U.S. patent number RE32,098 [Application Number 05/932,759] was granted by the patent office on 1986-03-25 for radioimmunoassay for measurement of thyroxine (t.sub.4) and triiodothyronine (t.sub.3) in blood serum.
This patent grant is currently assigned to Research and Education Institute, Inc.. Invention is credited to Inder J. Chopra.
United States Patent |
RE32,098 |
Chopra |
March 25, 1986 |
**Please see images for:
( Certificate of Correction ) ** |
Radioimmunoassay for measurement of thyroxine (T.sub.4) and
triiodothyronine (T.sub.3) in blood serum
Abstract
This invention relates to a highly accurate, rapid and simple
estimation of thyroxine (T.sub.4) directly from blood serum and
also relates to the accurate measurement of triiodo-L-thyronine
(T.sub.3) directly from blood serum. More specifically, the
invention relates to a rapid, specific and reliable
radioimmunoassay (RIA) technique for measurement of both T.sub.4
and T.sub.3 in unextracted serum. The method requires very small
amounts of serum, e.g., 25 microliters (.mu.l) to measure T.sub.4
concentration in nearly all specimens representing clinical states
of eu-, hypo- and hyperthyroidism, and 250 .mu.l to measure T.sub.3
concentrations in specimens representing most clinical states.
Inventors: |
Chopra; Inder J. (Los Angeles,
CA) |
Assignee: |
Research and Education Institute,
Inc. (Torrance, CA)
|
Family
ID: |
26951303 |
Appl.
No.: |
05/932,759 |
Filed: |
August 10, 1978 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
Reissue of: |
265586 |
Jun 23, 1972 |
03911096 |
Oct 7, 1975 |
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Current U.S.
Class: |
436/500; 222/409;
436/539; 436/540; 436/541; 436/542; 436/804; 436/817; 436/825;
436/826 |
Current CPC
Class: |
G01N
33/78 (20130101) |
Current International
Class: |
G01N
33/74 (20060101); G01N 33/78 (20060101); G01N
033/56 (); G01N 033/56 (); G01N 033/60 (); G01T
001/00 () |
Field of
Search: |
;424/1 ;23/23B |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Chopra, Clin. Res. 19(1):125(1971). .
Lieblich and Utiger, J. Clin. Invest. 50:60a(1971). .
Mitsuma et al., J. Clin. Endocrinol. Metab. 33:364-367(1971). .
Chopra et al., J. Clin. Invest. 50:2033-2041(1971). .
Larsen, Metab. 20:976-980(1971). .
Mitsuma et al., J. Clin. Invest. 50:2679-2688(1971). .
Liebich and Utiger, J. Clin, Invest. 51:157-166(1972). .
Green et al., Science 175:1378-1380(1972). .
Chopra et al., J. Lab. Clin. Med. 80:729-739(1972). .
Weber, G. (1952) Biochem. J., 51, 145 and 51, 155. .
Laurence, D. J. R. (1952) Biochem. J., 51, 168. .
Wolff, J. et al., (1961), J. Clinical Investigation, 40, 1373.
.
Osorio, C. (1962), J. Physiol., 163, 151. .
Ingbar, S. H. (1963), J. Clinical Investigation, 42, 143. .
Stryer, L. (1965), J. of Molecular Biology, 13, 482. .
Gally, J. A. et al. (1965), Biochim. Biophys. Acta, 94, 175. .
McClure, W. O. et al. (1966), Biochemistry, vol. 5, No. 6, 1908.
.
McClure, W. O. et al. (1967), Biochemistry, vol. 6, No. 2, 559.
.
Edelman, G. M. et al. (1968), Accounts Chemical Research, 1,
65..
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Primary Examiner: Nucker; Christine M.
Attorney, Agent or Firm: Drucker & Sommers
Government Interests
The invention described herein was made in the course of work under
a grant or award from the Department of Health, Education and
Welfare.
Claims
I claim:
1. A method of measurement of the concentration of a particular
thyroid hormone selected from the group consisting of thyroxine
(T.sub.4) and triodo-L-thyronine (T.sub.3) in unextracted human
serum, which comprises:
A: adding to a measured quantity of unextracted human serum,
a. a blocking agent .Iadd.selected from the group consisting
8-anilino-1-napthalene sulfonic acid and
3-(4-anilino-1-naphthylazo) 2,7 naphthalene disulfonic acid, and
present .Iaddend.in an amount sufficient to displace essentially
all of said particular thyroid hormone to be measured from
thyroxine-binding globulin (TGB),
b. buffering ions to buffer said serum to a pH of between about 6.8
to about 9.6,
c. radioactive thyroid hormone, of the type to be measured, in an
amount which will give a measurable counting rate of either
antibody bound or free radioactivity after reaction equilibrium has
been reached as set forth in Step B below; and
d. an antibody in sufficient quantity to bind a significant
quantity of said radioactive thyroid hormone in the absence of any
of the particular nonradioactive thyroid hormone to be
measured,
B: allowing reaction of both particular thyroid hormone, to be
measured, and said radioactive thyroid hormone, with said antibody
to proceed substantially to equilibrium to thereby produce antibody
bound radioactive thyroid hormone;
C: separating said antibody bound radioactive thyroid hormone, to
be measured from said free radioactive thyroid hormone;
D: measuring the quantity of radioactive thyroid hormone selected
from antibody bound radioactive thyroid hormone and free
radioactive thyroid hormone;
E: preparing a standard curve with known amounts of the particular
thyroid hormone to be measured, and
F: correlating the quantity of radioactive thyroid hormone measured
with a known amount of said particular thyroid hormone read from
said standard curve.
2. The method of claim 1 wherein said thyroid hormone to be
measured is thyroxine, said blocking agent is
8-anilino-1-napthalene disulfonic acid, said blocking agent being
added to said human serum in quantities of at least about 4 ug/ul
serum.
