Note: Descriptions are shown in the official language in which they were submitted.
~Z~3~7
BACKGROUND_OF THE INVENTION
This invention relates to the detection of the presence
and concentration of unbound species, e.g., hormones, in a liquid
sample such as serum. More particularly, it relates to a proce-
dure well suited for the detection of free species of the type
which are capable of reversibly binding with protein in samples
containing the free species, the binding protein, and a concen-
tration of species-protein complex.
The "competitive assay" technique for measuring the
concentration of various biologically active substances for di-
agnostic purposes is now well established. The technique involves
a reaction system wherein antibody specific to the species to be
determined is incubated together with the test sample and an ali-
quot of a distinguishable ana~ogue of the sample. The analogue
and natural species compete for sites of attachment to the anti-
body, and after separation of the antibody from the remainder of
the reaction system, the antibody or supernatant may be assayed
for analogue. The amount of analogue associated with the anti-
body is an inverse function of the concentration of the test
species in the sample.
Two common methods of performing such competitive deter-
minations are known as "liquid phase radioimmunoassay" and "solid
phase radioimmunoassay". In each system the immunogenic substance
which is bound to an antibody must be removed from unbound immuo-
genic substance in order to make a measurement of the amount of
labeled analogue on the antibody. From this measurement, the
amount of immunogenic substance in the sample is determined.
In "liquid" phase radioimmunoassay systems, the anti-
body, labeled analogue, and immunogenic substance to be determined
are incubated in solution. A number of antibody binding sites are
,3~
'I
112~397
1 available and reaction time is rapid. To separate the antibody
complexed immunogenic substance from the uncomplexed immunogenic
substance, the antibody immunogenic complex is precipitated,
centrifuged, and the supernatant is decanted.
"Solid phase" radioimmunoassay systems avoid the preci-
pitation step. The antibody is bound to a glass chip or inside
surface of a tube. Centrifugation of the glass chips or decan-
tation of the coated tubes will separate the antibody complexed
immunogenic substance from the uncomplexed immunogenic substance.
However, the reaction time is relatively slow because a number of
available active sites on the antibody is blocked by the chip or
tube.
Hormones such as those excreted from the thyroid, testes,
ovaries, adrenal cortex, and other mammalian glands are often
transported through the circulatory system in association with
hormone binding proteins or globulins. Often, it is of diagnostic
significance to be able to determine the presence and concentra-
tion of free hormone, as opposed to protein-bound hormone or total
hormone. For e~ample, thyroxine ~T41 in the bloodstream exists
in a dynamic equilibrium as a species bound to a transport protein,
Cthyroxine-binding globulin, albumin, or prealbumin~ and, in a
small fraction ~potentially 0.1% or less), as an unbound substance
~"free" or unbound T4~. Free T4 ~s thought to be the active
species of the thyroid hormone system. Unfortunately, free-T4
assay methodology has beenltedious, complicated, expensive, and
error-prone because of the difficulty in standardization of
materials and in measuring minute quantites of unbound hormone in
the presence of overwhelming amounts of hormone relatively loosely
bound to protein. The competitive assay procedure noted above is
not capable of determining free species concentrations.
--3--
1;~3~7
1 Of the methods for measuring free T4 and other hormones
which exist in vivo as protein complexes, the dialysis membrane
method affords a high degree of accuracy. A dialysis bag con-
taining known quantities of serum to be tested and labled hormone
is suspended in buffer. The labled hormone distributes itself
between the free and bound states in the same proportions as the
serum hormone. The mass of labled hormone added must be extremely
small so as to avoid seriously perturbing the system, yet the
count rate must be high so that detection of small amounts is
feasible. In the case of radioactive lables, this requires that
labled hormone of very high specific activity be used. After a
suitable period (about 24 hours), the tagged and natural hormone
not bound to protein diffuse through the semipermeable dialysis
bag while hormone bound to protein ~molecular weight greater than
20,000~ remains within the dialysis bag. To determine the quant-
ity of free hormone present in the serum, one assays an aliquot
of the buffer for labled hormone. As a result of the assay, the
fraction of labled hormone that crossed into the buffer may be
calculated, and the fraction of total hormone that exists in the
free state inferred. To convert this fraction to mass units
(e.g,, ng/dl) of free hormone, a total hormone assay must also be
run on the sample.
While this system can give meaningful results in free
T4 and other free hormone assays, it is poorly suited for routine
use. Two tests must be run, and the accuracy of the final result
can be compromised by either procedure. Rather large amounts of
radioactivity must be employed per test. Interfering impurities
in the labeled hormone can cause special problems, and long incu-
bation times are required. Only serum can be used, and it must
be extremely fresh. Furthermore, the collection and standardiza-
tion of all the materials necessary for the assay is not a routine
(13~7
1 matter. Thus, the application of the procedure has been limited
due to the high cost and labor intensity of the method and the
skilled personnel required.
Another method of free hormone assay involves reaction
kinetics and requires two separate tests. Each test measures a
kinetic curve related to how fast an antibody captures hormone,
e.g., T4, away from the opposing pull of the primary binder such
as thyroxine binding globulin.
In such an assay system, inaccuracies results from
several pathologic conditions. If more protein ~inding sites are
present than usual, the effective attraction of the globulin for
the hormone will be greater and the rate of binding to antibody
will decrease. In the case of thyroxine analysis, this would give
the appearance that the sample had little T4 when in fact there
might be a high level of T4, but all bound.
SUMMARY OF THE INVENTION
The instant invention provides a process, reagent, and
test set for the direct assay of unbound hormones and other such
species. The assay may be performed in the presence of serum
proteins and the bound hormone.
In accordance with the invention, semipermeable micro-
capsules containing antibody complementary to the hormone or
,; -
V~7
1 c.h~r s?~ci~ to ~e detected are incu~ted ~ith both a
distirg~ishable analogue of the hormone and the test sample.
