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Description  |
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This invention relates to clinical techniques for immunological
determinations and more particularly to radioimmunoassay by manual and
preferably by automated technique.
In the aforementioned copending application and in the copending
application Ser. No. 33,098, filed Apr. 29, 1970, now U.S. Pat. No.
3,793,445 entitled "Reagent for Immunological Determinations", description
is made of a device and technique for immunological determinations of a
fluid system in which use is made of a measured volume of dried,
insoluble, yet highly hydrophilic gel particles in which there are
entrapped one or more binding proteins or combinations of binding proteins
and radioactive tag material. The dry gel particles are characterized by
pores of a size that permit entry of low molecular weight compounds into
the intragel volume but insufficient to permit entry of large molecular
weight components which remain in the extra gel volume, thereby to achieve
a desirable fractionation which excludes high molecular weight
interferences otherwise present to interfere with blood or serum assays.
Application of liquid sample to the measured volume of dry gel particles is
determined by water regain of the gel thereby to provide a pipetless
method of sample application based on use of highly reproducible
dry-volume aliquots of the dried or lyophilized gel particles.
The dry gel particles can be preloaded with radioactive tagged hormone and
standard hormone either during preparation of the dry gel particles, or by
after treatment of the dry gel particles previously provided with the
entrapped antibody.
The dry antibody-gel reagent is stable under widely diversified atmospheric
conditions to enable storage over long periods of time without deviation
or loss in binding activity. Thus, there is described a radioimmunoassay
system which provides for automatic exclusion of high molecular weight
interferences; elimination of the need to pipet or centrifuge the sample
to be tested; long term stability of binding reagents stored under ambient
conditions; and a reliable and accurate method of assay which has been
successfully applied to the assay of angiotensin I and insulin.
The aforementioned copending applications are addressed to an assay system
and device which is characterized by manual operation in a single use,
with the device being discarded after the assay has been completed.
However, it is desirable and it is an object of this invention to provide
a system which is capable of automation for multiple use without loss in
binding activity of the antibody gel particles thereby to afford
reproducible results which makes the system for radioimmunoassay much more
attractive, at least from the standpoint of cost, time, inventory and
results.
Proteins having a binding capacity for certain specific smaller molecules
and which can be readily immobilized in the polymer gel are illustrated by
various protein globulins and antibodies.
Gel systems having such globulins or antibodies entrapped therein can be
used effectively for analyzing for a variety of antigens or haptenes, such
as drugs, polypeptide hormones and steroid hormones. Antigens and haptenes
susceptible of being assayed in a system of the type described include,
for example, angiotensin, insulin, growth hormone, gonadotropic hormone,
parathyroid hormone, glucagon, cortisol, protoglandin, corticosteroids,
cyclic fatty acid hormones and estrogens, and drugs such as digoxin,
thyrosin, morphene, digitalis, and the like. By way of example, specific
corticosteroid binding globulins, immobilized by entrapment in the gel
system, permit assay of corticosteroid hormones in addition to the many
polypeptide hormones. 7S gamma-globulin (150,000 m.w.) can be used to
assay for angiotension (1,000 m.w.).
BRIEF DESCRIPTION OF THE DRAWING
The FIGURE shows a sectional view of a device with which to practice the
method of the invention.
The invention will hereinafter be described in greater detail by reference
to a specific example.
EXAMPLE 1
A gel, in which dilute anti-angiotensin immune serum containing 7S gamma
globulin (m.w. 150,000) is entrapped in a gel matrix having a pore size to
enable diffusion of angiotensin (m.w. 1,000) for assay may be prepared as
follows:
8 grams of acrylamide and 1 gram of N,N'-methylenebis-acrylamide is
dissolved to a final volume of 36 ml in sodium phosphate buffer solution
(0.1 M, pH 7.4) containing a suitable dilution of antiserum. To the
resulting solution 0.1 ml of a suspension of 100 mg of riboflavin in 20 ml
of distilled water and 0.02 ml of N,N,N',N-tetramethyl-ethylenediamine is
added, followed by 0.2 mg of sodium hydrosulfite. The mixture, in a
suitable vessel, is stoppered and agitated and then is exposed to light
from an ordinary tungsten light bulb to induce polymerization.
Polymerization is complete in 5-15 minutes. The reaction vessel is
continuously chilled in an ice water bath, as needed, to prevent the heat
generated by the exothermic polymerization reaction from adversely
affecting the binding protein.
The antibody gel is fragmented by pushing through a No. 40 mesh brass
screen. The gel particles are collected on a No. 60 mesh screen and washed
with 5 liters of distilled-deionized water. The gel particles sediment
rapidly and are stiff enough to tolerate moderate flow rates (0.5 to 2.0
ml/min) in a chromatographic column without plugging. After sieving, the
gel particles are equiliberated with 0.1 M pH 7.4 phosphate buffer. The
gel particles are now ready for the drying procedure.
