Note: Descriptions are shown in the official language in which they were submitted.
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BACKGRQU~D OF THE INVENTION
This invention relates to diagnostic tests for deter-
mining the level of thyroid hormone within a body fluid. In
another aspect, this invention relates to an improved test for
measuring total thyroid hormone in a sample of serum. In
another aspect, this invention relates to a novel radioimmun-
oassay for thyroid hormone.
Various diagnostic tests are known for determining
thyroid function. These tests include the basal metabolism
test, the thyroid uptake test, various colorimetrlc and chemical
procedures for determining the level of thyroxine iodine in the
blood and a test commonly referred to as the T-3 uptake test
which measures the unsaturated binding capacity of thyrobinding
globulin and other thyroxine binding proteins within a serum
sample. Perhaps the most commonly used test available is the
diagnostic test which utilizes radioisotopically labelled
hormone to determine the level of thyroid hormone thyroxine
(C15HllI4N04)present in serum. This test, commonly referred to
as T-4 assay, measures the total quantity of hormone within a
sample of blood serum. The most commonly used T-4 assay which
determines the level of thyroxine within a sample of blood ~erum,
utilizes the technique of competitive protein binding. To carry
out the T-4 assay, it is necessary first to release the thyroid
hormone thyroxine from endogenous thyroxine binding proteins
present in a serum sample. After this, a known quantity of
thyroxine binding -;~
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protein (generally thyroid hormone bindincJ globulin) and a
tracer quantity of radioactively labelled thyroid hormone are
added to -the thyroxine obtained from the sample. The thyro-
xine including the endogenous thyroxine obtained from thesample and the radioactively labelled thyroxine compete for
binding sites on the known quanti-ty of thyroxine binding pro-
tein. After the competitive binding step/ the free or unbound
thyroxine is separated from the thyroxine bound to the thyro-
xine binding proteins and the relative quantity of thyroxine
in the original sample is determined by counting the radio-
activity of either the free thyroxine or the bound thyroxine
in a scintillation well counter.
Recently the competitive protein binding assays for
thyroid hormone have been modified to include the use of spe-
cific antibodies as the thyroxine bi,nding protein instead of
the thyroxine binding globulins. Such procedures are called
radioimmunoassays of thyroid hormone.
In general, the methodology of -the T-4 competitive
protein binding assay and the radioimmunoassay is quite simi-
lar. In both procedures, it is necessary first to releasethyroxine from endogenous thyroxine binding proteins present
in the serum and to alter -the thyroxine binding proteins in
such a way that their further participation in the reaction
is prohibited. In the conventional T-4 competitive protein binding
assays, this has been accomplished by initially treating the
serum sample with organic solvents such as ethyl alcohol to
denature the thyroid hormone binding protein. Such a procedure
is described in U.S. Paten-t No. 3,666,854. Other chemical
methods have been utilized which include the treatment of the
sample with inor~anic chemicals such as alkaline solutions.
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However, the use of alkaline extraction solutions as cornmerclal
test kit components is not desirable because such solutions
exhi~it instabili-ty during storage and use. rlore specifically,
such solutions rapidly absorb CO2 w~ich results in a lowering
o~ the pH thereo~. Another method includes the treatment o~ the
sample with.an acid solution to effect separation between the
thyroid hormone and the thyroid hormone binding protein and
thereafter contacting the solution with an inorganic crystal-
line sorbent such as magnesium silicate which sorbs the free ;~
normone only. Such a method is descri.bed in U.S. Patent
3,776,698. Generally, when acid extractions have been utilized in
competitive protein binding assays, the precipitated endogenous
proteins are physically removed from the resulting endogenous
thyroid horrnone prior to proceeding with subsequent steps in
the test procedure which occur at higher pH's. This is due
mainly to the fact that acid precipitated or denatured proteins
are known to be resolubilized or renatured in solutions of
higher pH. Thus acid denaturation is believed to be "reversible".
Indeed a commercially available so-called normalized T-4 type
20 test sold under the trademark oE "Quantisorb-125" by Abbott
~aboratories utilizes this above described phenomenon of re-
naturation of acid denatured proteins as an essential step
thereof.
