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BACKGROUND OF THE INVENTION
The invention herein relates to measurement of the concentration of
antigenic substances, most usually in biological fluids, and to assessing
the immunologic competence of a subject organism. More specifically, the
invention employs perturbed angular correlation spectroscopy (PAC) to
monitor the interaction of antibody/antigen-sensitized vesicles loaded
with a gamma-emitting cation with antibodies for the subject
antigen/antibodies.
Techniques for immunoassay of specific substances have undergone extensive
expansion in the last 10 to 15 years. The concentration of antigen is most
commonly sought in biological fluids, for example, blood, urine and spinal
fluid, although there is no theoretical reason why antigen concentration
could not be measured in the context of any fluid medium, biological or
not. Radio immunoassay (RIA) is an extremely sensitive and reasonably
specific technique and has found a large scope of use (Thorell et al
1978); however, it requires separation of bound from unbound antigen in
order to assess the results. Spin immunoassay (SIA) has also been employed
(Leute, et al. 1972, Hsia et al 1973, Wei et al. 1975); it obviates the
need for separation, but is relatively insensitive. An additional
technique, using enzyme mediated reactions as the measure of the extent of
the antigen-antibody reaction has also recently been employed. Again,
ordinarily, separation of the bound material is required, although this is
not always the case. The present invention, as employed in the measurement
of the concentration of the antigenic substances, offers a method with
sensitivity comparable to RIA, which does not require separation of the
bound antigen.
In addition to measurement of antigen concentration, there is considerable
interest in determining the general competence of the immune system of an
organism. This is desirable, for example, in order to assess the effects
of various environmental conditions on the immune system (Tengerdy, et al.
1972, 1973; Bramen, et al. 1973; Thomas, et al. 1973; Nulsen, et al. 1974;
Kripke, et al. 1976). In addition, it is frequently desirable deliberately
to manipulate the immune system, and to monitor these manipulations.
Protocols and drugs are, for example, employed to immunosuppress
recipients of organ transplants to prevent rejection (Maugh, 1980; Trotta,
et al. 1981) and thus it is necessary to measure the effectiveness of
suppressing immune response. Conversely, assays of immunological
competence are necessary to assess the effectiveness of attempts to
potentiate the immune system, employing, for example, adjuvants which may
help control malignant growth (Schnipper, et al. 1980; Taniguchi, et al.
1981). The present method, in view of its ability to quantify
immunological response, is an aid in designing and administering these
drugs and protocols which affect the immune system.
The present invention takes advantage of the ability of complement to
mediate the lysis of liposomes sensitized with antigen that bind to
antibody.
It is known that liposomes loaded with a complexed gamma-emitting cation,
usually In-111 linked to a chelator such as nitrilotriacetic acid (NTA)
can be assayed for integrity by the use of perturbed angular correlation
spectroscopy, (PAC), which generates a factor related to the tumbling rate
of the gamma emitter. When the gamma emitter is enclosed in the vesicle,
the tumbling rate is greater than when it is freed into a biological
fluid, because of the ability of the gamma-emitting cation to associate
itself with other proteins found in the fluid. The tumbling frequency is
thereby slowed. Accordingly, it has been shown that PAC can be used as a
measure of the extent of lysis of liposomes containing such gamma
emitters. See, for example, Mauk, M. R. and Gamble, R. C. Proc. Natl.
Acad. Sci. (USA) 76: 765-769 (1979).
In the present invention, this technique is used to follow that lysis of
the "sensitized" vesicles which is due to specific binding of the vesicle
containing an antigenic surface to the antibody correlated with it. When
such binding occurs, complement mediates lysis. Accordingly, this
technique serves as a method to assess the antigen-antibody reaction in
any suitable context.
SUMMARY OF THE INVENTION
Briefly, the present invention comprises a method of determining the
concentration of an antigenic substance in a sample, which method
comprises mixing together:
(a) the sample;
(b) antibody to said antigenic substance;
(c) vesicles loaded with a gamma-emitting cation, and including on their
surface an entity competitive with the antigenic substance for the
antibody; and
(d) complement; and measuring the time integrated pertubation factor,
G.sub.22 (.infin.), associated with the gamma-emitting cation.
