U.S. patent number 3,961,894 [Application Number 05/462,882] was granted by the patent office on 1976-06-08 for test for determination of triiodothyronine.
This patent grant is currently assigned to Yissum Research Development Company. Invention is credited to Amirav Gordon, Jack Gross.
United States Patent |
3,961,894 |
Gordon , et al. |
June 8, 1976 |
Test for determination of triiodothyronine
Abstract
A radioimmunoassay method for the determination of
triiodothyronine in human and animal fluids and tissues,
particularly in serum. The method involves the separation of
triiodothyronine from its binding proteins using a dextran gel
column followed by competitive binding between the freed
triiodothyronine and radioactive labeled triiodothyronine which
remain bound to the gel column for triiodothyronine-specific
anti-serum. Bound and free labeled triiodothyronine are separated
by washing the column and their ratio is determined based on the
amount of radioactivity remaining in the column to that originally
added thereto.
Inventors: |
Gordon; Amirav (Jerusalem,
IL), Gross; Jack (Jerusalem, IL) |
Assignee: |
Yissum Research Development
Company (Jerusalem, IL)
|
Family
ID: |
11047085 |
Appl.
No.: |
05/462,882 |
Filed: |
April 22, 1974 |
Foreign Application Priority Data
Current U.S.
Class: |
436/500; 250/303;
436/542; 436/810; 436/541; 436/804; 436/825 |
Current CPC
Class: |
G01N
33/78 (20130101); Y10S 436/804 (20130101); Y10S
436/825 (20130101); Y10S 436/81 (20130101) |
Current International
Class: |
G01N
33/74 (20060101); G01N 33/78 (20060101); G01N
033/16 (); G21H 005/02 () |
Field of
Search: |
;23/23B,230.6
;424/1,12,1.5 ;250/303,304 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Surks et al., J. Clin. Invest., vol. 51, pp. 3104-3113 (1972).
.
Alexander et al., Clin. Chem., vol. 20, pp. 1353-1361
(1974)..
|
Primary Examiner: Wolk; Morris O.
Assistant Examiner: Hagan; Timothy W.
Attorney, Agent or Firm: Klawitter; Andrew L.
Claims
We claim:
1. A method for the in vitro determination of triiodothyronine in a
biological fluid sample comprising the steps of:
a. adding to a column containing a crosslinked dextran gel at a pH
of at least about 10.5 or less than about 3 a predetermined
quantity of said fluid sample and a quantity of radioactive labeled
triiodothyronine,
b. washing said column with an aqueous solution having a pH of
between about 6 and 9,
c. adding to said column a predetermined quantity of antibody to
triiodothyronine,
d. incubating said column,
e. washing said column with an aqueous solution to substantially
remove the antibody-bound-triiodothyronine,
f. determining the ratio of radioactive labeled triiodothyronine
retained in said column after step (e) to that added in step (a),
and
g. comparing the ratio determined in step (f) to ratios obtained
using standard samples containing known amounts of
triiodothyronine.
2. A method as in claim 1 wherein said predetermined quantity of
said fluid sample and said quantity of radioactive labeled
triiodothyronine added to said column in step (a) are first
combined and equilibrated before introduction into said column of
dextran gel.
3. A method as in claim 2 wherein said predetermined quantity of
antibody of triiodothyronine added to said column in step (c) is in
the form of anti-serum prepared in a suitable laboratory animal
against a conjugate of triiodothyronine and a suitable carrier.
4. A method as in claim 2 wherein said dextran gel is cross-linked
with epichlorohydrin and has a water regain of from about 1 to 5
grams per gram of dry gel.
5. A method as in claim 4 wherein the amount of radioactivity
contained in said column is measured once after step (c) but before
step (e) and again after step (e).
6. A method as in claim 1 wherein said column in step (a) is at a
pH of at least 10.5.
7. A method as in claim 1 wherein the quantity of radioactive
labeled triiodothyronine added to said column in step (a) is
predetermined and the amount of radioactivity contained in said
column is measured after step (e).
8. A method as in claim 1 wherein the radioactivity contained in
said column is measured before and after step (e).
9. A method as in claim 1 wherein said aqueous solution used in
step (e) has a pH of between about 6 and 9.
10. A method as in claim 9 wherein said aqueous solution contains a
phosphate buffer.
11. A method as in claim 1 wherein said biological fluid sample is
serum.
Description
The present invention relates to a novel method and device for the
determination of triiodothyronine, designated hereinafter as
T.sub.3. More particularly, the present invention relates to a
novel quantitative specific radioimmunoassay for the determination
of T.sub.3 in human and animal fluids and tissues, and especially
in human sera. Other and further features of the present invention
will become apparent hereinafter.
