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.

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.

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.

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.