CLIN. CHEM. 32/11, 2034-2039 (1986)

2034 CLINICAL CHEMISTRY, Vol. 32, No. 11, 1986

Choice of Immunoglobulin G Purification Method in Assays for Antibodies tothe Thyrotropin ReceptorRosalind S. Brown, Louis P. KertHes, and Richard E. Kleinmann’

Activity of autoantibodies to the thyrotropin receptor in theserum of patients with active Graves’s disease was com-pared when the patients’ lgG was purified by three differentprocedures: ammonium sulfate precipitation (I), a modifiedbatch diethylaminoethyl cellulose method (II), and affinitychromatography on Protein A-Sepharose CL-4B (III). IgGextracted by I was significantly less potent in inhibitingbinding of 1251-labeled thyroid membranes than that preparedby either II or Ill, and was significantly less effective than II instimulating adenyl cyclase activity in thyroid membrane.Thyroglobulin, a serum protein whose concentration is in-creased in patients with various thyroid diseases, was copre-cipitated in amounts sufficient to significantly inhibit bindingonly when method I was used, but not with either of the othertwo procedures. Evidently method I is inferior to either of theother two when used for purification of autoantibodies to thethyrotropin receptor. Method II used in this study, being fasterand more economical than I and of equivalent efficacy, is afeasible alternative method for clinical use.

AdditIonal Keyphrases: autoantibodies Graves’s diseasesample preparation . anion-exchange and affinity chromatogra-phy ‘ thyroglobulin

The thyrotoxicosis of Graves’s disease is thought to be

caused by autoantibodies to the thyrotropin (TSH) receptor,which mediate their thyroid-stimulating effect through acti-vation of adenyl cyclase in thyroid membranes (1��.3).2 Of thenumerous assays currently available for their measure-ment, the two most frequently used clinically are theassessment of inhibition of ‘�I-labeled bovine (b) TSHbinding (TSH-binding-inhibitory immunoglobulins, Thfl)(4-8), and stimulation of thyroid-membrane adenyl cyclase(thyroid-stimulating immunoglobulins, TSI) (9-11). In gen-eral, the technique used to separate IgG from serum for usein these assays has received little attention; yet, for opti-mum sensitivity and specificity, high purity may be ofutmost importance. Furthermore, the antibody may beinactivated during purification.

Precipitation with ammonium sulfate, for example, isused by many investigators (4-7), even though non-IgGserum proteins may be co-precipitated to a variable degreeand cause nonspecific interference in the TBII assay (12).

Loss of activity of TSH receptor autoantibody has also been

Departments of Pediatrics and Medicine, University of Massa-chusetts Medical Center and the University of MassachusettsMedical School, Worcester, MA.

Address correspondence to Department of Pediatrics, Universityof Massachusetts Medical Center, 55 Lake Avenue North, Worces-ter, MA 01605.

1C�j’j�nt address: West Virginia University Medical Center,Charleston, WV.

2Nons�d abbreviations: TSH, thyrotropin; TBII, TSH-bind-ing-inhibitory IgGs; TSI, thyroid-stimulating IgGs; DEAE-C, dieth-ylanunoethyl cellulose; BSA, bovine serum albumin; b, bovine.

Received May 27, 1986; accepted August 15, 1986.

reported when this method has been used, probably owingprecipitation during the dialysis step (13).

In an effort to obtain a more highly purified and biologically active IgG preparation, other investigators hayturned to either affinity chromatography with use of staphylococcal Protein A-Sepharose CL-4B (14) or the anionexchange resin diethylaminoethyl cellulose (DEAE-C) (15)Unfortunately, these methods have not been systematicallcompared to determine which is the better (in terms of TSreceptor autoantibody activity of the product). Nor is iknown to what extent serum proteins such as hemoglobin othyroglobulin (the latter known to be increased in variothyroid diseases) are included in the product and whethetheir presence is sufficient to interfere in assays for TSHreceptor autoantibodies.

We have modified the batch DEAE method of Baumstarket al. (16) to give the simple, relatively rapid, and economi-cal procedure for IgG extraction described here, which isparticularly suited to the purification of a large number of

serum samples. Here we compare the IgG so purified withthat prepared by ammonium sulfate precipitation and byProtein A, by determining the activity of the TSH receptor

autoantibodies and the IgG purity. We have also performedstudies to evaluate the possible interference in the TBIIassay of thyroglobulin and hemoglobin.