~he analogue may be introduced into the ~icrocapsules prior to
the incubation in quantities such that the antibody is sa~urated
at the hormone binding sites. The microcapsule walls co~prise
'membranes separating the test sample from the antibody, and have
!l a permeability sufficient to allow passage of free species andits analogue, but insufficient to permit passage of the antibody,
'! natural proteins, or protein-bound hormone. ~7hen a test sample
l,is added to an aliquot of the microcapsules, free hormone dif-
ijfuses through the membranes and competes with its analogue forsites of attachment on the anti~ody. Thus, the distribution of
analogue between the antibody and the remainder of the reaction
¦¦system becomes indicative of the amount of free hormone origi-
nally present in the sample. This procedure combines the
~linherent accuracy of a conventional liquid phase radioimmunoassay
! and the sim~licity and convenience of a solid phase system.
Next, the antibody toaether with its bound hormone (ana
bound analogue) is separated from the free species, and either
the antibody or the remainder of the reaction system is assayed
for analogue. The separation may be conducted by inducing an
osmotic change so that the capsules collapse and unbound hormones
migrate out of the capsules. This can be accomplished, for
example, by adding serum albumin or polyethyleneimine to the
system which promotes efflux of intracapsular liquid.
Alternatively, free species may simply be washed from the micro-
capsules. Results are interpreted by comparing the assay of
analogue content to a standard, such as a curve of free hormone
concentration vs. radioactive count, fluorescent intensity, or
other marker characteristic used to indicate the presence of the
analogue.
llZ~397
1 The assay may be used to detect the presence and/or
concentration of free thyroxine, tri-iodothyronine, neonatal
thyroxin, testosterone, cortisol, other steroid hormones, and
other substances which reversibly bind with protein. The assay
may also be used to detect species which do not bind to protein
to any significant degree, e.g., drugs such as digoxin. Essent-
ially any such material may be determined provided a complement-
ary binding substance and a distinguishable analogue of the
material is available or can be produced.
The assay may be routinely conducted using a test set
comprising analogue, microcapsules containing antibody standards
and blanks containing predetermined concentrations of the subject
species, and a reagent for removing unbound species and analogue
from the capsules after completion of the incubation. For qual-
ity control, the solution in the microcapsule may be colored. A
colored supernatant, after the capsules have been settled out or
sedimented via centrifugation, would be indicative of microcap-
sule breakage and possible leakage of antibody. Unlike convention-
al assays, this assay avoids loss of clinical correlation with
diagnosed conditions over a broad range of concentrations of
protein, hormone, and interfering substances.
Objects of the invention are to provide a rapid simple,
and reproductible method of detecting the presence and concentrat-
tion of species unbound to protein in a liquid sample, a reagent
use in such assays, and a test se~ suitable for rapidly and con-
veniently conducting such assays. Another object is to provide
a method of determining free T4 in samples extracted from human
blood and containing protein bound T4.
Another o~ject is to provide a competitive process for
the determination of an i~munogenic substance which combines the
~2U397
1 advantage~ of the solid phase radioimmunoassay system and the
advantages of the liquid phase radioimmunoassay system. A further
object is to provide a highly reliable process for the determin-
ation of the amount of digoxin in sexum. These and other objects
and features of the invention will be apparent from the descrip-
tion which follows,
BRIEF DESCRIPTION OF THE DRAWINGS
Fig, 1 illustrates a standard curve made in accordance
with the procedure of the invention. It consists of a plot of
counts per minute ~x 103~ on a y-axis linear scale vs. free-T4
concentration in ng/dl on an x-axis log scale (see Table 1 for
data~;
Fig. 2 illustrates a second standard curve for a free
T4 assay of the invention in cpm ~linearl v. ng/dl free-T4 (log
scale~. Data for this figure is found in Table 2;
Fig. 3 is a graph of cpm from T4 (125I) bound to T4
antibody contained within semipermeable microcapsules immersed in
standard solutions of free T4 VS. time. As serum T4 replaces T4
~125I~ at T4 antibody binding sites, the cpm remaining in assoc-
2~ iation with the antibody decreases. Note that the rate of replace-
ment is largely linear to two hours of incubation at 37C;
Fig. 4 graphically illustrates the correlation of
results ~etween the dialysis assay technique and the microencap-
sulation system of the invention;
Fig. 5 graph~cally illustrates the normal range of free-
T4 as established by the microencapsulated assay system;
Fig, 6 graphically illustrates the correlation between
kinetic radioimmunoassay and the microencapsulated type assay
system of the invention; and
Fig. 7 shows a digoxin stardard curve as counts per
minute Ccpm~ bound to antibody, as plotted on the linear scale, vs.
112~3~
1 digoxin in nanograms/milliliter on the log scale of 2 cycle semilog
paper ~data from Table 5);
Fig. 8 shows digoxin as % bound (relative) vs. digoxin
concentration, Percent bound (relative) is calculated as % bound
(relative) = mean cpm bound/means cp~ bound of O ng/dl digoxin
standard x 100 ~data from Table 8); and
Fig. 9 is a diagram showing the retention of antibody
and free passage of immunogenic substance in microcapsules formed
in accordance with the present invention.
DESCRIPTION OF THE PREFERRED EMBODIMENT
The process of the invention requires that the sample
containing the species to be detected be incubated with antibody
complementary to the species (or other substance capable of rever-
sibly binding with the species~ and a distinguishable analogue of
the species, and that-:the sample and antibody be separated by a
semipermeable membrane or membranes that exclude the passage of
high molecular weight proteins.
Antibodies to selected species may be produced in
accordance with well-known techniques involving injection of the
hormone into laboratory animals, possibly together with an adju-
vant, and subsequently extracting and purifying the produced anti-
bodies. Many such antibodies are available commercially. Many
distinguishable analogues of species detectable by the p~ocess of
the invention are also commercially available or can be made using
known techniques~ The term "distinguishable analogue" as used
herein, refers to a molecule which has antibody binding properties
similar to and preferably identical to the species sought to be
detected, and which i5 characterized by a property which allows a
measure of its concentration to be readily obtained~ Preferred
analogues compr~se a sample of the species to be detected tagged
~,
,,~ .
