Plasma immunoreactive TSH: Spurious elevation due to antibodies to bovine TSH which cross-react with human TSH

Plasma

Immunoreactive TSH: Spurious Elevation Due to Antibodies to Bovine TSH Which Cross-React With Human TSH Lawrence

A patient with thyroid elevated bTSH

carcinoma

and nonsuppressible

and human

techniques. throughout

TSH

Antibodies

treated were

and subunits

for

revealed

and CTSH-@

unreactive.

displaced

more

than

who received

appeared

the binding

TSH.”

using specific one year

of the antibodies

two populations

which bound only bTSH. Both antibodies hTSH-@

by thyroidectomy

demonstrated

present

this period. Characterization

hormones

Michael A. Baron, and Arthur

levels of plasma “immunoreactive

IhTSH) were

A. Frohman,

antigenic

determinants

by gel filtration

Evidence for suppression by demonstrating

on the beta subunits of plasma

multiple

radioimmunoassay binding

of bovine (LTSH)

exhibited

to TSH of the IgG class which bound and radioimmunoelectrophoretic

of bTSH

was

greater

than

that

oi hTSH

of antibodies,

one of which bound both bTSH and hTSH and the other

to be directed toward antigenic sites on the beta subunit of TSH as both

of intact

TSH from

antibodies

whereas

a peak of immunoreactivity

of TSH secretion by thyroxine

administration

that the level of plasma “immunoreactive

B

OVINE THYROTROPIN (bTSH) is used as a diagnostic agent for the evaluation of autonomously functioning thyroid nodules and an adjunct in the detection or treatment of metastatic functioning thyroid carcinoma. The injection of bTSH in humans would be expected to produce an immune response and evidence for the development of anti-bTSH antibodies has been reported.‘.2 The hTSH RIA is often performed by a double antibody precipitation technique in which the radioiodinated hTSH bound to rabbit anti-hTSH antibody is coprecipitated by an anti-rabbit immunoglobulin serum. With this method the presence of bTSH antibodies cross-reacting with hTSH could compete for the radioiodinated hTSH, decrease the radioactivity in the species specific immunoprecipitate, and thereby lead to an apparent elevation of TSH level. The recognition of such a falsely elevated TSH level is of importance in patients with thyroid carcinoma in whom effective suppression of endogenous TSH secretion constitutes a major component of therapy and has been shown to be of importance in the long-term prognosis.3 With hTSH RIA methods where the unbound radioiodinated hTSH is precipitated, the presence of cross-reacting

the alpha subunits

of the antibody to hTSH occurred

of the two species. Documentation

in which

injections

Antibodies

with respect to the binding of human and bovine glycoprotein

The binding studies suggest that the cross-reactivity

achieved

and

6. Schneider

TSH”

of a true elevation

co-eluting

with

virtually

of plasma hTSH was

(‘Z61]-hTSH could be shown.

in the presence of interfering

did not change in response

were

on the basis of common

antibody was obtained

to TRH administration.

antibodies will lead to a falsely lowered TSH which is also misleading during suppression therapy. The present report describes studies performed in a patient with thyroid carcinoma in whom repeated injections of bTSH resulted in the development of cross-reacting antibodies to hTSH which led to a falsely elevated “plasma TSH” level. Experiments were performed to quantitate, characterize and temporally monitor the antibodies as well as to assess endogenous TSH secretion in the presence of circulating antibodies. MATERIALS The patient, a 27-yr-old,

AND

METHODS

white male received external radiation to

the tonsils and nasopharynx

region as a child.4 A ““Tc

revealed a localized region of decreased radionuclide was not initially detected by palpation. (normal: &6)

and T4 was 7.7 &dl

Plasma TSH (normal:

tomy disclosed a 1.6 cm papillary-follicular

scintigram

uptake which was 3.4 pU/ml

5-l 1). A thyroidec-

carcinoma with a single

metastasis to a regional lymph node. Ten days postoperatively seven daily injections of IO units of bTSH

were given. A “‘I neck and chest

scan revealed multiple cervical lymph node metastases, 100 mCi “‘I was given, and L-thyroxine,

0.2 mg/d,

Two months postoperatively rg/dl.

TSH

Ten months postoperatively,

was begun. was 1.0 @U/ml

triiodothyronine

and T4 9.3

(T3)

was sub-

stituted for thyroxine for three weeks and then discontinued. daily injections of bTSH

Three

were given prior to a repeat scan. On this

occasion the patient experienced swelling, erythema,

and pruritis at

the injection sites. The scan again revealed cervical metastases and

From the Division of Endocrinology and Metabolism, Department of Medicine Michael Reese Medical Center and the University of Chicago, Pritzker School of Medicine, Chicago. III. Receivedfor publication December 3. 1981. Supported in part by USPHS Grants AM-18722 and CA-21 518. and by the Michael Reese Medical Research Institute. ABS is the recipient of a USPHS Research Career Development Award AM 00103. Address reprint requests to Arthur B. Schneider, M.D.. Division of Endocrinology. Michael Reese Hospital & Medical Center, 29th Street & Ellis Avenue, Chicago, Ill. 60616. 0 I982 by Grune & Stratton, Inc. 0026-0495/82/3108~014$01.00/0

834

three additional bTSH

injections and 50 mCi “‘I were administered.

Severe local reactions at the sites of TSH

injections were noted.

Tyroxine, 0.2 mg/d was reinstituted and the following month plasma TSH was 8.6 pU/ml

and T4 was 9.5 pg/dl.

to 0.3 mg per day and three months later plasma TSH was 18.3 rU/ml

Thyroxine

(1S mo

was increased

postoperatively)

with a corresponding T4 of 12.9 pg/dl.

It was at this time that he was referred to us. A TSH stimulation test (500 pg, iv) was performed while receiving thyroxine (TRH

study 1)

and subsequently after thyroid therapy had been discontinued for 17 days (TRH

study 2).

A neck and chest scan at this time

was

considered negative and the patient resumed thyroxine therapy. The

patient has subsequently had no other clinical evidence recurrence and has continued on thyroxine suppression.

of tumor

Metabolism, Vol. 3 1, No. 8 (August), 1982

835

SPURIOUS TSH ELEVATION DUE TO ANTIBODIES

Hormone Preparations

Table 1. Binding of ‘*sl-TSH by Human Serum TSH Antibodies

and Antisera

Using Double Antibody

Purified hTSH, rabbit anti-hTSH serum, and HCG were provided by the NIH, NIAMDD Pituitary Hormone Distribution Program. The hTSH reference preparation used as standard in the RIA was No. 60/38, obtained from the Medical Research Council, Mill Hill, England. This preparation contained an immunopotency of 5 U/mg of the NIH hTSH preparation. bTSH, bTSH-o, bTSH/3, and bLH-/3 were provided by Dr. John Pierce; hLH, bLH, and their a subunits by Dr. Leo Reichert; hTSH-a and hTSH-@ by Dr. Albert Parlow; hCG-a and hCG+3 by Drs. Griff Ross and Robert Canfield; anti-HCG-cu serum by Dr. Griff Ross, and rabbit antihIgG serum by Drs. John and Margaret Goldman.

Precipitation

Reactants 1*51-hTSH

Secondary

Soundt

Incubatwn*

Primary Incubation*

“sl-hTSH + NRS

SaR

4.3

‘%hTSH

SaR

3.7

SaR

2.4

‘*‘I-hTSH + RaTSH

SaR

19.9

‘?-hTSH

SaR

14.4

+ NRS + Control Serum

1%)

flOpI) ‘251-hTSH + NRS + “High TSH” Serum (10 ~1) + RaTSH + “High TSH”

Serum

Radioimmunoassay

Procedures

“sl-hTSH + High TSH” Serum

All hormones and subunits were radioiodinated with Na12’I by the lactoperoxidase method’ and purified successively on Sephadex G-25 and G-100 columns. Double antibody RIAs were performed by standard technique@ as previously described. The hTSH RIA as modified’ was sensitive to OS- 1.O ~U/ml. For those assays in which the patient’s serum was used as the source of the TSH antibodies, all dilutions were made in 2% normal human serum and rabbit antihIgG serum was used for immunoprecipitation with the exception of the experiment in Table 1 where goat anti-hIgG serum was used.

‘251-hTSH + RaTSH + “High TSH”

GaH

29.8

SaR + GaH

37.7

Serum Abbreviations:

NRS: Normal Rabbit Serum (2 ~1) RaTSH: Rabbit anti-human TSH serum. 1: 100,000

dilution

in

NRS

SaR: Sheep anti-rabbit IgG, sufficient to precipitate completely NRS GaH: Goat anti-human IgG, sufficient to precipitate completely Control Serum or “High TSH” Serum “High TSH Serum”: Patient’s serum of g/24/75

which was initially

interpreted as containing 24 @J/ml of TSH.

Immunoelectrophoresis

‘Primary incubation was in a total volume of 200 ~1 for 48 hr at 4 C;

lmmunoelectrophoresis was performed on standard microscope slides using 1.8% agarose and 0.02 M barbital-acetate buffer, pH 7.5. Antibodies to human serum IgG, IgA, and IgM were purchased from Meloy, Springfield, Va.

Sephadex

Chromatography

Plasma samples (1.0 ml) were gel filtered on a 0.9 x 80 cm Sephadex G-100 column using 0.05 M phosphate buffer, pH 7.5, containing 0.2% bovine serum albumin. The column was calibrated with “‘I-human thyroglobulin, “‘1-hTSH, and Na “‘1 in quantities insufficient to interfere with subsequent RIA procedures. Fractions of I .O ml were collected and stored at 4C until assayed for hTSH. RESULTS

Detection, Quantitation, hTSH Antibody

and Characterization

of

Evidence that the elevated TSH values which developed in the patient’s plasma during therapy was artifactual and due to the presence of an antibody to hTSH is shown in Table 1 where the binding of 12’I-hTSH to the patient’s antibodies was evaluated directly. Binding of ‘251-hTSH to rabbit anti-hTSH serum (19.9%) was reduced by 25% (to 14.4%) in the presence of the patient’s plasma. When the patient’s plasma was used alone as the first antibody and precipitated with goat anti-hIgG serum, 29.8% of the ‘251-hTSH was bound. This increased to 37.7% in the presence of both the patient’s plasma and rabbit antihTSH and the corresponding second antibodies. Nonspecific binding of “‘I-hTSH was unaltered by normal plasma and reduced slightly by the patient’s plasma. It should be noted that this experiment was performed using only 10 ~1 of the patient’s plasma rather than the

secondary incubation was for 24 hr at 4 C. tMean of duplicate determinations.

100 ~1 used for the TSH RIA because of the prohibilively larger quantity of the second antiobdy which would be required for complete immunoprecipitation. Radioimmunoelectrophcresis of the patient’s serum and control serum is shown in Fig. 1. The control serum did not bind lz51-hTSH. The patient’s antibody to ‘251-hTSH was present in the IgG fraction but not in the IgM or IgA fractions. Therefore a double antibody system employing anti-hIgG serum was sufficient for further evaluation of the anti-hTSH antibodies. A comparison of the binding of ‘251-hTSH and “‘1-bTSH to the patient’s serum obtained 15 and 17 mo after the initial bTSH injection is shown in Fig. 2. The binding of ‘251-hTSH to the sera never exceeded 60% at the highest concentration tested (1:7.5) and the serum with the higher titer bound 50% of 12’I-hTSH at a final dilution of 1:20. The binding of ‘251-bTSH by the sera was considerably greater, with virtually complete binding present at a dilution of 1:15 and with a 1:700 dilution required for 50% binding. Time Course of the Antibody

Activity

The chronologic sequence of bTSH injections, “apparent” plasma TSH values, and binding of “‘1-hTSH and ‘*‘I-bTSH to serum are shown in Fig. 3. The binding studies were performed at a final serum dilution of 1:125. At this dilution, no binding was noted five weeks following the first bTSH injection. Peak binding occurred 16 mo after the initial and 6 mo after the second series of bTSH injections, following

838

FROHMAN,

NI

Pt

NI

Pt

w

NI

Pt

NI

BARON. AND SCHNEIDER

Pt

w

Fig. 1. Radioimmunoelectrophoretic characterization of hTSH antibodies in the patient’s serum. “61-hTSH was preincubated with patient’s serum (Ptl or normal control serum (NI) for 24 hr following which the mixtures were placed in the right and left wells, respectively, of each of four immunoelectrophoresis slides. After completion of electrophoresis, antisera to whole human serum (HS), immunoglobulin G (IgG), immunoglobulin A (IgAl and immunoglobulin M (IgM) were placed in the individual troughs from left to right, repectively. The precipitate lines formed at 24 hr are shown in the lower panel. The entire slab was then covered with x-ray film and the image formed is shown in the upper panel. The location of the radioactivity (“‘I-hTSH) seen in the upper panels corresponds to the position of IgG and is only precipitated by antiserum to whole serum and to IgG.

which a decrease in the binding capacity occurred for both bTSH and hTSH. Binding of ‘*‘I-hTSH could no longer be detected at this dilution 21 mo postoperatively whereas binding of 12’I-bTSH was still present. The “apparent” hTSH values rose progressively over a 15 mo period despite the suppression doses of L-

thyroxine and peaked at approximately the same time as did the anti-TSH titer. A subsequent rise in immunoreactive TSH values occurred at 21 mo in response to short term discontinuation of L-thyroxine and was unassociated with an increase in bTSH or hTSH binding capacity.

SPURIOUS TSH ELEVATION DUE TO ANTIBODIES

100-r

Fig. 2. Dilution curve of the patient’s anti-TSH serum using ‘2r’l-hTSH and ‘261-bTSH.Incubations were in a total volume of 150 &I for 48 hr at 4 C following which goat anti-hlgG (100 al) was added and the incubation continued to another 24 hr. All dilutions beyond 1:5D were made in 2% normal human serum. Serum from 15 mo (circles) and 17 mo (triangles) postoperatively were used.

Immunologic

o-

Characterization of the TSH Antibody

placement of ‘251-hTSH and 12’I-bTSH by homologous and heterologous glycoprotein hormones and subunits is shown in Table 2. Greater displacement of ‘251hTSH occurred with hTSH-/3 and bTSH-/3 than with the a-subunit or other glycoprotein hormones from either species. Significant displacement of ‘2SI-bTSH occurred only with bTSH-0. In addition, ‘251-hTSH-cx did not bind (~1.2%) to the patient’s serum whereas slight binding (5.5%) of ‘*‘I-hTSH-P was observed.

The relative binding affinities of the anti-TSH serum PCWhTSH and bTSH were assessed using ‘*‘IhTSH and “‘1-bTSH. Displacement of ‘*‘I-hTSH (Fig. 4, left panel) occurred at lower concentrations of bTSH than of hTSH (50% displacement: 3.8 ng/ml versus 8.5 ng/ml). bTSH was also strikingly more potent in displacing ‘*‘I-bTSH from the antibody than was hTSH (Fig. 4, right panel). In addition, hTSH was capable of displacing only 30% of the antibody-bound ‘*‘I-bTSH, even at the highest concentration tested (lpg/ml, or 2,000 x the concentration of bTSH required for similar displacement). The relative dis-

I

Evaluation of Endogenous TSH Secretion in the Presence of TSH Antibodies Two methods of assessing the presence and secretory pattern of endogenous TSH were used. In the first, the

L-THYROXINE

4

10

1;

POST-OPERATtVE

16

li MONTH

22

25

Fig. 3. Time course of the development and disappearance of antibodies to TSH. The two series of bTSH injections are indicated by the arrows, binding of ‘?-bTSH and “?-hTSH by closed and open circles. respectively, and the hTSH immunoreactivity in serum by triangles. Binding studies with ‘?-bTSH and ‘*?hTSH were performed at a final serum dilution of 1:125.

