Neutralizing and Non-neutralizing Antibodies

to Bovine Thyroid-Stimulating Hormone and its Subunits

GIDONN. BEALL, INDER J. CHOPRA,DAVID H. SOLOMONJOHNG. PIERCE, and JAMESS. CORNELL

From the Department of Medicine, Harbor General Hospital, Torrance,California 90509 and the Departments of Medicine and Biochemistry,UCLASchool of Medicine, Los Angeles, California 90024

A B S T R A C T To test the possibility that the long-acting thyroid stimulator (LATS) might representan immune complex either of thyroid-stimulating hor-mone (TSH) with anti-TSH or of a subunit of TSHwith an appropriate antibody, we immunized rabbitswith bovine TSH (bTSH), bLH (luteinizing hor-mone), and their a and 8 subunits (bTSHa andbTSHP). Binding, neutralizing, and nonneutralizingantibodies were demonstrated in the antisera obtained.First, antisera to TSH, TSHP, and TSHa all bound["1I]TSH and ["'I]TSHa. Anti-bTSH,8 antiserabound [PI]bTSHP better than did anti-TSH sera,while the binding of [zI]bTSH was similar with bothtypes of antiserum. Second, the thyroid-stimulatingactivity (McKenzie bioassay) of TSH could be neu-tralized by incubation with various dilutions of anti-TSH or anti-TSHfi. Finally, when incubation mixturescontaining TSH and dilutions of anti-TSH,8 antiserathat only partially neutralized TSH were treated withan antiserum against rabbit immunoglobulins to pre-cipitate immune complexes, the bioassay response ofthe TSH was abolished. This phenomenon was notobserved when antiserum to the intact hormone wassubstituted in the incubation mixture. The removal ofTSH biological activity from a mixture of TSH andanti-bTSHP by addition of an anti-immunoglobulinindicated that biologically active immune complexeswere formed between TSH and anti-TSH,3 but notbetween TSH and anti-TSH. The time-course of thebioactivity and several other characteristics of thesecomplexes differentiate them from LATS.

Dr. Chopra is the recipient of U. S. Public HealthService NIH Research Career Development Award 1 KO4 AM-70225.

Received for publication 13 July 1973 and in revisedform 10 August 1973.

INTRODUCTIONBovine thyroid-stimulating hormone (bTSH) 1 has re-cently been shown to consist of two different subunitsdesignated TSHa and TSH#. The subunits are sepa-rated by gel filtration in ammonium bicarbonate solu-tion after treatment with 1.0 M propionic acid (1).Neither subunit is, by itself, a thyroid stimulator.Similar subunits have also been isolated from lutein-izing hormone (LH) (2, 3). The a-chains of LHand TSH have identical or nearly identical amino acidsequences and they will substitute for one another inreconstituting biologically active LH or TSH with theal)propriate f-chain (1, 4, 5).

Antisera to human or bovine TSH incubated in vitrowith appropriate amounts of either bTSH or hTSHcan produce a thyroid-stimulating material that insome studies has been found to be more active at 8 hthan at 2 h in the McKenzie bioassay, thus resemblingthe long-acting thyroid stimulator (LATS) ratherthan TSH (6, 7). We thought it important to investi-gate further the possibility that LATS might representan immune complex of either TSH-anti-TSH or a sub-unit of TSH with an appropriate antibody. Conse-quently, we prepared rabbit antisera to purified bTSHand its subunits (8). In preliminary experiments, wefound that sera from rabbits immunized with bTSH,8contaminated with bTSH or with purified bTSH alonecaused thyroid stimulation in the mouse. This bio-logical activity of the undiluted serum disappeared afterseveral weeks storage at -20°C. We then studied the

'Abbreviations used in this paper: a, alpha subunit; fi,beta subunit (after TSH or LH); b, bovine; BSA, bovineserum albumin; GARGG,goat anti-rabbit gamma globulin;h, human; LATS, long-acting thyroid stimulator; LH,luteinizing hormone; PBS, phosphate buffered saline; TSH,thyroid-stimulating hormone.

The Journal of Clinical Investigation Volume 52 December 1973* 2979-2985 2979

ability of these antisera to combine with and neutralizebTSH. Similar studies were also done with antisera tohighly purified preparations of bTSH subunits.

METHODSAnimals. Healthy New Zealand white rabbits were im-

munized. Equal numbers of both sexes were used. Theirweights at the beginning of the study ranged from 1.4 to3.0 kg.

Hormone preparations. The preparations of bTSH usedto make subunits had a potency of 30-40 U/mg. Two dif-ferent groups of animals were immunized. The subunits ofbTSH used for the first group (experiment I) containedsignificant thyroid-stimulating activity in both the Mc-Kenzie assay and by measurement of the uptake of 32Pinto chick thyroids. (The activity of the bTSHp subunitwas 6%o of the original bTSH activity in the McKenzieassay, and the bTSHa was found to contain 10-15% ofthe original TSH activity in the chick assay.) Contamina-tion of the subunit preparations with bTSH is the mostprobable cause of the residual activity. This results fromincomplete separation of the subunits with subsequent re-combination to form intact bTSH (5). The subunits ofLH were prepared by countercurrent distribution (1).Their potency was approximately 0.04 X N.I.H.-LH-S1standard for the a-subunit and 0.08 for the ,8-subunit.

In experiment II, highly purified subunits prepared bya second gel filtration were used for. immunization. ThesebTSH,8 and bTSHa subunits contained no detectable thy-roid-stimulating activity when assayed at a concentrationof 500 ng/mouse. They could be recombined to producebTSH with approximately 25% of the original activity(5).

Immunization. In experiment I, 23 rabbits were used.Six were immunized with bTSH,8, three with bTSHa,three with bLHa, five with bTSH (Thytropar, ArmourPharmaceutical Co., Chicago, Ill.), and six with bLH,8. Allof the rabbits were injected with antigen on days 1, 10,20, 32, and 42. The protein was dissolved in 1.0 ml ofphosphate-buffered saline (PBS) (0.14 M NaCl, 0.01 Msodium phosphate buffer, pH 7.3) or, in the case of bTSH#subunits, 0.012 M glycine-NaOH, pH 9.5. The solutionswere then homogenized in an equal volume of completeFreund's adjuvant, and a total of 2 ml of homogenate wasinjected into two subcutaneous sites on the back. The firstinjection of the subunits was 200 ,ug. Subsequently, 100-iLginjections were alternated with 200 jug. The first injectionofbTSH was 330 /Ag. Subsequently, 165-/Ag injections werealternated with 330 Aig. After the fifth immunization, theexperiment was continued only with the animals immunizedwith bTSHI3 and bTSH, the former receiving 200 ,ugand the latter 6 mg/injection on days 56, 66, 76, and 97.The animals were bled on days 0, 32, 52, 66, 76, 83, 90,104, 111, 118, and 175. The binding of [HI]bTSH and[1"I]bTSHfi was assessed with the day 52 serum.

As will be described in the following paper, some ofthese sera contained thyroid-stimulating activity. Thatactivity disappeared after storage at -200 C. Such serafrom bleedings on days 32, 52, and 66 were used for theneutralization studies.

For experiment II, 21 rabbits were immunized, 6 withbTSHa, 6 with bTSHB, 4 with bTSH (the purified prep-aration used to make the subunits), and 5 with bovineserum albumin (BSA). Immunizations were performedon days 1, 10, 20, 30, 40, 50, and 65 with the same technique

described for experiment I. The animals received either100 /Ag of the subunits, 200 jug of bTSH, or 500 ,ug BSAat each immunization. Bleedings were obtained on days0, 28, 40, 47, 61, and 77. The studies on neutralization andbinding of bTSH were all performed with serum obtainedon day 77.

Thyroid-stimulating activity. The McKenzie bioassaywas performed as previously described (9). Groups of sixappropriately prepared mice were used for each bioassay.Each mouse received 0.5 ml of test material intravenously,and bleedings were obtained at 0, 2, and 8 h. Thyroid-stimulating activity of TSH is presented as the responseindex at 2 h (the percent of the zero hour value). Statis-tical comparisons were made using the logarithm of theresponses. Group means were compared by Student's ttest.

Binding of ["2I1]bTSH or [`4I]bTSHt3 by rabbit antisera.Appropriate dilutions of rabbit sera were incubated 24 hwith trace quantities of the polypeptides labeled with "Iby the method of Greenwood, Hunter, and Glover (10).Specific activities always exceeded 200 gGCi/tg. The labeledpolypeptide was obtained as a single peak from SephadexG-75 gel filtration. To separate globulin-bound from freehormone or subunit, goat anti-rabbit gammaglobulin (GAR-GG) was added in antibody excess (11, 12). Control tubeswithout rabbit antisera were routinely included. Five con-centrations of each antiserum were assayed. The resultantbinding (percent of maximum) was plotted against amountof antiserum, and the amount needed for 50% binding wasread from the curve.

These studies were performed with serum from the 52nd-day bleeding of six rabbits immunized with bTSH,3, fourwith bTSHa, four with bTSH, and one with LHfi inexperiment I, and the 77th-day bleeding of six rabbitsimmunized with bTSHp, six with bTSHa, four withbTSH, and five with BSA in experiment II.

Effects of antiscra onl the thyroid-stimulating activity ofbTSH. Antisera were combined with bTSH in a rigidlyprescribed manner so that final antiserum concentrationsof 1: 33 to 1: 2,700 (vol/vol) and final bTSH concen-trations of 5 mU/ml were used. To duplicate tubes, suf-ficient rabbit antiserum to give a desired final concentrationin 5.0 ml was brought to 0.05 ml with normal rabbit serumand added to 25 mUbTSH dissolved in 0.1 ml 2%o BSA.This antiserum-TSH mixture was then brought to a volumeof 1.0 ml with 2%o BSA and incubated for 2 h at roomtemperature. GARGG, 0.2 ml, was then added to one ofthe tubes, and the volume of both tubes was brought to5.0 ml by the addition of more 2%o BSA. After anotherincubation for 30 min at room temperature, the sampleswere stored overnight at 4VC and centrifuged in the cold,and the supernates were decanted. The supernates, re-warmed to room temperature, were then bioassayed in mice.A sufficient number of concentrations of each antiserumwas incubated with TSH to permit construction of -adose-response curve varying from <2 to > 98% neutral-ization. The residual TSH was quantitated by comparisonwith a standard TSH bioassay curve. Neutralization of50%o of the 5 mU/ml bTSH was read from the curve.

Effects of dilution of the TSH-anti-TSH complexes.In the experiments described in the previous paragraph,the antiserum-TSH mixtures were prepared and incubatedin a 1.0 ml volume for subsequent dilution to 5.0 ml.Thus, ratios of antiserum and TSH were varied but thefinal dilution (from 1.0 ml to 5.0 ml) was constant. Toassess the possible effect of dilution of the complexes inthe mouse, complexes formed at a constant TSH to anti-

2980 Beall, Chopra, Solomon, Pierce, and Cornell

0.01 0.1Al OFANTISERUM

FIGURE 1 Binding of [5I]bTSH,8 by the rabbit antiseraof experiment I obtained at day 52. The group mean andSEM are shown for each point. The anti-bTSH# binds['I]bTSH,8 much more avidly than does the anti-bTSH.

serum ratio were diluted variably. These diluted complexeswere then subjected to McKenzie bioassay.

RESULTSBinding of iodinated TSH and its subunits by the

antisera. The bTSHP antisera of experiment I showedconsiderable specificity for [1"I]bTSHfi. Such antiserabound this subunit much more avidly than did antiseraraised to bTSHa or bTSH (Fig. 1). In contrast, thebinding of intact ['mI]bTSH was similar with anti-bTSHa, anti-bTSH, and anti-bTSHjP. 50% of thetracer was bound by similar quantities of all threetypes of antisera although the anti-bTSH,8 antiserumbound slightly more TSH when limited amounts ofantibody were present (Fig. 2).

The antisera from experiment II were less potentthan the experiment I antisera in their ability to bind

100a * ANTI-bTSH1GZ 0-0 ANTI-bTSH

80 _ ANTI-bTSH aEm; h60|ANTI-bLH-

U)60.0

C401--zU 20 -

IL

0.001 0.01 0.1 1.0Aul OF ANTISERUM

FIGURE 2 Binding of [5I]bTSH by rabbit antisera ofexperiment I obtained at day 52. Percent bound peptideis plotted against the microliters of antiserum added.Mean±+SEM of each group is shown. Antisera to eitherbTSH or its subunits bind bTSH equally well.

TABLE IAntiserum Required to Bind 50% of Tracer ['251]bTSH

(mean +SEM)

Experiment

Antiserum No. I No. II

Anti-bTSH 4 0.31+0.22 4 1.32+ 1.23Anti-bTSHj# 5 0.08+0.02 5* 2.78+0.89Anti-bTSHa 3 0.16+0.09 4* 3.14+41.73

* In addition, one anti-bTSH,8 serum and two anti-bTSHasera did not bind 50% of the tracer even when 100 ,l of anti-serUm was added to the 1.0 ml incubation mixture.

['2'I]bTSH (Table I). Although the mean binding ofthe anti-bTSH sera exceeded mean binding by anti-subunit sera, the spread of values was large, and thedifferences were not significant. Antisera to LH andBSAdid not bind ['I]bTSH.

Neutralization of TSH by the antisera. Almost allof the antisera to bTSH and bTSHfi subunits werehighly effective in neutralizing the biological activityof bovine TSH. 1 ml of the most potent-anti-bTSHiPserum (#29) was capable of neutralizing over 10,000mU of bTSH (Table II). The activity of individualantisera varied widely. The least active sera neutral-ized less than 25 mUbTSH/ml of antiserum.

TABLE I I.1Intiserum Required to Neutralize 50%X of 5 mU/ml b TSH

Experiment

Rabbit I Rabbit II

isi/mi i/mi

anti-bTSH,63 0.46 38 17.0

29 0.22 42 3.513 0.46 43 4.613 0.46 43 4.6

14 + 16 0.60 45 *46 4.847 2.4

mean+SEM 0.44+0.08 6.5+4.5

anti-bTSH12 0.55 39 3.827 * 48 1.619 2.00 49 12.026 4.40 50 3.9

mean+SEM 2.32+ 1.22 5.4+2.3

* Concentrations up to 30 Al/mlsignificant amounts of bTSH.

(1:33) did not neutralize

Neutralizing and Nonneutralizing Antibodies to bTSH and its Subunits 2981

Anti-bTSH sera had greater neutralizing potencythan did the anti-bTSH,8 sera. The neutralizing activ-ity, like the ['I]bTSH binding activity, was greater inthe sera from experiment I than the sera from ex-periment II.

Biologic neutralizing potency and binding of [iZI]-bTSH tracer may be related phenomena. The Spear-man rank-order correlation coefficient (r) for correla-tion of 50% neutralization with 50% binding was+ 0.73 (P < 0.05) in experiment I but only + 0.47(NS) for experiment IL.

Nonneutralizing antibodies. When incubation mix-tures containing TSH and dilutions of anti-bTSHjOsera that only partially neutralized bTSH were treatedwith GARGGto precipitate immune complexes, thebioassay response of the supernate for TSH was furtherneutralized. Since free TSH was unaffected by GARGG,we ascribe this difference to nonneutralizing antibodiesto bTSH which combined with TSH but did notneutralize its biological activity. These nonneutral-izing antibodies were demonstrated in almost all of theanimals immunized with bTSH8 and none of thoseimmunized with bTSH. Figs. 3 and 4 present examplesof this increased neutralization by GARGGadded toan anti-bTSH,8 serum and the absence

E COMPLEXEDbTSHZ FREE bTSH

6.05.04.03.0

2.0

DE 1.0I(I).-D 0.5

0.37 1.1 3.3 10.0ANTISERUM(,x)

FIGURE 3 Anti-bTSH,8 serum (#38) phbTSH. The height of the bars indicates arnbiological activity remaining after incubatamounts of antiserum indicated. Open barsbTSH. Cross-hatched bars indicate amourcomplexed to rabbit IgG and neutralizedaddition of GARGG.

5.04.0

3.0

2.0

E0.

.0 0.5

IF

0.37 1.1 3.3 10.0 30.0ANTISERUM(,u )

FIGURE 4 Anti-bTSH serum (#19) plus 5 mU/mlbTSH. Open bars indicate free bTSH (bioassay resultafter addition of GARGG). Increasing amounts of anti-bTSH neutralize increasing amounts of TSH. GARGGdoes not further reduce the biological activity.

of this effect with an anti-TSH serum. The amounts of bTSH-anti-bTSHP complex neutralized and thus demonstrated bythe addition of GARGGvaried widely. Addition ofGARGGneutralized from 0 to > 98% of the ac-tivity of the 5 mU/ml TSH present.

This nonneutralizing activity of anti-bTSHP wasmost marked in the sera with the least neutralizingpotency. In fact, the two activities were significantlynegatively correlated (r = - 0.74). This relationshipdid not explain the lack of nonneutralizing antibodiesin the anti-bTSH sera. These antisera had neutralizingactivity in the same range as the anti-bTSHP sera,so the striking difference between antisera to bTSHand bTSHP was not related to differences in neutral-izing potency. These observations are summarized inTable III.