1 with a radioactive atom: for example, thyroid hormones may be
conveniently tagged with 125I and can then be quantitated by
measuring gamma radiation. However, it is contemplated that other
types of analogs may be employed, so long as the analoaue has a
molecular weiqht and resulting dimensions well below those of
natural proteins and the antibody used. Thus, analogues may be
produced bytagging a sample of the species to be detected with a
relatively low molecular weight enzyme, fluorescent moiety, or
other moiety which enables quantitative measurement of the con-
centration of the analogue by physical or chemical means.
To practice the assay, the antibody and analogue must
be separated from the sample by one or more membranes having a
permeability sufficient to preclude the passage of antibodies and
natural proteins (,which uniformly have a molecular weight in
excess of 20,000 daltons), but sufficient to allo~ free passage of
the species to be detected and its analogue. In a preferred
embodiment of the invention, the membranes take the form of semi-
permeable microcapsules containing the antibody and analogue.
--10--
~Z~3~
1 It is noteworthy that the semipermeable microcapsules
used have a permeability similar to the dialysis membranes de-
scribed above. However, the semipermeable microcapsule wall is
hundreds of times thinner than conventional dialysis membranes,and
its available surface area is orders of magnitude larger per unit
weight. Free-T4 or other free hormones freely enter the micro-
capsule and displaces, in proportion to its concentration, labled
T4 from the T4 antibody. Thus, an equilibrium of free T4 and
labled T4 is approached within the capsule during the incubation
period.
Suitable methods of encapsulating biological materials
in membranes having the foregoing permeability properties are
disclosed in detail in Canadian patent number 1,075,179; Canadian
patent application serial number 333,335 filed August 3, 1979, all
to F. Lim et al., and in Canadian patent application serial number
348,524 (March 26, 1980) to F. Lim. The presently preferred
method of producing such microcapsules produces semipermeable
polyamide membranés by an interfacial polycondensation technique.
Mutually immisible solvents or solvent systems are selected, e.g.
~0 water and a cyclohexane based solvent, and one monomer of a
complementary pair which form a copolymer is dissolved in the
water together with the material to be encapsulated. The aqueous
solvent containing the material to be encapsulated is then
emulsified within the other solvent to form a plurality of discrete
droplets. The second, complementary monomer is next added to the
continuous phase of the emulsion to initiate polymerization about
the droplets at the phase boundary. Membrane permeability and
uniformity of polymer depositions are controlled by varying the
affinity of the continuous phase of the emulsion for the encapsu-
30. lated monomer during the course of poly-
--11--
B
~i2~3~7
1 ~eriz2tic~ 2~.~ ky cent-ollins the concertr2tion of the r~2ctirg
monomers and the duration of the polymerization.
. , .
In one a~proach, the continuous phase at the outset is
a solvent or solvent system having a relatively high affinity for
the encapsulated monomer so that, in a first stage of polymeriza-
tion, a relatively thick polymer network is produced about the
¦¦droplets- Thereafter, the continuous phase is altered such that
¦¦its affinity for the first monomer is decreased, e.g., by
lidiluting the continuous phase with a second solvent or by
llreplacing the continuous phase with a fresh solvent. Upon the
addition of second monomer, further polymerization occurs pre-
jiferentially within the initially deposited polymer network,
patching macroporous defects and resulting in uniform capsule
membranes which allow diffusion of solutes below a certain mole-
cular weight.
In another approach, the continuous phase at the outset
is selected to have a low affinity for the encapsulated monomer
so that thin, relatively dense membranes form in a first stage of
polymerization. Thereafter, the affinity of the continuous phase
for the encapsulated monomer is increased to draw further quan-
tities of monomer through the membrane and to deposit a second
outer layer of insoluble polymer.
When the discontinous aqueous droplet phase is buffered
to provide a compatible environment for labile biological
materials such as an antibody, the encapsulation can be conducted
in a manner to preserve a large percentage of the labile
material' 5 biological activity. The operability of the encap-
sulated material is also preserved by adding second monomer to
the continuous phase in increments over the duration of the poly-
~lZ~397
1 ~erizati~n ~o th~t i~ concentration at any oiven time is relati-
vely low and the antibody is not expo~ed to high concentrations
of potentially destr~ctive s~bstances.
!l In a preferred reaction system, aqueous droplets con-
taining a diamine, a high ~olecular weight filler material, and
Il the antibody are prod~ced in a continuous phase of cyclohexane
jlwhose affinity for the monomer dissolved in the droplet phase is
!! modified by the addition of chloroform as a diluent. The addi-
lltion of a diacid halide to the system results in the formation
¦¦ of semipermeable polyamide microcapsules. This microencap-
sulation approach is effective for producing membranes having an
upper limit of permeability in the 2000-30,000 dalton molecular
,weight range. Thus, the capsules can easily be engineered to
¦¦permit the diffusion of many hormones.
To conduct the assay, the test sample is mixed with
~microcapsules of the type described and incubated, preferably at
~about 37C. Prior to the incubation, the capsules may be
suspended in a solution of the species analogue of sufficient
concentration to load the antibody with a detectably amount of
the analogue concentration. However, the assay can be conducted
by adding the analogue to the reaction system d~ring or after the
incubation with serum. Protein in the sample and protein-species
complexes cannot traverse the microcapsule wall.
Next, the antibody is separated from the remainder of
the reaction system (optionally exclusive of the microcapsule
membranes) and`either the antibody or the remainder of the system
is assayed for analogue. In a preferred method of making the
separation, a high molecular weight hydrophilic material such as
a solution of polyethylenimine or serum albumin is added to the
3S~
1 extracapsular volume. This induces an osmolality increase result-
ing in collapse of the microcapsules and migration of intracap-
sular unbound hormone and its analogue into the supernatent.