3. The method of claim 1 wherein said buffering ions are barbital
ions, and said pH of said serum is about 8.6.
4. The method of claim 1 wherein said radioactive thyroid hormone
is initially present in the order of 10,000 counts per minutes.
5. The method of claim 1 wherein said antibody bound radioactive
thyroid hormone after reaction, gives a counting rate of from about
2000-6000 counts per minute.
6. The method of claim 1 wherein said antibody bound radioactive
thyroid hormone, to be measured, is separated from said free
radioactive thyroid hormone by precipation of said antibody bound
radioactive thyroid hormone with a second antibody.
7. The method of claim 1 wherein said antibody bound radioactive
thyroid hormone, to be measured, is separated from said free
radioactive thyroid hormone by precipation of said antibody bound
radioactive thyroid hormone with gamma globulin.
8. The method of claim 1 wherein said antibody bound radioactive
thyroid hormone, to be measured, is separated from said free
radioactive thyroid hormone by precipation of said antibody bound
radioactive thyroid hormone with charcoal suspension.
9. The method of claim 1 wherein said antibody bound radioactive
thyroid hormone, to be measured, is separated from said free
radioactive thyroid hormone by precipation of said antibody bound
radioactive thyroid hormone with polyethylene glycol.
10. The method of claim 1 wherein said thyroid hormone to be
measured is T.sub.4, said blocking agent is 2.7 napthalene
disulfonic acid, said blocking agent being added to said human
serum in quantities of at least about 2 ug/ul serum.
11. The method of claim 1 wherein said thyroid hormone to be
measured is T.sub.3, said blocking agent is 2,7 napthalene
disulfonic acid and said blocking agent is added to said human
serum in quantities of at least about 1 ug/ul serum.
Description
BACKGROUND OF THE INVENTION
In a normal human serum, about 99.97% of thyroxine (T.sub.4) and
99.7% of triiodothyronine (T.sub.3) present, is bound to proteins
such as thyroxine-binding globulin (TBG), prealbumin (TBPA) and
albumin; the remaining 0.03% of T.sub.4 and 0.3% of T.sub.3 is
present as unbound (dialyzable or free) hormones. The serum
concentration of thyroid hormones, among other facts such as
thyroidal secretion and degradation, is also dependent on serum
concentration of TBG; other binding proteins in serum are
relatively less important since their binding affinity for thyroid
hormones is far less than that of TBG. The concentration of TBG is
elevated in conditions such as pregnancy, estrogen treatment or a
genetic abnormality; serum total T.sub.4 (and T.sub.3) is increased
in these situations. Conversely, serum TBG concentration is
decreased during treatment with androgens or due to a genetic
deficiency of TBG. This is associated with subnormal concentrations
of thyroid hormones. However, in either of the above-mentioned
situations of altered TBG concentration, the patient remains
eumetabolic and concentrations of free T.sub.4 and (and T.sub.3)
and that of total T.sub.4 (and T.sub.3) corrected for TBG
abnormality are within the range of normal subjects.
Until a few years ago, serum T.sub.4 concentration was assessed
indirectly by measurements of organic (protein bound or butanol
extractable) iodine. However, these measurements were frequently
erroneous because of iodide contamination in the laboratory or of
administration of iodide containing drugs to the patients. In order
to overcome these problems, a competitive-protein binding assay
(CPBA) for T.sub.4 was introduced by Murphy and Pattee (1). This
procedure employs the principal of saturation analysis, and
quantitates T.sub.4, relatively specifically, by measurement of
displacement of radioactive T.sub.4 from T.sub.4 -binding sites on
TBG. It allows measurements of T.sub.4 in a range of 3-20 ug %
using a butanol-ethanol extract of serum. In the Murphy-Pattee
method, one must first extract thyroxine from the blood serum with
alcohol or butanol-ethanol; this introduces error since the
extraction procedure itself results in the extraction of varying
amounts of thyroxine. The drying of the extract is also time
consuming, and must be accomplished before one commences the
measurement of T.sub.4.
The Murphy-Pattee procedure thus leaves one with a dried extract
containing T.sub.4 as well as much non-thyroxins, such as lipids.
To the tubes containing the dried extract, one then adds a 1/32
diluted human serum containing radioactive T.sub.4 (T.sub.4 *). The
T.sub.4 in the dried extract competes with T.sub.4 * for binding
sites on TBG. After equilibrium is reached one separates the
unbound T.sub.4 by means of an ion exchange resin and measures the
amount of radioactive counts (of T.sub.4 *) remaining bound to TBG.
The quantitation of T.sub.4 is accomplished by reading from a
standard curve, prepared simultaneously with known amounts of
T.sub.4.
The disadvantages of the Murphy-Pattee method are that it requires
an extraction procedure; it is not sensitive enough and it is
cumbersome for processing a large number of samples.
A radioimmunoassay (RIA) has been previously developed and
reported, by the applicant herein in conjunction with others, which
employs a highly specific antibody to T.sub.4 for its subsequent
measurement instead of utilizing TBG from human serum. In this
method, a rabbit anti-thyroglobulin antiserum is employed as the
T.sub.4 binding protein. Further, in this method, the T.sub.4 must
be first extracted as in the Murphy-Pattee method but the method is
more sensitive than Murphy-Pattee. This RIA method of applicant
herein is reported, in detail, in Journal of Clinical
Endrocrinology, 33:865, 1971, incorporated herein by this
reference. In general, the precision, reproducibility and
practicality of this RIA is comparable to those of competitive
protein binding assay (CPBA) using serum TBG as the T.sub.4 binding
protein.
