Encapsulated scintillators for measuring the concentration of tritiated solutes

Microencapsulation of solid phase scintillators in gels selectively permeable to diffusible radioactive label. These encapsulated scintillators are used to monitor the concentration of radioactive-tagged subtances in fluid systems.

BACKGROUND OF THE INVENTION 
The present invention relates to a new and novel microencapsulation of 
solid phase scintillators in gels that are permeable to selected 
diffusible labels carrying radioactive sources capable of activating the 
encapsulated scintillator. These microencapsulated scintillators have been 
used to develop a method for dynamically monitoring the free concentration 
of diffusible, radioactive-tagged substances in fluid systems. 
In biological systems, it is frequently important to quantify uptake, 
binding, or release of soluble substances by colloidal or macroscopic 
phases such as cells, sub-cellular particles, or macromolecules. Examples 
include cellular uptakes of various drugs, the release of cellular 
catabolites into the blood stream, blood serum levels of various drugs, 
enzymes, and hormones, and the presence or absence of specific 
immunoglobulins in the serum of a patient. 
Generally, uptake or release is measured either directly by assaying the 
macroscopic phase after separation from its normal bathing medium, or 
indirectly by determining the free concentration of the solute. Although a 
wide variety of analytical techniques (see, for example, S. Ramos, S. 
Schuldiner, and H. R. Kaback, Methods in Enzymology, Volume IV, part F, 
pages 680-688; Academic Press, New York 1979) are currently used to obtain 
these measurements, no single procedure is applicable in every instance. 
Most procedures are either relatively insensitive, exhibit poort time 
resolution, or are applicable to a relatively limited group of solutes. 
SUMMARY OF THE INVENTION 
The present invention relies upon the fact that certain radiolabels, most 
notably tritium, release beta radiation which has a very limited range. 
For example, the tritium beta emission has a range of only about 7 
micrometers in water. Consequently, microscopic particles of a 
scintillating material need only be microencapsulated with a thin (more 
than 7 .mu.m ) coating to exclude all exciting radiation from tritium 
bound to material which cannot penetrate the encapsulation, i.e., a 
scintillator may be totally shielded from a tritiated source that is not 
freely diffusible. If the coating, is however, permeable to small 
tritiated molecules but not to larger objects, i.e., the macrophase, then 
the scintillator will be excited only by the small molecules which are 
free in the solution and are able to pass through the encapsulation. The 
advantage of using microscopic scintillator particles is that the 
scintillator particles and macroscopic phase may be very intimately mixed 
in the solution. Furthermore, the surface area to volume ratio of the 
scintillator particles can be made very large if the scintillator is 
finely divided; this improves the scintillator detection efficiency to low 
energy beta radiation. 
The tritium isotope has the desirable characteristics of decaying via the 
emission of a low energy electron (E.sub.ma =18.6 keV; mean weighted 
energy=6.3 keV) and of being incorporated into many organic compounds. 
Other Beta emitters may also be used such as carbon-14, sulphur-35, and 
phosphorous-32. However, the thickness of the gel encapsulation must be 
increased with increased Beta energy, and therefore low energy Beta 
emitters, such as tritium, are preferred. As the emitted Beta electron 
from tritium is responsible for the resultant photon emission of the 
scintillator, the range, (that is the distance required between the 
tritium atoms and the target surface, which reduces the energy deposited 
beyond the surface to some nominal fraction of the energy emitted) 
appropriate to the tritium Beta-ray spectrum is approximately 1 micrometer 
in materials with the density of water. Thus, an effective aqueous shield 
to Beta radiation need only be a few micrometers thick. 
In order to provide that a tritiated molecule sequestered by the 
macrophase--e.g. colloidal phase, cells, vesicles, or macromolecules--is 
unable to excite a scintillator bead, it is desirable that the 
scintillator beads be segregated from the macrophase being monitored. 
We have discovered that segregation of the macrophase from the scintillator 
can be accomplished by providing a selective permeable coating about the 
scintillator. These coatings may be composed of suitable hydrophilic 
materials capable of encapsulating the scintillator such as, for example, 
coatings of carbohydrate origin such as gelatin or agarose, or various 
gels such as polyacrylamide gel. Other materials may also be used to 
encapsulate the scintillator, for example, non-hydrophilic material, or 
hydrophobic materials may also be utilized depending upon the test medium. 
For the purpose of this discussion, all selective permeable coatings will 
be referred to as "gels". 