The result is a packed pellet of encapsulated antibody which,
after an optional centrifuge treatment, may be isolated by aspir-
ating or decanting the supernatent. Alternatively, the separation
may be conducted by washing free species and analogue from the
capsule.
Tables 1 and 2, and corresponding Figs. 1 and 2 of t~e
drawing disclose the results of assays embodying the invention
designed to detect the presence of free T4 using free T4 antibody,
125I labled thyroxine as an analogue, and solutions of known free
T4 concentration. Tests of unkno~ns run in parallel with the
procedure used to gather this data may be interpreted by refer-
ence to the standard curves.
~;1
3~7
TABLE 1
Free ~4 Assay Standard Curve
I Free T4 CPM MEAN CPM
0.5 ng/dl 57097 57,285
57472
I! 1. 3 ng/dl 54256 54 ' 839
Il 54717
Il 3.0 ng/dl 50960 50,953
I; 50946 - I
i! 5,0 ng/dl 48487 48,302
II 48117
Il . I
117-7 ng/dl 45982 46,025
II 46067
I¦TOTAL COUNT
¦~ T4 ~125I) ADDED 86,403
incubation 2 hours, 37C
~iZV3~7
; TABLE 2
Free T~ Assay Standard Curve
~l AVG. CPM
jj0.1 ng/dl 61219
60398 60933
61183
¦¦ 0.5 ng/dl 58020
55618 56675
56389
¦jl.0 ng/dl 52096
52671 52824
53705
2.0 ng/dl 48483
50126 49536
49971
4,0 ng/dl 44853
. . 45108 44732
44235
6.0 ng/dl 42008
41344 41456
41015
TOTAL COUNT
T4 (125 I~ ADDED 86,903
2 hour incubation, 37C
~ZV397
1 The invention will he further understood from the
following, non limiting example.
Preparation of Microcapsules
Hexanediamine carbonate (pH = 8.5 + 0.1) solution is
prepared by mixing 17.7 ml 1,6 hexanediamine with 32 ml of water,
and bubbling CO2 through the solution for about 1 hour or until
the pH level is reached. Terephthaloyl chloride (TCl) solution is
prepared by adding 20 g TCl in 200 ml of organic solvent consisting
of 4 parts cyclohexane and 1 part chloroform. TCl is dissolved
by stirring vigorously, and the solution is then centrifuged for
10 minutes at 2600 rpm. Any precipitate is discarded.
750 ml cyclohexane are mixed with 125 ml SP~-85*
~emulsifier, fatty acid ester or sorbitan) in a 2-liter mixer
e~uipped with a magnetic stirring bar. While stirring, a mixed
solution made from one ml antiserum to thyroxine (4~ in phosphate
, buffered saline, R. F. Laboratories, Houston, Texas, or Radioassay
Systems Laboratories, Carson, California), 25 ml of polyvinyl
pyrrolidone - 4% bovine serum albumin, and 30 ml of hexanediamine
carbonate solution is added to the cyclohexane. When droplets of
the desired size have been produced, 70 ml TCl solution are added.
Thirty seconds later, 37.5 ml of TCl are added. Sixty seconds
later, 25 ml of chloroform are added. Three additional 25 ml
aliquots of chloroform are added at 30 second intervals.
The microcapsules are recovered by centrifuging the
two-phase reaction system, decanting the supernatant, and mixing
the capsules with TWEEN-20* (polyoxyethylene derivative of fatty
acid partial ester of sorbitol anhydride - emulsifier buffered
with NaHCO3) and phosphate buffered saline. The capsules
*Trade Mark
-17-
~,~
~lZV3~
1 retain the polyvinylpyrrolidone and bovine serum albumin filler
materials, as well as the thyroxine antibody.
Saturation of Antibody with Analogue
Microcapsules made in accordance with the foregoing
procedure may be loaded with 125I labled thyroxine (Cambridge
Nuclear Corporation, Billerica, Massachusetts~ by the following
steps.
1. Add to each of 100 standard tubes 0.5 ml of micro-
capsu~ suspension and 1.0 microCurie of T4 (125I~ (high specific
activity of 5-6000 micro Ci per microgram). Allow to incubate at
37C for at least thirty (30) minutes.
2. Wash the microcapsules with twice their volume of
phosphate buffered saline (0.15 M NaCl, pH = 7.5, 0.015 M phos-
phate buffer~.
3. Centrifuge at 20Q0 xg for 15 minutes and decant
supernatant.
4. Repeat steps 2 and 3 twice.
5. Dilute microcapsule suspension ~ith 1.6 times their
volume of the phosphate buffered saline disclosed above. Total
volume equals 80 ml. 0.8 ml of microcapsule suspension are used
per test; thus, 100 tests may be conducted with the capsules.
Test Procedure
1~ Place 25 microliter test samples and 5 samples of
known free T4 concentration in separate tubes. In the standard
curve from Table 1, concentrations of 0.5, 1.3, 3,0, 5.0 and 7.7
ng% of free T4 were used, but any series of free T4 concentrations
may be adapted according to well-known experimental techniques. A
control tube containing 25 microliters of saline may also be
included as a further check on assay accuracy.
2~ Pipette 800 microliter of T4 (1 5I) pre-saturated
microcapsules (supplied as such) into each tube.
-18-
.. .. .
~l~V397
1 3) Vortex each tube and incubate for 120 minutes at
37C.
4) After incubation, add 1.0 ml of 2.0% polyethyle
neimine (m.w. 40-50 thousandl in phosphate buffered saline to
each tube.
5) Incubate for an additional 20 minutes.
6~ Decant supernatants.
7) Count each tube for one minute in a gamma counter.
Calculation of Results
1~ Each time an assay is run for determination of un- -
known free-T4 concentration in a sample~s), standards to prepare
the standardscurve should be run.
2~ Upon completion of the assay, a standard curve such
as shown in Fig. 2 or 3 is prepared using values obtained from
the standards which were assayed concurrently with unknownsamples.