SUMMARY OF THE INVENTION
I have found that I can achieve significantly improved measurement
of T.sub.4 and T.sub.3 directly from unextracted serum by
displacing T.sub.4 and T.sub.3 from serum thyroxine (or T.sub.3)
binding globulin (hereinafter generically referred to as TBG by
means of a foreign compound (i.e., one not itself a thyroid
hormone) which compound not only has the capability of preventing
completely the binding of radioactive T.sub.4 and radioactive
T.sub.3 (T.sub.4 * or T.sub.3 *, respectively) or T.sub.4 and
T.sub.3 to TBG, respectively, but does not inhibit in any way the
reaction of T.sub.4 and T.sub.3 with T.sub.4 -antibodies used as
the horome binding proteins (instead of TBG). Foreign compounds
falling in this category will be termed herein, and in the claims
simply as "blocking agent" or "blocking agents".
The novel approach adopted herein is believed to be broadly new and
is not dependent upon the use of a particular blocking agent. Many
different types of blocking agents have been proven to have the
desired qualities aforementioned, e.g.,
8-anilino-1-napthalene-sulfonic acid (ANS),
3-(4-anilino-1-napthylazo) 2,7-napthalene disulfonic acid (ANNDS),
2,4 6-trinitro benzene sulfonic acid (TNBS), napthalene sulfonic
acid, Thimerosal, 5-5-diphenyl 2-thiohydantain, Doxepin Hcl,
Diazepam, sodium salicylate, prochlorperazine, halofenate (MK-185)
Merck, Sharpe and Dohme.
Further, the inhibition of the binding of T.sub.4 (or T.sub.3) to
thyroxine binding prealbumin (TBPA) is accomplished by the setting
up of the assay in a suitably buffered medium, e.g., containing
barbital ions, e.g., barbital buffer, and having a pH of between
about 6.8-9.6. T.sub.4 binding by prealbumin is also affected by
the presence of compounds such as ANS, or sodium salicylate.
Thus, with respect to the measurement of T.sub.4 by my novel RIA,
the process involves only the following few steps: (1) incubation
for one hour, of human serum, in the presence of radioactive
T.sub.4, T.sub.4 antibody, and one of the blocking agents mentioned
above or others in a barbital or other suitable buffer - to thereby
displace T.sub.4 bound to TBG in the serum and make it available
for reaction with T.sub.4 antibody (and thus measurable by RIA
techniques) while minimizing or completely inhibiting the binding
of the added radioactive T.sub.4 to TBG. Finally, the radioactive
T.sub.4 bound to the antibody is separated from free radioactive
T.sub.4. This may be accomplished in various ways, e.g., by use of
a "second antibody", e.g., goat anti-rabbit gammaglobulin which
precipitates the antibody bound radioactivity which may then be
counted. A standard curve is prepared with known amounts of T.sub.4
and unknowns are read off the curve. Using the just-described RIA,
it is feasible to quantitate T.sub.4 in a wide range (1 to 40 ug %)
using only 15 to 25 ul of unextracted serum.
Measurement of T.sub.3 in serum has been more involved and
difficult than that of T.sub.4. On an average, T.sub.3 is present
in normal serum in concentrations about 1/70 that of T.sub.4.
However, the importance of its quantitation in serum has been
highlighted by recent suggestions that T.sub.3 may be the
predominant biologically active thyroid hormone and that T.sub.4
exerts its biological effect only after prior conversion to T.sub.3
in the body. Several instances of hyperthyroidism due to elevated
serum T.sub.3 (and normal serum T.sub.4) have been described. A
popular method for measurement of T.sub.3 involves three steps,
i.e., extraction of thyronines from serum by column chromatography,
separation of T.sub.4 from T.sub.3 by paper chromatography and
finally quantitation of T.sub.3 by competitive protein binding
assay using TBG in a manner similar to that for T.sub.4 measurement
by Murphy-Pattee. However, chromatographic separation of T.sub.4
from T.sub.3 may not always be complete. It may also be associated
with in vitro conversion of T.sub.4 to T.sub.3, thereby leading to
inappropriate high values. These problems have been circumvented by
the use of a radioimmunoassay (RIA) method for quantitating T.sub.3
in unextracted serum by a method similar to that above-described
for T.sub.4 assay.
Thus, improvements in accurate measurement of serum T.sub.3 by RIA
requires, according to this invention, adequate blocking of
thyronine-binding globulin (TBG) in serum, by means of the same
blocking agents heretofore enumerated. The approach to T.sub.3
measurement is precisely the same as for T.sub.4 measurement; the
improvement in the T.sub.3 RIA being due primarily to the use of
blocking compounds such as ANS, which block T.sub.3 binding sites.
ANS, when used in a concentration of 1 ug/ul. test serum, displaces
nearly all T.sub.3 bound to TBG and prevents completely the binding
of radioactive T.sub.3 to TBG. ANS has negligible affinity for
T.sub.3 -binding sites on the rabbit antiserum used in this RIA
(which antiserum was produced by immunization with T.sub.3
-enriched thyroglobulin). The T.sub.3 -antibody may be precipated
with a "second antibody" and the bound radioactive T.sub.3 is
separated from free T.sub.3, and counted by previously known
methods, to provide an accurate measurement of T.sub.3.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an example of the standard curve relating T.sub.4 (ng) to
percent radioactive T.sub.4 precipitated;
FIG. 2 illustrates two more standard curves indicating the
substantially completely blocking of TBG by the particular blocking
agent utilized, viz. ANS; and
FIG. 3 is an example of the standard curve relating to T.sub.3 (ng)
to percent radioactive T.sub.3 precipitated
DESCRIPTION OF THE PREFERRED EMBODIMENTS
A. T.sub.4 Assay
In general, the method requires the incubation for about one hour
of human serum, in the presence of a barbital or other suitable
buffer and in the presence of T.sub.4 antibody, T.sub.4 *, and a
blocking agent present in sufficient quantity to displace T.sub.4
bound to TBG in the serum and make it available for reaction with
the said T.sub.4 antibody. One can readily assess the amount of any
particular blocking agent required to meet the foregoing condition.