Simplistically, one embodiment of our procedure allows for a finely divided 
scintillator to be encapsulated as microbeads in a selectively permeable 
gel. The selective permeability of the gel is achieved by selecting a gel 
pore size (in accordance, for example, with the procedure of L. Ornstein, 
Annals of New York Academy of Science, 121:321-349, the disclosure of 
which is hereby incorporated) large enough to allow for diffusion of the 
tritiated solute in solution through the pore while excluding larger 
molecules--including the tritiated solute when complexed with another 
macrophase--from exciting the encapsulated scintillator. As a result, the 
scintillation light output continuously monitors the free concentration of 
the tritiated species with no contribution from solute that is not freely 
diffusible. 
The microencapsulated beads of the present invention may be used in the 
determination of substances in many fluids such as body or aqueous fluids. 
As an example, consider a determination based upon general 
radioimmunoassay techniques known in the art. For example, if a test was 
required to determine the concentration of a given drug in blood serum, 
antibodies, including monoclonal antibodies or antibodies prepared by 
other means, to the drug would be prepared, isolated and purified. This 
antibody would then be mixed with the blood serum, and encapsulated 
scintillators, made according to the present invention would be added. The 
pore size of the gel encapsulation would be chosen small enough to exclude 
antibody drug molecule complexes but not antibody with drug molecules 
attached. If the blood serum contained the drug, the antibody would bind 
with the drug to form an antigen-antibody complex. When tritiated-labelled 
drug is then added, the drug would not be bound to the antibody, and would 
be free to diffuse through the pores of the gel coating to excite the 
scintillator. If the blood serum does not contain any of the drug, the 
antibody would bind to a subsequent addition of tritiated-labelled drug, 
and the scintillator would not be excited. Furthermore, by serial 
dilutions of a known antigen standard amount, a standardized curve may be 
obtained from which the titer of bound antibody in a sample can be 
calculated. 
Alternately, the blood serum or other body fluid may be pretreated to 
purify any antigen present by removing possible interferring substances 
which might cause anomalies with the test. For example, in the data 
presented in Table 1, the test media is an aqueous solution, and to 
achieve similar sensitivities in samples of blood serum, interfering 
substances such as albumin, immunoglobulins, and other protein fractions 
should be removed. This would be accomplished by conventional separation 
techniques, such as precipitation, or chromatrographic techniques. Other 
body fluids, for example saliva or urine, might undergo similar 
separation, i.e. purification, pretreatment. If, on the other hand, the 
gel was chosen to be compatible with the test media, no pretreatment would 
be necessary. 
Various scintillation means have previously been used to determine the 
presence of various immunospecific substances. 
U.S. Pat. No. 4,000,252 for an "Immunoscintillation Cell" discloses an 
immunoscintillation composition used in a solid phase radioimmunoassay. In 
this immunoassay, a mixture of labeled and unlabeled antigens is 
introduced into a cell containing solid, insolubilized or coated 
scintillators and solid or insolubilized antibodies which selectively 
retain a portion of the labeled and unlabeled antigens. In one embodiment 
of the invention, scintillators and antibodies are chemically bound to an 
insoluble substrate; in another embodiment, the phosphor and the 
antibodies are maintained separately by means of a protective material. In 
both embodiments, the radioactive energy released by the labeled antigen 
excites the scintillators releasing burst of photons which are measured by 
appropriate means. Studies have shown that the counted or measured photons 
are proportional to the concentration of labeled antigens bound to the 
antibodies. 
As shown in FIG. 2 of the Immunoscintillation Cell patent, the 
scintillators are embedded within the sides of a tube, or, in FIG. 4, 
within plastic balls. This "protection" of the scintillators is not for 
the purpose of maintaining any particular separation between the 
scintillator and the radioactive label located elsewhere in the system, 
but rather for the purpose of preventing the scintillators from 
contaminating each other and for preventing the scintillators from being 
solubilized from the carrier solvent. It differs, most importantly, from 
the present invention by not utilizing a selectively permeable 
scintillator coating. 
Similarly, U.S. Pat. No. 4,271,139 presents a "Scintillation Proximity 
Assay" as an improvement over conventional immunoassay latex fixation 
tests. Briefly, the purpose of the technique disclosed in this patent is 
to measure the degree of linking or proximity of one type of latex 
particle (type A) to polystyrene scintillant particles (type B). This 
invention takes advantage of the limited range of the particular radiation 
employed (about 1 .mu.m) in aqueous media emitted from a tritiated type A 
particle. When type A and type B particles are "tagged" with antigen and 
antibody, respectively, the amount of particle aggregation resulting from 
the concentration of antigen (or antibody) present can be determined using 
liquid scintillation counting equipment; if the type A and B particles are 
separated, i.e., no immunomediated aggregation, the radiation from the 
type A particle is attenuated in liquid and never reaches the scintillator 
type B particles to release photons.