Two fundamentally different drying procedures have been employed including
(1) lyophilization and (2) dehydration in acetone, ethanol, or other low
boiling alcohol, followed by air drying. Neither method was associated
with loss of antibody activity. However, ethanolic dehydration is
preferred because of its simplicity and rapidity of drying. For this
purpose, the gel particles were suspended in 5 volumes of 95% ethanol and
allowed to settle. The supernatant solution was discarded and the ethanol
treatment repeated one or more times. The ethanol dehydrated gel particles
were then allowed to dry in room air by spreading on a nylon net.
Storage stability has been demonstrated by hermetically sealing the
lyophilized gel particles, with the entrapped antibody activity, at
temperatures of 37.degree.C, 25.degree.C, 4.degree.C and -20.degree.C for
over two years without detectable loss of antibody activity when the
entrapped protein is in the form of anti-insulin introduced by the way of
antibody sera for the anti-angiotensin in the described example. Dried gel
particles containing anti-angiotensin antibody prepared in accordance with
this example has also demonstrated storage stability, without loss of
antibody activity for over six months. As indicated above, rabbit antisera
for use against angiotensin I and guinea pig antisera for use against
insulin can be substituted for the antisera in producing the antibody gel
particles of this example.
EXAMPLE 2
Evaluation device:
In its simplest form, an evaluation device for radioimmunoassay, as
described in the aforementioned copending application, is illustrated in
the form of a syringe 10, such as a 1 ml tuberculin syringe, having a
syringe needle 12 at one end and a plunger 14 operative in an elongate
barrel at the other end for drawing fluid for unidirectional flow through
the needle 12 into the body portion 16 of the syringe in response to the
generation of volume conditions upon withdrawal of the plunger.
Adjacent the needle end portion, the syringe is subdivided by a porous
barrier 18 into a calibrated portion 20 packed with a column of prescribed
volume of the dry gel particles of Example 1, while the plunger or other
withdrawal means communicates with the portion beyond the barrier 18.
In the described device, the barrier is in the form of a nylon fabric or
net having openings insufficient to permit passage of the gel particles.
In the illustrated modification, 33 mg of dry gel particles are packed in
the volume of 0.14 ml enclosed by the porous barrier made up of about
two-thirds intra gel volume and about one-third extra gel volume, with the
dry gel particles having a water regain of 100 mg water per 33 mg gel
particles.
EXAMPLE 3
Method of use:
In manual use, the needle 10 is inserted in the sample (serum, plasma or
whole blood drawn directly from a vein) and the plunger is pulled back to
draw some of the liquid sample into the syringe 10 in an amount to at
least fill the volume 20 below the barrier 18 and preferably in an amount
to at least cover the net.
As the sample is drawn into the syringe, the highly hydrophilic gel
particles 22 absorb a predictable and reproducible portion of low
molecular weight component in the sample by rehydration. To prevent
coagulation, when testing fresh whole blood or plasma, it is desirable to
incorporate a coagulant into the gel recipe from which the dry gel
particles are formed.
The size of the sample that is taken up by the particles depends upon the
water regain value of the gel as well as the amount of particles present
in the column, measured by weight or by volume. As a result, sample size
can automatically be controlled by the amount and water regain value of
the dried gel particles thereby to make the test reproducible and
accurate, independent of the amount of liquid sample that is drawn into
the syringe.
After the sample has been drawn into the syringe, the void volume (extra
gel volume of the miniature-syringe chromatographic column) is flushed
out, as with a buffer solution, such as a 0.1 M phosphate buffer or any
other physiological solution. The flushing liquid should be employed in an
amount corresponding to at least one void or extra gel volume but it is
preferred to make use of more than one and preferably two to five void
volumes in order to insure the removal of sample component otherwise
remaining in the extra gel volume. The low molecular weight polypeptide
hormone (or other antigen or haptene) remains substantially entrapped
within the intra gel space during this rapid column wash step for removing
high molecular weight interference. Thus high molecular weight
interferences are removed with the wash solution used to flush the column
of gel particles.
After the flushing step, a competing radioactive "tag" hormone of known
concentration is drawn into the syringe in an amount to at least fill the
void volume and preferably in an amount greater than one void volume.
The unit is then incubated for a specific period of time, which may range
from 30 seconds to 30 hours, at room temperature. It is not necessary for
the radio competitive binding assay to go completely to equilibrium before
final separation of the bound from the free radioactivity.