Materials and Methods

Sera

We stored, at -20 #{176}C,20 serum samples obtained from 18patients with Graves’s disease, selected without consciousbias, who were thyrotoxic at the time of examination; wealso pooled serum samples from 10 patients with activeGraves’s disease. The clinical diagnosis of Graves’s diseasehad been based on the finding of a diffusely enlarged thyroidgland, and clinical and chemical evidence of hyperthyroid-

iBm. Most of the patients also had ophthalmopathy orabnormally high titers of antithyroid antibodies, or both.

Control sera obtained from 17 ostensibly healthy labora-tory and office personnel with no family history of thyroiddisease were handled similarly. We used the same sera foreach of the different methods of IgG extraction so thatresults could be compared.

lgG Purification

Anion-exchange extraction with DEAF-cellulose. Thebatch DEAE method of Baumstark et al. (16) was used, with

the following modifications. DEAE was pre-equilibrated to a

pH of 7.5 rather than 6.5 and the osmolarity of serum to bepurified was first decreased by diafiltration. These modifica-tions obviated the need to adjust the pH and reduce theosmolarity of each IgG preparation before evaluation in theTBII or TSI assay. By reducing the osmolarity in this way,we aimed to avoid the nonspecific TSH binding inhibitionobserved when the osmolarity is greater than 50 mmol/L(17) and to minimize the amount of DEAE-C required.

CLINICAL CHEMISTRY, Vol. 32, No. 11, 1986 2035

DEAE-C rather than DEAE-Sephadex was used because theformer had been shown previously (15) to yield IgG prepara-ions with good activity of TSH receptor autoantibodies.

Pre-swollen, microgranular DEAE-C (Whatman Ltd.,Clifton, NJ 07014) was pro-equilibrated with 10 mmol/L1’ris HC1, pH 7.5 (“Tris buffer”). Suspended at 1 g (dryweight) per 5 mL final volume, the DEAE-C could be storedfor at least a month at 4#{176}Cwithout loss of activity. Todecrease the osmolarity, we diafiltered serum four timesagainsttheTrisbufferinaratioofl mLofserumto6mLofTris buffer. We used a rotor (JS 3.0; Beckman Instruments,Inc., Palo Alto, CA 84304) that could hold 42 ultrafiltrationcones (“Centriflo Membrane” cones, 244-CF5O; AmiconCorp., Lexington, MA 02173; nominal molecular mass cutoffof 50000 Da). The diafiltered serum from 1 mL of startingmaterial was then added to the sedimented DEAE from 1mL of the pre-equilibrated suspension and incubated withconstant rotation in a “Roto-Mix” (Fisher Scientific Co.,Pittsburgh, PA 15219) at 4#{176}Cfor 1 h. The mixture wascentrifuged at 2500 x g for 15 mm and the supernate, whichcontained the partly purified IgG, was again incubated(same conditions) with fresh DEAE-C. To avoid unnecessarydilution of the final IgG product, we made no attempt torecover the IgG that was trapped nonspecifically and lost onthe DEAE. When necessary, we added enough 2 mol/L NaClsolution to the colorless, clear supernatant solution to give afinal concentration of 50 mmol/L [this buffer, 10 mmol ofTris HC1 and 50 mmol of NaCl per liter, pH 7.5, is hereafterreferred to as ‘�Fris-salt buffer”]. Occasional supernates thatremained cloudy despite centrifugation at 100 000 x g wereclarified by ifitration through 0.22-sm pore-size filters (Mil-lex-GS; Millipore Corp., Bedford, MA, 01730).

Ammonium sulfate precipitation. We added ice-cold am-momum sulfate, 3.75 mmol/L, to 1 mL of serum, to give afinal concentration of 1.6 mol/L, vortex-mixed, and incubat-ed the mixture with constant stirring for 1 h at 4#{176}C.Aftercentrifugation at 2000 x g for 10 miii, we washed theprecipitate three times in 1.6 molIL ammonium sulfate,dissolved it in 100 mmolJL Ti-is HC1 buffer, pH 7.5, anddialyzed this against the Ti-is-salt buffer for 24 h at 4#{176}C,oruntil no precipitate formed on addition of barium chloride.