Thus, by way of example only, the ability of ANS to displace
T.sub.4 has been demonstrated by its displacement of radioactive
T.sub.4 from TBG in a CPBA system, (1) supra. 100 ug of ANS was
found to displace 18 nanograms (ng) T.sub.4. Thus, when 100 ug of
ANS is added 25 ul of serum, it will displace all T.sub.4 up to a
concentration of 72 ug per 100 ml. of serum. Even though the upper
concentrations of T.sub.4 found are well below 72 ug/100 ml, an
excess of ANS, e.g., 150 ug added to 25 ul of serum, may be and is
used, in the instant assay procedure. In general, then, it is well
within the skill of the art, to determine the required minimal
quantity of blocking agent required for the assay procedure.
Further examples of the displacement ability of other blocking
agents are set forth in Table I below, the first compound set forth
in Table I being ANS for ready comparison.
TABLE I ______________________________________ AMOUNT T.sub.4
BLOCKING AGENT DISPLACED FROM TBG
______________________________________ (100 ug utilized except
where noted otherwise) 1. ANS 18 2. ANNDS (50 ug) >20 3. TNBS
7.1 4. .alpha.Napthalene Sulfonic Acid 3.0 5. 5,5 diphenyl-2
thiohydantoin 6.1 6. Doxepin HCl 8.2 7. Diazepam 6.3 8.
Prochlorperazine 7.2 9. Dilantin .RTM. 5.7 10. Thimerosal (1 mg.)
8.4 11. Sodium Salicylate (10 mg.) 6.1 12. Halofenate* (MK-185) 8.4
13. Chlorpromazine HCl ((50 ug) 5.6
______________________________________ *Proprietary compound
supplied by Merck, Sharp & Dohme
The amount of T.sub.4 * added is determined by that quantity
required to give to the assay tube, in which the assay is being
conducted, a measurable counting rate, after reaction with the
T.sub.4 antibody. The amount of radioactive T.sub.4 to be added is
not critical and may vary over a wide range depending upon the
sensitivity of the counting equipment. One may, for example,
utilize sufficient radioactive T.sub.4 initially, to cause a
counting rate of 2000-6000 CPM (counts per minute) to result from
measurement of the T.sub.4 *-antibody precipitate. One may also
desire to measure the free radioactive T.sub.4, in which event, the
counting rate of the free radioactive T.sub.4 should be preferably
at this range. Of course, as the counting equipment becomes more
sensitive, the amount of radioactive T.sub.4 * to be added may be
further reduced.
Preparation of antibodies specific to T.sub.4 is known and will be
described in some detail hereafter. The amount of T.sub.4 -antibody
added, to a predetermined quantity of human serum, e.g., 25 ul is
that quantity having the ability to bind substantial quantities of
T.sub.4 *, e.g., from about 20-60% of radioactive T.sub.4, in the
absence of any non-radioactive T.sub.4.
The contents of the assay tubes ae buffered, preferably by barbital
ions, to a pH in the range of from about 6.8 to 9.6, with a pH of
about 8.6 being preferred.
Incubation of the contents of the assay tubes, containing measured
amounts of T.sub.4 *, T.sub.4 antibody, and blocking agent in a
measured amount of serum, takes place over a period of an hour or
so at room temperature and for five minutes at 4.degree. C. During
this period of incubation, competitive reactions between T.sub.4 *
and T.sub.4, on the one hand, with T.sub.4 antibody on the other,
takes place, and near equilibrium of these competing reaction is
attained.
The radioactive T.sub.4 bound to antibody (antibody bound
radioactivity) is then separated from the free radioactive T.sub.4
by anyone of a number of methods. For example, a charcoal
suspension when added to the final reaction mixture, after the end
of the incubation period, adsorbs the free radioacitivity. The
reaction mixture, containing the charcoal, is centrifuged and the
charcoal settles. One may count either the bound radioactivity in
the supernatant, or the free radioactivity in the charcoal.
Alternatively, one may add a polyethylene glycol (Carbowax 6000) to
the reaction mixture. Polyethylene glycol will precipitate
gammaglobulins, and when added to the reaction mixture, will
precipitate the antibody bound radioactivity, and may be measured
as indicated above.
The method of precipitation of antibody bound radioactivity
presently peferred, involves the use of a second antibody which
will precipitate the antibody bound radioactivity. After a suitable
period of incubation, e.g., 24 hours at 4.degree. C., the antibody
bound radioactivity may then be counted. Measurement of T.sub.4
concentration is made by correlating the antibody found
radioactivity with a standard curve previously prepared.
B. T.sub.3 Assay
In general, the method of measuring T.sub.3 requires the incubation
for about 20 hours of human serum, in the presence of a barbital or
other suitable buffer and in the presence of T.sub.3 antibody,
T.sub.3 *, and a blocking agent, present in sufficient quantity to
displace all T.sub.3 bound to TBG in the serum and make it
available for reaction with said T.sub.3 antibody. It has been
demonstrated that TBG competes with T.sub.3 antibody for
radioactive T.sub.3 as well as for non-radioactive (or stable)
T.sub.3. The concentration of blocking agent required to prevent
this competition or interference from TBG is readily determined. In
general, the binding of T.sub.3 to TBG is much weaker than is
T.sub.4 to TBG and the amount of blocking agent required is less
per given amount of human serum than is required for the T.sub.4
assay. Thus, for example, 1 ug/ul serum of ANS is utilized in a
T.sub.3 assay as compared with 4-6 ug/ul ANS for a T.sub.4 assay.