Triiodothyronine or T.sub.3 is now considered to be the active form
of thyroid hormone, and therefore, measurements of the quantity of
T.sub.3 in the serum reflects the thyroidal status more closely
than other thyroid function determinations. Furthermore, conditions
have been described in which the only thyroid hormone abnormality
was an increase in the level of T.sub.3, a condition described as
T.sub.3 Thyrotoxicosis.
There are known various methods for the quantitative determination
of T.sub.3 in biological materials, but these are quite
timeconsuming and complicated. According to the present invention
there is provided a simple, comparatively rapid and reproducible
radioimmunoassay for the specific determination of T.sub.3. The
interference of T.sub.4 (thyroxine) is so small as to be
practically negligible.
The novel method of the present invention may be carried out using
a test device which comprises essentially a cylindrical tubular
body 11 having a fixed geometry and terminating at one end in a
tapered tip portion 13. The body 11 is formed of polypropylene or
other suitable material and is provided with valve means, such as a
friction fit cap 12, which fits over and removably closes the tip
portion 13. A quantity of cross linked dextran gel 14, which will
be more completely described hereinafter, is retained between two
porous polyethylene discs 15 and 19 in the lower portion of the
body 11. A buffer or the reaction mixture 16 may be retained in the
upper portion of the body 11 as shown. The upper end of the body 11
may be formed with an outwardly projecting flange 17 for ease of
holding the body 11 in an upright position in a suitable rack and a
removable cap 18 is provided for sealing the upper end of the body
11 and thereby preventing the column from drying out during storage
or shipment.
As noted above, the basic chemical constituent of the test device
of the present invention is the column 14 of cross linked dextran
gel retained in the lower end of the body 11. A preferred dextran
gel is that known commercially as Sephadex. This material is a
cross linked polysaccharide having the ability to act as a
molecular sieve, i.e. it retains molecules in various degrees
depending upon their size and to some extent their charge. The use
of this dextran gel technique as a molecular sieve is often called
gel filtration. Sephadex G-25, a type of dextran gel which excludes
materials of greater than 2000 to 3000 molecular weight from the
grains, is insoluble in water and salt solutions and is very stable
in alkalies and weak acids. Such a substance is preferable for use
in the present invention. Sephadex G-15 and G-10 can also be
adapted to serve the same purpose.
The test device of the present invention may advantageously be
prepared by initially hydrating the dry dextran gel with an
appropriate buffer (as will be described hereinafter). About 450
mg. (on a dry basis) of gel 14 is placed on the lower one third of
the body 11 between porous polyethylene discs 15 and 19. The body
11 illustrated has an inside diameter of about 13 mm. and a total
length of about 7.6 cm., and the discs 15 and 19 have a diameter of
13 mm. The gel 14 occupies a volume of about 2.5 ml. While the
above materials and sizes are merely exemplary, it has been found
that such a geometry ideally fits the counting well of certain
commercial radiation counting instruments, such as the Gammacord
gamma counter.
As far as the method of the present invention is concerned, it has
been found that the previously described test device, coupled with
the utilization of a small quantity of sample, such as serum,
results in a thyroid function test device and method which has
several unexpected and important advantages. By a small quantity of
serum sample, it is meant a sample size which may range from about
0.1 ml. to about 0.5 ml. By using a small serum sample in
combination with an amount of radioactive tagged thyroid hormone
appropriate for the test system being utilized, a test is effected
which has all the advantages previously mentioned, i.e. a single
reaction device and counting vessel, universal application and so
forth.
The method according to the present invention, which is specific
for the quantitative determination of T.sub.3, is a modification of
an isotope dilution technique.
According to the present invention, the method comprises:
a. Admixing at the top of the column a predetermined quantity of
serum or other biological fluid and a predetermined quantity of
radioactive labeled T.sub.3 made up in a solution having either pH
higher than 10.5 or lower than pH 3.
b. Equilibrating the mixture for a predetermined period of
time;
c. Introducing the equilibrated mixture into the column of dextran
gel prepared in a suitable solution having a pH less than 3 or more
than 10.5;
d. Washing out substantially all the proteins by means of a liquid
(preferably a buffer) of a pH between 6 - 9;
e. Introducing into the column a predetermined quantity of T.sub.3
- specific anti-serum, adapted to bind part of the total T.sub.3
content of the column;
f. Making a count of the radioactivity of the column;
g. Incubating the anti-serum and the T.sub.3 in the column for a
predetermined period of time;
h. Washing out the anti-serum-bound T.sub.3 by means of a suitable
buffer;
i. Making a further count of the radioactivity of the column;
j. Determining the T.sub.3 content of the sample from a previously
prepared calibration curve.