Another approach for releasing the thyroxine from r
the endogenous thyroxine binding protein is by heat denatura-
tion. Heat denaturation of the serurl~ protein provides good recovery
of the thyroid hormone with extraction efficiencies approach-
ing about 100%. However, this method is time-consuming in
that it
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requires a minimum of 15 minutes in a boiling ~ater bath for
complete destruction of binding proteins.
Likewise, when carrying out tlle radioimmunoassay for
thyroxine, it is firs~ necessary to release the thyroxine from
endogenous thyroxine binding proteins present ~ithin the serum
sample and to alter the thyroxine binding proteins in such a
way that their further participation in the reaction is pro-
hibited. In the radioim~unoassay, this is most commonly accom-
plished to a degree by blocking the thyroxine binding sites on
endogenous proteins with chemicals such as 8-anilino-l-napllt]lalen
sulfonic acid, diphenylhydantoin, salicylates, or thimerosal.
l~o~ever, the use of such blocking agents in radioimmulloassay has
posed problems. For example, it has been shown that the quantity
of these blocking agents necessary to occupy all sites may be
related to the level of endogenous thyrobinding protein whicl
can vary significantly in individual serum samples.
Thus, in both the conventional T-4 competitive protein
binding assay and the radioimmunoassay, the thyroxine must
initially be extracted from native proteins and thereafter bound
to specific ~roteins, and then the free hormone separated from
the protein bound hormone. The final separation has been
accomplished by sorption of the free hormone such as to resins~
charcoal or inorganic crystalline sorbents. The conventional
resins include ion exchange resins such as the ion exchanger
having strongly basic amino or quaternary ammonium ~roups such
as disclosed in U.S. Paten~ No. 3,414,383. These organic ion
~xchange resins can be either in loose form or incorporated in
polyurethyane sponges as disclosed in U.S. Patellt No. 3,206,602,
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or enclosed in porous bags or the like. Other conventional
methods include a selective sorption of the Eree hormone by
charcoal which has been coated with suitable proteins or other
polymers, or the use of molecular sieves such as Sephadex.
The use of the inorganic crystalline sorbent materials are dis- -;
closed in U.S. Patent ~o. 3,666 j854 and U.S. Patent No. 3,776,
698. In general, the methods which rely on sorption systems to
separate the free hormone from the hormone bound to the protein
are dependent upon protein concentration in the assay system,
and some systems may require adjustment of the protein level
for valid results. In addition, some of the sorbents such as
the resins are temperature sensltive necessitating correction
oE assay values obtained in working conditions where the
temperatures are variable. In addition, the sorbents are in
general time dependent and careful timing during the sorption
process is necessary in order to obtain reproducible results.
Antibody-bound hormone may be separated from the free
fraction by precipitation of the specific protein. One common
approach used to precipitate the bound fraction is the double
antibody technique which, due to a second incubation period,iis
time-consuming. Another means of sepaxation is chemical precip-
itation of the bound fraction with either high molecular ~eight
polymers or salts. In order to precipitate the minute amounts
of gamma globulin present, exogenous gamma globulin is conven-
tionally added to the assay system prior to separation of the
bound and free fractions.
A problem which has been encountered is the phenomenon
of non-specific binding occurring during the assay. Non-speciEic
binding stated simply is the binding of the free hormone
including
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the free radioactivel~ labelled hormone with naterials other
than the thyrobinding protein in the conventional T-4 assay or
the antibody in the radioimmunoassay. The inability to assess
the degree of non-specific binding occurring in the absence of
the hinding protein is a disadvantage of those assay systems in
which binding protein and radioactively labelled hormone are
employed as a single reagent.
In general, a thyroid hormone assay is needed which
will initially extract all or substantiallv all of the tilyroid
hormone from the endogenous serum sample in a reproducible
manner, will not rely on blocking agents or solid sorbents to
separate the bound from the free hormone after -the competitive
~inding step, but wil.l effect -the separations quickly and
efficiently in a reproducible manner wherein a low but repro-
ducible anount of non-specific binding will occur during each
test and which can be carried out in a single test tu~ without
the requirement of intermedia-te decanting of test solution(s)
thereform.
