1 micro mole vehicle lipid in one ml reaction buffer
containing 20 my citric acid, 35 my disodium phosphate,
108 my Nail, and l my DATA adjusted to pi 6.5 with 1 N
Noah. The reaction was stirred under a stream of argon
for 14 hours at room temperature. The vehicles were
twice pelleted by centrifugation at 20,000 G for 20
minutes to remove unrequited proteins.
The pelleted and resuspended reagent vehicles
were assayed for beta-galactosidase activity using the
~395~3~
I
chromogenic substrate ortho-nitrophenol-3-D-
galactopyranoside (nitrophenyl galactoside). The
relative specific activity values shown in Table I
represent, respectively, 54 percent and 64 percent of
the total beta-galactosidase activity added to the
coupling reaction mixture that was found to be
associated with the vehicles. The data indicate greater
enzyme coupling reactivity toward the vehicle
preparation containing the higher concentration of
phosphatidylglycerol (PUG), apparently resulting from the
greater charge attraction between the negatively charged
phosphatidylglycerol surface groups and the positively
charged enzyme.
TABLE I
15 PUG B-Gal Couplingsignal-to-noise
Efficiency ratio
10% 393 54 I
20% 431 64 4.8
The specific binding activity of the two
reagent preparations toward human Gig adsorbed on a
polypropylene solid support was measured to determine
roughly the relative amounts of Fob' carried on each of
the two vehicle reagents. Polypropylene tubes were
coated with human Gig according to known procedures. An
Alcott containing 1 nanomole of each vehicle
preparation was added to a coated tube, and the
suspension was incubated for a half hour at room
temperature in a phosphate-buffered saline buffer, pi
7.4. As a control, the two vehicle preparations were
also incubated with tubes coated only with bovine serum
albumin (USA). After incubation, the tubes were washed
one time with a low-salt buy f or and the enzyme activity
color development) associated with each of tune tubes
was determined. The signal-to-noise ratio for each of
the two vehicle preparations was calculated by dividing
the enzyme activity obtained for the Gig coated tubes by
~.~3958%
-30-
that for the tubes coated only with BRA. As seen in
Table I, a greater signal-to-noise ratio, presumably
attributable to a greater surface concentration of Fob
molecules, was associated with the vehicle preparation
containing less phosphatidylglycerol.
The data in Table I suggest that an increased
negative charge on the surface of lipid vehicles favors
beta-galactosidase coupling, resulting in enzyme
coupling efficiencies of up to about 60 percent. The
lo data also indicate that nanomolar amounts of the
liposome reagent are sufficient to produce a strong
signal-to-noise ratio in a binding assay, and that the
ratio of enzyme and ligand molecules in tune vehicles can
be adjusted to produce an optimal signal-to-noise ratio.
EXAMPLE IV
Preparation of a Reagent
availing Different Ligand to Enzyme ratios
Reagent vehicles having different ratios of
surface attached enzyme and ligand molecules were
prepared, and their immunospecific binding to
erythrocytes examined.
Lipid vehicles containing MPB-PE thiol-reactive
surface groups were prepared in accordance with the
method described in example I.
Rabbit anti-human Gig antibodies were obtained
in a form purified according to standard methods.
Reduced Fob' fragments, and beta-galactosidase were
provided in accordance with Example III. Vehicle
preparations, containing about l micro mole of vehicle
lipid, were reacted with selected amounts of reduced
Fob' and beta-galactosidase, as indicated in Table II.
The reactant concentrations of Fob' were 0.75, lo or
1.25 my per ml (column lo, and those of
beta-galactosidase, were 0.1, 0.3 or 0.5 my per ml
(column 2) for each Fob' concentration. The coupling
reactions were carried out at pi 6.8 under a stream of
Jo I
argon gas for 12 hours at room temperature, similar to
what has been described above. The vehicles were twice
washed to remove unrequited Fall and beta-galactosidase,
and the protein concentration associated with each
vehicle preparation was determine according to the
method of Lowry (reference 8). Column 3 in Table II
shows the measured protein concentrations, expressed in
micrograms of protein per micro mole vehicle
phospholipid. The data show that, for each reactant
concentration of jab' r increasing amounts of
beta-galactosidase resulted in increasing amounts of
protein covalently coupled to the vehicles. That the
increased reagent protein concentration is attributable
to increased amounts of coupled beta-galactosidase can
be seen from the data in column 4 in Table II, showing
specific activities of beta-galactosidase (expressed as
arbitrary beta-galactosidase activity units per
micro mole of vehicle lipid). It is interesting to note
that the amount of beta-galactosidase coupled to the
vehicle lipids was relatively independent of the initial
reactant concentration of Fob' in the Ebb' concentration
range shown (column 4).
The binding of the 9 different reagent
preparations to IgG-coated erythrocytes was determined
in accordance with the method described in Example III.