The ability of ANS to displace T.sub.3 has been demonstrated by its
displacement of radioactive T.sub.3 from TBG. 10 ug. of ANS was
found to displace 11.2 nanograms (ng) T.sub.3. Thus, when 200 ug.
of ANS is added to 250 ul of serum, it may displace all T.sub.3 up
to a concentration of 88 ug T.sub.3 per 100 ml. of serum. Even
though the upper concentrations of T.sub.3 found are well below 2.5
ug/100 ml, an excess of ANS, e.g., 250 ug is normally added to 250
ul of serum in the instant assay procedure. In general, then, it is
well within the skill of the art, to determine the required minimal
quantity of blocking agent required for the assay procedure. The
procedure for determining the minimal quantity for ANS is set forth
in Example 2 hereof. Further, examples of the displacement ability
of other blocking agents are set forth in Table II below, the first
compound set forth in Table II being ANS for ready comparison.
TABLE II ______________________________________ AMOUNT T.sub.3
DISPLACED FROM BLOCKING AGENT TBG (ng.)
______________________________________ (10 ug utilized except where
noted otherwise) 1. ANS 11.2 2. ANNDS 10.4 3. TNBS (100 ug.) 7.0 4.
.alpha.Napthalene Sulfonic acid (100 ug) 7.0 5. 5,5 diphenyl-2
thiohydantoin 7.6 6. Doxepin HCl 10.8 7. Diazepam 7.0 8.
Prochlorperazine 7.0 9. Thimerosal (100 ug.) 8.0 10. Sodium
Salicylate (1 mg) 11.0 11. Halofenate* (MK-185) 5.2 12.
Chlorpromazine HCl (25 ug) 6.6
______________________________________ *Proprietary compound
supplied by Merck, Sharp & Dohme.
The amount of T.sub.3 * added is determined by that quantity
required to give to the assay tube, in which the assay is being
conducted, a measurable counting rate, after reaction with the
T.sub.3 antibody. The amount of radioactive T.sub.3 to be added is
not critical and may vary over a wide range depending upon the
sensitivity of the counting equipment. One may, for example,
utilize sufficient radioactive T.sub.3 initially, to cause a
counting rate of 2000--6000 CPM (counts per minute) to result from
measurement of the T.sub.3 *-antibody precipitate. One may also
measure the free radioactive T.sub.3, in which event, the counting
rate of the free radioactive T.sub.3 should be preferably at this
range. Of course, as the counting equipment becomes more sensitive,
the amount of radioactive T.sub.3 to be added may be further
reduced.
Preparation of T.sub.3 antibodies specific to T.sub.3 is known and
will be described in some detail hereafter. The amount of T.sub.3
antibody added, to a predetermined quantity of human serum, e.g.,
250 ul, is that quantity having the ability to bind from about
20-60% of radioactive T.sub.3 added in the absence of any
non-radioactive T.sub.3.
The contents of the assay tubes are buffered, preferably by
Barbital ions, to a pH in the range of from about 6.8 to 9.6, with
a pH of about 8.6 being preferred.
Incubation of the contents of the assay tubes, containing measured
amounts of T.sub.3, T.sub.3 antibody, and blocking agent in a
measured amount of serum, takes place over a period of 20 hours or
so, at 4.degree. C. During this period of incubation, competitive
reactions between T.sub.3 * and T.sub.3, on the one hand, with
T.sub.3 antibody on the other, takes place, and near equilibrium of
these competing reactions is attained.
The radioactive T.sub.3 bound to antibody (antibody bound
radioactivity) is then separated from the free radioactive T.sub.3
by anyone of a number of methods. For example, a charcoal
suspension when added to the final reaction mixtures, after the end
of the incubation period, adsorbs the free radioactivity. The
reaction mixture, containing the charcoal, is centrifuged and the
charcoal settles. One may count either the bound radioactivity in
the supernatant, or the free radioactivity in the charcoal.
Alternatively, one may add a polyethylene glycol (Carbowax 6000) to
the reaction mixture. Polyethylene glycol will precipitate
gammaglobulins, and when added to the reaction mixture, will
precipitate the antibody bound radioactivity, and may be measured
as indicated above.
The method of precipitation of antibody bound radioactivity
presently preferred, involves the use of a second antibody which
will precipitate the antibody bound radioactivity. The antibody
bound radioactivity is then counted and measurement of T.sub.3
concentration is made by correlating the antibody bound
radioactivity with a standard curve previously prepared.
C. Specific Examples
EXAMPLE 1
The following example illustrates the use of ANS as a blocking
agent in the measurement of T.sub.4 at a variety of concentrations,
and illustrates, as well, the making of a standard curve, to allow
accurate correlation of the unknown T.sub.4 concentrations with the
standard curve. This example serves as the basis for a paper to be
shortly published in Journal of Clinical Endochrinology, 34: 938,
1972 under the title "A Rapid Radioimmunoassay For Measurement of
Thyroxine in Unextracted Serum".
The reagents employed are:
1. T.sub.4 -binding antiserum or T.sub.4 antibody: The serum used
was obtained from a rabbit immunized with normal human
thyroglobulin (Tg), as described by Chopra et al., J. Clinical
Endocrinology 32:299. It was used in a final dilution of 1:300; in
this dilution it bound approximately 50% of a tracer amount of
radiactive T.sub.4.
2. Radioactive (.sup.125 I) T.sub.4 (SA 50-75 uc/ug) in 50%
propylene glycol was obtained from Abbott Laboratories, North
Chicgo, Illinois.
3. ANS was obtained from K & K Laboratories, Hollywood,
Calif.
4. Reagent grade Na-L-T.sub.4 (non radioactive T.sub.4) was
obtained from Mann Research Laboratories, New York. It was weighed
and dissolved in 0.1M NaOH and diluted to desired concentrations in
0.075M barbital buffer, pH 8.6, containing 2% normal rabbit serum
(NRS). The NRS is employed as a carrier protein to render the
antibody precipitate visible, in the final step, but is not present
in sufficient quantity to interfere with the T.sub.4
measurement.