SHORT STATE~IENT OF THE INVENTION
According to the invention there is provided a
method of measuring the level of thyroid hormone in a sample
of serum containing endogenous thyroid hormone and endogenous
thyroid hormone binding protein com?rising: (a) admixing said
serum sample with an effective amount of an aqueous acid solu-
tion sufficient to separate said thyroid hormone from said
thyroid hormone binding protein; (b) adjusting the pH of the
resulting mixture to a value in the range of from about 7.0 to
about 8.5 in the absence of blocking asents while adding thereto
a known amoun-t of radioacti.vely labelled thyroid hormone and a
known amount of thyroid hormone binding antibodies and allow-
ing the resulting solution to equilibrate- (c) thorouqhly ad-
mixing the resulting equilibrated mixture from step (b) wi-th
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an aqueous solution of a water soluble sulfate which contains
sufficient sulfate such that the resulting mixture has a sul-
fate concentration between about 20 and 30 weight percent to
thereby result in precipi-tation of said thyroid hormone binding
protein material containing thyroid hormone bound thereto
and leave free thyroid hormone in the solution; (d) separating
the precipitated material from said solution; and (e) counting
with a scintillation counter one of (1) the free radioactively
labelled thyroid hormone in said resulting solution, and ~2)
the bound radioactively labelled thyroid hormone bound to said
thyroid hormone binding antibodies in said precipitate.
In accordance with one embodiment of the subject in-
vention, a radioimmunoassay for thyroid hormone is provided
which includes the initial extraction of endogenous thyroid
hormone from sample serum with an acid reagent to result in an
effective inactivation of endogenous thyroid hormone binding
proteins, the subsequent adjustment of the pH to a high level
suitable for competitive binding (without removing the endoge-
nous thyroid hormone binding protein therefrom) and the
addition of a tracer quantity of radioactively labelled thyroid
hormone and a known
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quantity of thyroid hormone antibodies and thereafter allowing
the resulting solution to equilibrate and competitive binding
to occur, and then separation of the bound moiety from the ~ree
moiety by the addi~ion of an aqueous sulfate salt solution whic~
causes precipitation of the bound moiety; and thereafter counting
the prccipitated bound moiety or ~]~e supernatant containing the
free ~oiety in a scintillation well counter. This procedure is
carried out in a single tube without intermediate decanting
steps.
In accordance with another embodiment of the subject
invention, I have found that the separation of free from bound
thyroid hormone fractions which rcsnlts ~rom the competitivc
binding step in a thyroxine assay syst.em can be efficiently and
reproducibly effected by a manner whereby the non-specific bind
ing in the system is monitored at a lo~ constant level if the
bound fraction is precipitated with an aqueous sulfate salt
solution to which a minor but effective amount of added animal
serum has been previously added.
DETAILED DESCRIPTION OF T}IE INV~lTIO~
. ~
The assay of the subject invention was developed in
an effort to provide a "single tube" thyroid hormone assay which
would not utilize blocking agents nor an external solid phase
sorption step and require a minimum of manipulation by the ~.
laboratory technician.
First, I have discovered tha~ endogcnous thyroid
hormone binding protein can be initially inactivatcd and thereby
scparated from endogenous thyroid hormone by acid treatment~ and
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thereafter the pl-l of the resulting mixture can be raised and
thyroid hormone antibodies added thereto to competitively bind :
with the endogenous thyroid hormone without interference from
the endogenous protein. This is quite surprising in view of
the fact that acid inac-tivated thyroid hormone binding protein
is known to renature at higher pH's and interfere with competi- ~.
tive binding between thyroid hormone and exogenous thyroid
hormone binding protein by actually entering into the competitive
binding reaction. Thus, I have found that a radioimmunoassay
for thyroid hormone can be carried out immediately after acid
separation of endogenous thyroid hormone from endogenous thyroid
hormone binding protein after a single upward pH adjustment of ;^~
the mixture and without first having to remove the endogenous
thyroid hormone bindiny protein from the mixture.
Secondly, as stated above, most sorbents are dependent
upon the total protein concentration in the mixture such that
slight variations in the protein concen-tration of the sample can
affect the percentage uptake of the sorbent. In addition, an
effective thyroxine radioimmunoassay must have a low reproducible
non-specific binding of the thyroid hormone which in essence is
a low but uniform degree of binding of the thyroid hormone to
constituents other than the antibody.