Thereafter, a buffered wash solution, which may be the same as that used to
flush the column in the previous step, is passed through the column. This
operates to separate unbound hormones from bound hormones. The column is
flushed with sufficient volume for a length of time substantially to
complete the separation step. The total amount of tagged radioactive
hormone used will equal the bound plus the unbound. The radioactivity
bound to the binding sites in the column can be measured directly or it
can be determined indirectly by measuring the radioactivity of the wash
solution. Use can be made of standard values sufficient to construct a
standard working curve having the per cent bound as the abscissa and the
log scale of the standard as the base, whereby the unknown in the sample
can be read directly from the curve, in accordance with the standard
procedures of current radioimmunoassay.
The described technique and materials are capable of a number of
modifications and ramifications.
The formulation of the gel of which the particles are formed is not
significant since use can be made of any gel forming material which is
capable of being reduced into porous, insoluble, highly hydrophilic
particles of controlled pore size. Thus, other highly hydrophilic,
insoluble gels capable of the desired pore size and drying or
lyophilization can be used but it is preferred to make use of the
polyacrylamide gels of the type described wherein an acrylamide monomer
and cross linking agent are combined with sufficient cross linking agent
to stabilize the pore size and support the monomer not only to provide the
desired pore size but to give the gel the density desired for rapid
sedimentation in particulate form and to provide sufficient rigidity to
the gel particles.
As described in the aforementioned application, the ratio of one part by
weight of cross linking agent to 4 to 10 parts by weight monomer is
sufficient to give a gel having a total gel polymer concentration in the
range of 12-35%.
The binding globulin, antibody or protein entrapped within the dry gel
particles can be varied, depending upon the hormone, drug or other
material to be assayed, such as angiotensin, insulin, growth hormone,
gonadotropic hormone, parathyroid hormone, cortisol, digoxin, digitalis or
thyroxin.
The water regain value of the gel particles determines how much sample
reaches the antibody binding protein contained in the intra gel
compartment. The water regain of the gel will depend somewhat on the per
cent polymer and per cent cross linking agent and more particularly on the
thoroughness of the drying process. The gel reagents of the type described
can be dried so that water regain will not change under the conditions of
temperature and humidity used in storage. Other degrees of dryness and
water regain can be used, but are not convenient, because of changes in
hydration that occur with changes in storage temperature and humidity. The
particle size as well as the form and shape of the dry gel particles can
be varied depending upon the size and shape of the test device.
Instead of a syringe, other containers can be employed in which a known
volume or weight of dry gel particles can be retained and through which
the liquid sample, flush solution and tag solution can be transported for
engagement with the gel particles in the amounts previously described. In
the event that a device is used in which the gel particles are retained as
a column in an intermediate section of known volume, then barriers such as
a nylon net should be provided at opposite ends to confine the particles
into the chromatographic column while enabling the various fluids to pass
therethrough into and out of the column of particles.
The porous barrier functions automatically to separate bound from unbound
radioactivity which occurs during the flushing of the column. This porous
structure functions also an an anticonvection barrier, which discourages
mixing of any solution sequestered outside the gel particle compartment.
Since the volume of gel particles is known and the water regain is
constant, the amount of sample taken up by the gel particles will be the
same independent of the size of the sample or the amount introduced into
the test unit. The same applies with respect to the fluid solution used to
flush the particles for removal of material in the extra gel volume and
the amount of radioactive tag material to which the gel particles are
ultimately exposed. Thus many of the steps critical to prior immunoassays
have been eliminated with equal or greater reproducibility and accuracy of
test results. Since the antibody is insolubilized and immobilized within
the gel particles, conventional centrifugation and washing steps, normally
employed in current procedures, can be eliminated in their entirety in the
preparation and use of a test unit of the type described.
Similarly, since the unknown and tag material taken up depends on such
known values as volume and water regain, the critical steps of pipetting
and the numerous errors associated therewith are eliminated thereby to
increase the accuracy of the analysis.
By immobilizing the antibody binding reagent in the gel, the centrifugation
step of conventional RIA can be eliminated. Furthermore, by engaging the
antibody binding protein in a gel polymer matrix, high molecular weight
interference of RIA can be excluded. Such high molecular weight
interferences which are found in some but not all biological samples, are
of at least two types, namely, (1) proteolytic enzymes, which can degrade
polypeptide hormone, and (2) endogenous antibody, which can bind
polypeptide hormone. Endogenous antibody that binds insulin is frequently
found in patients treated with injections of bovine or porcine insulin.
These high weight molecular interferences are too large to penetrate into
the polymer matrix of the gel particles and are sequestered in the extra
gel space, where they can be removed by washing the gel column.