The invention also comprises a method for measuring the concentration of an
antigenic substance in a sample, which method comprises:
(a) mixing the sample with gamma-emitting cation loaded, antigen-sensitized
vesicles, antibody to the antigenic substance, and complement; and
(b) measuring the time integrated pertubation factor G.sub.22 (.infin.)
associating with the gamma-emitting cation.
Still further the invention comprises a method for determining the
immunological response in vivo in a subject vertebrate, which method
comprises:
(a) injecting a subject vertebrate with antigen sensitized vesicles loaded
with a gamma-emitting cation; and
(b) measuring the time integrated pertubation factor for the gamma-emitting
the cation.
In one aspect, the invention relates to a method of measuring the
concentration of an antigenic substance in a fluid sample commonly a
biological fluid. In the method, the antigenic substance in the sample
will be allowed to compete with sensitized vesicles for binding with the
appropriate antibody. The vesicles contain a gamma-emitting cation, so
that the extent of disruption of the vesicles may be measured by applying
perturbed angular correlation spectroscopy to measure the tumbling rate of
the gamma emitting cation. In biological fluids, encapsulated cations have
higher tumbling rates than those which are released into the surrounding
fluids. The higher the concentration of the antigenic substance in the
sample to be assayed, the more successful it will be in preventing the
binding of the sensitized vesicles to antibody and thus in preventing the
relase of the gamma emitting cation into solution. Thus, the magnitude of
the decline in tumbling rate will depend on the ability of the measured
antigen to prevent the disruption of the sensitized vesicles.
Accordingly, in this aspect, the invention comprises mixing, with the
sample to be analyzed: (1) vesicles loaded with a gamma-emitting cation,
and sensitized with a substance competitive with the antigenic substance
to be measured; (2) antibody to the antigen to be measured, and (3)
complement.
The extent of change in the time integrated pertubation factor (G.sub.22
(.infin.)) as measured by PAC is then a measure of the amount of antigen
present in the sample.
In another aspect, the invention concerns a method for assessing the
immunological response of a subject vertebrate organism by utilizing the
ability of the antibodies formed by the subject system to precipitate the
lysis of the above-described labeled vesicles.
The advantages of the method of the present invention are several fold. It
is sensitive, specific, and does not require separation of bound and
unbound antigen. It can be applied in vivo, and is relatively
non-invasive, in that it requires the injection of only small amounts of
label, and does not require the withdrawal of appreciable samples of the
subject's own fluids.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
As used herein, "gamma-ray perturbed angular correlation spectroscopy
(PAC)" refers to a technique which utilizes a gammaray coincidence
spectrometer to monitor changes in the rotational correlation time of a
gamma emitter by measuring a "time integrated pertubation factor",
G.sub.22 (.infin.). This technique is described by Meares, C. F., et al.
Proc. Natl. Acad. Sci., 69: 3718 (1972), incorporated herein by reference.
The G.sub.22 (.infin.) value is high when a high tumbling rate is
experienced by the measured species, and decreases as the tumbling rate
diminishes.
"Antigen" includes substances which are capable of eliciting an antibody
response, and substances which bind to the antibodies formed. It is
understood that while antibody response may be elicited by a
macromolecule, the entire surface of this molecule may not be required for
binding to occur. "Antigen" here includes both the binding site above, and
the entire hapten.
"Vesicles" refers to liposomes which are constructed by standard means
known in the art from phospholipids, and other components appropriate to
the particular usage intended. Means of constructing both large
unilamellar vesicles (LUV) and small unilamellar vesicles (SUV) are
well-known in the art. SUVs may be constructed, for example, by the method
of Mauk, M. R. et al, Proc. Natl. Acad. Sci., 76: 765 (1970), incorporated
herein by reference, by probe sonication of a lipid mixture containing,
for example, distearoyl phosphatidylcholine (DSPC) and cholesterol in
phosphate buffered saline. LUVs may be prepared for example, according to
the method of Deamer, D. et al, Biochem. Biophys. Acta 443: 629 (1976),
incorporated herein by reference. In this method, the DSPC: cholesterol
mixture is dissolved in an ether-ethanol mixture and aspirated into an
aqueous solution with an infusion pump.
Preferably the walls of the unilamellar vesicles are self-aligned layers of
L-.alpha.-disteraroyl phosphatidylcholine and/or L-.alpha.-dipalmitoyl
phosphatidylcholine or similar lipid substances. The walls of the vehicles
can also be formed from soybean phospholipid, egg yolk lecithin and
L-.alpha.-dimyristoyl phosphatidylcholine.