31 Counts per minute (cpm~ for each value can be
plotted in the linear scale of 2 cycle semilog graph paper versus
free-T4 concentration in nanogram percent on the log scale.
An alternative to plotting cpm v. free T4 concentration
is to plot percent bound ~relative) v. free T4 concentration.
--19--
~12~
1 ,nis can ~e acco~plished by calculating the percent bound
trelati~e) for each standard, control, or unknown and plotting
these values on two cycle semiloa paper in a manner similar to
i that described previously for cpm. Percent bound (relative) is
calculated as follows: percent bound (relative) = cpm bound/mean
cpm bound of standard of lowest free T4 concentration.
il Figure 4 is a graph of free T4 concentration in ng%
of about 200 test sa~ples, each of which were assayed by the 1,
I! method of this invention and the dialysis method. As shown,
llthere is a high degree of correlation between the two test
Ijmethods.
!! Figure S is a graph of frequency of a given free T4
concentration vs. free T4 concentration based on some 200 test
j~samples assayed in accordance with the procedure set forth above.
Il I
Figure 6 is a graph of free T4 concentration in ng% of
about 100 test samples, each of which were assayed by the method
¦¦of this invention and the kinetic radioassay technique. As
shown, there is a high degree of correlation between the two test
methods. ,
Table 3 shows the consistency of the results intra
assay.
397
TA~LE 3
_ntra Assay Variation (Values in n~/dl)
!i Level A (1.2) Level B (2.0) Level C (5.3)
1.5 '~f.3 3.6
' 1.3 2.1 4.2
1.1 2.2 4.2
1.1 2.1 3.3
1.2 2.1 3-3
Ij I
' !
1.3 2.0 3.9
" 1.3 1:7 4.1
1.4 2.1 3.7
1.2 2.5 3.5
j 1.3 1.8 3.5
Il !
1.3 2.2 4.4
!1 1.3 2.1 3-9
1 6 2 3 4 6
1 2 2 5 4 6
1.3 2.3 4.4
1.2 2.1 4.2
1.2 2.1 3.4
1.3 2.3 3.8
1.5 2.1 4.2
1.4 ~ 2.2 4.6
~lZ~39~ ~
"
1.4 2.1 3.7
1.4 2.1 4.0
1.4 2.1 4.0
1.4 2.3 4.3
1.4 2.3 4.0
1.6 2.4 4.0 i
1! 1.3 2.0 4.2
il !
1.2 2.1 4.2
2.4 4.5
il I
;~Coefficient of Variation Number x + S.D. C.V.
!t
!1 A 28 1.34 + .13 9.6%
¦~ B 29 2.2 + .17 7.7%
C 29 4.1 + .3~ 9.3%
~able 4 shows the consistency of results, interassay.
~iZV3~7
1 TABLE 4
Interassay Variation: ~values in ng~dl)
Test ~ Level A (1.2) Level B ~2.0) Level C (5.3)
1 1.3 2.4 4.6
1.2 1.8 4.4
2 1.4 1.5 4.6
1.5 2.6 4.7
1.2 2.0 4.0
1.3 2.0 3,7
1.4 2.5 3.8
1.2 1.9 4.0
3 1.3 2.2 3,9
4 1,4 2.0 3.2
1.2 2.1 4.0
1.5 1.8 4,2
6 1.3 2.Q 4.2
1.2 2.0 4.5
7 1.3 2.1 3.5
1.35 2.5 3.7
1.1 1.7 4.0
1.2 2.0 3.5
1.4 4.2
1.4 4.0
-23-
~r
V39t7
Co2fficient of Variation ~'ur~ber x + S.D. C.V.
_
A 20 1 . 32 + . 11 8 . 41%
~j B 18 2.1 + . 27 13%
C 20 4 . 0 + . 4 lQ%
li !
Il i
Il .
Il ~
Il I
Il !
!l i
1~ 1
Il . ' ~
--24--
~2~397
Digoxin Assay
A proc~dure for encapsulating antibody specific to
l dia~xin is set forth below.
! The reag~nts use~and their suppliers are listed belo~:
il
l Sodi~m Bicarbonate
Cyclohexane
Chloroform All Fisher Chemical
Sodium Chloride
Sodium Phosphate, monobasic
Sodium Phosphate, dibasic
1,6-Hexanediamine
Span-85
Tween-20 Ruger Chemical, New Jersey
Anti-Digoxin Rabbit Antiserum Arnel Products, Brooklyn,
New York
Bovine Serum Albumin
Comassie Brilliant 81ue R Sigma Chemical
Polyvinylpyrrolidone-40 Aldrich Chemical
Terephthaloyl Chloride Eastman Kodak
The quantity of each ingredient is as follows:
1,6 Hexanediamine Carbonate 30 ml
Phosphate Buffered Saline 40 ml
Polyvinylpyrrolidone 40/Comassie Blue 25 ml
Anti-Digoxin Rabbit Antiserum 5 ml
Cyclohexane, ACS 750 ml
SPAN-85 ~ 125 ml
Terephthaloyl Chloride Soluti~n 107.5 ml
Chloroform 100 ml
~l~V3~7
1 Tween-20 ~ash Solution Q.S. 200 ml
Phosphate Buffe~ed Saline Q.S. 8 1
Procedure: Rinse all glassware in distilled water prior to use.
1. Place 2 liter glass mixer in hood on magnetic
stirrer,
2. On bench adjacent to hood, set up microscope.
3. In 250 ml glass graduated cylinder, carefully mea-
sure 125 ml SPAN-85.
4. Pour measure SPAN-85 into glass mixer in hood.
5. Measure 750 ml cyclohexane in a 1000 ml glass grad-
uated cylinder. Pour into glass mixer in hood.