The thyroid hormone radioimmunoassay of the subject
invention utilizes a salt precipitation step to separate the
bound hormone from the free hormone in solution. Carrier or
adjuvant proteins are conventionally added to such mixtures
before the addition of sulfate in order to effect and expedite
precipitation. However, I have found that the adjuvant protein
can be mixed with the sulfate prior to in-troduc-tion to the assay
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mixture. Using this combination as a precipitant, I have found
that a constant, reproducible non~specific binding as well as
an acceptable spread between the hypothyroid and hyperthyroid
samples will result. Furthermore, the use of this combination
as precipitant permits the use of a wide range of volumes of the
serum sample, and allows the assav of serum samples containing
a wide range of protein concentration.
The subject invention will be described in detail in
relation to radioimmunoassay for thyroxine, even thou~h one
having ordinary skill in the art can easily adapt my novel pro-
cedure to assay triiodothyronine and other thyronines.
Before the endogenous serum thyroxine can be assayed
utilizing the improved salt precipitation step of the subject
invention, the hormone must be efficientlv and reproducibly
extracted from a serum sample. In addition, all endogenous pro-
tein which could possibly bind thyroxine during the several -
steps of the assay must be completely inactivated. In accordance
with one embodiment of the subject invention, the endogenous
thyroxine is extracted from the serum sample with an acid solu-
tion. The acid solution is generally maintained at a nH within
the range of from about l.0 to 2.2. The preferable pH range is
from about 1.0 to about 2Ø Any stable acid solution which is
nondeletrious to the thyroid hormone can be used in the scope
of the subject invention. For example, aqueous solutions of
HCl, H2SO4, H3PO4 and the like can be used within the scope of
the subject invention. HCl is the preferred acid. These materi-
als can be buffered with suitable buffering agents, e.g., salts
of weak acids at a concentration of about 0.02 to 0.07 l~l. Also,
the ionic strength of the acid solution can be maintained by
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the presence of suitable materials such as neutral sal-ts, e.g.,
NaCl, KCl and the like at a concentration of 0.02 to 0.07 molar. ~,
In general, at least about 10 and preferably about 20
volumes of the acid extraction reagent is combined with each
volume of the serum sample. The pH of the resulting mixture
should be in the range of from about 1.3 to about 3Ø Complete
inactivation of the endogenous thyroxine binding proteins is
accomplished immediately, even though the inactive proteins .
remain in solution. For example, the serum is added to the
acid extraction solution contained within a vial, and the vial
is shaken for a few seconds (generally 10 to 15 seconds) to
admix thoroughly the serum and the acid extraction solution.
This will allow time for the acid solution -to break the bonds
~etween the thyroxine and the thyroxine binding protein and to
inactivate completely the thyroxine binding protein.
Now that the resulting solution contains all of the
endogenous thyroxine therewithin, and substantially all of the
thyroxine binding protein has been deactivated, it is necessary
to adjust the pH of the solution upwardly to a suitable pH in
which competitive binding of the thyroid hormone and thyroid
antibody can take place while adding to the solution a tracer
quantity of radioactively labelled thyroid hormone (T-4) and a
known,quantity of antiserum containing the thyroid hormone anti-
bodies. In accordance with a preferred embodiment of this in-
vention, the radioactive thvroid hormone followed by the anti-
serum is added to the acid solution containing the unknown
quantity of endogenous thyroid hormone. More specifically,
the solution
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contailling the radioacti~e thyroxine can cor.l~rise a solution
having a plll~ithin thc range of about 7.6 to 9.0 and preferal~ly
about 8.3, and l~ill contaill a tracer ~uantity of radioactively
labelled thyroicl 1lOT~One, and preferably a bu~fer to maintain
the pll of the solution. A suitable SUCh SO1UtiO11 COmPriSeS
0.04 ~ sodium bar~ital carrier W]liC]I is adjusted to a p~l of 8.3
by the addition of hydrochloric acid ~hich contains the tracer
quantity of T-~ 125I
j Any radioactive isotope of iodine, tritium, or carbon
10 !,can be used. It is preferred that a hormone be utilized l~hic]
jlis labelled Wit]l 125I. The buffered solution containing the
¦,tracer quantity o~ radioactively labclled ~hyroid hormone
litllyroxine, which is at least 20 and prcfer.lbly about 40 volumcs
! greater than the serum sample, is added to the acid solution an-l
llthe con~ents are thereafter thoroughly mixed by sl~aking.