Affinity chromatography with Protein A-Sepharose CL-4B. One milliliter of serum was allowed to percolate slowlythrough a 1-mL column of Protein A-Sepharose CL-4B(Pharmacia Fine Chemicals, Piscataway, NJ 08854) at22#{176}C.We washed the column with 10 mL of Tris buffer toremove any nonspecifically adsorbed protein, then specifi-cally eluted the IgG by adding sodium acetate, 100 mmol/L,in 1 mol/L NaC1 solution, pH 4.0. After rapidly correcting�he pH of the eluate to 7.5 with NaOH (200 rnniol/L), we�lia1yzed the solution against Tris-salt buffer for 24 h at 4#{176}C.

�)ther Procedures

TBII assay. As required, IgG solutions that had been�itored at -20 #{176}Cfor as long as three weeks were allowed to�haw at room temperature. Protein content was estimated�olorimetrically (Bio-Rad Protein Assay; Bio-Rad Labora-�ories, Richmond, CA 94804) with an IgG standard and�orrected to the desired concentration by addition of Ti-is-�alt buffer. We then added 1.1 g/L bovine serum albumin�BSA) solution, 1 mL per 10 mL of IgG solution, so that the�lnal solution contained 0.1 g of BSA per liter (“Tris-salt-�SA”).

The TBII assay, one of the more commonly used assays forIISH receptor autoantibody activity, was performed as de-

scribed previously (18), except that we added 100 Kallikreinunits of aprotinin (Sigma Chemical Co., St. Louis, MO63178) per liter to the buffer to minimize degradation of thelabeled hormone (19). In brief, we preincubated 200 �L ofIgG (in Tris or Ti-is-salt buffer containing BSA, 0.1 g/L) in 12x 100 mm polycarbonate tubes at 37#{176}Cwith 50 �L of acrude human thyroid membrane extract (6-12 mg equiva-lents) in 10 mjnol/L Ti-is HC1 buffer containing 0.1 g of BSAand 0.25 mol of sucrose per liter. After 10 miii, we added 50�uL (18-24 pg) of receptor-purified lmI�labeled bTSH, andcontinued the incubation for 50 miii. The reaction wasstopped by the addition of 1 mL of ice-cold Ti-is or Ti-is-saltbuffer containing BSA; bound and unbound fractions wereseparated by centrifugation at 27000 x g in a refrigeratedcentrifuge. The total radioactivity bound to membranes wasmeasured in a standard gamma-counter (3% counting er-ror). All samples were studied in duplicate. Specific bindingof �I-labeled bTSH in the presence of IgG from subjectswith Graves’s disease was compared with that in the pres-ence of normal IgG evaluated in the same assay. Similarly,IgG preparations extracted from the same sera by each ofthe above methods were always compared in the sameassay.

TSI assay. IgG samples from six more patients withGraves’s disease and a pooled serum sample obtained from10 other patients with active Graves’s disease were evaluat-ed for the ability of their IgG to stimulate thyroid-mem-brane adenyl cyclase. Another widely used test for TSHreceptor autoantibody that assesses bioactivity directly, thisassay has been described in detail previously (10). In brief,

the generation by human thyroid membranes of cyclic AMPfrom II32PIATP is assessed in the presence of test IgG andexpressed as the percentage above the mean obtained in thepresence of IgG for three normal subjects tested in the sameassay run.

IgG measurement. IgG was estimated by fluoroimmunoas-say (Immuno-Fluor IgG; Bio-Rad Laboratories).

Statistical evaluation. The significant of the differencebetween groups was evaluated by Student’s paired t-test forIgG extracted from the same sara. When results fromdifferent sera were compared, we used Student’s unpaired t-

test. Correlation coefficients were determined by the methodof least squares. Unless indicated otherwise, results areexpressed as mean ± SD.