The radioactively-induced scintillation may be detected with the apparatus 
similar to that illustrated in FIG. 1. Several milliliters of a mixture of 
the macrophase to be monitored, a tritiated substrate, and the gel-bead 
slurry prepared in accordance with Example 2 were contained (to a depth of 
1-2 mm) in a polished lucite cylinder, which was coupled with optical 
grease to the face plate of an RCA 8850 photomultiplier tube. The 
photomultiplier tube was operated at -2000 volts (sufficient to give a 
photoelectron gain of about 8.times.10.sup.6), and the output current from 
the tube was read on an ampmeter (in the case of the system actually used, 
output was read directly on a Keithley 610C electrometer). Typical dark 
currents, that is background "noise", for the photomultiplier, after dark 
adaptation, were about 10.sup.-9 Amperes. The slurry was continuously 
stirred by a motor driven comb cut from a thin sheet of Teflon, and fitted 
on a long shaft sufficient to allow the motor to be located at a distance 
sufficient so the motor fields did not affect the photomultiplier. The 
entire photomultiplier-cylinder-motor apparatus was enclosed in a 
light-tight box with openings limited for tubing allowing the addition of 
substrate by injection. 
An alternative, and more sensitive, means for recording the output current 
from the photomultiplier tube, such as the pulse height 
discriminator-counter system is indicated by phantom lines in FIG. 1. By 
such a means it would be possible to directly omit the background noise, 
or dark current, being emitted from the tube. 
Other modifications could also be made, and are well within the skills of 
the manufacturer of such instrumentation. The mixing, for example, could 
be accomplished by means other than the motor-shaft-comb combination, that 
is equal mixing may be accomplished by using magnetic stirrers or other 
devices. Also, the cylinder does not necessarily need to be of lucite, for 
example, equally useful cylinders may be composed of other 
non-scintillator materials such as silica. 
The scintillator and slurry preparations used to demonstrate the 
feasibility of the method were made as described in the following 
examples, which are given for the purpose of more clearly illustrating the 
invention. 
EXAMPLE 1 
SCINTILLATOR PREATION 
Two grams of Polyvinyltouluene based NE 102 plastic scintillator 
microspheres of 1-10 um diameter (Nuclear Enterprises, Inc., San Carlos, 
Calif.) were suspended in 20 ml of 3% Triton X-100 surfactant by vortexing 
and gentle sonication. The detergent was required because the beads are 
difficult to wet. The detergent was then removed by five or six water 
washes. For each wash, the beads were sedimented by centrifugation at 
6000.times.g for 10 minutes. After each sedimentation, the supernatant was 
discarded and the beads re-suspended in 30 ml of distilled water. After 
the final wash, the re-suspended beads were allowed to settle overnight, 
and the supernatant was carefully removed. The resultant slurry was 
roughly 10% beads by volume. 
EXAMPLE 2 
ENCAPSULATION 
The following quantities were used, for each ml of settled bead slurry, to 
produce a polyacrylamide gel: 300 mg acrylamide and 1.5 mg 
N,N'-methylene-bis-acrylamide were dissolved in the slurry. Polymerization 
was catalyzed by the addition of 10 ul of 10% (w/v) ammonium persulfate 
solution and 2 .mu.l of N,N,N',N-tetramethyl-ethylenediamine (TEMED). The 
polymerized gel was cut into pieces, suspended in distilled water, and 
then sheared in a blender for several minutes to a uniform consistency of 
fine particles. Microscopic examination of the product revealed 
irregularly shaped bits of gel (mean size 0.1 mm) encasing clumps of 
scintillator. the scintillator to gel volume ratio was about 10%. The gel 
particles were allowed to settle after the supernatant was discarded, and 
the final slurry was refrigerated until used. 
Although the gel used to provide the microencapsulation of the scintillator 
was polyacrylamide, other materials may also be used such as gelatin, 
agarose, etc. The requirements for the coating being the ability to 
encapsulate with well defined pore sizes and therefore being permeable to 
the label but not to the macrophase. The reason polyacrylamide was chosen, 
in fact, was becuase it has been extensively characterized how to obtain 
various pore sizes with this material. 