~ Next, a solution containing the antithyroxine scrum
¦ is added to the resulting solution. The antiserum can contai~l
i a suitable buffer such as sodium barbital and can generally havc
I',a p~l within the range from about 7.6 to 9Ø The volume of tl~c
I'antiserum solution added to the test mixture can be the same as
¦that used to deliver the ~adioact;~e isotope labelled thyroid
hormone to the test mixture. After the thyroxine antiserum solu
tion has been added to the solution containing the extracted
!Ithyroid hormone and the radioactive ~uantity of thyroid hormone,
I,the resulting mixture is thoroughly mixed. The resulting solu-
tion will have a p~T in the range of from about 7.0 to 8.5 and
preferably about 7.~-8.4 and is incubated at ~oom temperature
from 30 to about 60 ~inutes to allol~ the formation of the
thyroxine-antibody com~lexes. Since the antibody ~ill bind both
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the radioactive thyroxine and serum thyroxine e~ually well, the
amowlt of radioactive thyroxine reco~ered ~ill reflect the con-
centration of thyroxine in the original sample. As is well
~nown, the antiserum used in this step should llave a high speci~
ficity for thyroxine. Furthermore, as previously noted, the
inactivated thyroid hormone binding protein in the mixture sur-
prisingly does not interfere with the competitive binding betl~een
the thyroid hormone and the antibody.
Once the solution is equilibrated sucll that the
competitive binding between the thyroxine antibodies and
thyroxine is complete, the antibodics containing bound hormone
are precipitated in accordance with the improved salt precipita-
tion step of the subject invention. The precipitant solution
whicll is used in the scope of the subject invention comprises
an aqueous solution of a water soluble sulfate salt which will
ef~ectively precipitate proteinaceous materials without deleteri-
ously ~recipitating free hormone from solution. Examples of
suitable sulfate salts which can be used are the alkali metal
sulfates such as sodium and potassium sulfate, ammonium sulfate,
and zinc sulfate. Because of the solubility range, availability
and convenience, the most preferred salt is ammonium sulfate.
The concentration of the ammonium sulfatc can vary according to
the amount of aqueous precipitant fluid which is desired to be
used. Generally, the concentration in the precipitant solution
should be SUCII that when it is admixed Wit]l the ~hyroxine-anti-
body containing solution, the resulting concentration o~ sulfate
will be in the range of about 20 to 30% by weight and pTeferably
in the range of about 23 to 27% by weight and most prefcrably
about 23 to 24% by ~eight. I have found that the concentration
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of the sulfate in the precipitant solution can conveniently
range from about 30 to about 40% by weight and preferably
from about 33 to 39% by weight thereof.
In addition to the sulfate, I have found it necessary
to add a minor but effective amount of animal serum as adjuvant
to the antibody-thyroxine containing solution at the time of ;
precipitation in order to obtain a low but reproducible order of
non-specific binding and a marked distinction in thyroxine
levels of hypothyroid and hyperthyroid serum samples. Further-
more, the use of the above combination permits the use of a
wide range of sample volume (i.e., from about 2 to about 50
microliters), and allows the assay of serum samples containing
a wide range of protein concentration. In general, the amount
of added serum which is necessary to accomplish this result is
a volume greater than the initial volume of the serum sample
being tested. Generally, any type of animal serum can be uti- `
lized as an adjuvant in this manner even though I have found
some variation in the quantity of serum which must be used
which is directly related to the specie of animal from which
the serum is obtained. In general, when utilizing a seru,n
sample of 10 microliters or less and when using bovine serum,
sheep serum or human serum as the source of adjuvant protein,
the volume of this added carrier protein must be greater than
about 4 times the initial volume of the serum sample being
tested and preferably it is greater than about 6 times the
volume of the sample being tested and can be a much larger
volume, e.g., from 12 to 20 times the volume of the initial
sample. In essence, once the total protein in the solution
reaches about 60 microliters then a constant non-specific
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binding and an acceptable percent spread is obtained and .-~
further amounts of protein added either from the sample or
otherwise will not deletriously effect the analyses. I have
found that approximately twice as much rabbit serum is needed
than the above bovine, sheep or human serum and about one-half
as much horse serum is needed than the bovine, sheep or human
serum. The serum is preferably contained within the concentra-
ted sulEate precipi-tant solution which as s-tated above generally
has a concentration of about 30 to 40~ by weight and preferably
of about 33 to 39~ by weight.
To separate the free from the bound fractions in the
test solution, a sufficient quantity of the precipitant solu-
tion is added to the sample solution containing the thyroxine
.~rld antibody to result in a final concentration o~ the sulfate
between abou-t 20 and 30 weight percent thereof as set forth
above and to result in a sufficient concentration of the adju- `
vant carrier protein within the ranges as set forth above. The
~ixture should be thoroughly admixed, for example, by covering
the tube in which it is contained and inverting it from 5 to
20 times. The precipitate of the antibody containing the bound
hormone together with the adjuvant proteins forms immediately
at the lower sulfate concentration and the resulting mixture
should be centrifuged until the precipitated proteinaceous
material forms a small button within the bottom of the vessel.
Thereafter, either the free fraction in the super-
natant fluid or bound fraction in the precipitate should be
counted in a scintillation well counter. It is preferred to
count the bound fraction. The reading of the scintillation
counter is compared to the total number of counts contained
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in the amount of tracer radioisotopically labelled thyroid
hormone which was initially added. The percent antibody bound
values are thus obtained. ~he percent antibody bound values
are then correlated with standard values obtained by measurin~
percent antibody bound of standard samples containing known
amounts of thyroid hormone to thereby determine the amount of
thyroid hormone within the sample in a manner well known in
the art.
The minor but effective amount of adjuvant serum pro-
tein is necessary in order to provide an assay which can uti-
llze various volumes of serum and which has constant non-spe-
cific binding and also has an acceptable recovery range between ;
high and low thyroxine values. I-t is believed tha-t the prin-
ciple active ingredient within the serum which effects the
desired but unexpected results i9 yamma globulin. Generally,
most animal serum contains between about 0.5 and 2 grams gamma
globulin per 100 milliliters serum while -the total protein in
the serum is about 5 to 8 grams per 100 milliliters serum. It
is noted at this point, while it is believed that gamma globu-
lin is the active ingredient in the serum for this purpose,
because of convenience and availability, it is preferred to
utilize the entire serum sample. In general, when utilizing
bovine, human or sheep serum in quantities of 4 times the volume
of the initial serum sample or less, fluctuating, non-reprodu-
cible values of non-specific binding have been determined in
addition to an unacceptable percent spread between the high and
low thyroxine containing serum samples. However, when using
such serum in volumes greater than about 4 times the initial
volume of the serum, a plateau is reached where substantially
constant non-specific binding is obtained and an excellent
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spread in r~covery values is also obtained. This plateau
is well defined at about 6 volumes greater than the volume oE
the ini-tial sample and continues with no variation with added
quantities of protein up to 12 volumes and more based upon
original volume of the sample serum.
The following examples are given to better facili-
tate the understanding of this invention and to show some
specific preferred embodiments of the subject invention and
are therefore not intended to limit the scope of the claimed
10 invention. !,
EX~lPLE I
This example illustrates -the assay reproducibility
of the novel thyroxine assay of the subject invention. The
specific assay comprised the use of the following reactant
solutions-
(a) The extractant solution consisted of
a solution containing ~.025 N HCl; and
0.05 ~ KCl;
(b) The T-4 I reagent solution contained
a tracer quantity of radioiso-tope T-4
in a 0.04 molar sodium barbital solution
which contained sufficient HCl to render
a pH thereof of 8.3;
(c) The T-4 antiserum contained thyroxine
antibodies (formed in a xabbit) and con-
tained within O~r);l ~1 sodium barbital
solution which had been adjusted with
HCl to render a final pH of 8.3-
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. ~d) The precipitant solution consisted of an
aqueous solution containing 37% by weight
ammonium sulfate and containing bovine
serum at a concentration of 4 volumes .