The steps of the radioimmunossay (RIA) precedure follows:
In 10.times.75 mm disposable glass tubes, the various regents were
added in the following order to yield a final volume of 0.5 ml:
1. Three hundred ul of 0.075 M barbital buffer which contained 2%
NRS, 150 ug ANS and approximately 10,000 CPM (counts per minute) of
.sup.125 I-T.sub.4 (.about.0.2 ng T.sub.4). The stock solution of
ANS (50 mg per 100 ml of buffer) was made fresh before every
assay.
2. One hundred ul of various dilutions of T.sub.4, i.e. 0.5 ng/ml
to 200 ng/ml. were employed to yield 0.05 to 20 mg T.sub.4 for a 10
to 14 point standard curve (see FIG. 1).
In the case of the unknown, 25 ul of serum was employed, followed
by 75 ul of .075 barbital buffer containing 2% NRS but without
additional ANS or .sup.125 I-T.sub.4. The standard curve and
unknowns were assayed at least in duplicate.
3. To all tubes, 100 ul of a 1:60 dilution of T.sub.4 antibody was
added.
Steps 1 and 3 were conveniently and accurately performed using an
automatic pipettor (Repipet 1.0 ml, Lab Industries, Berkeley,
California). All tubes were briefly swirled after steps 2 and 3.
The tubes were then incubated for 1 hr at room temperature and 5
min at 4C. Pilot experiments had indicated that a state of
near-equilibrium was reached during this period of incubation.
4. To precipitate .sup.125 I-T.sub.4 bound to antibody,
approximately 40-50 ul of a previously titered goat antirabbit
.gamma.-globulin was added and tubes were reincubated overnight
(.about.20 hr) at 4C. The details of separation of bound from free
radioactivity, correction for nonspecific binding or trapping or
.sup.125 I-T.sub.4 in the precipitate and plotting of standard
curves have been described recently in a RIA for triiodothyronine
(T.sub.3). Chopra et al. Radioimmunoassay for measurement of
triodothyronine in human serum, J. Clinic Investg. 50:2033, Oct.
1971. The results are set forth below:
FIG. 1 shows the typical standard curve obtained. The curve is
essentially linear between 0.3 to 10 ng, allowing measurement of
serum T.sub.4 over the range of 1.2 to 40 ug per 100 ml when 25 ul
serum was assayed. The index of precision (.lambda.) was 0.063 in
this and another standard curve.
FIG. 2 shows a comparison of standard curves obtained in barbital
buffer and in the presence of 25 ul of T.sub.4 -free serum. The two
standard curves were nearly superimposable, indicating that TBG in
the serum was adequately blocked under the assay conditions
used.
Table III illustrates the results of serum T.sub.4 as determined by
the RIA described in this example and those obtained by CPBA in
sera from euthyroid subjects and patients with or without thyroid
functional abnormalities.
TABLE III ______________________________________ Comparison of
estimates of serum T.sub.4 concentration by this RIA and CPBA.
Serum T.sub.4 ug/100 ml Source of sera No. CPBA RIA
______________________________________ Euthyroid 40 7.51 .+-. 0.31*
8.33 .+-. 0.38 Hyperthyroid 40 19.9 .+-. 0.83 24.6 .+-. 1.39
Hypothyroid 7 3.34 .+-. 0.13 3.45 .+-. 0.36 Estrogen-treated 8 12.7
.+-. 0.61 13.2 .+-. 0.79 ______________________________________
*Mean .+-. SEM
In euthyroid individuals the mean serum T.sub.4 by this RIA was
10.9% higher than that by CPBA. This difference was statistically
significant. It is attributable, in part, to losses in T.sub.4
during extraction of serum in CPBA, since the recovery of
radioactive T.sub.4 in the butanol-ethanol extraction averaged
88%.
Precision of T.sub.4 measurements by the RIA, described herein, was
assessed by comparing the duplicates within assays. The mean value
for percent departure of duplicates from their mean in 85 sera was
4.32.+-.0.38.
Reproducibility of estimates of serum T.sub.4 by RIA was studied by
comparison of T.sub.4 concentration in 10 sera measured in
duplicate in different assays. The mean value for percent departure
of duplicates from their mean was 7.1.+-.1.22. The working time
involved in setting up an assay comprised of 94 tubes was only 116
min. This includes 56 min. spent in pipetting standards and test
sera which would be common to all methods.
The RIA proposed here is adequately sensitive, precise and
reproducible. The requirement of only 50 ul of sample for duplicate
determinations of serum T.sub.4 over a range of 1.2 to 40 ug per
100 ml, in one attempt, not only makes the assay useful for routine
clinical purposes but also for measurement of T.sub.4 in serum of
infants and small experimental animals where sample availability
may be limited. Since T.sub.4 -binding antisera (a T.sub.4
antibody) can be raised quite regularly when rabbits are immunized
with Tg in Freund's adjuvant, this RIA appears to be a very
practical method for measurement of serum T.sub.4. The practicality
of RIA is further emphasized by the simplicity of the procedure
described here as well as the short working time involved.
The specificity of the T.sub.4 -binding antiserum is also quite
acceptable.
EXAMPLES 2-13
The procedure of Example I may be followed, except that the
following compounds listed below may be employed instead of ANS.
The amounts used will be proportional to the blocking power of the
compounds as listed in Table I, taking into account solubility
considerations. Thus, 2 ug ANNDS per ul of serum can be
successfully employed. The results should be comparable to those of
Example I.