Although the scintillators following this treatment may appear as single, 
double, or multiple hydrophobic or hydrophilic scintillator beads within 
an encapsulating material, the ideal microencapsulated scintillator is a 
single particle surrounded by a layer of the encapsulating material. While 
single, double, or multiple encapsulations of scintillator may be used in 
the method of the present invention, provided the pores of such are large 
enough to allow for the diffusion of the labelled, unbound component, the 
single encapsulated scintillator is favored because of economic, both cost 
and reactivity, factors. Further, single encapsulated scintillator has an 
advantageous time response by reducing the thickness of gel through which 
the labelled compound must diffuse. Also, in order to achieve 
compatability with the unknown component, each scintillator may be coated 
with more than one layer of material, for example by coating the 
scintillator using glycophase coating technology common in chromatography 
preparation. 
The data obtainable by our method of preparing a radioimmunoassay using 
microencapsulated scintillator according to our invention, and made in 
accordance with Examples 1 and 2, are shown in Table 1 in which each 
indicated run is the mean value of two separate runs. In each instance, 
the tritiated compound used as the labeled compound is tritiated 
chlorpromazine, and the antibody is chlorpromazine antibody prepared in 
accordance with accepted practice. 
The test media in this instance is water, not body fluid, the substitution 
of which would cause the sensitivity (CPM) of the test to be lower, but 
still acceptable, because of inteferring substances normally contained in 
various body fluids; this decrease in sensitivity would be overcome by an 
increase in the number of encapsulated scintillators. 
TABLE 1 
______________________________________ 
Run Bead [.sup.3 H] 
H.sub.2 O 
[AB] CPM 
______________________________________ 
1 -- 100.lambda. 
0.4 ml -- 14 
2 50.lambda. 
-- 0.45 ml -- 17 
3 50.lambda. 
100.lambda. 
0.35 ml -- 251,296 
4 -- 100.lambda. 
0.4 ml -- 15 
5 50.lambda. 
-- 0.45 ml -- 18 
6 50.lambda. 
100.lambda. 
0.35 ml -- 234,499 
7 100.lambda. 
100.lambda. 
0.25 ml 100 11,947 
______________________________________ 
[.sup.3 H] Refers to the concentration of tritiumlabelled antigen; 
100.lambda. being the equivalent of approximately 3 micrograms of antigen 
[AB] Refers to the concentration of affinity purified antibody to 
chloropramazine; 100.lambda. being equivalent to approximately 100 
micrograms of antibody. 
Bead refers to the concentration of gel encapsulated beads made in 
accordance with Example 2; 50.lambda. being equivalent to approximately 10 
milligrams of beads. 
The data contained in Table 1 clearly establishes that when either the 
label (runs 1 and 4) or the microencapsulated scintillator (runs 2 and 5) 
is present alone in the system, only background counts (dark current) are 
registered. On the other hand, when both components are present together 
(runs 3 and 6) the labelled compound is able to diffuse through the 
coating about the scintillator, and the resulting photo emission is 
increased to a much greater number. When, in addition to the 
microencapsulated scintillator and the tritiated label, chlorpromazine 
antibody is also added to the system, (run 7) a substantial decrease in 
counts is observed. This clearly indicates that the immune complex formed 
between the labelled substrate and the antibody does not penetrate the 
scintillator coating. 
The feasibility of using a immunoassay system for unknowns based upon our 
invention is clearly established. Unknown titers could be detected by 
establishing a standard curve in the usual manner with varying 
concentrations of the target substance determining the unknown titer from 
the curve relative to the counts observed. 
As discussed previously, our invention may be used to determine the levels 
of various drugs, hormones, and enzymes in blood serum or other body 
fluids such as saliva or urine. The encapsulated scintillator beads may 
also be used to conduct rate uptake studies such as described in our 
publication: "A Method For Rapid, Continuous Monitoring Of Solute Uptake 
and Binding", Biochemistry 1982, 21,3239, the disclosure of which is 
hereby incorporated in toto. The encapsulated scintillator beads may also 
be used in receptor binder assays, for example, estrogens, acetylcholine, 
opiate peptides, and others. 
From the foregoing description, one skilled in the art can easily ascertain 
the essential characteristics of this invention and without departing from 
the spirit and scope thereof can make various changes and/or modifications 
to the invention for adapting it to various usages and conditions. Thus, 
while we have illustrated and described the preferred embodiment of our 
invention, it is to be understood that this is capable of variation and 
modification, and we therefore do not wish to be limited to the precise 
terms set forth, but desire to avail ourselves of such changes and 
alterations which fall within the purview of the following claims: