U.S. patent number 3,928,553 [Application Number 05/445,115] was granted by the patent office on 1975-12-23 for radioimmunoassay method for triiodothyronine and thyroxine.
This patent grant is currently assigned to New York University. Invention is credited to Charles S. Hollander.
United States Patent |
3,928,553 |
Hollander |
December 23, 1975 |
**Please see images for:
( Certificate of Correction ) ** |
Radioimmunoassay method for triiodothyronine and thyroxine
Abstract
This invention relates to a radioimmunoassay method for
triiodothyronine or thyroxine or triiodothyronine and thyroxine
present in unextracted serum containing thyroxine binding
prealbumin and thyroxine binding globulin which comprises: A.
adding to unextracted serum, a sufficient amount of a binding
inhibitory agent to substantially inhibit binding of
triiodothyronine or thyroxine or triiodothyronine and thyroxine to
the thyroxine binding prealbumin; B. adding to the serum an amount
of an isotopically labelled triiodothyronine or thyroxine or
triiodothyronine and thyroxine sufficient for detection; C. adding
to the serum, a sufficient amount of a binding inhibitory agent to
substantially inhibit binding of triiodothyronine or thyroxine or
triiodothyronine and thyroxine to the thyroxine binding globulin;
D. adding to the serum an amount of antiserum containing antibody
for triiodothyronine or thyroxine or triiodothyronine and thyroxine
which is approximately proportionate to the titer; E. incubating
the serum at a temperature and for a period of time which is
sufficient to provide substantial equilibration of the antibody
bound and unbound triiodothyronine or thyroxine or triiodothyronine
and thyroxine; F. separating the unbound triiodothyronine or
thyroxine or triiodothyronine and thyroxine from the bound
triiodothyronine or thyroxine or triiodothyronine and thyroxine
while substantially preventing re-equilibration of antibody bound
triiodothyronine or thyroxine or triiodothyronine and thyroxine;
and G. measuring the adsorbed unbound triiodothyronine or thyroxine
or triiodothyronine and thyroxine and the bound triiodothyronine or
thyroxine or triiodothyronine and thyroxine.
Inventors: |
Hollander; Charles S. (New
York, NY) |
Assignee: |
New York University (New York,
NY)
|
Family
ID: |
26930584 |
Appl.
No.: |
05/445,115 |
Filed: |
February 25, 1974 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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237342 |
Mar 23, 1972 |
|
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|
|
Current U.S.
Class: |
436/500; 436/529;
436/541; 436/804; 436/826; 436/524; 436/539; 436/542; 436/825 |
Current CPC
Class: |
G01N
33/78 (20130101); Y10S 436/804 (20130101); Y10S
436/826 (20130101); Y10S 436/825 (20130101) |
Current International
Class: |
G01N
33/74 (20060101); G01N 33/78 (20060101); A61K
043/00 (); G01T 033/00 (); G01T 001/16 (); G21H
005/02 () |
Field of
Search: |
;23/23B ;250/303
;424/1,12 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Padgett; Benjamin R.
Assistant Examiner: Nucker; Christine M.
Attorney, Agent or Firm: Pennie & Edmonds
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation in part of my earlier filed
application Ser. No. 237,342, filed Mar. 23, 1972, and now
abandoned.
Claims
I claim:
1. A radioimmunoassay method for triiodothyronine or thyroxine or
triidodthyronine and thyroxine present in unextracted serum
containing thyroxine binding pre-albumin and thyroxine binding
globulin which comprises:
a. adding to unextracted serum, a sufficient amount of a binding
inhibitory agent to substantially inhibit binding of
triiodothyronine or thyroxine or triiodothyronine and thyroxine to
the thyroxine binding prealbumin;
b. adding to the serum an amount of an isotopically labelled
triiodothyronine or thyroxine or triiodothyronine and thyroxine
sufficient for detection;
c. adding to the serum, a sufficient amount of a binding inhibitory
agent to substantially inhibit binding of triiodothyronine or
thyroxine or triiodothyronine and thyroxine to the thyroxine
binding globulin;
d. adding to the serum an amount of antiserum containing antibody
for triiodothyronine or thyroxine or triiodothyronine and thyroxine
which is approximately proportionate to the titer;
e. incubating the serum at a temperature and for a period of time
which is sufficient to provide substantial equilibration of the
antibody bound and unbound triiodothyronine or thyroxine or
triiodothyronine and thyroxine;
f. separating the unbound triiodothyronine or thyroxine or
triiodothyronine and thyroxine from the bound triiodothyronine or
thyroxine or triiodothyronine and thyroxine while substantially
preventing re-equilibration of antibody bound triiodothyronine or
thyroxine or triiodothyronine and thyroxine; and
g. measuring the adsorbed unbound triiodothyronine or thyroxine or
triiodothyronine and thyroxine and the bound triiodothyronine or
thyroxine or triiodothyronine and thyroxine.
2. The method of claim 1 wherein the binding inhibitory agent for
thyroxine binding pre-albumin is barbituric acid, a derivative of
barbituric acid or a salicylate.
3. The method of claim 2 wherein the derivative of barbituric acid
is sodium barbital.
4. The method of claim 1, wherein the binding inhibitory agent for
thyroxine binding globulin is dilantin, a salicylate, methiolate,
tetrachlorothyronine or 8-anilino-1-napthalene sulfonic acid.
5. The method of claim 1 wherein the isotopically labelled
triiodothyronine is triiodothyronine .sup.125 I.
6. The method of claim 1 wherein the isotopically labelled
thryroxine is thyroxine .sup.131 I.
7. The method of claim 1 wherein the serum is maintained at a pH of
about 8 during incubation.
8. The method of claim 1 wherein incubation is carried out at about
37.degree.C for a period of about 1 to 2 hours.
9. The method of claim 1 wherein separation of unbound from bound
triiodothyronine or thyroxine or triiodothyronine and thyronine is
carried out by adsorbtion of the unbound product on dextran coated
activated carbon added to the incubation mixture about ten minutes
after the mixture has been cooled to a temperature of about
4.degree.C.
10. The method of claim 9 wherein the adsorbed unbound product is
separated from the adsorbed product by centrifugation of
filtration.
11. A radioimmunoassay method for triiodothyronine or thyroxine or
triiodothyronine and thyroxine present in extracted serum in which
thyroxine binding pre-albumin and thyroxine binding globulin are
substantially absent, which comprises:
a. adding to the serum an amount of an isotopically labelled
triiodothyronine or thyroxine or triiodothyronine and thyroxine
sufficient for detection;
b. adding to the serum an amount of antiserum containing antibody
for triiodothryronine or thyroxine or triiodothyronine and
thyroxine which is approximately proportionate to the titer;
c. incubating the serum at a temperature and for a period of time
which is sufficient to provide substantial equilibration of the
antibody bound and unbound triiodothyronine or thyroxine or
triiodothyronine and thyroxine;
d. separating the unbound triiodothyronine or thyroxine or
triiodothyronine and thyroxine from the bound triiodothyronine or
thyroxine or triiodothyronine and thyroxine; and
e. measuring the adsorbed unbound triiodothyronine or thyroxine or
triiodothyronine and thyroxine and the bound triiodothyronine or
thyroxine or triiodothyronine and thyroxine.
12. A radioimmunoassay method for triiodothyronine or thyroxine or
triiodothyronine and thyroxine present in unextracted serum
containing thyroxine binding pre-albumin and thyroxine binding
globulin whic comprises
a. adding to unextracted serum a mixture comprising
1. a sufficient amount of a binding inhibitory agent to
substantially inhibit binding of triiodothyronine and thyroxine to
the thyroxine binding pre-albumin;
2. an amount of an isotopically labelled triiodothyronine or
thyroxine or triiodothyronine and thyroxine sufficient for
detection; and
3. a sufficient amount of a binding inhibitory agent to
substantially inhibit binding of triiodothyronine or thyroxine or
triiodothyronine and thyroxine to the thyroxine binding
globulin;
b. adding to the serum an amount of antiserum containing antibody
for triiodothyronine or thyroxine or triiodothyronine and thyroxine
which is approximately equal to the titer;
c. incubating the serum at a temperature and for a period of time
which is sufficient to provide substantial equilibration of the
antibody bound and unbound triiodothyronine or thyroxine or
triiodothyronine and thyroxine;
d. separating the unbound triiodothyronine or thyroxine or
triiodothyronine and thyroxine from the bound triiodothyronine or
thyroxine or triiodothyronine and thyroxine while substantially
preventing re-equilibration of antibody bound triiodothyronine or
thyroxine or triiodothyronine and thyroxine; and
e. measuring the adsorbed unbound triiodothyronine or thyroxine or
triiodothyronine and thyroxine and the bound triiodothyronine or
thyroxine or triiodothyronine and thyroxine.