FROHMAN,

838

, -‘n 0

8

AND

SCHNEIDER

100

pt. SERUM 1~125 9*/T= 125X-hTSH, .22 3900

cpmltube 80

60

60

*

125X-bTSH, 3300 cpm/tube Pt. SERUM

l:1000

Be/T=.26

< 0

BARON,

40

40

bTSH 20

\

0 _I

I

IO

100

NG/ML

.I

I

IO

1000

NCVhfL

Fig. 4. Comparison of the binding affinity of the patient’s serum for bTSH and hTSH. The left panel compares the displacement of ‘2s1-hTSHby bTSH and hTSH while the right panel shows the same comparison using ‘261-bTSH.B/B, represents the ‘zsI-TSH cpm bound to antibody in the presence of unlabeled hormone divided by the ‘261-TSHcpm bound to antibody in the absence of unlabeled hormone.

pattern

of “TSH immunoreactivity” on Sephadex gel filtration was examined while the patient was receiving 0.3 mg/day of thyroxine, and during a period when thyroxine had been discontinued for three weeks. As shown in Fig. 5, the “TSH immunoreactivity” while receiving thyroxine (upper panel) was restricted to the void volume, the location of IgG, and was attributed to Table 2. Relative Potencies of Human and Bovine Glycoprotein Hormones and Subunits in Displacing hTSH and bTSH from Patient’s Serum Radiolabelled

De.placing Hormone

HOmlOlX

or SubunIt

Relative Displacement* 1 .o

hTSH

‘=I-hTSH

10.001

h-glycoproteln cu-subunit hCG,

10.00

hCG-/3

hTSH-8

0.02

bTSH

1.6

b-glycoprotein

0.0

1

DISCUSSION 1

a-subunit

‘251-bTSH

bLH

0.005

bTSH-fl

0.03

bTSH

1.0

h-glycoprotein a-subunit


1


1

hLH hTSH-0 b-glycoproteln

0.005

a-subunit

*Calculated the hormone

bTSH-B

0.21

bLH

0.003

by a comparison homologous

of the 50%

to the radiolabelled

displacement TSH.

the presence of TSH antibodies. After thyroxine withdrawal (middle panel) a second peak of immunoreactivity was also present at the position of ‘251-hTSH and represented endogenous plasma TSH. The elution pattern of immunoreactivity in a subject with primary hypothyroidism is shown for comparison in the lower panel. The question of whether TSH secretion was effectively suppressed was evaluated by TRH administration. The results (Fig. 6) show that during the first TRH test (performed while the patient was receiving thyroxine) no change occurred in the “immunoreactive TSH” level whereas during the second test (after discontinuation of thyroxine) “immunoreactive TSH” levels rose abruptly after the injection of TRH.

dose to that for

The rarity of “unsuppressible TSH” and the history of allergic reactions to the second series of bTSH injections prompted the search for antibodies to bTSH which might cross-react with hTSH. Since bTSH is biologically active in humans, it is not surprising that there is a considerable degree of structural similarity between bTSH and hTSH.8,9 Accordingly, cross-reactivity of anit-bTSH antibodies with hTSH would be expected and has been reported.“,” Displacement studies with bTSH and hTSH showed that two populations of anti-TSH antibodies were present: one, which reacted only with bTSH and the other, of lower titer, which reacted with hTSH almost

SPURIOUS TSH ELEVATION DUE TO ANTIBODIES

‘=I-hTG

839

‘PSI-hTSH

as well as with bTSH. The apparent plasma TSH values tended to parallel the levels of anti-hTSH antibodies and became undetectable when the titer of anti-hTSH antibodies had dropped to barely detectable levels, despite the persistence of anti-bTSH antibodies. We characterized the antiserum with respect to its cross-reactivity with other glycoprotein hormones and subunits in order to determine the basis for the cross-reactivity with hTSH. The antibodies which bound hTSH did not react with the isolated human a-subunit alone or as present in hCG. However, evidence was present for binding the TSH-P: unlabelled hTSH-@ displaced [“‘I]-hTSH from the antibody and [“‘I]-hTSH-0 bound to the antibody. These findings suggest that the antibodies are directed primarily toward antigenic sites on the P-subunit of bTSH and that the 90% identity in the sequence of the human and bovine &subunits4 form the basis for the cross-reactivity. This conclusion is consistent with the observations that; (1) the majority of glycoprotein hormone antibodies tend to be directed toward the hormone specific subunit, and (2) antibodies directed against the (Ysubunit do not show interspecies cross-reactivity despite structural homology nearly comparable to that of the @-subunit.‘2s’3 The presence of antibodies which bind bTSH has been reported in the serum of patients with thyroid cancer and has been attributed to prior injections of bTSH for diagnostic and therapeutic purposes.2l’4 In one instance there was an apparent cross-reactivity with hTSH.” We have recently studied a second patient with cross-reacting antibodies given bTSH 7 and 19 yr previously who had constant antibody titers. The presence of anti-TSH antibodies in the serum of