6. Place cover on mixer.
7. Turn on magnetic stirrer.
8. Measure: 30 ml Hexanedianmine Carbonate; 30 ml PBS;
25 ml 15~ P~P/Comassie Blue/4~ BSA; 5 ml Antibody; mix the 40 ml
PBS with the 5 ml Antibody in a 50 graduated cylinder.
~ . Put a 2-inch stir bar with spin ring in a 400 ml
beaker. Place on magnetic stirrer.
lQ. To beaker add 30 ml hexanedianmine. Start magnetic
stirrer.
11. To Hexanediamine in ~eaker add in the following
specified order: 25 ml 15% PVP/Comassie Blue/4% BSA; 35 ml PBS/
Antibody solution. Let mix for 2 min. Let solution mix for
three ~3~ min.
~26-
llZV3~ ~
,l 12. ~hile solution ic miYing, ~easure one 70 ml por-
tion and one 37.5 ml portion of TCL in separate 100 ml glass gra- ¦
duated cylinders. Cover ~ith watch glass and set in hood.
I ~easure Iour ~4J 2~ ml por~ions or cnioroform in separate 25 ml
¦ glass grad~ated cylinders. Cover with watch glasses and set
¦ inside hood.
~ 13. Through side ar~ of vial, add contents of 400 ml
¦ beaker to glass mixer in hood.
14. As rapidly as possible, with a disposable glass 1
ml pipette, take a sample of the solution in the mixer and put it
on the m~croscope slide. Check to determine that the droplets
are of an acceptable size. (10-80 microns in diameter)
15. When the droplets are of acceptable size T=0, add
measured 70 ml of TCL through side arm of mixer. At T=30,
exactly 30 seconds later, add second 37.5 ml portion of TC~ to
mixer through side arm. Let mix exactly 60 seconds.
16. At 60 seconds (T=90) add first 25 ml portion of
chloroform. ~ix 30 seconds. (T-120) Add ~econd measured 25 ml
chI~roform, mix 30 seconds (T-150). Add third measured 25 ml
chloroform. Hix 30 seconds ~Tz180). Add fourth measured 25 ml
chloroform. Mix exactly 30 seconds ~T=210"). Stop mixer.
17. Pour contents of mixer into two 1 liter plastic
centrifuge bottles which have been rinsed in distilled water 3
times. Centrifuge at 500 RPM for three minutes.
2~ 18. Carefully decant supernatant.
19. To each bottle add approximately 50 ml of Tween-20
solution (i.e. approximately the ~ame volume Iween-20 as
capsules). Mix well with stir bar retriever for about 5 min.
1 ~1Z{~3~7 1
20. Add approximately 10-15 mls Pss to each ~ttle.
S~ir hell.
epea~ sLe~ 2~ im~s.
22. Add 400 mls PBS. Mix well.
1 23. Balance and centrifuge bottles for 20 min. at 3000
RP~. Aspirate supernatant. Add approximately 800 ml PBS to each
bottle. Stir well and cap.
24. Repeat step 23, 10 times. After final aspiration
add 100 ml of PES to each bottle and combine contents of both
bottles. Shake well. Pour into 500 ml glass sraduated cylinder
and Q.S. to 500 ml with PBS.
¦ 25. Store in 1 litre glass reagent bottle at 4C.
¦ The Tween-20 wash solution is prepared as follows:
Procedure: Can be made day before use
1. Weigh 6.06 g sodium bicarbonate on triple beam
balance.
2. Carefully put 6.06 g sodium bicarbonate into 250
ml volumetric flask containing stir bar.
¦ 3. Add approximately 200 ml purified water and stir on
magnetic stirrer until sodium bicarbonate is dissolved.
4. Remove stir bar and Q.S. to 250 ml with purified
water.
5. Carefully pour into 500 ml Erlenmeyer flask con-
taining stir bar.
I
I - 2~ -
i
112V397
1 6. Carefully measure 25~ ml Tween-20 in a 250 ml gra-
duated cylinder.
7. Add to Erlenmeyer flask containing sodium bicar-
bonate solution.
8. Mix on magnetic stirrer until completely mixed
(approximately 1 hour).
9. Store tightly sealed in polyethylene 1 L bottle
with screw cap at approximately 25C.
The terephthaloyl chloride solution is made as follows:
Should be made day of use. DO NOT REFRIGERATE.
1. Weight of terephthaloyl chloride ~TCIl is recorded
on bottle containing TCI. Multiply weight of TCI is grams by ten
to calculate volume in milliters cyclohexane/chloroform solution
to add to TCI.
Calculations:
g. TCI x 10 = ml total volume cyclohexane/chloroform
solution. Be sure total volume ifi sufficient for procedure being
followed.
2. The cyclohexane/chloroform solution is four parts
cyclohexane and, part chloroform. Divide the total volume cyclo-
hexane/chloroform solution (Step 1 above~ by five to calculate the
volume chloroform. Multiply the chloroform volume by four to
determine the cyclohexane volume.
Calculations:
(a~ ml total volume - 5 = ml chloroform.
cyclohexane/chloroform
(b) ml chloroform x 4 = ml cyclohexane.
3. Carefully measure the calculated volumes cyclohexane
and chloroform in graduated cylinders. Combine in Erlenmeyer
flask. Swirl gently to mix. Cover with watch glass. Put in fume
hood,
-29-
l~ZV397
1 4. Put magnetic stirrer in fume hood~
5. Put bottle containing TCI on magnetic stirrer.
Open bottle and as quickly as possible add a magnetic stir bar
and cyclohexane/chloroform solution. Replace cap on bottle.
6. Stir on magnetic stirrer until all TCI is dissolved.
It may be necessary to tip bottle to dissolve any TCI around top
o~ bottle.
7. As quickly as possible, equally fill as many 200 ml
glass centrifuge bottles as necessary with TCI solution and cap.
Centrifuge 10 min. at 2600 rpm in room temperature centrifuge
8. Pour supernatant in 500 ml amber bottles and seal
well.