Results

TBII Activity

Preliminary studies to determine the optimum proteinconcentration at which to compare IgG from patients withGraves’s disease and normal IgG involved DEAE-extractedIgG in Tris-BSA buffer (Figure 1). As previously studied byothers (12), IgG from both sources significantly inhibitedbinding of �I-labeled bTSH, although the effect withnormal IgG was observed only at 0.2 mg of protein or moreper 200 pL. IgG from patients with Graves’s disease hadsignificantly greater TBII activity at all concentrationstested but the difference between Graves’s and normal wasgreatest when the protein did not exceed 0.8 mg per 200 1zL.We arbitrarily chose 0.4mg of protein (i.e., 200 �tL of a 2 kg!L solution) for use in further studies, to minimize interfer-ence of normal IgG, maximize the difference between nor-mal and Graves’s disease IgG, and avoid the apparentpositive cooperativity we observed with some samples of IgGfrom patients with Graves’s disease, at lower concentra-tions. A further advantage of this concentration was the

Qr NORMALS, Nr4

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30

25

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lgG ADDED (mg)

Fig. 1. Mean (± SD) binding of lseI.Iabeled bTSH to human thyroidmembranes In presence of IgG obtained from patients with Graves’sdisease compared with that from control subjectsAssay performed withTrls-BSA without added NaCI. gO obtained from patientswith Gravees disease caused significantly more binding Inhibition than normalIgG at all concentratIons tested, but the relative difference was greatest at 0.8mgat protein per sample

ease with which IgG samples could be prepared from 1 mL ofserum or less.

Figure 2 compares the TBII activity of IgG from 10patients with Graves’s disease and eight control subjects’IgG, as extracted by the three methods. TBH activity ofDEAE-purified IgG from Graves’s disease patients wassignificantly greater than when ammonium sulfate precipi-

tation was used (p <0.01) and was equivalent to results withProtein A. On the other hand, nonspecific interference bynormal IgG was least when IgG was prepared with ProteinA. The ratio of specific binding of 1saI�labeled bTSH in thepresence of Graves’s disease IgG to the specific binding inthe presence of normal IgG was equivalent for the DEAE-Cand Protein A methods (84.9 ± 4.2% vs 83.9 ± 5.6%).Similarly (Figure 3), TBU activity of individual IgG samplesextracted from the same sera by either DEAE or Protein Awas significantly correlated (r = 0.763, p <0.001), stronglysuggesting that each was acting on the same characteristicsof the imniunoglobulin. Interestingly, TSH binding-inhibi-tory activity IgG prepared from the same specimens ofserum with ammonium sulfate precipitation did not differfrom normal when studied at this protein concentration (seeDiscussion).

IgG Content

To evaluate the purity of these preparations, we estimat-ed IgG concentrations by fluoroimmunoassay and expressedthese as a percentage of the total protein. By DEAE the %IgG (83.7 ± 9.2%, n = 20) was significantly greater thanobtained by ammonium sulfate precipitation (71.4 ± 13.6%,

80 CJContrcls

�Graves’ DIsease (10)

(10) #{149}�� 60 (tO) -�

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Fig. 2. Mean (± SEM) specific binding of lml�labeIed bTSH to humanthyroid membranes in presence of 0.4 mg of lgG extracted from thesame sara by three different methodsNumbers Inparentheses referto the number of serum samples tested. Whenboth DEAE cellulose and ProteinA methods were used, gO from patients wIthGravess disease caused significantly greater inhibition of ‘#{176}#{176}I-IabeledbTSHbindingthandidnormal 9G. Incontrastusing the same serum sample, we foundno differencebetween patientsand normal individuals when their IgG wasextracted by ammonium sulfate precipitation. p <0.001

n = 20, p <0.005) and less than that obtained with ProteinA (93.5 ± 7.2%, n = 20, p <0.001). Two separate experi-ments with DEAE yielded 86.3 ± 2.5 (n = 8) and 81.9 ± 2.8(n = 12) % IgG, showing good reproducibility.

Inhibition of 1251-Labeled bTSH Binding (TBI) by Non-lgG Proteins in Serum

IgG purity and the specific binding of lmI�labeled bTSHwere significantly correlated (Figure 4); i.e., the greater thepurity of normal IgG, the less the inhibition of 1�I-labeledbTSH binding. This suggested that part of the inhibition ofTSH binding observed in the presence of normal IgG wasdue to contamination with non-IgG protein. Preliminary

studies indicated that thyroglobulin in excess of 20 �&g (i.e.,0.2 mL of a 100 mg/L solution) caused significant inhibitionof TSH binding. Hemoglobin also had a slight effect, butonly when 0.2 mg or more was added. Transfer-in, a majorcontaminant of DEAE-C extraction (20), had no TBU activi-ty. To elucidate whether increased serum concentrations ofthyroglobulin, as found in some patients with Graves’sdisease, cancer, or goiter (21), might be coextracted with IgGand interfere in TBII assays, we added 0.2 mg (i.e., 0.2 mL ofa 1 g/L solution) of porcine thyroglobulin (Sigma) to normalsara and extracted the IgG by each of the three methods.The possible effect of hemoglobin was studied similarly. Asindicated in Figure 5, which represents the combined resultsof three separate experiments, addition of thyroglobulunslightly but significantly (p <0.005) inhibited TSH bindingonly when IgG was extracted by ammonium sulfate precipi-tation. Hemoglobin tended to slightly increase 1�I-labeledbTSH binding, particularly with ainmonium sulfate precipi-tation, but the effect was not significantly different from thebehavior of the control.

TSI Activity

TSI activity of IgG prepared by ammonium sulfate precip-itation and by DEAE-C extraction was compared in an

2036 CLINICAL CHEMISTRY, Vol. 32, No. 11, 1986

r- .763

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1g. 3. Specific binding of 1251-labeled bTSH to human thyroid mem-ranes in the presence of the same control and Graves’s disease lgGreparation extracted from serum by DEAE-C compared with staphylo-occal Protein A treatment (SPA)inear regression analysis of the data points: y = 1.3x - 10

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ig. 4. Specific binding of 1251-labeled bTSH to human thyroid mem-wanes in presence of normal IgG in relation to the purity of the IgGireparationxtraction method: DEAE (0), ammonium sulfate (A), Protein A (0). Linear�gression analysis of data points: y = 0.36x + 42.0

Ammonium

Sulfate

DEAE-

Cellulose

CLINICAL CHEMISTRY, Vol. 32, No. 11, 1986 2037

% of Specific Binding(DEAE)

�ControlThyroglobulin

- Hemoglobin

6)

61 16)

2oL 1910An�im C�SO

Fig. 5. Mean (± SEM) specific binding of 1�I-$abeIed bTSH to humanthyroid membranes in presence of normal IgG extracted by threemethods and studied under three different conditions (left to right):control; porcine thyroglobulin, 0.2 mg, added prior to extraction;hemoglobin, 0.2 mg, added prior to extractionNumbers in parentheses refer to the number of sara tested, the same beingutibzed in each method. Addition of thyroglobulin significantly inhibited the binding(p <0.005) only when ammonium sulfate was used to extract 19G. Addition ofhemoglobin prior to extraction did not significantly affect binding. p <0.005

additional three normal individuals, six patients with un-treated Graves’s disease, and the pooled serum sample from

10 patients with active Graves’s disease. The TSI for thenormal IgG prepared by ammonium sulfate precipitation

and DEAE-extraction was not significantly different (1.53 ±

1.5 vs -1.3 ± 2.4, respectively), although the value in the

DEAE-C extracted group was consistently lower. In con-trast, TSI activity of IgG from patients with Graves’s diseasewas significantly greater (p <0.05) when IgG was extractedby DEAE-C than by ammonium sulfate precipitation (Fig-

ure 6).

Discussion

We conclude that the method of IgG extraction is of0 utmost importance in determining both the sensitivity and

specificity of assays of autoantibodies to TSH receptor. Ofthe three methods tested, ammomum sulfate precipitationwas clearly inferior to either DEAE-C extraction or ProteinA, both for specific inhibition of TSH binding in samplesfrom patients with Graves’s disease and for purity of prepa-

Fig. 6. StimulatIon of human thyroid-membrane adenyl cyclase by lgGprepared from the same sera (from patients with Graves’s disease) bytwo different methodslgG extracted by ammonium sulfate precipitation (0) was significantly less potent(p <0.05) than that purified by DEAE-cellulose (#{149})in this bioassay for TSHreceptor autoantibodies

2038 CLINICAL CHEMISTRY, Vol. 32, No. 11, 1986

ration, and was also inferior to DEAE-C in terms of TSI

activity. On the other hand, the sensitivity obtained withDEAE-C purification was equivalent to that with Protein Adespite the slightly lesser purity of the former preparations.Indeed, inhibition of TSH binding by IgG from Graves’sdisease, prepared with DEAE-C, was actually greater thanthat prepared with Protein A-probably because the DEAE-C method used in the present study was gentler and did notrequire dialysis. Smith et a!. (13) showed previously thatautoantibody activity toward the TSH receptor, as deter-mined by the long-acting thyroid stimulator assay, can befound in the precipitate that forms during dialysis; it isprobable in our studies that some TBII activity was lostanalogously during Protein A treatment and ainmoniumsulfate precipitation. Furthermore, the acid utilized to eluteIgG from the column of Protein A may have caused variabledamage to the binding site for the IgGs that inhibit TSHbinding. In support of this possibility, we observed markedlyincreased potency of IgG from subjects with Graves’s disease

