43

CZinica Chimica Acta, 70 (1976) 43-60 0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

CCA 7670

A SIMPLE AND RAPID THYROXINE RADIOIMMUNOASSAY (T,-RIA) IN UNEXTRACTED HUMAN SERUM; A COMPARISON OF T,-RIA AND T4 DISPLACEMENT ASSAY, T,(D) *, IN NORMAL AND PATHOLOGIC SERA

B.N. PREMACHANDRA Immuno-endocrinology

and 1.1. IBRAHIM ** Research Department,

Veterans Administration

Hospital, Jefferson

Barracks, and Washington Uniuersity, St. Louis, Missouri (U.S.A.) (Received July 18,1975)

Summary A simple, rapid and accurate thyroxine radioimmunoassay (T,-RIA) in unextracted serum or plasma has been described, and for comparison T4 determinations have also been made by a T,(D) procedure using Abbott Tetrasorb kits. T,-RIA procedure basically involved denaturation of serum to dissociate T4protein bond, and T4 released was allowed to react with [‘251]T4-labeled T4 antiserum elicited by immunizing rabbits against bovine thyroglobulin. The displaced unbound [ ‘*‘I] T4 was rapidly taken up by an anionic resin sponge within 15 min and this sponge [ ‘*‘I] T4 uptake was linearly related to T4 present in standards or serum. The denaturation of serum effected by trichloroacetic acidsodium hydroxide permitted virtually 100% T4 extraction recovery in normal, pregnancy, hypo- and hyperthyroid sera whereas 72.9~87.6% T4 recovery from normal serum (and with large individual differences) was noted with lower alcohols in T,(D) procedure. Cumbersome and/or tedious steps such as pre-extraction, centrifugation, time consuming bound and unbound hormone separation procedures, etc. are obviated in T,-RIA and the entire assay can be completed in the same tube in approximately an hour. These attributes along with increased sensitivity and specificity and the need for only microamounts of test sera (25-50 ~1) in T,-RIA offer distinct advantages over T,(D) procedures, and in simplicity excel even other T4-RIAs. T,-RIA values in physiological and pathological states were highly correlated (r = 0.97) with T,(D) measurements and no differences between these two techniques were found. The reported discrepancies between T,-RIA and T,(D) measurements in human sera and some * Nomenclature suggested by the American Thyroid Association [ll. Both Tq-RIA techniques and competitive protein binding or thyroxine (displacement), Tq(D), assays requiring no antibody are based on the same principle involving isotopic displacement. In this communication, however, merely for convenience, Tq(D) techniques requiring no antibody are referred to as Tq(D) assays, whereas those Tq(D) procedures requiring antibody are referred to as T4-RIA. ** Present address: The Radiobiology Division, Atomic Energy Establishment, Cairo. Egypt.

of the reasons for attributing these inconsistencies errors and variations are discussed.

to probable

methodological

Introduction The virtues of a radioimmuno~say (RIA) over other methods for measurement of blood hormones are well recognized, and the various aspects of RIA techniques have been adequately dealt with in numerous recent publications [ 2-6 3. In the case of thyroxine, an immunoassay utilizing T1, binding thyroglobulin antibodies was reported briefly by Ibrahim and Premachandra [7] ; the procedural details were basically akin to those described by Murphy [8] for T4(D) assay, the main difference being that actively induced thyroglobulin antibodies, instead of thyroid hormone binding globulin (TBG), served as the site for isotopic displacement. An essentially similar T4 immunoassay was described in detail by Chopra et al. [9] and in their technique free and bound hormone was separated by using a second antibody instead of resin. However, these T4 immunoassays attracted no unusual interest partly because Tq can be satisfactorily measured by the existing T,(D) techniques requiring no antibody, and partly because T,-RIA methods did not offer decidedly superior advantages over the widely used T,(D) procedure. The recent development of RIA techniques for measuring T4 without preextraction of serum [lO,ll] offers certain advantages over T,(D) procedures and has rekindled interest in T4 immunoassay [12-201. These advantages, however, are offset by the need to use often tedious steps for separation of free and bound hormone, and hence T,-RIA techniques have not been readily susceptible for routine use in clinical laboratories. An immunoassay for thyroxine has, therefore, been developed which obviates all cumbersome steps (centrifugation, pre-ext~ction, time consuming separation procedures, etc.) and permits completion of the entire assay in the same tube in about an hour. Materials and methods T4 antibodies were generated in rabbits immunized against bovine thyroglobulin (1%) as previously described [Zl] .T4 ~ti~rurn was mixed with [lz51 J T4 (Abbott Laboratories) at a concentration of 5 ~g/lOO ml and left in the refrigerator overnight for complete equilibration [22]. A 75fold dilution of the antiserum (l/157 final dilution in the assay) was found appropriate for the assay: To 10 ml labeled * antiserum, 6 ml propylene glycol and 2 ml phenol (1%) were added to assure stability and sterility and was followed by the addition of 732 ml of phosphate buffer (0.2 M). T4 labeling concentration amounted to 0.667 ng/ml or 0,335 ngjtube. Labeled T4 antiserum proved stable until 2 weeks (period tested) as noted by the superimposibility of standard curves. Thyronine free plasma or serum was prepared as described previously [ 221. For preparing T4 standards, T4 free acid (Sigma) was dried overnight at 4O”C, ap* “Labeled” serum or antiserum merely indicates that they have been premixed with tracer [ 12 5 I] Tq.