13. The method of claim 12 wherein the binding inhibitory agent for
thyroxine binding pre-albumin is barbituric acid, a derivative of
barbituric acid or a salicylate.
14. The method of claim 13 wherein the derivative of barbituric
acid is sodium barbital.
15. The method of claim 12, wherein the binding inhibitory agent
for thyroxine binding globulin is dilantin, a salicylate,
merthiolate, tetrachlorothyronine or 8-anilino-1-naphthalene
sulfonic acid.
16. The method of claim 12, wherein the isotopically labelled
triiodothyronine is triiodothyronine .sup.125 I.
17. The method of claim 12, wherein the isotopically labelled
thyroxine is thyroxine .sup.131 I.
18. The method of claim 12, wherein the serum is maintained at a pH
of about 8 during incubation.
19. The method of claim 12, wherein incubation is carried out at
about 37.degree.C for a period of about 1 to 2 hours.
20. The method of claim 12, wherein separation of unbound from
bound triiodothyronine or thyroxine or triiodothyronine and
thyronine is carried out by adsorbtion of the unbound product on
dextran coated activated carbon added to the incubation mixture
about ten minutes after the mixture has been cooled to a
temperature of about 20 C.
21. The method of claim 20, wherein the adsorbed unbound product is
separated from the adsorbed product by centrifugation or
filtration.
Description
BACKGROUND OF THE INVENTION
I. Field of the invention
This invention is related to the field of radioimmunoassay methods
in general, and more specifically, to radioimmunoassay methods for
triiodothyronine and thyroxine.
II. Description of the Prior Art
Radioimmunoassay methods are known for determining the amount of
the thyroid hormones, triiodothyronine and thyroxine. These
hormones regulate the rate of metabolic processes in the body.
Measuring the amount of triiodothyronine and/or thyroxine permits
detection of hyperthyroidism and hypothyroidism and monitoring the
therapy for these conditions.
In the method disclosed by Ekinds et al., Proc. Int. Thyroid Conf.,
6th, 1970 138 (Abstr.), a preliminary extraction from serum and a
paper chromatographic separation are performed prior to
radioimmunoassay. This procedure is time consuming, inaccurate and
does not measure thyroxine
Utiger et al., J. Clin. Invest., Vol. 51, 1972, and Larson,
Metabolism, Vol. 20, 1971, have disclosed radioimmunoassay methods
for triiodothyronine which require two and onehalf to three days
for completion and do not measure thyroxine.
The method of Solomon et al., J. Clin. Invest., Vol. 50, 1971, is
limited in usefulness because it does not detect triiodothyronine
in serum from hypothyroid patients, requires the use of hypothyroid
sheep serum, takes two days to complete and does not measure
thyroxine.
The method of Mayberry et al., J. Clin., Endocrinol. Metab., Vol.
31, 1970, requires at least two days for completion and does not
measure thyroxine.
Chopra et al., J. Clin. Endocrinol. Metab, Vol. 32, 1971, have
disclosed a radioimmunoassay method for thyroxine which, however,
does not measure triiodothyronine, requires a timeconsuming
extraction and takes one and a half to two days for completion.
SUMMARY OF THE INVENTION
A radioimmunoassay method has been discovered with permits, if
desired, simultaneous measurement of triiodothyronine and thyroxine
or selective measurement of either of these hormones.