______________________________________ EXAMPLE COMPOUND
______________________________________ 2 ANNDS 3 TNBS 4
.alpha.Napthalene Sulfonic Acid 5 5,5 diphenyl-2 thiohydantoin 6
Doxepin HCl 7 Diazepam 8 Prochlorperazine 9 Dilantin .RTM. 10
Thimerosal 11 Sodium Salicylate 12 Halofenate* (MK-185) 13
Chlorpromazine HCl ______________________________________
*Proprietary compound supplied by Merck, Sharp & Dohme.
EXAMPLE 14
The following example illustrates the use of ANS as a blocking
agent in the measurement of T.sub.3 at a variety of concentrations,
and illustrates, as well, the making of a standard curve, to allow
accurate correlation of the unknown T.sub.4 concentrations with the
standard curve. This example serves as the basis for a paper to be
shortly published in the Journal of Laboratory and Clinical
Investigation under the title: An Improved Radioimmunoassay of
Triiodothyronine in Serum.
The reagents employed are:
1. T.sub.3 -thyroglobulin conjugate was first prepared in order to
make T.sub.3 antibody. The procedure was as follows:
L-triiodothyronine (T.sub.3) was conjugated to human thyroglobulin
(Tg) by a modification of the method of Oliver et al.,: The
measurement of digitoxin in human serum by radioimmunoassay, J.
Clin. Invest. 47:1035-1042, 1968. The method of preparation of
human Tg was the same as described earlier. Chopra et al.:
Production of antibodies specifically binding triiodothyronine and
thyroxine, J. Clin. Endocr. 32:299-308, 1970. To 100 mg. of Tg in 2
ml. of phosphate buffered saline (0.14 M sodium chloride, 0.01 M
sodium phosphate, pH 7.5, PBS), was added 10 mg. of Na-1-T.sub.3
(Mann Research Laboratories, New York) dissolved in 2 ml. of
dimethyl formamide and 20 mg. of 1-cyclo-hexyl-3
(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate
(Morpho-CDI, Aldrich Chemical Co., Inc., Milwaukee, Wis.). The
solution was kept at room temperature in the dark with occasional
stirring for 18 hours. The reaction mixture was then dialyzed
against 3 changes of normal saline, each time using 4 l for 24
hours at 4.degree. C. The conjugate was stored frozen (-10.degree.
C.). Pilot experiments using radioactive T.sub.3 indicated that 60%
of the T.sub.3 was incorporated into the conjugate.
2. T.sub.3 -binding antiserum was then prepared. The serum used in
RIA was obtained from a New Zealand rabbit immunized with 8
injections of the aforedescribed Tg-T.sub.3 conjugate, 1 to 2 mg
each, emulsified in 1 ml. of complete Freund's adjuvant, at 2-3
week intervals. One hundred ul of a 1:400 dilution of antiserum was
used in a final reaction mixture of 1 ml. At this final dilution of
1:4000 it bound 33% of a tracer amount (0.1 ng) of radioactive
T.sub.3.
3. In order to make the standard curve (FIG. 3) hypo-thyroid sheep
serum was employed. This sheep serum was obtained from an adult
sheep 6 week after a surgical total thyroidectomy. Total T.sub.4 in
this sheep serum, as measured by a sensitive RIA for T.sub.4, was
less than 1.0 ug per 100 ml. T.sub.4 -binding capacity of the
thyroxine-binding globulin (TBG) of sheep serum was 17.5 ug per 100
ml.
4. Radioiodinated (.sup.125 I) T.sub.3 (SA 90-100 uci/ug was
obtained from Industrial Nuclear Co., Inc., St. Louis, Mo.
5. Reagent grade Na-1-T.sub.3 (non-radioactive) was obtained from
Mann Research Labs., New York. It was dissolved and diluted to a
concentration of 100 ug/ml in .01M NaOH containing 20% propylene
glycol. Dilutions for use in standard curve, i.e., 0.1 ng/ml to 100
ng/ml T.sub.3, were made in 0.075M barbital buffer, pH 8.6,
containing 1% normal rabbit serum and 0.1% sodium azide. Hereafter,
this diluent is referred to as barbital buffer.
6. ANS was obtained from K&K Laboratories, Hollywood, Calif.
The steps of the radioimmunoassay procedure follow--In 10.times.75
mm disposble glass culture tubes, the various reagents were added
in the following order: (a) Barbital buffer, volume to adjust to a
final volume of 1 ml; (b) ANS, 250 ug (100 ul of a solution
containing 2.5 mg/ml); (c) 250 ul of hypothyroid sheep serum in the
standards and an equal volume of test serum in all other tubes; (d)
various volumes of four dilutions, e.g., 0.1 ng/ml, 1.0 ng/ml. 10.0
ng/ml and 100 ng/ml, of non-radioactive T.sub.3 to provide 10 pg to
10 ng T.sub.3 in tubes for a standard curve (FIG. 3); (e) 100 ul of
1:400 dilution of T.sub.3 -binding rabbit serum; (f) approximately
7000 cpm of T.sub.3 -.sup.125 I (.about.0.1 to 0.15 ng T.sub.3) in
100 ul of barbital buffer.
After a brief mixing, the tubes were incubated at 4.degree. C. for
24 hours. To precipitate T.sub.3 -.sup.125 I bound to rabbit
anti-T.sub.3, 75 ul of a previously tiltered goat anti-rabbit
.gamma. -globulin was added, and the tubes reincubated at
40.degree. C. for 20-24 hour. The details of subsequent separation
of bound from free radioactivity, correction for nonspecific
binding or trapping of T.sub.3 -.sup.125 I in the precipitate and
plotting of the standard curve have been described previously.
Chopra et al.: Radioimmunoassay for measurement of triiodothyronine
in human serum, J. Clin. Invest. 50:2033-2041, 1971.