The calibration curve is prepared as set out in the above
procedure, with the only difference that known samples of T.sub.3
and T.sub.3.sup.125 I are mixed and a calibration curve is
prepared.
A small quantity of serum is sufficient for the quantitative
determination according to the present invention. An example of
such determination is described henceforth:
0.3 ml. of serum is admixed at the top of the column with 0.7 ml.
of T.sub.3.sup.125 I in 0.1 N NaOH and left for a period of about
15 minutes in order to attain an equilibrium between the protein
bound T.sub.3 and the added radioactive T.sub.3. After this period
of time the mixture is introduced by gravity flow into the dextran
gel of the column which was prepared in 0.1 N NaOH. The
triiodothyronine is adsorbed on the gel and after this the serum
protein is eluted in a substantially quantitative manner with 4 ml.
of 0.1 N phosphate buffer of pH 7.4. While the protein is eluted,
over 98% of the T.sub.3 and T.sub.3.sup.125 I remains on the
column.
A quantity of 0.5 ml. of dilute T.sub.3 -anti-serum is pipetted
onto the column and allowed to drain into the incubate within the
column for 2 hours. The quantity of anti-serum is chosen so as to
bind a part of the T.sub.3 only. During the 2 hour incubation
period, the first count of radioactivity is taken. At the end of
this period the anti-serum-bound T.sub.3 is washed out by means of
4 ml. of the same buffer and the second count of the radioactivity
is taken. The quantity of T.sub.3 in the serum is read off from the
calibration curve, which is prepared beforehand. This calibration
curve is prepared by means of a substantially identical procedure;
only instead of the unknown serum there is taken a standard
solution of T.sub.3. The entire test takes only about 3 hours. The
standard curve is rather constant, the variance of the slope of 6
separate curves being 6.6%. By such means the value of T.sub.3 in a
Euthyroid sera was found to be 168 mg./ 100cc. whereas sera from
hypothyroid patients gave distinctly lower values and sera from
hyperthyroid patients gave distinctly higher values.
The anti-sera in this example were prepared in rabbits against a
conjugate of bovine serum albumin and purified T.sub.3 according to
the following procedure:
1. T.sub.3 was cleaned by purification in 2N HCl according to the
method of Gross T. and P.H. Rivers R; Biochemical Journal 53, 645
(1953).
2. T.sub.3 was conjugated to bovine serum albumin and antiserum
against this conjugate was prepared according to the method
described by Gharib H., et al.; J. Clin Endocr. 31, 709 (1970).
However, T.sub.3 may be conjugated to other carrier polymer such as
Human Serum albumin, hemocyanin (Keyhole Limpet), poly L-Lysine, or
others. These conjugates are equally effective in eliciting proper
anti-T.sub.3 anti-serum in suitable animals.
3. The titer of anti-serum used in the test was determined as the
dilution of anti-serum sufficient to remove 30% of the hormone
(T.sub.3) when tested against a standard serum containing 5 ng/ml.
T.sub.3.
The following results were obtained:
Status T.sub.3 range in mg%
__________________________________________________________________________
Hypothyroid (12 patients) 66 - 110 Euthyroid (19 patients) 130 -
281 Hyperthyroid (23 patients) 291 - 2047
__________________________________________________________________________
Reproducibility Determined by the Calibration Curves Parameters and
T.sub.3 Values of a Control Serum Calibration Curve (*B/F vs. Ln
T.sub.3 concentration)
__________________________________________________________________________
Coefficient Exp. Slope Intercept Correlation T.sub.3 mg% in control
serum
__________________________________________________________________________
104 -3.77 9.39 0.988 167 104A -3.36 9.10 0.986 159 105 -3.33 9.03
0.986 204 110 -3.21 8.93 0.986 153 111 -3.12 8.78 0.984 168 112
-3.28 8.85 0.996 160 Mean .+-. SD -3.34.+-..22 9.01.+-..21
0.988.+-..004 168.+-.18
__________________________________________________________________________
Bound (T.sub.3) *B/F = Free (T.sub.3)
As T.sub.4 is present in serum in a much larger quantity than
T.sub.3, it was important to determine whether and to what extent
the presence of T.sub.4 interferes with the determination of
T.sub.3. A standard serum was loaded with T.sub.4 to levels
exceeding 40 mg% and T.sub.3 was determined as a function of the
increase of the content of T.sub.4. The level of interference was
0.11% and this can be neglected.
* * * * *