Cholesterol, various carbohydrate analogues of cholesterol, and other
additives can also be added to the phospholipid vesicle walls. For
example, L-.alpha.-phosphatidyl ethanolamine, L-.alpha.-
phosphatidyl-L-serine, dicetyl phosphate, and stearylamine. An ionophore
is also present in the vesicle wall.
"Sensitized vesicles" refers to vesicles which incorporate into their
construction a substance which is competitive with the antigen to be
measured for antibody, i.e. which incorporate an antigenic substance at
their surface. Such sensitized vesicles are constructed, as more
particularly set forth hereinbelow, by including in the initial
construction mixture, besides the basic structural phospholipid, a small
percentage of the sensitizing substance or antigen.
"Loaded" vesicles refers to vesicles which have enclosed, within the
envelope they create, a material which will be released if the envelope is
broken. In the context of the present invention, vesicles are typically
loaded with a gamma-emitting cation, preferably Indium-111, which is
preferably bound to a chelating agent. The material to be loaded is
conventionally either included in the reaction mixture from which the
vesicles are originally formed or is subsequently added and incorporated
by the already formed vesicles. This process is frequently aided by the
presence in the vesicles of an ionophore.
The chelator within the vesicle preferably is nitrilotriacetic acid (NTA).
However, other chelators for the cations may be used. Where the cations
are polyvalent metal ions, polyamino carboxylic acid chelators for such
ions may be employed, such as ethylenediaminetetraacetic acid (EDTA),
diethylenetriaminepentaacetic acid, diaminocyclohexanetetraacetic acid and
iminodiacetic acid.
The cations should be radioactive tracers with two gamma rays emitted in
succession, desirably bivalent or trivalent, for example, .sup.111 In.
In addition to the ionophore mentioned above, ionophores generally are
useful, and include polyethers:-lasalocid A (X-537A), 5-bromo derivative
of lasalocid; cyclic depsipeptides: beauvericin; cyclic peptides: DE-CYL-2
and valinomycin; and antifungal toxins:avenaciolide.
The preferred ionophore (a generic term intended to imply compounds which
are ion-loving or ion attracting) [6S-[6.alpha.(2S*, 3S*),
8.beta.(R*),9.beta.(R*),9.beta.,
11.alpha.]]-5-(methylamino)-2-oxo-2-(1H-pyrrol2-yl)ethyl]-1,7-dioxaspiro[5
.5]undec-2yl]methyl]-4-benzoxaxolecarboxylic acid, hereinafter referred to
as ionophore A23187, has been used to complex and carry divalent cations
across natural and artificial lipid membranes, Hyono, A., Hendriks, Th.,
Daemen, F. J. M., and Bonting, S. L. (1975) Biochim. Biophys. Acta., 389,
34-46; Sarkadi, B., Szasz, I., and Gardos, G. (1976) J. Membrane Biol. 26,
357-370; LaBelle, E. F. and Racker, E. (1977) J. Membrane Biol., 31,
301-315; Pfeiffer, D. R. Taylor, R. W. and Lardy, H. A. (1978) Ann. N. Y.
Acad. Sci., 307, 402-423. Evidence also exists that A23187 can form
complexes with trivalent cations, e.g., La.sup.+3, Pfeiffer, D. R., Reed,
P. W., and Lardy, H. A. (1974) Biochemistry, 13, 4007-4014.
METHOD OF THE INVENTION
A. Determining Concentration of Antigenic Substance in Biological Fluid
In the aspect of the invention which utilizes complement mediated lysis to
measure the concentration of an antigenic substance, biological fluids are
withdrawn and measured in vitro.
Biological fluids which are acceptable for use as samples for such
measurements include, for example, blood, serum, urine, spinal fluid, or
even less frequently measured fluids such as saliva, or gastric juices.
The source of such fluids may be any organism which produces fluids which
contain, possibly, the substance sought to be analyzed. Accordingly, the
organism may be vertebrate or invertebrate, plant or animal, although the
most important applications, of course, apply to vertebrates. In addition,
the method can be used with respect to fluids of non-biological origin, so
long as additional protein or other binding agents are supplied which will
have the effect of decreasing the tumbling rate of the gamma-emitting
cation released from the vesicles during the assay. Thus, a plain aqueous
solution may be used, if, for example, serum albumin is added so as to
provide such a binding agent.