The results are set forth below:
FIG. 3 shows a typical standard curve obtained in the presence of
250 ul sheep serum and 250 ug ANS. The threshold was 50 pg in this
assay and varied between 30 and 50 pg in other assays,
corresponding to a T.sub.3 concentration of 12 and 20 ng/100 ml,
respectively.
Adequacy of the amount of ANS employed in RIA--It has been
previously demonstrated that T.sub.3 -binding proteins in serum,
such as TBG, interfere in RIA of T.sub.3 by competing with T.sub.3
-binding antibody for radioactive (and stable) T.sub.3. The
concentration of ANS required to prevent this interference was
determined by adding 25 to 500 ug ANS to tubes containing the
typical reaction mixture but no stable T.sub.3 ; the proportion of
T.sub.3 -.sup.125 I bound to T.sub.3 -binding antibody was compared
to that bound to antibody in the absence of sheep serum and ANS.
Radioactivity bound to antibody in the presence of 200 ug of ANS
was only slightly less (95-98%) than that bound to antibody in
plain buffer, indicating thereby an almost complete neutralization
of the inhibiting effect of sheet serum TBG. An excess of ANS,
i.e., 250 ug. was employed in the final RIA procedure.
Serum T.sub.3 concentration in health and disease--Table IV
presents the data on serum T.sub.3 concentration in 148 subjects of
whom 96 were healthy and euthyroid, 30 hyperthyroid, 12 hypothyroid
and 10 euthyroid with elevated serum TBG either due to estrogen
treatment or to a genetic abnormality.
TABLE IV ______________________________________ Serum T.sub.3
concentration in health and disease. Source of Sera No. Serum
T.sub.3 (ng/100 ml) ______________________________________
Euthyroid 96 112.8 .+-. 3.3 (45-216)* Hyperthyroid 30 490.7 .+-.
42.3 (176-1120) Hypothyroid 12 40.1 .+-. 7.6 (<12-104) Euthyroid
with elevated 10 157.6 .+-. 31.2 (50-353) serum TBG
______________________________________ *Mean .+-. S.E.M.
(range)
In the euthyroid subjects, serum T.sub.3 varied from 45 to 216 ng
per 100 ml with mean.+-.S.E.M. of 112.8.+-.3.29 ng per 100 ml. The
serum T.sub.3 concentration of 30 hyperthyroid patients was
490.7.+-.42.3 ng/100 ml. In 12 hypothyroid subjects, serum T.sub.3
ranged from 12 to 104 ng/100 ml (mean, 40.1.+-.7.65); serum TSH as
measured by an RIA.sup.2 in eight of these patients ranged between
62.5 and 280 uu/ml (normal range 1 to 10 uu/ml). In 10 sera from
euthyroid subjects with high serum TBG, T.sub.3 was 157.6.+-.31.2
ng/100 ml; serum T.sub.4 in these sera, measured by the m ethod of
Murphy et al: (The determination of thyroxine by competititive
protein binding analysis employing an anion exchange resin and
radiothyroxine, J. Lab. Clin. Med. 66:161-167, 1965) was
14.8.+-.1.42 ug per 100 ml, range 10- 22.5 (normal 4-11). The
maximal T.sub.4 -binding capacity of TBG as measured in serum of 7
of these subjects by the method of Inada and Sterling (Inada, M.
and Sterling, K.: Thyroxine transport in thyrotoxicosis and
hypothyroidism, J. Clin. Invest. 46:1422-1450, 1967,) ranged
between 40.7 and 56.3 ug per 100 ml (mean 46.9).
This procedure is sensitive enough to allow reliable measurements
of serum T.sub.3 not only in hyperthyroid patients but also in
euthyroid and hypothyroid subjects. While this may be attributed in
part to the use of a T.sub.3 -binding antiserum which usually
allows detection of 0.03 ng of T.sub.3 in comparison to 0.1 ng
T.sub.3 detected by the antiserum used previously known, the major
improvement in sensitivity is a result of the use of nonthyroid
hormone blocking agent, e.g. ANS. Also, ANS has some advantages
over compounds, such as Dilantin and tetrachlorthyronine including
better solubility at pH of RIA, lower cost and ready
availability.
A mean serum T.sub.3 concentration of 113 ng per 100 ml in
euthyroid subjects obtained by the use of the present method is
comparable to 120, 110 and 105 ng per 100 ml observed by some other
investigators, using another RIA of T.sub.3. However, it is
believed that the difference in the mean normal serum T.sub.3 of
138 ng per 100 ml reported by these investigators, (Mitsuma et
al.,: Radioimmunoassay of triiodothyronine in unextracted human
serum. J. Clin. Endocr. 33:364-367, 1971) and 113 ng per 100 ml
obtained by this procedure is indicative of random sample
variation.
It is concluded that measurement of serum T.sub.3 by RIA affords an
adequate separation of hyperthyroid and hypothyroid patients from
normal subjects.
EXAMPLES 15-26
The procedure of Example 14 would be followed except that the below
listed compounds will be employed instead of 250 ug ANS,
respectively. The amounts used will be proportional to the blocking
power of the compounds as listed in Table II, taking into account
solubility considerations. Thus, 1 ug ANNDS per ul of serum can be
successfully employed. The results would be comparable to those
obtained in Example 14.
______________________________________ EXAMPLE COMPOUND
______________________________________ 15 ANS 16 ANNDS 17 TNBS 18
.alpha.Napthalene Sulfonic acid 19 5,5 diphenyl-2 thiohydantoin 20
Doxepin HCl 21 Diazepam 22 Prochlorperazine 23 Thimerosal 24 Sodium
Salicylate 25 Halofenate* (MK-185) 26 Chlorpromazine HCl
______________________________________ *Proprietary compound
supplied by Merck, Sharp & Dohme.
While various modifications of the invention have been herein
described, various modifications of this invention will become
apparent to those skilled in the art, and the scope of the
invention is to be determined by the claims which follow.
* * * * *