Sensitized (IgG-coated) red cells, after incubation with
a vehicle reagent were separated by centrifugation at
low speed and washed in a low-salt solution, made
isotonic with sucrose, to remove nonspecifically bound
reagent. Enzyme activity associated with the red blood
cells was used to determine the percent of vehicle
reagent which bound to the cells. The values, which are
shown in column 5 in Table lit confirm that liposomes
having treater immunospecific binding capacity can be
prepared by coupling greater amounts of ligand to tile
vehicle surfaces, and that the amount of Lund bound
-32-
depends both on the initial reactant concentration of
ligand, and on the relative reactant concentrations of
ligand and enzyme.
TABLE II
1 2 3 4 5
Reagent
Fob' B-Gal Reagent Reagent Binding
Protein B-Gal to Cells
0.75 0.1 330 51 36
0.3 3~0 1~4 20
0.5 545 2g6 25
1.0 0.1 355 29 I
r 0.3 439 120 30
0.5 500 265 31
151.25 0.1 370 50
a o. 3 450 140 52
0.5 565 246 27
Example V
Enzyme immunoassay to detect human Siberia
20 antibodies on sensitized erythrocytes.
, _ .
This example illustrates use of the liposome
reagent of the invention in an enzyme immunoassay to
detect the presence of anti-subgroup antibodies on human
erythrocytes, and in particular, anti-D, anti-Jka, and
anti-Fya Gig antibodies carried on sensitized
erythrocytes.
To prepare the liposome reagent, lipid vehicles
containing MPB-PE were prepared in accordance with
Example I. Immunopurified anti-human Gig antibodies
were prepared and treated as described above to produce
Fob' fragments, each of which contains at least 1 they'll
group. These fragments (0.75 my per ml), and
beta-galactosidase, (0.4 my per ml) were reacted with
tube lipid vehicles ~1.0 micro mole of phospholipid per
ml) for 18 hours at room temperature, pi 6.8. The
reagent was separated from unworked protein by
.3~58~
-33-
centrifuqation and resuspended in a suitable reaction
buffer.
Erythrocyte samples typed according to either
D, Foe or Jka subgroup type were used. To sensitize
each cell type, freshly washed cells were subdivided and
each sample incubated with one of a series of two-fold
serial dilutions of the appropriate anti-D, anti-Fya, or
anti-Jka typing sofa at 37~C or 30 minutes. The cells
were washed three times Whitehall a saline solution. D, Foe,
and Jka positive control cells were washed three times
with the above saline solution and resuspended in
phosphate buffered saline. The sensitized cells were
treated with liposome surface reagent, (S nanomoles
liposomes per 5 x 107 cells) for 30 minutes at room
temperature, with rocking every few minutes. The cells
were subsequently washed 5 times with saline/BSA,
resuspended in 10% sucrose containing the enzyme
substrate nitrophenyl g31actoside, and incubated for 10
minutes at room temperature. Cells were pelleted and
the enzyme activity determined by measuring the
spectrophotometric absorption of the supernatant at 405
no.
A linear relationship between the reagent
enzyme activity observed ~supern3tant absorption) and
number of surface specific Gig molecules (antisera
dilution) was observed over a wide dilution range for
all three cell types. The immunoassay was between about
8 and 32 times more sensitive, in terms of the minimum
number of erythrocyte-bound antibodies which were
detectable, than a standard anti-slobin agglutination
test which is used commonly for the determination of
erythrocyte subgroup antigens.
Example VI
Enzyme immune for determination of Rubella
antibodies
A lipid vehicle surface reagent was prepared
3~582
essentially according to the method described in Example
IV. Specifically, 0.75 my per ml of immunopurified
anti-human IyG Fob' fragments and 0.4 go per ml of
beta-galactosidase were reacted with lipid vehicles
containing MPB-PE, under the reaction ondi~ions
described in Example IV.
A control sample containing a known amount of
rubella antibody was prepared in 4 different sample
concentrations, namely, an undiluted sample and 1:5,
1:25 and 1:125 serial dilutions thereof. The antibody
was then reacted with solid support discs coated with
Rubella antigens (Cords laboratories) for about I
minutes at room temperature. The support discs were
washed with saline/BSA, then placed in 0.5 ml of a high
salt solution containing 250 my Nail, 100 my phosphate,
and a 25 micro liter Alec of the liposome surface
reagent (0 1 micro moles per my The liposome reagent
was incubated with the support Pro about 2 hours at room
temperature. The support discs were then washed two
times with saline/BSA, and Ike enzyme activity
associated with the discs determined according to the
method noted above.
Table III shows the relative enzyme activity,
expressed in units per ml, associated with each of the
I different-concentration samples indicated in the table.
The data show increasing levels of enzyme activity
associated with increasing amounts of Rubella antibody
added to a solid support. The negative control serum
contained no Rubella antibody.
Table III
. _ .
Antibo dilutionB-&al. active (Odyssey)
1:0 0-49
1:5 0.38
1:~5 ~.26
1:12~ 0.16
negative control 0.15
~;~39582
While the invention has been described with
particular reference to specific examples, it will be
understood that these examples are in no way intended to
limit the scope of the invention. Various changes and
modifications may be made without departing from the
spirit of the invention.