45

propriate amounts in 0.25% albumin solution were then added to thyronine free plasma to provide final T4 concentrations of 5, 10, 15 and 20 pg/lOO ml. Trichloroacetic acid/sodium hydroxide (TCA-NaOH, sodium trichloroacetate, pH 12) was prepared by adding 3 parts of sodium hydroxide (0.5 N) to 1 part trichloroacetic acid (20%). T,-R IA

Principle of the technique consists in denaturing T4 protein binding sites by TCA-NaOH to dissociate tightly bound T+ T4 liberated from the native protein binding sites reacts with [ ‘*‘I] T,-labeled T4 antiserum and displaces [‘*‘I] T4 which is taken up by the resin sponge. This isotopic displacement (sponge [ 12’1]T4 uptake) is linearly related to T4 present in serum or standards. Procedure (a) 50 ~1 serum or T4 standards are pipetted into polypropylene tubes (15 mm X 86 mm). (b) 0.2 ml TCA-NaOH solution is added in each tube accompa-

nied by 2 min mixing in an automatic shaker. (c) 0.5 ml [ 1251]T4 -labeled antiT4 serum is then added followed by a further 2 min mixing, and the haptenantibody mixture is allowed to equilibrate at 37°C for 1 h (pH of the final reaction mixture = 7.2). After equilibration the tubes are allowed to stand at room temperature for approximately 15 min. (d) An anionic resin sponge * (Abbott Laboratories) is introduced into each tube and squeezed with a plastic plunger (Abbott Laboratories). The radioactivity in six tubes is counted to represent initial ac tiuity. (e) Exactly at 15 min, sponge incubation is terminated by aspirating excess fluid using a hollow plastic aspirator (Abbott) connected to a vacuum source through a flexible tubing. The sponges are then washed 3 times by filling the tube with distilled water, squeezing the sponge and aspirating the fluid each time. The washing procedure removes only bound [‘*‘I] T,+ The radioactivity in the sponge is subsequently determined (final activity). The sponge uptake (96) of radioactivity (final activity/initial activity) X 100 is linearly related to thyroxine present in standard or test serum up to a concentration of 20 pg T,/lOO ml. T4 extraction recovery studies were based on the rationale that once T,-protein bond is effectively destroyed by TCA-NaOH, unbound T4 would be readily abstracted by the resin sponge. Serum was labeled with [‘*‘I] T4 and the experimental procedure was simulated as closely as possible to that of the assay described before except for the addition of the antibody. In control experiments labeled test serum was replaced by [‘*‘I] T4-labeled gamma globulin or [‘*‘I] TJabeled buffer. In other investigations, to determine whether or not T4 recovery would be affected by protein concentration, extraction efficiency of T4 was carried out in solutions containing various levels of gamma globulin (412 g/100 ml). The procedure consisted in pipetting of 50 ~1 [‘*‘I] TJabeled serum, 50 ~1 [ 1251]T4-labeled gamma globulin (496, 8% or 12% solution) or 50 /..d * The resin sponge (supplied by Abbott Laboratories) comprises a polyurethane foam of intercommunicating cell type containing approximately 15-70 parts by weight of a strong base quaternary ammonium anionexchange resin (in the form of chloride salt) per 100 parts by weight of polyurethane matrix. The anion-exchange capacity is about 4 milli-equivalents/g of dry resin. All sponges contain exactly the same amount of finely divided resin. The precision of the assay attests to the claims of the manufacturer.

46

[‘*‘I J T,-labeled buffer in tubes followed by the addition of 0.2 ml TCA-NaOH mixture. After 2 min mixing, 0.5 ml phosphate buffer (fortified with 8% immune globulin * in some instances) was added, mixed and [“‘I]T4 uptake determined at various intervals (5 min-1 h) to study T4-extraction recovery. In the [lz51] T4 extraction recovery procedure just described sponge uptake was determined by dividing final activity in the relatively dry sponge by initial activity in the wet sponge. Since a wet medium does absorb low energy radiation it is natural to expect attenuation effects with the low energy “‘1 gamma emitter. To examine absolute serum [ ‘*‘1]T4 extraction recovery the following procedure was used: In sets of 20 tubes, 50 ~1 f1251]T4 labeled serum, labeled gamma globulin or labeled buffer was pipetted followed by addition of 0.3 ml TCA-NaOH. After mixing, 0.5 ml buffer was added. Sponges were inserted in each tube and 5 min later they were transferred to new sets of tubes to rid of residual radioactivity on the wall of the original tubes used for inserting sponges. Ten tubes (con~ning sponges) from each group were dried overnight at 110°C (approx. 18 h) until all liquid in the sponge was evaporated as shown by the lack of increase in radioactivity counts with further drying. This radioactivity in dry sponges represented initial radioactivity without radiation attenuation due to the wet medium, and was the same in all 10 tubes in each series. Since identical volume of labeled solution was taken in all 20 tubes in each series the initial radioactivity as represented by 10 tubes in each series also represented initial radioactivity in the remaining 10 tubes not used for initial activity determination. Sponges in these latter tubes (not used in initial activity determination) after lo-min incubation at room temperature were washed as described before and dried at 110°C for 1 h before radioactivity determination. Absolute ‘*‘IIT uptake (76) = (washed dry sponge radioactivity/dry sponge inisponge 1 tial radioactivity) X 100. For an evaluation and comparison of thyroxine immunoassay, T4 determinations were also made by a commercially available technique (Abbott Tetrasorb) based on the principle of competitive protein binding analysis. The method used to study T4 extraction recovery in this T,(D) procedure has been noted in the Abbott bulletin and also described by us previously [22]. Results T4 standard curve

When percentage sponge [ 1251]T4 uptake was plotted against T4 standards, a linear dose response curve was obtained (Fig. 1). The narrow range in standard deviation throughout the dose response curve as well as the highly significant F value obtained for linearity (F < 0.005) is a reflection of the precision of the assay. Also, standard curves constructed in thyronine free serum or plasma were superimposable. Methodologic A.

investigations

Equilibration * Gamulin

of labeled

(Dow Chemical Co.).

of various factors on standard curve T4 antiserum

with

T4 in the reaction

mixture

47

Fig. 1. Standard T4 dose-response curve. Dots and bars represent the mean. and range of values, respectively. Each dot represents the mean of 20 determinations over a 2 week period using different batches of labeled antiserum on different occasions. Broken line represents regression through the means. Linearity of dose response relationship was shown to be highly significant (F < 0.005).