The biological activity detected in the mixtures ofantiserum with bTSH was greater at 2 h than at 8.This was true of the nonneutralized bTSH bound torabbit gammaglobulin as well as the free bTSH. TableIV documents these observations. For comparison, the2 h/8 h ratio of the response indices for LATS isapproximately 0.7, and that for bTSH is 1.4 (13).

30.0 Effects of dilution of the TSH-anti-TSH complexes.Dilution of preformed complexes did not result in the

us 5 mU/ml release of biologically active TSH (Table V).iount of TSHtion with the DISCUSSION

indicate free The heterogeneity of antibodies has been repeatedlyits of bTSHonly by the documented and requires no special emphasis. To some

extent, such heterogeneity may be a reflection of the

2982 Beall, Chopra, Solomon, Pierce, and Cornell

TABLE I I IFrequency of Neutralizing and Binding Activity in Rabbit

Antisera to bTSHand bTSH#

Antibody activity

NumberNumber Number with non-

Total binding neutralizing neutralizingAntisera number TSH TSH antibodies*

Anti-bTSH# (impure) 4 4 4 3Anti-bTSH (Thytropar) 4 4 3 0Anti-bTSH,6 (free of bTSH) 6 5 5 5Anti-bTSH (purified) 4 4 4 0

*P < 0.01 for X2 comparing bTSH with bTSH#.

methods used to demonstrate the antibody. Thus theantibodies we have described as binding ['I]bTSHor ["I]bTSH# undoubtedly encompass some or allof the antibodies we have designated as neutralizingor nonneutralizing, based on their biological activityin the presence of TSH.

The nonneutralizing antibodies combine with bTSHbut do not completely inhibit its biological activity.These soluble antigen-antibody complexes can be pre-cipitated or neutralized by the addition of GARGG.Since we have been able to describe this activity onlyin terms of the bioassay results, quantitation of theantibody is difficult and not easily comparable with thebinding activity.

There are several possibilities as to the nature ofneutralizing and nonneutralizing antibodies. The mostobvious possibility is that the neutralizing antibodycombines with a hormonally active portion of bTSHwhile nonneutralizing antibody combines elsewhere.Neutralization could also be caused by precipitation orby high affinity antibodies that produce conforma-tional changes in the TSH. The former seems unlikelysince workers interested in radioimmunoassay of TSHhave found that it is difficult to achieve complete pre-cipitation. Another possibility is that the apparent dif-ferences in antibodies, neutralizing vs. nonneutralizing,

TABLE IVTime-Course of Biological Actitity of bTSH-

Antiserum Mixtures

Response indexAssays 2 h/8 h

n mean (range)

anti-bTSHj plus bTSH 30 1.5 (0.8-1.8)anti-bTSH,8 plus bTSH plus GARGG 25 1.6 (1.1-1.9)anti-bTSH plus bTSH 13 1.4 (1.2-2.0)anti-bTSH plus bTSH plus GARGG 13 1.4 (1.1-1.6)

may represent varied rates of clearance of immunecomplexes in the bioassay mice.

More bTSH# is bound by anti-bTSHP than byanti-bTSH. These same antisera bind bTSH similarly.This difference supports the idea th-at the differing in-cidence of nonneutralizing antibodies in bTSH#- vs.bTSH-immunized groups is due to the antigen used.The results of experiment II, using highly purifiedP-subunits, were similar in this respect to those ofexperiment I, using P-subunits contaminated withbTSH. In this context, the contamination seems in-sufficient to have affected the results. The increasedbinding and neutralizing activity of experiment I anti-sera as compared to experiment II antisera might bedue to the subunit contamination with bTSH in ex-periment I, but a more likely explanation is that thepurification procedure used in experiment II partly de-stroyed the antigenicity of the subunits.

Nonneutralizing antibodies to TSH form antigen-antibody complexes in vitro that are biologically activethyroid stimulators in the mouse. The biological ac-

TABLE VFailure to Produce Thyroid-Stimulating Activity by

Diluting Neutralized Complexes

2-h Response IndexWithout WithGARGG GARGG

mean hISEAf

bTSH, 5. 0 mU/ml 1,315±2221.67 1,00541100.56 548±43

anti-bTSH#, 1.1 JI/mlplus bTSH, 5 mU/ml 370±35 190± 7anti-bTSHO, 3.3 jJ/mlplus bTSH, 5 mU/ml 149±20 190±21same, diluted 1:3 114± 16 157426same, diluted 1:9 78± 7 103 ± 6

Neutralizing and Nonneutralizing Antibodies to bTSH and its Subunits 2983

tivity, however, is short-acting (2-h activity greaterthan 8-h). One of the reasons for immunizing rabbitswith TSH and its subunits was to investigate furtherthe possibility that LATS might represent some formof an immune complex of IgG antibody with a hor-monally active antigen. Several groups have previouslystudied TSH antisera in various combinations withTSH. McKenzie and Fishman noted that an anti-bTSHthey used had a thyrotropic effect on the mouse thy-roid (14). Hoffmann, Mason, Good, Hetzel, and Fer-guson found long-acting thyroid stimulating activity(8-h greater than 3-h activity) when bTSH or myxe-dematous human serum was mixed with diluted anti-bTSH (6). They proposed that immune complexeshad been formed during the incubation period. Aftercombining hTSH and diluted anti-hTSH, Meek alsodetected long-acting thyroid-stimulating activity whichhe termed "LATS" (7). In contrast we have foundwhen gamma globulin-bound TSH was injected intobioassay mice the thyroid-stimulating activity wasgreater at 2 h than at 8 h. We are unable to reconcilethe differences in these observations. Hoffmann et al.did not attempt and Meek was unsuccessful in pre-cipitating the alleged complex with GARGG(probablybecause insufficient GARGGwas added). All threegroups have raised antisera in a similar fashion withroughly equivalent amounts of antigen and duration ofimmunization. Incubations were also similar althoughwe were always careful to keep the total amount ofrabbit serum constant by adding normal rabbit serum.Such a step seems critical only if GARGGis to beadded. Hoffmann et al. used intraperitoneal injectionsof uncentrifuged material in the bioassay mice, whereaswe and Meek centrifuged and injected the supernateintravenously. Despite these differences it seems clearthat each group has produced TSH-anti-TSH com-plexes in vitro capable of thyroid-stimulating activity.Should we infer that LATS is a similar complex? Wedo not feel that the fact that Hoffmann et al. andMeek have demonstrated a long-acting thyroid stimu-lating activity with TSH-anti-TSH complexes arguesvery strongly for the concept. The time of peak thyroidstimulation is probably not a completely reliable basisfor the differentiation of thyroid stimulators. LATScan be converted from long-acting to short-acting bycleaving the gamma globulin with papain (15). It islikely that other procedures that affect such thingsas the size, persistence in the circulation, immune elim-ination, and binding characteristics of thyroid stimu-lators also alter their time-course of action. It isnotable that the TSH-anti-TSH or TSH-anti-TSHpcomplexes formed in vitro are still inhibited by anti-TSH. Numerous investigators have noticed that anti-TSH antisera do not neutralize LATS (6, 7, 14, 16-

18); this has also been our experience. On the otherhand TSH is always neutralized if enough antiserumis added. In addition, ethanol extraction of serum de-stroys LATS but does not extract TSH, LATS per-sists in the serum of hypophysectomized patients (19),and we have been unable to liberate any thyroactivematerial from LATS by dialysis or affinity chromatog-raphy (20). Dissociation of LATS with low pH andNaSCN has been attempted by ourselves and others.The results have been difficult to interpret since LATSactivity is generally decreased by any such treatmentbut no TSH-like material has been recovered fromcolumn-eluates or dialysates. Finally, in recent, alsounpublished, experiments we have not found any evi-dence for binding of hTSH by LATS-IgG nor of bind-ing of bTSH or its subunits by LATS-IgG or its frag-ments produced by cleavage with papain.

Present evidence continues to indicate that LATSis an ordinary IgG and not an immune complex, de-spite the interesting knowledge that anti-bTSH3 seracan form nonneutralized complexes with TSH.

ACKNOWLEDGMENTSOur thanks go to Mr. Raymond Ajugwo, Mrs. Ruey S. Ho,Mr. Jeffrey Keene, Mrs. Natalie Limberg, and Miss TiiuReino for laboratory assistance and to Mrs. Hilda Dorn-fest for typing the manuscript.

This work was supported by grants from U. S. PublicHealth Service (AM-13126, C-2290, and GM-364) andThe John A. Hartford Foundation, Inc.

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Neutralizing and Nonneutralizing Antibodies to bTSH and its Subunits 2985