The device which has heretofore been described, and described in the
copending application, has been manually applied for single assays, after
which the device is discarded. It would, of course, be desirable to be
able to make multiple use of the device, without loss of binding activity,
and it would be still more desirable to be able to automate the operation
for multiple use thereby to effect considerable savings in time and
materials, as well as reproducibility of the results.
It has been found, in accordance with the practice of this invention, that
the binding sites of the antibody gel particles in the chromatographic
column of the test device can be returned to their original state to
enable re-use as a test device, without loss of binding site, and that
this can be achieved again and again in a simple and efficient manner for
either manual or automated radioimmunoassay systems.
Unbound radioactivity can be washed from the column, in the manner
previously described, with a variety of physiological solutions at neutral
pH for separation of unbound radioactivity and unbound hormones from the
system. Bound radioactivity can be detached from the binding sites to
enable elution from the antibody gel particles in the column by an acid or
hyperthermic wash solution.
The unbound radioactivity and hormone washed from the column or the bound
radioactivity eluted from the antibody gel particles can be made the
subject of analysis, as by gamma or liquid scintillation type detectors,
to arrive at the assay value for the previous sample.
Following elution of the bound radioactivity, the column of gel particles
is washed with a buffer for removal of the acid or hyperthermic reagents.
This is followed by washing with an organic solvent which is at least
partially miscible with water and which is highly volatile and which forms
a constant boiling mixture with water to effect removal of the buffer and
to enable rapid dehydration and collapse of the antibody gel particles.
Upon evaporation of the organic solvent these gel particles have been
returned to their original dry state. Collapse of the gel particles, which
occurs with solvent drying, is preferred because it prevents entrapment of
large amounts of air which effect reproducibility of the quantity of
sample or water regain and/or is released as tiny bubbles on rehydration.
For detachment of the bound radioactivity and bound hormones from the
binding sites of the antibody gel particles in the column, it is preferred
to make use of an aqueous acidic medium having a pH of less than 2.5 and
preferably an aqueous solution of hydrochloric acid. The acidic solution
is introduced into the gel column in an amount corresponding to at least
one gel volume and preferably more than one gel volume but less than
three.
Removal of the acidic medium with the detached radioactivity and hormones
is effected by washing with a buffer solution, such as an 0.1 M phosphate
buffer having a pH of about 7.4 but other suitable buffers can be used as
previously described. The amount of buffer passed through the column
should be sufficient to rinse the acid from the column of gel particles,
usually one to five volumes will be sufficient.
The organic solvent is employed primarily to effect removal of aqueous
medium and to permit rapid drying and collapse of the gel particles in the
column. For this purpose, it is desirable to make use of an organic
solvent which does not denature the binding activity of the entrapped
antibody and which forms a constant boiling mixture with aqueous medium to
effect water removal and in which the organic solvent is highly volatile
to facilitate rapid removal and permit rapid drying of the gel particles.
Representative of suitable organic solvents are acetone, methyl alcohol,
ethyl alcohol, low boiling esters, and the like.
Solvent removal and accelerated drying is effected by passing air through
the column at ambient temperature or at a temperature slightly above
ambient but below the temperature which would destroy any of the antibody
binding sites. The stream of drying air accelerates volatilization of the
organic solvent for rapid and complete removal.
The thus regenerated antibody gel particles in the column are returned to
their original state including collapsed structure for re-use of the test
device without the need for any recalibration.
It will be apparent from the foregoing description that, in the
regeneration as well as in the use of the device, the materials are caused
to flow through the device in one direction. Such column-chromatographic,
unidirectional flowing stream configuration of analysis and regeneration
enables complete automation of the use and regeneration of the device
merely by the addition of a precision syringe or proportionating pump
operatively connected with the ingoing end of the device to force the
passage of the various materials in the desired volumes therethrough; a
detection means communicating with the outgoing end of the device to test
for unbound radioactivity or bound radioactivity in the materials that are
eluted from the column; and a processor-controller for sequencing the
materials for flow through the device.
An automated system of the type described provides for highly reproducible
applications of samples, reagents, wash solutions, solvents and air to the
column of antibody gel particles. The ability to exclude high molecular
weight interferences and to work with highly stable pre-packed antibody
gel reagent is advantageous for both manual and automated approaches. Gel
antibody RIA does not require dilution of plasma to reduce or eliminate
high molecular weight interferences. Thus reaction kinetics need not be
slowed by reason of dilution in the system.
Manual methods of radioimmunoassay (RIA) have generally called for
incubation of several hours in order for the system to reach equilibrium.
However, in the described automated system, it is no longer necessary to
reach equilibrium before carrying out the separation of bound from unbound
hormone. Response times of 5 to 10 minutes are achievable with automation
with a test device of the type described.
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