In the method of the invention, the antigenic substance to be measured will
be determined by its ability to compete for antibody with sensitized
vesicles containing the gamma emitting label. Accordingly, vesicles must
be constructed which contain in their surface, molecules which are
competitive for antibody with this antigenic substance. Ordinarily, the
antigenic substance, itself, would be incorporated into the vesicles in
making this construction. For example, N-2, 4-dinitrophenyl-E-amino
caprolate (DNP-cap) is an antigenic substance whose concentration can be
measured. In that case, the vesicles would be sensitized by utilizing in
their construction, E-dinitrophenyl amino-caproyl phosphoethanolamine
(DNP-cap-PE). The DNP-cap will thereby be incorporated into the surface of
the vesicles. The vesicles and the antigen will thus be attracted into the
same antibody.
Antibodies can be obtained in a variety of ways. Ordinarily, these are
obtained by producing antiserum to the antigen to be measured using mice
or rabbits. Often, such antisera are commercially available for commonly
encountered antigens. Further refinements can, of course, be made in
particular cases by, for example, preparing monoclonal antibodies entirely
specific to the desired antigen. These techniques are known in the art,
and are applicable to the present method.
Complement may be obtained from any appropriate vertebrate source and is
also generally commercially available. For example, guinea pig complement
can be obtained from GIBCO.
In the method of the invention, a small quantity of sample is mixed with
appropriately sensitized vesicles, appropriate antibodies, and complement.
Ordinarily, vesicles, antibody, and complement are added in sequence,
although any order is satisfactory. If the sample to be tested does not
have sufficient cation binding materials contained in it, additional such
materials in the form, for example, of serum albumin must also be added.
The G.sub.22 (.infin.) is measured after incubation has effected lysis of
the vesicles. Appropriate conditions of pH and salt concentration must
also be maintained.
In a typical assay, a few microliters of heat inactivated serum (if binders
are lacking in the sample), a similar quantity of vesicles,
antibody-containing serum, and guinea pig complement are added in
sequence. The solution is incubated for about ten minutes to one hour at
about 30.degree. to 40.degree. , and the value of G.sub.22 (.infin.)
determined by PAC spectroscopy. The incubation conditions, will, of
course, vary with the specific assay used, however, in general it is found
that approximately 37.degree. C. or biological temperature, is desirable,
and the lysis takes place within approximately thirty minutes.
The lysis of the loaded sensitized vesicles can be confirmed by subsequent
addition of isopropanol to test the complete lysis of any remaining
vesicles to provide a control standard against which the measured G.sub.22
(.infin.) can be compared.
B. In vivo Assessment of the Immune System
Due to the use of a high-energy gamma-ray radiocation and the highly
sensitive spectroscopy for monitoring the tumbling rate of radiocation in
different environments, the assay could also be used to assess
immunological responsivenes in vivo. Visicle sensitized with antigen can
be injected directly in vivo in the presence of antibodies, complement
will mediate the lysis of the vesicles in circulation. This releases the
encapsulated radiocation which will immediately bind to the circulating
protein. This binding will slow down the tumbling rate of the radiocation
and decrease G.sub.22 (.infin.) as monitored by PAC spectroassay. This
change in G.sub.22 (.infin.) can be used to quantify the amount of
antibodies present in the system.
The following examples are intended to illustrate but not limit the
invention:
PREPARATION A
Preparation of DNP-cap Sensitized LUVs
20 .mu.mole DSPC, 12.5 .mu.mole of cholesterol, 0.1 .mu.mole of A-23187 (a
commercially available ionophore) and 0.2 .mu.mole DNA-cap-PE were
dissolved in 16 ml ether-ethanol (4:1 by volume). The lipid solution was
aspirated in a glass syringe into the aqueous phase at 0.25 ml per minute
with the aid of an infusion pump (Sage instruments). The aqueous phase
consisted of 4 ml phosphate saline buffer (PBS) pH 7.4, and 10 mM
nitrolotriacetic acid (NTA) and was maintained at 80.degree. C. At the
conclusion of the injection, the vesicle suspension was removed and
filtered through a 1.0 .mu.m nucleopore polycarbonate filter. The NTA
external to these vesicles was then removed by column chromatography using
Sephadex G-50.