The effects of progressively increasing periods of equilibration at room temperature (26°C) of antigen * - antibody reaction on T,-RIA are shown in Fig. 2. Increasing equilibration until 4 h correspondingly increased sponge

Fig. 2. Effect of varying periods of equilibration at room temperature of T4 antiirum with T4 standards in tbe reaction mixture on sponge [ r *5X1T4 uptake. Approximately 95% equilibration was attained at all T4 concentrations in 1 h. * It should perhaps be pointed out that T4 is not an antigen in the strict sense. It can induce antibodies only when it is covalently linked to a protein and thus T4 is really a hapteo.

48 [ ‘2sI] T4 uptake at all T4 standard concentrations, the maximum effect noted at 20 pg Tq. After 4 h, sponge [ 1251]T4 uptake corresponding to various T4 concentrations was the same as that noted at 4 h indicating attainment of complete antigen-antibody equilibration. In reference to the values noted at 4 h, 97%, 95%, 94% and 95% equilibration, respectively, was achieved at 5,10,15 and 20 pg T4 concentrations 1 h after antigen-~tibody incubation; similar results were also apparent in normal, hypo- and hyperthyroid sera. In contrast to the room temperature observations, virtually complete equilibration at all T4 concentrations was noted at 15 min at 45” C, and at 30 min at 37°C thus indicating that higher temperatures facilitate hastening of antigenantibody equilibration. It is also of significance that after various intervals of exposure of T4 antiserum at 37-45”C, the blank values were the same indicating that prolonged equilibration even at relatively high temperatures do not compromise the integrity of labeled immune serum as shown by the absence of spontaneous release of [ ‘2SI]T4 from [ 12’I]T4-labeled antiserum. B. Variation in interval of resin sponge incubation in the assay medium In order to determine the optimal time interval essential for separating bound and unbound [ 1251]T.+, resin sponges were introduced into completely equilibrated T4 antiserum-T, system in standards at various intervals starting from 5 min to 2 h and [‘2SI]T4 uptake was determined. Sponge [iz51]T4 uptake increased sharply within about 15 min after sponge incubation and was proportional to various T4 standards; the uptake slowed down considerably from 30 min onwards (Fig. 3). A comparison of the difference in sponge

Fig. 3. Effect of varying intervals of resin sponge incubation in the assay medium (containing T4 standaxds) on [*2s11 T4 uptake. Sponge abstraction of unbound [ I 2 5 II T4 was completed within 15 min (arrow) as can be noted in the initial steep part of the curve. Increase of Cl 25Kl T4 uptake beyond 15 min w&4 largely representative of resin sponge induced dissociation of [ 1 2 5 II T4 the magnitude of which was approximately the same at zero and at all T4 concentrations.

49

[ ‘251]T4 uptake corresponding to O-20 /_.igT4 between 0 and 15 and between 15 and 120 min dr~ati~ally illustra~s the fact that while there was 203% increase in the first 15 min (100 X (51.5 - 17.0)/17 = 203%), no such increases were noted between 15 and 120 min. It is also interesting and significant to note that between 15 and 120 min, the magnitude of change occurring in blank, i.e. zero Tq standard (approx. 18 units) was approximately the same as that occurring at all T, concentrations strongly suggesting that after 15-min incubation the slow gradual release of f ‘*‘I] T4 from immune serum was strictly induced by the sponge and was not due to T4 present in the system. Whatever unbound [’ 251] T4 that was present in the medium as a result of antigen-antibody equilibration was rapidly taken up by the sponge in the first 15 mm as shown by the steep part of the curve. C. Duration of mixing of reagents on T,-RIA No differences in T4 values were noted when the duration of the mixing of the reagents in the automatic shaker was varied from 1 to 30 min. D. Effect of elapsed period after TCA-N~OH addition on T,-RIA In order to determine if there are any residual effects of TCA-NaOH on T4RIA, T4 standards in replicates were extracted with TCA-NaOH for 2 min and then left at room temperature for 0, 30, 60 and 120 min. At the end of these time intervals labeled T, antiserum was added and the assay was completed. The values for zero and T, standards at all these intervals were the same thus showing that in human serum TCA-NaOH releases T, from T4 binding proteins virtually instant~eously. E. Effect of moderate excess of TCA-NaOH on T,-RIA A two-fold excess of TCA-NaOH extractant had no deleterious labeled antibody or the assay.

effect on the

Serum-T, extraction efficiency of TCA-NaOH versus 95% ethanol When resin sponges were left in the extraction medium for 15 min, about 94% of [ ‘251] T4 radioactivity was taken up by the sponge (Table I). A slight increase, up to 98%, was apparent when sponges were left for 1 h in the extracting medium. These results show that it takes about 15 min for the sponges to get properly equilibrated in the surrounding medium and to abstract bulk of the available unbound [ 12’1] Tq. The results also indicate that if [ 12sI] T4 is present in a clear medium (buffer), sponge abstraction of radioactivity would be instant~eous as seen in the 5-min values (Table I). On the other hand, if the medium contains protein and other substances, then there is some delay in complete abstraction of radioactivity. This is readily apparent as shown in the medium containing non-T, binding protein such as gamma globulin. Therefore, the observation of 87% sponge [ 1251]T4 uptake 5 min after incubation does not suggest that only 87% of bound [ 12sI] T4 has been released from denatured T4 binding sites. If there is 94% abstraction of available radioactivity (15-min value) by a secondary T, binding site such as the resin, it would, therefore, be reasonable to assume that in the presence of T4 antibody, virtually all unbound

50

T4 present in the system (after treatment of serum by TCA-NaOH) would react with it almost instantaneously. [ “‘1 J T4 uptake values at 15 min after sponge incubation were the same in buffer as well as in various sera indicating the excellent ability of TCA-NaOH to completely remove T4 from T4 binding proteins (this also shows that TBG, TBPA and albumin do not interfere in the assay). When the buffer used in extraction efficiency procedure was fortified with 8% immunoglobulin (to simulate assay conditions as closely as possible) similar results were obtained (Table I). In other investigations, virtually 100% [12sI]T4 extraction recovery was noted when 4%, 8% and 12% human gamma globulin labeled with [’ “11 T4 was treated with TCA-NaOH, thereby demonstrating that T4 extraction efficiency is not affected by plasma protein concentration. In the absence of TCA-NaOH, i.e. no protein denaturation, sponge [125I]T4 uptake was only l/3 of that noted when serum was extracted with TCA-NaOH. Absolute [‘251]T4 recovery from [ ‘251]T4labeled serum, labeled gamma globulin and labeled buffer was 91.3 (91-96), 92.7 (92-96), and 99.3% (96-lOl), respectively (Table II). With serum and gamma globulin the maximum noted was 96%. The failure to note 100% recovery of [ 12sI]T4 from [ 12’I]T4 labeled serum does not indicate the attainment of maximum [ 12sI]T4 exchange capacity of the resin sponge, rather the failure can partly be attributed to the mechanical aspects of the sponge washing procedure, i.e. with repeated washing one can demonstrate a very small but progressive loss of abstracted radioactivity from TABLE I COMPARISON OF TCA-NaOH AND 95% ETHANOL SERA

ON T4 EXTRACTION

EFFICIENCY

IN HUMAN

Lower half of the table represents intervariation in (TCA-NaOH) T4 extraction efficiency. T4 extraction efficiency based on recovery of [i2511T4 from [ I2 6I]T4- labeled senrm. TCA-NaOH extraction efficiency of T4 has been noted at various intervals in serum and in controls for valid comparisons and conclusions (see text). Figwes represent means k SD. Number of samples in parentheses. Extraction of [ 12611~~ from [ 12sIlT4-labeled sera and controls

-.--l---Normal (12) NormaI (6) ** Hypothyroid (12) Hypothyroid (6) * * Hyperthyroid (12) Hyperthyroid (6) ** PxegnancY (12) Pregnancy (6) * *

T4 Extraction efficiency

---

-..___-

TCA-NaOH * 5 min __._____87.2 + 1.4 88.1 * 1.0 87.6 * 2.1 87.4 + 1.2 88.3 ? 3.6 88.7 * 3.6 35.5 f 2.3 87.0 f 1.4

Ethanol 10 min

15min

91.0 92.3 91.0 91.7 92.6 93.0 90.9 92.2

94.0 95.0 94.5 93.4 94.4 95.2 92.6 93.3

* 1.6 * 0.9 + 1.6 f 1.0 f 2.2 f 2.8 f 1.8 f 1.2

f 1.0 * 1.5 f 1.0 It 1.8 * 1.7 + 2.3 f 1.8 + 1.1

30 min ___-_ 96.9 +- 1.3 96.3 t 1.7 96.7 t 1.2 97.4 * 1.1 96.9 f 1.1 96.8 f 0.6 95.8 f 1.5 96.1 f 0.8

60 min 98.1 98.8 98.7 98.8 98.3 98.4 98.0 97.8

t 0.8 + 0.8 f 1.1 * 1.0 f 1.1 f 1.7 f 0.8 f 1.3

78.5 + 3.6 81.8 t 4.0 79.0 + 3.4 80.1 f 4.5

87.3 f 1.9 91.7 t 1.9 93.4 c 1.5 96.4 f 1.1 98.0 f 1.1 Pooled human serum (12***) Controls 88.2 * 2.3 91.0 * 1.3 93.9 f 1.2 96.0 t 1.1 98.3 * 0.7 Gamma gfobrdin (12* * * ) 94.9 f 1.7 95.4 t 2.2 96.3 f 2.2 96.1 f 1.9 98.7 f 2.2 Buffer (12***f -* Sponge [ 12 5 I] T4 uptake (%) at various intervals after sponge incubation in the extracting medium. ** The values obtained in these samples denoted (TCA-NaOH)T4 extraction efficiency Using immUuOgIobUlin-enriched buffer instead of plain buffer (see text). *** Number of estimations in the same sample carried out at different times.

51

TABLE

II

FROM [‘*51]T4 ABSOLUTE RECOVERY OF [ ‘*51]T4 AFTER TCA-NaOH EXTRACTION (REPRESENTATIVE All counting

rates represent

Labeled substance

Final [‘251]T4 activity in sponge (cpm)

ORCONTROLS

Absolute recovery (%) * *

Immediately after washing

Washed * and dried

11060 100938

11640 104899

10548 96574

10632 97209

91.3 92.7

(91-96) (92-96)

7390

7653

7588

7640

99.8

(96-101)

drying

* Sponges used for washing were not the same as those and initial activity determination (see text). recovery

In parentheses:

SERUM

After drying

Before

* * Absolute

HUMAN

the mean of 10 replicates.

Initial [I * 5 I] T4 activity in the sponge containing labeled substance and extraction solvent (cpm)

Serum Gamma globulin Buffer

LABELED

ILLUSTRATION)

used for drymg;

Final [I * 51]T4 activity in washed dry sponge (%) = _ _~.~_ Initial activity in dry sponge

replicates

were used for drying

x 1oo

range noted in various experiments.

the sponge. This loss amounted to 2-3s in 3-5 washes. If one accounts for this loss and individual recoveries are corrected, the range in absolute recovery (%) in serum, gamma globulin and buffer would amount to 94-99, 95-99, and 99-104, respectively. [ l*‘I] T4 recoveries from 95% ethanol treated [‘*‘I] T4-labeled normal, pregnancy, hypo- and hyperthyroid sera were 78.5, 80.1, 81.8 and 79.0%, respectively (Table I). The inter-variation in extraction efficiency was the highest with pregnancy sera (SD. 4.5), and even greater variability has been noted in most recent investigations in certain thyroiditis sera. Sensitivity,

precision,

validity and accuracy of T4-RIA

Sensitivity For determination of sensitivity, standard deviation of 20 replicate measurements of sponge [‘*‘I] T, uptake corresponding to zero T4 concentration was computed. T4 concentration corresponding to sponge [‘*‘I] T4 uptake 2 standard deviations from zero was taken to represent the lower limit of the sensitivity of the assay and this amounted to 0.6 pg/lOO ml which was in close agreement with the value of 0.8 r.lg/lOO ml obtained experimentally (by testing 0.2 pg T4 increments). The sensitivity obtained in T,-RIA was higher than that noted in T,(D) procedure (approx 1.5 pg/lOO ml). Precision The index of precision =

daverage

of weighted

(h) variances of y corresponding to 0 and variousT4 Regression coefficient (slope)

was 0.13 for T,-RIA, a value believed dividual lambda values of y = d-)/b

standards

to reflect a high order of precision. Incorresponding to 0, 5,10,15 and

52

20 pg T4 standards were 0.11,0.14,0.13,0.11 and 0.15 respectively. The interassay precision of determinations in T, standards and in sera was also examined by computing coefficient of variance (C.V.) from 20 replicate determinations in successive assays using different batches of reagents including labeled T4 antiserum. Coefficients of variance (%) corresponding to 0, 5, 10, 15 and 20 ,ug T4 standards were 4.36, 3.91,2,63,1.75 and 2.08, respectively; whereas in normal, hyperthyroid and hypothyroid sera coefficients of variance were 5.90,5.07 and 16.13, respectively. Standard deviation of replicate T4 values in hypo- and hyperthyroid sera was approximately the same; hence division of SD. by a smaller mean T, value (as in hypothyroid sera) resulted in higher C.V. than in hyperthyroid sera. The coefficients of variance noted in normal, pregnancy, hypo- and hyperthyroid sera during intra-assay comparisons were 3.8, 3.0, 7.5 and 1.9%, respectively. Vulid~ty Specificity Effect of in vitro addition of T4 analogues and various other substances on T4-MA. Among T4 analogues of interest, T3 in high concentrations affected the test both in the presence as well as absence of thyroxine * but, T3 levels as encountered in normal or hyperthyroid subjects had no influence on T,-RIA (Table III). The acetic and propionic acid thyroid derivatives in high concentrations showed extensive cross reaction because of structural similarity to thyroxine, and DIT and thyroglobulin in large doses increased T, levels slightly, but the observations are only of academic interest as these substances in significant amounts are not known to be present in plasma. D-Thyroxine which does not occur naturally was as effective as L-thyroxine in the assay, showing the lack of formation of stereospecific antibodies to T4 hapten. Synthetic thyroprotein (iodinated casein) in rather large amounts affected T,-RIA thereby showing the crossreactivity of T4 antiserum to covalently linked Tq. No interference was noted when iodine rich radiographic substances or various sensitized and nonsensitized gamma globulin preparations were used in the assay. A variety of substances known to affect T4-plasma protein interaction were without effect on T,-RIA. These included salicylate (Arthropan), barbiturates, penicillin, goitrogenic, hypolipemic and anticonvulsant drugs. Dilantin, even in a concentration of 1 mg/ml did not affect T,-RIA whereas half this concentration (500 pgglml) has been shown to affect T,(D) [23] . T,-RIA was not interfered by the presence of various preservatives and antico~lants, steroids and various sulfa and antidiabetic drugs. Phenylbutazone in concentrations exceeding pharmacological doses had a slight effect on T,-RIA and increased plasma T4 concentration by about 7% (beyond the range of 95% probability of control T4 value) but was virtually without effect when added to thyronine free plasma.

* The lack of interference in the assay of the large number of diverse substances tested in thyronine free plasma is particularly reassuring because, with low affiity antibodies, any nonspecific effects due to low molecular weight and other substances would be more intntsive in the absence or presence of hormone in low concentration.

53

TABLE

III

SPECIFICITY

OF THYROXINE

RADIOIMMUNOASSAY

Substance

Amount

Effect

of addition

of test substanc

added In thyronine-free human plasma. sponge [ *2511T4 uptake (%)

wg/w

(Continued

on P. 54)

T4 Wg/lOO

ml)

16.4 f 0.95 t

7.3 f 0.47 t

x 103 x103 X lo3

11.4 17.6 16.9

7.4 7.1 1.1

x x x x x X x

17.4 16.9 29.2 40.6 18.4 18.0 32.5

8.2 9.2 26.9 38.3 10.0 9.3 16.9

21.8 17.8

13.0 8.7

x 10’ x 101 x 101

14.9 14.8 14.9

6.7 6.7 6.6

8 5 5 5

X102 x10-2* x 10-Z * x 10-2 *

16.8 16.4 15.2 17.1

7.1 7.4 7.3 7.8

5 5

x102 x 101

16.3 15.1

7.2 1.0

5 5

x101 x102

15.0 14.6

6.5 7.0

5 x 103 1.5 x 102

15.0 16.0

7.4 1.4

5 5

x102 x102

15.7 15.4

6.9 7.1

x101 x 10-l x 103 x102

14.7 16.5 16.1 15.7

7.1 6.8 7.4 7.5

6 2

X lo3 x102

15.3 14.6

7.6 7.0

5

x 101

15.8

7.1

1 5

x 103 x 101

15.5 14.8

7.4 7.3

None

Iodinated compounds Potassium iodide Iodoacetamide Hypaque sodium Thyroxine analogues 3-Iodo-D,L-tbyronine 3.5Diiodo-L-tyrosine 3.3’.5-Triiodothyropropionic acid 3,3:.5,5’-Tetraiodothyroacetic acid 3.3’,5-Triiodo-L-thyronine 3,3’,5-Triiodo-L-thyronine D-Thyroxine Thyroid and other iodoproteins Thyroprotein (iodinated casein) Bovine thyroglobulin Goitrogenic drugs Tapazole Thiourea Prowl-thiouracil Non-sensitized and sensitized gamma globulin Immune serum globulin (human) Rabbit gamma globulin (Antibodies Inc.) Goat anti-rabbit gamma globulin serum tt Goat anti-human thyrogIobuIin serum tt Anticoagulants Ammonium oxalate Heparin sodium Anti-diabetic drugs Phenformin . HCl Orinase Narcotic drugs Methadone . HCI Morphine Sulfa drugs Sodium sulphathiazole Sulphacetamide Anticonvulsants Diazepam (Valium) Diazepam Dilantin Dilantin Steroids Solu-Cortef (hydrocortisone) Decadron phosphate (dexamethasone) Hypolipemic drugs Clofibric acid Preservatives Sodium azide Thimerosal

In intact human plasm;

2 2 25 5 5 5 4 5 2.5 1

10-l 10’ 10-l 10-l 10-Z 1O-2 10-l

2.5 X lOI 1 x 10’ 10 10 10

2 50 1 5

54 TABLE

HI (Continued)

Substance

AmOUnt added

Effect

@g/ml)

of addition

of test substance

In thyronine-free

In intact human plasma T4

human plasma, sponge [I * 5 II Tq uptake (%)

@g/l00

ml) -

Miscellaneous Arthropan (choline salicylate) Sodium chloride Penicillin G Bacto complement (Difco) Adrenaline chloride Digoxin Phenyl butazone Phenobarbital --.* ** t tS

5 5 12.5 5 5 6.5 10 5

x102 x 103 x 103 ** x 10-l * x 101 X 10’ x 10’ x102

17.5 14.9 14.9 14.3 15.0 14.6 18.0 14.6

7.7 6.9 6.8 6.8 6.8 6.9 8.8 6.6

--~

ml/ml. units/ml. Mean +_S.D. Hyland Labs.

Effect of varyingptasma protein concentration on T,-MA. T4 standards were prepared in 4%, 8% and 12% lyophilized thyronine free plasma and standard curves at each protein concentration were constructed. The 3 standard curves TABLE

IV

TqRIA:

T4 RECOVERY

Sera

* AND EFFECT

OF DILUTION -____

Endogenous serum

T4 added exogenousiy

T4 w!z/100

flig/lOO ml)

ml)

A

** .-_-___ Serum thyroxine after exogenous T4 addition Wg/lOO ml)

B

c

5 10 5 10 5 10 5 10

12.7 17.8 8.1 13.2 19.2 24.2 16.4 22.2

Normal

7.7

Hypothyroid

3.5

Hyperthyroid

14.4

Pregnancy

11.8

Dilution factor

Serum 1, dilution in --

Recovery (%) C-A B -

x 100 _---

100 101 92 91 96 98 92 104

Serum 2, dilution in -.-

T4-free plasma

Buffer

T4-free plasma

(l/2) (l/4) (l/8)

12.4 12.0 12.8

12.0 12.0 9.6

31.4 30.0 32.0

31.6 29.4 31.2

Undiluted

12.3

12.0

30.8

30.8

1 1 1

:1 :3 :7

* Absolute rawvery (see text). ** All T4 values shown in the lower half of the table are corrected

Buffer

for appropriate

dilution.

55

were superimposable indicating that protein concentration in the ranges tested does not affect T,-RIA. Effect of dilution of serum on T,-RIA. T,-RIA determinations in 2 serum samples progressively subjected to &fold dilution in thyronine-free plasma or buffer are recorded in Table IV. Assay values were essentially the same in diluted and undiluted samples. In still other experiments, T4 values noted were similar regardless of whether 25 or 50 ~1 serum was used for the assay. The correspondence of values in diluted and undiluted sera is a reflection of the specificity of the technique and rules out nonspecific effects of buffer salts or other interfering substances. T4 levels in patients under T3 therapy. In 6 obese patients under T3 therapy T4 levels noted were in the hypothyroid range (3.7 - 0.8 E.cgTJlOO ml) as expected, indicating inhibition of TSH. By the same token these results also show that therapeutic doses of triiodothyronine do not affect T,-RIA. Accuracy The accuracy of the technique was examined by determining recovery of added T4 in the assay. Two levels of T4, viz. 5 and 10 pg/lOO ml were added to normal, pregnancy, hypo- and hyperthyroid sera. The added thyroxine was allowed to equilibrate with the serum overnight prior to the assay. At both levels

m

.! P

,

Fig. 4. Comparison of T4-RIA and T4(D) values in euthyroid, Horizontal bars represent means. No significant differences T4(D) were noted in any sera.

pregnancy, hype- and hyperthyroid sera. in mean T4 values between T4-RIA and

56

of added Te, there was virtually complete recovery in all sera (despite TBG variation), the range noted being 92-104% (Table IV). T4(D) and T4-RIA in various physiological and pathological states Serum thyroxine values @g/100 ml) as noted in the two techniques are shown in Fig. 4. The mean +- S.D. and the range in T,-RIA values in normal, pregnancy, hypo- and hyperthyroid sera were, respectively, 8.0 + 1.6 (5.512.4), 13.2 + 2.2 (7.4-17.7), 2.5 + 1.3 (O-5.5) and 18.7 4 4.8 (12.7-28.4, excluding T4 values in T3 toxicosis); the corresponding T,(D) values were 9.5 of: 1.7 (6.1-13.7), 14.8 +_ 2.3 (lO-18.9), 3.5 +_ 1.4 (0.5-7.1) and 19.1 + 5.5 (11.8-31.7, excluding T, levels in T3 toxicosis) in normal, pregnancy, hypoand hyperthyroid sera, respectively. The range in T, values in clinically euthyroid subjects as computed from the mean + 2 S.D. (4.8-11.2 pg T4 or 3.1-7.3 yg T.+I in T,-RIA and 6.1-12.9 pg T4 or 4.0-8.4 pgT,I in T,(D) assay) did not completely cover the span of T4 values in normal subjects in either T,-RIA or T,(D) as also noted by others [ 241. Excellent overall correlation between T4(D) and T,-RIA was noted \r = 0.97). Discussion A review of the results makes it clear that the RIA technique described for T4 determination is an accurate, sensitive, reliable and valid procedure, and thus satisfies the major criteria of an assay. Furthermore, cumbersome and/or time consuming steps such as pre-extraction, evaporation, centrifugation, column preparation, transfer of assay reactants for free and bound hormone separation, etc., are completely obviated. The assay can be completed in the same tube at room temperature in approximately 85 min, or even less if T4 - T4 antiserum equilibration is carried out at 37--45°C. These attributes of the new T,-RIA procedure, along with the increased sensiti~ty and specificity as well as the need for only microamounts of test sera, offer distinct advantages over T,(D) procedures, and in simplicity excel even other T4-RIAs. Various solvents have been used in T,-RIAs which do not require pre-extraction of serum. ANS (8-~ilino-l-naphthalene sulfonic acid), one of the more commonly used solvents to prevent T4 binding by thyroxine transport protein, reacts with T4 antiserum and hence the usable level has to be tested with each T4 antiserum. A more important objection to the use of ANS has been raised by Hesch et al. [25] who reported that with varying TBG concentration and T4, ANS has correspondingly differing effects on antiserum and this complicating effect has been suggested to explain the discrepancy between T,(D) and T,-RIA in some hyperthyroid sera (vide infra). Tetrachlorothyronine, another agent used to prevent binding of T4, varyingly crossreacts with T4 antiserum [ 111. Salicylate has also been used to displace T4 from TBG but its efficiency in effecting complete removal of Tq from TBG remains uncertain [26]. The various solvents used as noted above prevent binding as well as displace T4 from TBG by competitive binding reactions and there is some evidence to indicate that displacement reactions are never complete. In contrast, the virtual complete removal of T4 from serum effected by TCA-NaOH and its lack of effect (even in moderate excess) on T4 antiserum is reassuring.

Some investigators [10,12], but not others [15,17,25], have reported that in comparison to T,(D) values, T,-RIA is approximately 10% higher in euthyroid subjects and is very much higher (as much as 50%) in some thyrotoxic individuals. A consideration of the following aspects may aid in an evaluation of T,-RIA - T,(D) discrepancy. (a) In the reports where discrepant values between T,-RIA and T,(D) have been noted, T,-extraction recovery from serum by the T,(D) method is not always provided. In other investigations, a constant factor has been used to account for incomplete T,-extraction from serum by the T,(D) procedure. As T,-extraction from serum is variable with the usual solvents employed in T,(D) assay (Table I) it seems important to correct T,(D) values individually for T4-extraction recovery in critical comparisons with T4RIA. (b) For determining T4-extraction recovery in the T,(D) procedure, [ ‘251]T4 is almost exclusively used at the present time. With this low energy gamma emitter, minor but significant attenuation errors are introduced because in T4-extraction recovery estimations, the initial radioactivity is measured in serum whereas the final extracted radioactivity from serum is determined in ethanol or other lower alcohols. With identical [‘251] T4 concentration in ethanol, and in serum, counts per min in the former is 2-3s higher than in the latter. (c) Recent investigations show that with lower alcohols (T,(D) procedure) T4 binding substances in serum are coextracted into the aqueous phase which would overestimate T4-extraction recovery and hence underestimate T,(D) measurement [27,28]. Irvine [29] reported that T,(D) values may be underestimated by as much as 12%. (d) Minor variations in T4 standards, superimposed on other possible errors reviewed above, may become manifestly apparent during comparison of T,-RIA from one laboratory with T,(D) assay from another as interlaboratory comparison of T4 standards is seldom carried out. When viewed against these considerations, it seems unlikely that the reported small differences in T4 values (approx. 10%) between T,(D) and T,-RIA in euthyroid subjects are significant. On the other hand, the rather large discrepancy in T4 values between T,(D) and T,-RIA (T,-RIA approx. 50% higher) in some thyrotoxic individuals, as noted by Chopra [lo] and Beckers et al. [12], has been attributed to the pathological release of iodoproteins (containing some covalently linked T4) which may crossreact with T4 antiserum [lo] causing an elevation in T,-RIA whereas T,(D) assay would not be affected since covalently held T4 is not readily extracted with alcohols. The validity of this suggestion was also borne out in the present investigations where crossreaction between T4 antiserum and synthetic iodoprotein was demonstrated (Table III). Nevertheless, several investigators [ 11,15,16,17,19,20,25,30,31] have been unable to confirm significant discrepancies between T,(D) and T,-RIA values in hyperthyroid sera. The low specificity of T4 antiserum induced by thyroglobulin immunization [17], and poor recovery of T4 in T,(D) procedures particularly in samples with high T4 concentration [ 16,20,30], have also been suggested to reconcile T,-RIA - T,(D) discrepancy. To determine whether or not the different procedures used in separating free and bound hormone may contribute to T,(D) - T,-RIA discrepancy, serums from 23 hyperthyroid patients (including 1 case of T3 toxicosis) were sent to 3 laboratories for T,-RIA estimation and in each of the four RIA procedures

58 TABLE V SERUM THYROXINE CONCENTRATION &g/100 ml) IN HYPERTHYROID SURED BY Tq(D) AND T4-RIA IN VARIOUS LABORATORIES --_-_ .--__---.-TqRlA T4 CD) No.

Authors’s Lab.

Mayo b clinic Lab.

20.0 18.9 33.1 15.8 16.3 31.4 14.3 21.4 13.6 23.7 20.3 17.3 16.9 19.6 14.2 12.8 22.9 16.1 19.7 31.7 31.5 9.6 30.5

20.1 19.7 23.9 18.3 20.1 26.5

Abbott = WeIIesleyd Lab. HOSP. Lab. (Toronto)

1 2 3 4 5 6 7 a Q 10 11 12 13 14 15 16 17 18 19 20 21 22 23

-_____

Mean + SD. 20.5 i_ 6.7

14.3 25.0 14.6 19.3 18.1 19.7 32.0 18.0 13.0 13.2 20.0 13.4 19.0

Patterson f Coleman Lab.

A~tbors’~ Lab.

SUBJECTS AS MEA-

Pantex g Lab.

Nichol’s h Institute

__-.-

25.2 18.6 20.8 16.6 18.8 26.6 12.7 22.6 13.8 20.0 16.6 17.1 19.8 19.6 13.9 16.8 24.0 14.8 20.0 28.2 28.4 7.8 27.4

18.8 16.3 16.3 11.8 14.6 23.9 9.3 17.8 10.0 14.0 14.1 18.7 25.0 24.5 15.0 16.1 25.5 19.0 20.0 17.8 16.2 7.8 27.0

24.8 15.0 19.4 14.7 12.9 23.3 13.5 19.0 12.1 22.0 15.6 16.3 18.4 22.6 13.2 12.9 22.6 13.4 20.6 21.0 22.3 7.8 25.5

21.6 16.6 25.8 13.9 16.6 22.8 11.9 18.6 13.0 19.8 16.3 17.0 20.0 19.5 12.8 14.4 29.1 14.3 20.1 22.5 21.3 8.0 21.9

19.5 f 5.3

17.4 f 5.1

17.8 + 4.7

17.8 * 4.2

a T4 values individually corrected for T4-extraction recovery. b T4 values corrected using mean T4-extraction recovery value of 78%. C T4 values corrected using mean Tq-extraction recovery value of 79.6%. d T4 values uncorrected for T4-extraction recovery. e T4 extraction recovery facilitated by TCA-NaOH denaturation of protein and separation of bound and unbound labeled hormone using resin sponge. f T4 extraction recovery facilitated by heat denaturation of protein and separation of bound and unbound labeled hormone using charcoal. g T4 extraction recovery facilitated by blocking TBG binding of T4 with ANS and separation of bound and unbound labeled hormone using second antibody. h T4 extraction recovery facititated by blocking TBG binding of T4 by ANS and separation of bound and unbound Iabeled hormone using pofyethylene gIyco1.

(including our laboratory) a different technique was involved for separating bound and unbound hormone. Mean T4-RIA and T,(D) values (Table V) were similar and no significant differences either between T4-RIAs, or between T,(D) and any one of the T4-RIAs were noted. However, when T4 values in ~~d~u~d~a~ samples were critically examined considerable variation between techniques (in both T,(D) and T4-RIA) was noted. In the same individual samples, a maximum of 75% difference in T4 values was noted between T4-RIAs; between T,(D) procedures 89% difference was noted. By our assays, in a selected subgroup of hyperthyroid sera mean T,-RIA values exceeded T,(D) determinations by 23%,

59

findings consistent with that of Chopra [lo] and Beckers et al. [12]. On the other hand, in another subgroup of hyperthyroid sera mean T,(D) value was 28.8% higher than that noted in T,-RIA, and an increase in T,(D) over T,-RIA in subgroups has also been noted in another recent study [ 201. The failure to observe consistent discrepancies between T,(D) and any of the four T,-RIAs also suggests (i) that the reported T,-RIA - T,(D) discordance cannot be readily attributed to differences in specificity of antiserum unlike in the RIA of other hormones [ 321, (ii) that procedures used in separating bound and unbound hormone are unlikely to introduce a systematic artifact in T4RIA. In summary, the inconsistencies between T,(D) and T,-RIA in individual specimens appear likely due to methodological vagaries inherent in each of these techniques, a conclusion which is gaining increasing support [20,30]. In view of this, and in light of previously presented considerations, the finding of higher T,-RIA values over T,(D) in a subgroup of hyperthyroid subjects (and the converse) does not seem convincing. Acknowledgement The authors are indebted to various personnel at Abbott Laboratories who extended cooperation in this study, particularly to Drs. I.D. Smith and J.P. Miller for providing materials and equipment. It is also a pleasure to thank Drs. L. Cummins and L. Lin for useful comments in the course of this study. The authors are also indebted to numerous physicians and other scientific colleagues for providing serum specimens and supporting clinical diagnosis, and assistance particularly of Drs. W.L. Florsheim, H.W. Wahner, G.A. Hagen, V.V. Row, R. Volpe, I.B. Perlstein and R. Burstein, is gratefully acknowledged. It is also a pleasure to thank Dr. S. Lang for his valuable criticism in this study. Authors are also grateful to Drs. N.Y. Chiamori (Pantex Lab.) and A.L. Nichols (Nichols Inst.) as well as Mr. R.T. Dunn (Patterson Coleman Lab.) for performing T4-RIAs, and to Dr. N.S. Jiang and Mr. T. Hockert of the Mayo Clinic in carrying out T,(D) assays for comparative purposes.

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60 15 Larsen. P.R.. Dockalova, J.. Sipula, D. et al. (1973) J. Clin. Endocrinol. Metab. 37.117-182 16 Kubasik, N.P., Sine, H.E. and Murray, M.H. (1973) Clin. Chem. 19.1307-1308 17 Meinhold, H. and Wenzel. K.W. (1974) Horm. Metab. Res. 6,169-170 18 Werner, S.C.. Acebedo. G. and Radichevich, I. (1974) J. Clin. Endocrinol. Metab. 38. 493-495 19 Ratcliffe. W.A., Ratcliffe, J.G.. McBride, A.D. et al. (1974) Clin. Endocrinol. 3,481488 20 Hermann, J.. Rusche. H.J. and Kruskemper. H.L. (1974) Clin. Chim. Acta 54.69-79 21 Premachandra. B.N., Ray. A.K., Hirata. Y. et al. (1963) Endocrinology 73, 135-144 22 Premachandra. B.N. and Ibrahim. 1.1. (1975) Thyroid Hormone Metabolism (Harland, W.A. and Orr. J.S. eds.). PP. 281, Academic Press, New York 23 Siersbaek-Nielsen. K. (1967) Acta Med. Stand. 181.327-333 24 Brookeman. V.A. (1973) J. Nucl. Med. 14.660-668 25 Hesch, R.D.. Hufner, M., Muhlen. A. et al. (1974) Radioimmunoassay and Related Procedures in Medicine, Vol. II, pp. 161-176, Int. Atomic Energy Agency, Vienna 26 Larsen, P.R. (1971) Metabolism 20, 976-980 27 Goldie. D.J., Jennings, R.D. and McGowan, G.K. (1974) J. Clin. Pathol. 27, 74-82 28 Crombag. F.J.L.. D’Haene, E.G.M. and Tertoolen, J.F.W. (1973) Clin. Chim. Acta 46,345-349 29 Irvine, C.H.G. (1974) J. Clin. Endocrinol. Metab. 38.468475 30 Hehrmann, R. and Schneider, C. (1974) Proc. Eur. Thyroid Assoc. Meeting, PP. 221 Abstr. 31 Marsden, P., Facer. P., Acosta. M. et al. (1975) J. Clin. Pathol. 28, 608-612 32 Herington, A.C., Jacobs. L.S. and Daughaday, W.H. (1974) J. Clin. Endocrinol. Metab. 39. 257-262