Broadly, the method of this invention comprises:
a. adding to unextracted serum, a sufficient amount of a binding
inhibitory agent to substantially inhibit binding of
triiodothyronine or thyroxine or triiodothyronine and thyroxine to
the thyroxine binding prealbumin;
b. adding to the serum an amount of an isotopically labelled
triiodothyronine or thyroxine or triiodothyronine and thyroxine
sufficient for detection;
c. adding to the serum, a sufficient amount of a binding inhibitory
agent to substantially inhibit binding of triiodothyronine or
thyroxine or triiodothyronine and thyroxine to the thyroxine
binding globulin;
d. adding to the serum an amount of antiserum containing antibody
for triiodothyronine or thyroxine or triiodothyronine and thyroxine
which is approximately proportionate to the titer;
e. incubating the serum at a temperature and for a period of time
which is sufficient to provide substantial equilibration of the
antibody bound and unbound triiodothyronine or thyroxine or
triiodothyronine and thyroxine;
f. separating the unbound triiodothyronine or thyroxine or
triiodothyronine and thyroxine from the bound triiodothyronine
while substantially preventing re-equilibration of antibody bound
triiodothyronine or thyroxine or triiodothyronine and thyroxine;
and
g. measuring the adsorbed unbound triiodothyronine or thyroxine or
triiodothyronine and thyroxine and the bound triiodothyronine or
thyroxine or triiodothyronine and thyroxine.
This method provides a rapid, highly reproducible, accurate,
sensitive assay requiring relatively small amounts of serum, is
simple to perform and possess other advantages as will hereinafter
be evident to one skilled in the art.
BRIEF DESCRIPTION OF THE DRAWING
FIG. 1 illustrates a thyropin standard curve.
FIG. 2 illustrates a triiodothyronine standard curve.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The serum to be tested can be in the unextracted state in which
case there will be thyroxine binding pre-albumin and thyroxine
binding globulin present therein or the serum can be extracted by
known and conventional methods such as n-butanol extraction and
passage through anionic or cationic exchange columns in which case
these binding proteins will be absent.
When unextracted serum is employed, it is necessary to add
sufficient amounts of binding inhibitory agents to inhibit binding
of triiodothyronine and/or thyroxine to the binding proteins
contained in the serum. Some examples of agents which can be used
to inhibit the binding of triiodotrhyronine and/or thyroxine to
thyroxine binding pre-albumin include barbituric acid and its
derivatives (sodium barbital), the salicylates and many other
compositions as will be clearly obvious to one skilled in the art.
Sodium diethylmalonylurea has been found to be advantageous.
If reproducible results are desired, it is advantageous to add a
binding inhibitory agent which also acts as a buffering agent, or
to add a buffering agent as such. A pH of about 8 maintained by the
addition of sodium barbital has been found to be advantageous. Some
examples of agents which can be used to inhibit the binding of
triiodothyronine and/or thyroxine to thyroxine binding globulin
include dilantin, salicylates, merthiolate, tetrachlorothyronine
and numerous other compositions as will be clearly obvious to one
skilled in the art. 8-anilino-1-napthalene sulfonic acid has been
found to be advantageous.
The amounts of binding inhibitory agents to be added to the serum
which will be sufficient to inhibit the binding of triiodothyronine
and/or thyroxine to the binding proteins can be determined by
establishing standard curves for serum and buffer.
Examples of isotopically labelled triiodothyronine and thyroxine
include C.sup.14, tritium, I.sup.129 and I.sup.132. Because of
their relatively long half-lives and desirable counting properties,
triiodothyronine .sup.125 I and thyroxine .sup.131 I are especially
useful. The amounts of isotopically labelled hormones to be added
will be that which is sufficient to give detectable amounts of
radiation under the prevailing conditions.
Antiserum containing antibodies for either or both triiodothyronine
and thyroxine can be prepared by known and conventional methods.
For example, Mitsuma et al., J. Clin. Invest., Vol. 50, 1971, which
is incorporated by reference herein disclose methods whereby
triiodothyronine antibody can be advantageously prepared. The same
methods can also be used for preparing thyroxine antibody. The
amount of antiserum added to the serum will be approximately
proportionate to the titer which can be measured by known and
conventional methods, as for example, the method set forth in
Mitsuma et al., J. Clin. Invest., Vol. 50, 1971.
Incubation of the serum is made to take place at a temperature and
for a period of time which is sufficient to provide substantial
equilibration of the antibody bound and unbound triiodothyronine
and/or thyroxine. An incubation temperature of about 37.degree.C
for a period of from about 1 to 2 hours has been found to provide
good results. Other incubation temperatures and periods can also be
used as is clearly evident to those skilled in the art.
Separation of the unbound triiodothyronine and/or thyroxine from
the bound triiodothyronine and/or thyroxine can be carried out
according to known and conventional methods such as
electrophoresis, double antibody precipitation, salt precipitation
of the antibody, gel filtraiton, ion exchange resins, solid phase
antibody methods and adsorption on charcoal and silica.