percent;
(e) The standards utilized in running the
t~st were as follows:
1 microgram thyroxine per deciliter
6 micrograms thyroxine per deciliter
12 micrograms thyroxine per deciliter ~
1~ micrograms thyroxine per cleciliter .~:
Tlle above solutions ~ere uti.lized to assay fro~en
pools of hypothyroid serum, normal serum and hyperthyroid serum
by 11 different technicians,
The test procedure utilized included initially adding
10 microlit.ers o a serum sample ~or standaTd as the case may be~
to 200 microliters of the extraction solution, ~fter this step,
. the resulting solution was thoroughly admixed and then 400
microliters of the T~ 25I reagent were added to the resulting
solution and the solution agitated. After this, 400 microliters
of the T-~ antiserum solution we~e added and the resultillg solu-
tion thoroughly admixed, At this ~oint, the solu~ion was
incubated at room temperature for 45 or 60 minu~es, and at the
end of the incubation period, the precipitate in the sulfate
solution was resuspended and 2 milliliters of *his suspension
were added to each tube containing the test solution. This
resulted in 80 microllters of adjuvant serum bein~ added to each
origi.nal 10 microliter sample and a final sulfate concentration
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in each test solution of 23,7% by ~Jeight. The tubes ~ere cappcd
and each tubc inverted gently about 10 times,
Then within 20 minutes after the addition of tile
precipitant, the tubes ~.~ere centrifu~ed for 10 minutes at
1000-1500 gravities or 2000-2500 rpm and witllin 3 hours the
supernatant was discarded and the rcsultant button of precipitan
counted in a scintillation well counter. The results of the
runs are set forth in Table 1 below:
'~
Table
_e um Pool l-ly~othyroid Norr,lal l5ypcrt]lyroid
Number of Observations 21a 317 210
Number of Days 16 15 16
Number of ~ots 2 2 2
Number of Technicians 12 ll 12
~5ean (~g/dl) 3.2 7.9 14.1
Standard Deviation (~g/dl)0.28 0.30 0.47
Coefficient of Variation (~) 8.8 3.8 3.3
As sho~n from the table, the reproducibility of tlle
assay is excellent.
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EXAIPLE II
_ . .
The linearity of the radioimmunoassay of the subject
invention is demonstrated in this example utilizing -the assay
solutions described in Example I above. SpeciEically, the
endogenous thyroxine from 3 serum pools was initially measured
using the procedure set for-th in Example I above. Thereafter,
quantities of 5, 10/ 15 and 20 micrograms per deciliter of
crystalline thyroxine were added to 4 samples, respectively,
from each of the 3 serum pools and the resulting samples were
assayed in accordance with the procedure set forth in Example I
to illus-trate the uniformity of the percent recovery obtained
from running the -test. The results are set forth in Table 2
below:
TABLE 2
Measured Added Predicted Measured Recovered P~ecovered
Serum Endogenous Exogenous Total Total Exogenous Exogenous
_Pool T4(~lg/dl) T4(~g/dl) T4(~g/dl) T4(~g/dl) T4(Jg/dl) Percent
1 3.3 5 8.3 8.3 5.0 100
13.3 13.4 10.1 101
13.3 18.~ 15.1 101
23.3 23.6 20.3 102
2 3.6 5 8.6 8.7 5.1 102
13.6 13.5 9.9 99
18.6 18.8 15.2 101
23.6 24.0 20.4 102
3 3.0 5 8.0 7.9 4.9 98
13.0 12.9 9.9 99
18.0 18.1 15.1 101
23.0 23.0 20.0 100
The above results illustrate excellent recovery of the
added T-4 and linearity of the tes-t system.