9. Store, tightly sealed, at approximately 25C.
The procedure for preparing 15% PVP 40/Comassie Blue
with 4~ BSA is as follows:
Procedure: Make day of use. DO NOT REFRIGERATE.
1. Accurately weigh on triple beam balance 7.5 gm
Polyvinylpyrrolidone 40; 2 g BSA; and .1 g ~100 mg) Comassie Blue.
-30-
)397
2 . Put polyvinylpyrrolidone 40 into 50 ml glass beaker
~ ~ith stir bar.
!' 3. A~d 2pproximately 20 ml PBS. Put glass cover pl2te !
~ on beaker.
4. Stir on magnetic stirrer until dissolved.
5. Put .1 g Comassie Blue and 2 9 BSA into another
50 ml glass beaker.
6. Add approximately 20 ml P8S. Put glass cover plate
on beaker.
7. Stir and heat slightly using magnetic hot
plate/stirrer #10 with heating unit set on 2. Let stir until
dissolved (approximately 10 minutes).
8. Carefully add entire contents of beakers containing
polyvinylpyrrolidone 40 solution and Comassie Blue solution
¦¦ to a 5 ~1 glaLs volumetric flask with ~tir bar.
9. Mix combined solutions for 10 minutes on magnetic
stirrer. Remove stir bar.
I 10. Q.S. to 50 ml with PBS.
¦ 11. Adjust to pH - 7.5 ~ 0.5 with 1 N sodium hydroxide
, Orig. pH _ final pH Amt. lN NaOH used
12. Pilter solution with Nalgene disposable membrane
filter unit ~O.g5 u).
~ .
13. Store sealed in 60 ml polyethylene bottle with
¦ screw cap at approximately i50C.
. I
I -31-
~i2V3~7
1 The procedure for preparing phosphate buffered saline
is as follows:
Procedure: Can be made day before use.
1. In 1000 ml glass graduated cylinder, carefully
measure lQ00 ml phosphate buffered saline stock.
2. Pour into 20 L polyethylene carboy.
3, Measure, in 1000 ml glass graduated cylinder, 9000
ml deionized water and add to carboy containing phosphate buffer-
ed saline stock.
4. Stir using magnetic stir bar retriever.
5. Check pH. If necessary~ adjust pH to 7.5 + 0.05.
Final pH =
.
6. Store phosphate buffered saline in tightly sealed
20 liter polyethylene carboy. Store at approximately 25C until
used.
The procedure for making 1,6 Hexanediamine Carbonate
i i5 as follo~s:
Procedure: Can be made day before use.
1. Place bottle of 1,6 hexanediamine in 3 liter beaker.
Add enough tap water to the beaker to reach le~el of hexanediamine
in bottle.
2. Loosen cap of hexanediamine bottle,
3. Place beaker on magnetic stirrer~hot plate with heat
setting at 2 until hexanediamine is completely melted.
-32-
~,
v3~7
!! 4. In graduated glass 25 ml cylinder, accurately
¦I re~sure 17.7 ml hexanedia~ine. Pour carefully into 500 ml a~ber ¦
t.le.
¦ 5. ~ccurately measure 32 ml purified water in 50 ml
! glass graduated cylinder. Add to hexanediamine in amber bottle.
6. Bubble C02 through solution for approximately 1
hr. until pH - 8.5 ~ 0.1. ~inal pH
7. Seal amber vial and store at approximately 25C.
The microcapsule and principle of its operation are set
forth diagramatically in Fig. 9. As is shown in Fig. 9, antibody
g which is specific to digoxin is trapped within wall 10 of
microcapsule 12. Walls 10 of the microcapsule 12, however, have
openings 14 which are small enough to permit the free passage of
digoxin. Thus, digoxin 16 from the sample and labeled digoxin 1
are free to pass through the walls of the microcapsules and com-
pete with each other for sites on antibody 9. Any unbound
digoxin 16 or 18 can be washed from within the microcapsule after
incubation is completed.
As is ~et forth above, some of the digoxin i8 labeled
and it is the labeled digoxin that competes with the unlabeled
digoxin from the sample for sites on the antibody specific
¦ to digoxin. The preferred labeled digoxin i8 (125I) digoxin
¦ which may be obtained from New England Nuclear Corporation,
Billerica, Massachusetts.
Test Procedure
1. A 6ample or samples of serum to be tested is
obtained through procedure well ~nown to the art-In this example
controls are used.
- 33 -
1,
2(~3~7
il 2. Pi~et 0.1 ml (100~1) of each standard, control
or unknown patient serum sample into correspondingly labeled
! t~b~s containing 0.5 ml of digoxin antibody microczpsule
j suspension as prepared above.
3. Pipet 0.5 (500~ 1) of 125I labeled digoxin (in
phosphate-buffered saline) into each tube. Vortex for 3-4
seconds minimum.
4. Incubate all reaction tubes in a water bath (37Cl
for at least fifteen tl5) minutes. lAlternatively incubate all
reaction tubes at room temperature (20-25C) for at least thirty
(30) minutes.]
5. Add 0.5 ml (500~1) of wash solution to each tube.
Vortex for 3-4 seconds minimum.
6. Incubate all reaction tubes at room temperature
(20-25C) for five (5) minutes.
7. Centrifuge all tubes at 1600 xg minimum, for ten
(10) minutes, room temperature (20-25C), and decant supernatant
into an appropriate waste container catching the last drop on
blotter paper.
8. Count all tubes in a gamma counter, adjusted for
5I, for one (1) minute each.
9. Plot ~tandard curve of known amounts of digoxin v.
¦~ cpm an ead ynknown digoxin amounts ~rom this curve.
Reagents Used
The digoxin-antibody is encapsulated in semi-permeable
nylon microcapsules suspended in phosphate buffered saline as
~. ilZ0397
' pre?ared above. For quality control, the antibody microcapsules
j contain bl~e dye (Comassie Blue R250). A blue supernatant,
Il c~r ,he r~icroca~sules have settled out or sedimented via
¦! centrifugation, would be indicative of microcapsule breakage
S 11 and possible antibody leakage.