The function of an adsorbant is to rapidly and selectively adsorb
substantially all the unbound triiodothyronine and/or thyroxine to
the substantial exclusion of the bound triiodothyronine and/or
thyroxine. Some examples of adsorbants which can be used in the
method of this invention include talc, precipitated silica,
diatomaceous earth, styrene polymers and numerous other known and
conventional adsorbants. A mixture of dextran and activated carbon
has been found to be especially suitable.
Precaution must be taken during separation of the bound from the
unbound hormones to avoid re-equilibration of antibody bound
triiodothyronine and/or thyroxine. One method by which this can be
advantageously accomplished is to cool the incubation mixture prior
to and during addition of the adsorbant to a temperature at which
dissociation of the antibody bound triiodothyronine and/or
thyroxine is substantially prevented.
Separation of adsorbed unbound triiodothyronine and/or thyroxine
from the bound triiodothyronine can be quickly and conveniently
carried out by means of centrifugation or filtration.
Both bound and unbound triiodothyronine and thyroxine can be
measured according to known and convetional methods of isotopic
counting.
When extracted serum is employed, it is unnecessary to add binding
inhibitory agents to the serum as to the binding proteins,
thyroxine binding pre-albumin and thyroxine binding globulin have
been removed. In all other respects the method for assaying the
triidodthyronine and/or thyroxine content of extracted serum is the
same as that employed for the unextracted serum.
It is contemplated that when an unextracted serum is employed
containing thyroxine binding pre-albumin and thyroxine binding
globulin, the required amounts of binding inhibitory agents may be
added to the unextracted serum either sequentially with the
required amount of isotopically labelled triiodothyronine or
thyroxine or triiodothyronine and thyroxine, sufficient for
detection; or in the alternative these required binding inhibitory
agents may be premixed together with the required amount of
isotopically labelled triiodothyronine or thyroxine or
triiodothyronine and thyroxine, in a sufficient amount required for
detection and this mixture may subsequently be added to the
unextracted serum.
The following example further illustrates the method of this
invention.
EXAMPLE
Antiserum to triiodothyronine (T.sub.3) was produced in rabbits by
injection of a T.sub.3 -albumin conjugate prepared as described by
Gharib et al., J. Clin., Endocrinol. Metab., Vol. 33, 1971.
Antiserum to thyroxine (T.sub.4) was produced in analogous fashion.
The presence and titer of antibody was determined as described in
Mitsuma et al., J. Clin. Invest., Vol. 50, 1971. The antiserum
solution employed in this assay was prepared by mixing antiserum to
T.sub.3 and antiserum to T.sub.4 in the same vessel with barbital
diluent (.08M sodium barbital, 0.1 gm/100 ml bovine serum albumin,
pH 8.4) such that the anti-T.sub.3 antiserum was diluted 1 to 2000
and the anti-T.sub.4 antiserum 1 to 200.
Serum free of T.sub.3 and T.sub.4 (T.sub.3, T.sub.4 -free serum)
was prepared by incubating 100 ml of normal human serum with 20
grams of Norit A charcoal (Sigma Chemical Corp., St. Louis,
Missouri) for 24 hours at 4.degree.C. The resulting slurry was
centrifuged three times at 20,000 xg. This procedure removes over
99% of the T.sub.3 and T.sub.4 from serum, and does not affect
total protein concentration, pH or T.sub.4 -binding capacity.
Non-radioactive standards of T.sub.3 and T.sub.4 were obtained from
Sigma Chemical Corp. in the free acid form. T.sub.3 was dissolved
in ethanol; 1N HCl (2:1); T.sub.4 in 0.13M NaOH, 70% ethanol
solution. The standards were prepared in T.sub.3, T.sub.4 -free
serum.
T.sub.3 .sup.125 I and T.sub.4 .sup.131 I were obtained from the
Abbot Labs, North Chicago, Illinois. The isotope mixture used in
this assay was prepared by diluting both isotopes in barbital
diluent in the same vessel at a concentration of 30 pg T.sub.3
.sup.125 I and 50 pg of T.sub.4 .sup.131 I per 100 lambda of
diluent.
8-anilino-1-napthalene sulfonic acid (ANS; Eastman Kodak Corp.) was
disolved in barbital diluent. A concentration of 175 .mu.g/0.2 ml
was employed in assays in which serum or standards were diluted
1:4. In those cases involving serum samples or standards diluted
1:2, 350 .mu.g/0.2 ml was used.