- 20 -
,
~33Sl
E,~AMPLE III
This example is presented to illustrate the constant
reproducible plateau of non-specific binding of the hormone to
the antibody which is re~ched when the precipitant solution
contains bovine serum in excess of 4 volumes per volume of the
initial serum sample. In each instance, a solution containing
a known quantity of thyroid hormone ~a 10 microliter sample), a
tracer quantity of radioactively labelled thyroid hormone, and
a known quantity of antiserum wére contacted with different
precipitated solutions. Furthermore, the non-specific binding
for each such precipitant solution was determined by running . -
each tes-t but with the antiserum eliminated from the buffered
antiserum solution. The precipitant solution was varied bo-th
in concentration of the ammonium sulfate and the carrier protein
as illustrated in the table below and the non-specific binding
of three different serum standards were tested by eliminating
the antiserum from the antiserum buffer solution as described
in Example III. The results are set forth in Table 4.
- 21 -
~ . . . : .
13-322~
~Oq3351
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~ 33~1
E~A~IPLE IV
This example indicates the constant degree of non-
specific binding and excellent spread of recovery values
between hypothyroid and hyperthyroid serum samples when utili-
zing the test of the subject invention with the precipitant
fluid containing the minor but effective quantity of added
seru~ as adjuvant. ~lore specifically, the procedure se-t forth
in Example III was utilized to assay various serum standards . :
except that the concentration of the serum in the precipitant
solution was varied such that the resultant test solution would
carry 60, 70, 80, 90, 100 and 110 microliters of added serum
which is added with the precipitant to contact the test solution
containing the initial 10 microliters of the serum sample. As
will be noted when the carrier protein is present in the pre-
cipitant solution in quantities of 6 times the volume of initial
sample or more, the value of the non-specific binding stabili
zes; whereas when the quantity of the carrier protein is only
4 times that of the initial volume of the sample, the non-spe-
cific binding varies in an unpredictable manner. The results
are set forth in Tab~e 5 below:
- 23 -
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~3351
EXA~I~LE V
This example is presented to illustratc the specifici.ty
and sample serum protein independence of the radioimmunoassay of
t]~e subject invention. Specifically, tlle endogellous thyroxine
of different sample volumes of various sera containing elcvated
and decreased protein levels was measured usi.ng the test pro-
cedure and the reactant solutions described in Example I. The
results are set forth in Table 6 below:
335~
Table 6
Serum Measured Corrected*
Volume T4 to 10~l1
Type of Serum Sample (~ g/dl) (~g/dl)
Hyperthyroid 10 17.9 17.9
Total Protein, 7.3 g/dl 7-5 13.7 18.3
5.0 9.4 13.8
2.5 4.7 18.8
1.672.9 17.4
Hyperthyroid, 10 13.7 13.7
Total Protein, 7.5 g/dl 7-5 10.6 14.1
5.07.4 14.8
2.53.5 14.0
1.672.2 13.2
10 Alpha Globulins, 2.1 g/dl 10 15.4 15.4
(Elevated) 7-511.5 15.3
5.07.9 15.8
2.54.1 16.4
Albumin, 5.4 g/dl (Elevated) 10 7.7 7.7
5.04.0 8.0
3.32.5 7.5
2.51.8 7.2
Gamma Globulins, 4.6 g/dl 10 7.3 7.3
(Elevated) 5.03.6 7.2
3.32.5 7.5
2.5-1.7 6.~3
~ypothyroid 10 2.5 2.5
Total Protein, 8.2 g/dl 20 5.3 2.7
8.2 2.7
Total Protein, 4.5 g/dl 10 3.1 3.1
~Decreased) 20 6.7 3.4
30 10.4 3.5
Albumin, 0.9 g/dl 10 3.5 3.5
(Decreased) 20 7.2 3.6
30 10.4 3.5
Feline (10 Animals - 10 1.3 1.3
3 Determinations Each) 2q 3.6 1.6
5.4 1.8
Canine (10 Animals - 10 1.4 1.4
3 Determinations Each) 20 3.0 1.5
4.8 1.6
Equine (10 Animals - 10 0.8 0.8
3 Determinations Each) 20 2.2 1.1
3 8 1.3
* These values are corrected only to the volume of 10~l1 and
do not reflect a correction for non-specific binding. All
values for any serum would be equal if corrected for non-
specific binding.
- 26 -
,
.1 ~
.,
~335~
While this invention has been described in relation
to its preferred embodiments, it is to be understood that
various modifications thereof will be apparent to those skilled
in the art upon reading this specification, and it is intended
to cover such modifications as fall within the scope of the
appended claims.
- 27 -