PREPARATION B
Preparation of Loaded Sensitized Vesicles
0.1 mg of the vesicles as prepared in Preparation A were incubated with
InCl.sub.3 at 80.degree. C. for 45 minutes. Much of the In-111 was
incorporated into the vesicles, but any remaining In-111 was subsequently
complexed to EDTA and separated from the loaded vesicles by
chromatographing the mixture on a Sephadex G-50 column equilibrated with
PBS.
PREPARATION C
Vesicles Prepared for the Process of the Invention
Using the procedure set forth in Preparation A, the following vesicles were
prepared; the amounts given are mole ratios:
______________________________________
Abbreviation
DSPC Chol A-23187
DNPcap-PE
______________________________________
LUV/24 2 1.25 .005 .024
LUV/12 2 1.25 .005 .012
LUV/9 2 1.25 .005 .009
LUV/4 2 1.25 .005 .004
LUV/0 2 1.25 .005 0
LUV/20 2 1.25 .1 .020
LUV/10 2 0.20 .005 0.02
SUV 2 1.0 0.004 0.024
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EXAMPLE I
Measurement of Complement Mediated Lysis Using PAC
In a 10.times.75 mm glass tube, 100 .mu.l of heat inactivated calf serum,
50 .mu.l of vesicles prepared in Preparation C and loaded as described in
Preparation B, various amounts of anti-DNP-cap antiserum, and guinea pig
complement were added in sequence. The solution was then brought to a
total volume of 20 .mu.l with a solution of 0.15M NaCl, 1 mM MgCl.sub.2,
and 0.15 mM CaCl.sub.2, pH 7.4. After thirty minutes of incubation at
37.degree. C., the G.sub.22 (.infin.) values were determined, with results
as shown below.
Finally, all samples were treated with isopropanol to lyse any remaining
vesicles and the G.sub.22 (.infin.) redetermined.
______________________________________
Results
G.sub.22 (.infin.)
serum,
measured
after serum
30 min. +
Sample no serum 37.degree.
isopropanol
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Free .sup.111 IN-NTA Complex
0.70 0.19 0.18
.sup.111 IN-NTA complex loaded:
LUV/10 0.59 0.45 0.20
LUV/20 0.62 0.60 0.20
SUV 0.61 0.59 0.24
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As seen from these results, free In-111 binds to serum with appreciable
lowering of G.sub.22 (.infin.). This is evident also from column 3, where
isopropanol has freed all In-111 from the vesicles. The lowered values
after incubating with serum present indicates partical release from the
vesicles.
EXAMPLE II
Effect of Variables
Incubations were carried out generally as set forth in Example I, with the
moficiations below. The G.sub.22 (.infin.) consistently were measured
after thirty minutes at 37.degree. C.
A. LUVs treated with 5 .mu.l of anti-DNP-cap antiserum; and SUVs treated
with 25 .mu.l of anti-DNP-cap antiserum were incubated as in Example I
with varying amounts of complement. FIG. 1 shows the results of varying
the amount of complement from approximately 2 .mu.l to 100 .mu.l of
complement. It appears that 40 .mu.of complement is sufficient to maximize
the lytic effect under these conditions.
B. In a different experiment, LUVs treated with 20 .mu.l of complement and
SUVs treated with 50 .mu.l of complement were incubated with varying
volumes of antibody up to 5 .mu.l. Apparently, under these conditions, 2
.mu.l of antibody was sufficient to obtain the desired results. These
results are shown in FIG. 2.
C. In a third experiment, LUVs with varying DNP cap-PR concentrations,
incubated with 20 .mu.l of complement were used. The volume of antibody
required under these circumstances appears to be sufficient, regardless of
the composition of the vesicle, at approximately 1 .mu.l of added
antibody. These results are shown in FIG. 3.
EXAMPLE III
Calibration of the antigen concentration measurement
Incubation was carried out as set forth in Example I using LUV/24 with
samples of DNP-cap concentration of 10.sup.-5 M to 10.sup.-9 M
approximately. As shown in FIG. 4, 50% inhibition occurred in the range of
10.sup.-7 concentration of the antigen, when 5 .mu.l or 0.75 .mu.l of
antibody was employed.
Having fully described the invention, it is intended that it be limited
solely by the lawful scope of the appended claims.
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