The assay procedure was performed in quadruplicate in 10 .times. 75
mm disposable glass tubes by the sequential addition of 0.1 ml of
the unknown sample or standard (which had been diluted 1:2 or 1:4
in barbital diluent), 0.1 ml of the isotope solution, 0.2 ml of the
ANS solution and 0.1 ml of the antibody solution. The assay mixture
was agitated in a vortex mixer and incubated for 90 minutes at
37.degree.C in a gently shaking water bath. After incubation the
samples were allowed to cool for 10 minutes in a 4.degree.C cold
room where the separation was carried out by the addition of one ml
of a stock solution of dextran-charcoal. The stock solution was
prepared by mixing 5g of Norit A Charcoal and 0.5 gm of dextran
(M.W. 86,000; K and K Labs. Hollywood, California) in 600 ml of
barbital diluent and constantly stirring with a magnetic stirrer to
prevent settling during the addition procedure. The reaction
mixture was agitated in a vortex mixer incubated at 4.degree.C for
20 minutes, and centrifuged at 1000xg for 15 minutes. The
supernatant containing the antibody-bound hormone was decanted off
the hard charcoal pellet containing the free hormone. The two
fractions were then counted in a two-channel gamma counter (Nuclear
Chicago) equipped with a 300 sample automatic changer.
Barbital buffer was used to inhibit binding of T.sub.3 and T.sub.4
to thyroxine binding pre-albumin and ANS to inhibit binding of
T.sub.3 and T.sub.4 to thyroxine binding globulin (TBG). FIGS. 1
and 2 show typical standard curves for T.sub.4 and T.sub.3 prepared
in buffer and also in T.sub.3, T.sub.4 -free serum with and without
ANS. The solid dots in both FIG. 1 and 2 represent additions to
barbital diluent; open circles represent additions to T.sub.3,
T.sub.4 -free serum in the presence of ANS; open rectangles depict
additions of T.sub.3 or T.sub.4 to T.sub.3, T.sub.4 -free serum in
absence of ANS and the open triangles represent curves obtained by
dilutions of a hyperthyroid serum. The serum standard curves for
both T.sub.4 and T.sub.3 obtained with ANS are almost identical to
those obtained with buffer alone. Routinely a concentration of 175
.mu.g per tube was selected because it was found that ANS at a
concentration in excess of 150 .mu.g per tube successfully inhibits
TBG binding of T.sub.3 and T.sub.4 in hyperthyroid, normal,
pregnant and hyperthyroid subjects. The antigen-antibody reaction
reached equilibrium within 60 minutes at 37.degree.C. A 90 minute
incubation time which was found to fall well within the plateau of
the equilibrium curve was chosen. At 4.degree.C., the
dextran-charcoal binding of hormone reached equilibrium within 15
minutes. 20 minutes was therefore selected for the second
incubation.
Neither the anti-T.sub.3 nor the anti-T.sub.4 antisera used in this
assay showed measureable cross-reaction with monoiodotyrosine,
diiodotyrosine or ANS. The anti-T.sub.3 antiserum showed less than
1:5000 cross-reaction with chromatographically-purified T.sub.4 ;
the anti-T.sub.4 antiserum did not cross react with T.sub.3.
Recovery experiments performed by the addition of known amounts of
T.sub.3 and T.sub.4 to T.sub.3, T.sub.4 -free serum and normal
serum demonstrated a recovery of 99.4.+-.2.9% for T.sub.3 and
100.8.+-.1.4% for T.sub.4. The T.sub.4 values obtained in this
assay were compared with determinations on the identical specimens
by competitive protein binding analysis (Boston Medical
Laboratories). The table below summarizes the results obtained from
40 specimens, from 14 hypothyroid patients, 11 hyperthyroid
subjects and 15 normal subjects.
TABLE
__________________________________________________________________________
T.sub.3 T.sub.4 Thyroid Status No. Mean .+-. SEM Range Mean .+-.
SEM Range (ng/100ml) (.mu.g/100ml)
__________________________________________________________________________
Normal 15 134 .+-. 6.2 96-170 7.4 .+-. 0.5 3.9-9.8 Hyperthyroid 11
486 .+-. 77 320-1200 16.2 .+-. 1.1 12.0-22.0 Hypothyroid 14 67 .+-.