1251 L~beled Di~oxin
Each ml of solution contains 10~ 9 of 125I digoxin
of less than 4.2 ~ Ci.
Buffer Solution
1 0.015 M phosphate buffer, pH 7.5 containing 0.15 M
¦ sodium chloride 0.5~ Bovine Serum Albumin (BSA), 0.1% sodium
azide.
Wash Solution
~ Polyethlene glycol 6000, 20% 501ution in 0.5 M bar-
bital buffer, p~ 8.9 containing 0.15 M sodiu~ chloride.
Calculation of the Results
The results of this experiment a typical test are
shown in Table 3 and depicted graphically in Figs. 7 and 8.
¦ In Fig. 7 CPM is plotted on the linear scale of 2 cycle
¦ cemilog graph paper against the concentration of digoxin in ng~ml
¦ (nanograms/milliliter) on the log scale. An alternative to
plotting CPM vs. digoxin concentration is to plot % bound
(relative) vs. digoxin concentration (see Pig. 8). ~his can be
I accomplished by calculating the 4 bound (relative) for ea~ch stan-
~ dard, control,-or unknown, and plotting these values on two cycle
semilog paper in a manner similar to that described previously
for CPM. Per cent bound (relative) ~s calculated as follows: 4
bound (relative) = (Mean CP~ bound of 0 ng/ml digoxin standard~ x
100.
- 35 -
,. . .
12U~9~
i'
i! TA~LE 5
ii ng/ml CP~I Bound
i Digoxin 1 2
! Standards 0 16,888 17,136
0.5 14,464 14,473
1.0 12,279 12,192
2.0 9,690 9,717
4.0 7,344 7,445
Controls . 1 10,360 10,486
2 ~ 8,226 8,380
CPM Total = 26,780
Digoxin
Relative ~/~ Bound (CPM Value
Ilean Bound/CPM Bound)~; x 100 ng/ml
1 17,012 100.0
14,469 85.0
12,236 72.0
. 9,704 57.0
7,395 43.0
10,423 61.3 , 1.7
8,303 48.8 3.1
:~Bound = CPM Bound at 0 ng/ml digoxin
The assay system of the instant process has been found to be
quite precise: The intra assay variation is less than 7% and
¦the interassay variation is less than 9%.
Intra Assay Variation
The coefficients of variation, CV, for two control
samples, each sssayed twenty-five (25) times within one experiment
were found to be
~ZV3g7
i.ean Di~oxin ng/ml C-~
I~Control 1 1.54 4.9%
! ! Control 22.95 6.2%
I~InterAssay Variation
The coefficients of variation for two control samples,
each assayed three times in eighteen separate experiments were
found to be:
Mean Digoxin ngJml CV
IControl 1 1.50 7.3Z
Control 2 3.04 8.8%
Table 6 shows the extremely high specificity of this
assay for digoxin and to the exclusion of other potentially .
interfering substances.
¦ TABLE 6
jSpecificity
% Cross-reactivity of various biochemical compounds
with microencapsulated antidigoxin antisera
Compound Z Cross Reactivit~
Digoxin 100.00
Digitoxin 0.80
Progesterone 0.16
Cortisol . 0.10
Testosterone 0.08
Dehydroandrosterone sulfate 0.08
Cholesterol - 0.06
Quabain 0.02
3~7
1 I Quantative Recovery
The ~ tant procec~ ha~ been foun~ to be highlv
quantative, reflecting no less than 96r/~ recovery of the digoxin
,samples added.
I . ,
TABLE 7
IRecovery Study
Il Initial Total Measured
Digoxin Level Digoxin Digoxin Di~oxin
i ng/ml Added ng/ml ng/ml ZRecovery
i 0.64 0.5 1 14 1.13 99
Il 0.64 1.0 1.64 1.72 105
ll 0.64 2.0 2.64 2.70 102
0.64 4.0 4.64 4.45 96
Il i
¦IIt is of particular importance to note the high degree o
correlation of the results of the instant process with digoxin
determinations as made by other assay methods.
Table 8 expresses comparative test results obtained by the
process of this invention versus those of other assays.
The systems A and C represent liquid assay systems with
precipitating reagent separation, while system B utilizes solid
phase separation. As shown, t~ere is a 0.97 - 0.98 correlation
coefficient.
TABLE 8
AGREEMENT ~OR VARIOUS ASSAY PROCEDU~ES
Coefficients of-correlation for 135 samples assayed by System A,
System B, System C, and the Present System were:
n r* y
System A 135 0.98 1.12 x -0.1
System B 135 0.97 0.90 x -0.02
System C 135 0.97 0.96 x +0
Present System 135 0.97 0.95 x ~0.14
*r = coefficient of correlation
- 3~ -
3~ j
1 In vie~ of the foreooing, it is apparent that the assay
techr,igue disclosed herein may be used to determine the presence
ar.~ cv..c~-ntration Or any free species, provided that a complemen-
!! tarY substance ca~able of specific binding with the species and â !
distin~uishable analo~ue of the species are available. It is a
~further requirement that the molecular weight of the species and
of i~s analogue be sufficiently low so that it is feasible to
provide microcapsule memhranes which selectively allow diffusion
llof these substances while preventing passage of high molecular
llweiaht materials such as natural proteins. Fortunately, steroid
hormones, thyroid hormones, many drugs and other classes of
jlsubstances of clinical importance are characterized by molecular
!I dimensions far smaller than natural proteins.
!j The invention may be embodied in other specific forms
¦¦without departing from the spirit or essential characteristics
i! thereof. The present embodiments and example are therefore to
¦be considered in all aspects as illustrative and not restrictive
and the scope of the invention is ~ndicated by the appended
Iclaims rather than the foregoing description. A11 changes which
¦comë within the meaning and range of equivalency of the claims
are therefore intended to be embraced therein.