Pt. SERUM “OFF” THYROXINE

A 20

30

40 ML

HYPOTHYROID

50

SERUM

60

70

EFFLUENT

Fig. 5. Sephadex gel filtration patterns of the patient’s serum obtained in the presence and absence of thyroxine treatment. All samples were filtered through e 1 x 80 cm Sephadex G-100 column equilibrated with 0.05 M phosphosaline buffer, pH 7.4, containing 0.2% bovine serum albumin at a flow rate of 3 ml/hr. Top panel: Serum (1 ml) obtained while the patient was receiving thyroxine 0.3 mg/ day. Middle panel: Serum (1 ml) obtained after thyroxine had been discontinued for three weeks. Lower panel: Serum (1 ml) from a patient with primary hypothyroidism.

STUDY

No. 2 “OFF”

STUDY

No.1 “ON”

THYROXINE

8 Fig. 6. TSH secretory responses to TRH BOCIpg. iv) in the presence of circulating TSH antibodies. TRH studies were performed during thyroxine suppression treatment (Study No. 1I and three weeks after thyroxine had been discontinued (Study No. 2). The ordinate represents the calculated “immunoreactive TSH” values which do not reflect true TSH levels because of the presence of TSH antibodies.

3 2 2

80

:‘

20

% 1 ti

THYROXINE

IO

-10

0

IS

50

45

60

so

MINUTES

I20

150

180

FROHMAN,

840

four pituitary dwarfs has been reported.16 Interestingly, the serum of all four bound [“‘I]-hLH to a limited extent, as did the present patient’s serum (data not shown), suggesting the possibility of immunization with a heterologous and impure pituitary hormone preparation. A large molecular weight form of TSH was serendipitously found in normal volunteer by Spitz et al.” The serum was shown to have binding activity for TSH,‘* possibly due to antibodies (H. Hirsch, personal communication), although there was no history of previous TSH administration. Antibodies to TSH may have been present in two patients suspected of having immunologically reactive but biologically inactive TSH.‘9,20 One of these patients received a series of bTSH injections eight months prior to testing.20 In TSH screening programs for congenital hypothyroidism the potential effects of anti-TSH antibodies are an important consideration. Czernichow et al.” 14 and 7 motherand Gendrel et al. ** described newborn pairs, respectively, with spuriously elevated TSH due to transplacental passage of maternal immunoglobulins. Grendrel et al.” and Schaison et al.23 showed that immunoglobulins arose in the mothers as a result of rabbit tissue containing immunizations, and that the interference occurred with the precipitation step of the hTSH RIA. The problems with the TSH RIA documented here, and the possiblility of serious anaphylactic reactions support our opinion that bovine TSH injections are not clinically useful. When presented with a patient with cross-reactive antibodies, we have shown that a true elevation of plasma TSH can be demonstrated by Sephadex filtration. The effectiveness of thyroxine therapy in suppressing TSH can be assessed by TRH testing. The use of these procedures can solve the problems of managing patients with circulating antiTSH antibodies. ACKNOWLEDGMENT We are indebted to Drs. Albert Parlow. John Pierce, Leo Reichert, Jr., and Griff Ross for their generous gifts of glycoprotein hormones, subunits, and antisera. We acknowledge the excellent technical assistance of Willie Kiang and Kazimierz Kowalski.

REFERENCES 1. Hays MT, Solomon DH, Beall GN: Suppression of human thyroid function by antibodies to bovine thyrotropin. J Clin Endocrino) Metab 27: 154&l 549, I967 2. Greenspan FS, Lew W, Okerlund MD, et al: Falsely positive bovine TSH radioimmunoassay responses in sera from patients with thyroid cancer. J Clin Endocrinol Metab 38:1121-l 122, 1974 3. Mazzaferri EL, Young RL, Oertel JE, et al: Papillary thyroid carcinoma: the impact of therapy in 576 patients. Medicine 56:1711961977

BARON, AND SCHNEIDER

4. Favus MJ, Schneider AS, Stachura ME, et al: Thyroid cancer occurring as a late consequence of head and neck irradiation. N Engl J Med 294:1019-1025, 1976 5. Thorrell JI, Johansson BG: Enzymatic icdination of polypeptides with I*? to high specific activity. Biochem Biophys Acta 251:363-369, 1971 6. Frohman LA, Horton ES. Lebovitz HE: Growth hormone releasing action of a pseudomonas endotoxin (Piromen). Metabolism l6:57-67, 1967 7. Schneider AB, Favus MJ, Stachura ME, et al: Plasma thyroglobulin in detecting thyroid carcinoma after childhood head and neck irradiation. Ann Intern Med 86:29-34. 1977 8. Pierce JG, Faith MR, Guidice LC, et al: Structure and structure-function relationships in glycoprotein hormones, in Polypeptide Hormones: Molecular and Cellular Aspects. Ciba Foundation Symposium 4 1, Elsevier, Amsterdam, 1976, pp 225-250 9. Sairam MR. Li CH: Human pituitary thyrotropin: The primary structure of the a and fi subunits. Canad J Biochem 55:755760, 1977 IO. Werner SC, Seegal BC, Osserman EF: Immunologic and biologic characterization of antisera to beef thyrotropin preparations: J Clin Invest 40:92-104, 1961 1 I, Levy RP, McGuire WL, Heideman L: Quantitative studies of the reaction of bovine thyrotropin antiserum with human and bovine thyrotropin preparations: Proc Sot Exp Biol Med 110:598-600. I962 12. Vaitukaitis JL, Ross GT. Reichert LE. et al: Immunologic bases for within and between cross-reactivity of luteinizing hormone. Endocrinology91:1337-1342. 1972 13. Binoux M, Pierce JG, Odell WD: Radioimmunological characterization of human thyrotropin and its subunits: applications for the measurement of human TSH: J Clin Endocrinol Metab 38:674682, 1974 14. Greenspan FC, Lowenstein JM. West MN, Okerlund MD: lmmunoreactive material to bovine TSH in plasma from patients with thyroid cancer: J Clin Endocrinol Metab 35:79S-798, 1972 15. Sain A. Sham R, Singh A, et al: Erroneous thyroid-stimulating hormone radioimmunoassay results due to interfering antibovine thyroid-stimulating hormone antibodies. Amer J Clin Pathol 7 I :540--542, I979 16. Chaussain JL, Binet E, Job JC: Antibodies to human thyrotropin in the serum of certain hypopituitary dwarfs. Rev Europ Etudes Clin et Biol 17:95--99, 1972 17. Spitz IM, LeRoith D, Hirsch H, et al: Increased highmolecular-weight thyrotropin with impaired biologic activity in a euthyroid man. New Engl J Med 304:2788282, 198 I 18. Hirsch H. Spitz IM, LeRoith D: Characterization of large molecular weight human TSH. The Endocrine Society 63rd Annual Meeting, Abstract 421, I98 I 19. Belchetz PE: Idiopathic hypopituitarism with biologically inactive TSH: Proc Royal Sot Med 69:428-429.1976 20. Kreiger DT: Glandular end organ deficiency associated with secretion of biologically inactive pituitary peptides. J Clin Endocrinol Metab 38:964-975. 1974 21. Czernichow P, Vandalem JL, Hennen G: Transient Neonatal Hyperthyrotropinemia: A factitious syndrome due to the presence of heterophilic antibodies in the plasma of infants and their mothers. J Clin Endocrinol Metab 53:387-393, 1981 22. Gendrel D, Feinstein M-C. Grenier J, et al: Falsely elevated serum thyrotropin (TSH) in newborn infants: Transfer from mothers to infants of a factor interfering in the TSH radioimmunoassay. J Clin Endocrinol Metab 52:62-65, I98 I 23. Schaison G, Thomopoulos P, Moulias R, et al: False hyperthyrotropinemia induced by heterophilic antobodies against rabbit serum. J Clin Endocrinol Metab 53:20&202, 1981