2.2 52-78 1.3 .+-. 0.2 0.4-2.0
__________________________________________________________________________
The agreement was excellent (coefficient of correlation: 0.98).
T.sub.3 values obtained in the combined assay agreed closely with
data from the radioimmunoassay method disclosed in Mitsuma et al.,
J. Clin. Invest., Vol. 50, 1971 (coefficient of correlation 0.99).
Analysis of T.sub.3 and T.sub.4 in 5.lambda., 10.lambda.,
20.lambda., and 25.lambda. of hyperthyroid serum diluted to a
25.lambda. volume in T.sub.3, T.sub.4 -free serum yielded a curve
superimposable upon standard curves obtained by the addition of
T.sup.4 and T.sup.3 to T.sub.3, T.sub.4 -free serum (FIGS. 1 and
2). Intraassay reproducibility, assessed by measuring the identical
sample 33 times was 4.4% for T.sub.3 and 4.1% for T.sub.4.
Interassay variation, evaluated by determining 18 paired specimens
in two successive assays was 6.8% for T.sub.3 and 7.4% for
T.sub.4.
In 15 normal subjects mean T.sub.3 concentrations was 134.+-.6.2
ng/100 ml (mean .+-. S.E.M.) and mean T.sub.4 was 7.4.+-.0.5
.mu.g/100ml. In 11 patients with toxic diffuse goiter mean T.sub.3
was 486.+-.77 ng/100 ml and mean T.sub.4 was 16.2.+-.1.1 .mu.g/100
ml. 14 hypothyroid subjects had a serum T.sub.3 which ranged from
52 ng/100 ml to 78 ng/100 ml and serum T.sub.4 varied from 0.4
.mu.g/100 ml to 2.0 .mu.g/100 ml.
Tetrachlorothyronine, dilantin and salicylate have all been
employed to inhibit TBG binding to T.sub.3 in a T.sub.3
radioimmunoassay on unextracted serum and ANS has been utilized for
this purpose for the radioimmunoassay of T.sub.4. Purified
preparations of tetrachlorothyronine can be used for the T.sub.3
radioimmunoassay since they do not cross-react with the
anti-T.sub.3 antiserum. However certain prepartions of
tetrachlorothyronine do cross-react significantly with the
anti-T.sub.3 antiserum due to contamination with trichlorothyronine
and must be purified by gas-liquid chromatography prior to their
use in the assay. Moreover, even purified tetrachlorothyronine
possesses intrinsic cross-reactivity with anti-T.sub.4 antiserum
and cannot be applied to the combined T.sub.3, T.sub.4 assay. The
potential use of ANS and dilantin were therefore investigated for
this purpose. At the pH of this assay system, dilantin was not
soluble in concentrations high enough to block T.sub.4 binding to
TBG but ANS at concentrations in excess of 150 .mu.g/ tube proved
successful in inhibiting both T.sub.3 and T.sub.4 binding to TBG.
In those instances in which it was necessary to use a 1:2 dilution
of hypothyroid serum to achieve maximum sensitivity the ANS
concentration was doubled to 350 .mu.g/tube, and the standard curve
in T.sub.3, T.sub.4 -free serum was run at a 1:2 dilution. For
hyperthyroid sera suitable dilutions with T.sub.3, T.sub.4 -free
serum were employed. In order to improve the clinical convenience
of the assay, shorter incubation times than have been previously
reported were investigated. 90 minutes at 37.degree.C in a shaking
water bath was found to be sufficient for routine purposes since at
this temperature the anti-genantibody reaction reached equilibrium
after one hour. No signficant shift in favor of the free hormone
moiety was observed at this higher temperature, nor was conversion
of T.sub.4 to T.sub.3 found as a result of the heating. Binding of
the free hormone by dextran-charcoal reaches equilibrium at
4.degree.C after 15 minutes. The separation was performed at
4.degree.C since at higher temperatures all of the hormone
precipitated in an extremely brief and critical interval.
The values for T.sub.3 found in normal, hypothyroid and
hyperthyroid subjects are in good agreement with results published
in Mitsuma et al., J. Clin, Invest., Vol. 50, 1971 and with those
of others. The T.sub.4 determinations agree well with those found
by competitive protein binding analysis.
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