when the pH was adjusted immediately, as compared withwhen the pH was allowed to adjust slowly during subse-quent dialysis. Overall, the sensitivity of the procedure withDEAE-C was equivalent to that with Protein A because thegreater interference by normal IgG extracted with DEAE-Cmitigated the increased TBII activity of Graves’s diseaseIgG when prepared by this method. Our findings thussupport and extend the studies by de Bruin et al. (22), whodemonstrated that TBH activity may be lost when the

ammonium sulfate precipitation method is used. Note thatthe IgG (Graves’s disease) precipitated with ammoniumsulfate did not inhibit TSH binding significantly at theconcentration tested. However, we used only 0.4 mg of IgGper sample in the present study, whereas most investigatorsdoing ammonium sulfate precipitation have used 4 mg ofIgG protein or more (4-6).

Like Beall et al. (12), we found that part of the inhibitoryeffect of normal IgG in the TBH assay was from non-IgGcontaminants. Thus, TBII activity of normal IgG was leastafter staphylococcal Protein A treatment, which yieldedpreparations that contained >90% IgG. We considered itparticularly important to evaluate the possible interferenceby specific non-IgG proteins, especially hemoglobin andthyroglobulin. Not only is the concentration of thyroglobu-lin often increased in patients who have various thyroiddisorders (21) but also it reportedly inhibits binding of ��i-labeled bTSH (23) and stimulates mouse thyroid activity invivo (24). Furthermore, thyroglobulin is known to be precip-itated by ammonium sulfate (25). We observed a significanteffect of thyroglobulin only when ammonium sulfate precipi-

tation was used to extract IgG, but not with either DEAE-Cor Protein A treatment. This effect was observed with aslittle as 200 �tg of added thyroglobulin-an amount wellwithin the range that might be coprecipitated from the

serum of some patients with thyroid cancer goiter andGraves’s disease (21), if one uses the standard Smith andHall procedure for assessment of TBII (4), which involves 4mg of IgG protein in the assay. The lack of significant effectof hemoglobin in any of the three IgG purification methodsindicates that hemolysis does not interfere with the ob-served activity.

We conclude from these studies that, of the three IgGextraction procedures evaluated, the ammonium sulfateprecipitation method is clearly inferior and should not beused for purification of TSH receptor autoantibodies fromserum. DEAE-C and Protein A treatments appear to yield

IgG of equivalent potency, despite the fact that the formei

preparations are less pure. We conclude further that thEmodified DEAE-C method as described in this report is afeasible alternative to Protein A for clinical use-beingmore economical, faster, and not requiring column chroma.tography. In fact, subsequent work in our laboratory hasshown that the procedure can be simplified further bysubstituting overnight dialysis of serum for the diallltratiostep described. Although we have evaluated the influence othe IgG extraction method on just two TSH receptor autoantibody activities, the conclusions drawn here are probabiequally applicable to other characteristics of this importan

IgG population.

We thank Dr. J. Pierce, Department of Biochemistry, Universiof California, and the Pituitary Hormone Distribution Program cithe NIAMDD for their generous gifts of highly purified TSH for useas tracer. This work was supported by a grant from the Charles H�Hood Foundation and NIH Grants AM33796, AM18919, andS07RR5712.

References

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2. Bech K. Immunological aspects of Graves’ disease and impor.tance of thyroid stimulating immunoglobulins. Acts Endocrinol(Copenhagen) 1983;254:7-38.3. Burman KD, Baker JR Jr. Immune mechanisms in Gravesdisease. Endocr Rev 1985;6:183-232.

4. Mukhtar ER, Smith BR, Pyle GA, et al. Relation of thyroid.stimulating immunoglobulins to thyroid function and effects 01surgery, radioiodine and antithyroid drugs. Lancet 1975;i:713-5.

5. O’Donnell J, Trokoudes K, Silverberg J, et al. Thyrotropindisplacement activity of serum immunoglobulins from patientswith Graves’ disease. J Clin Endocrinol Metab 1978;46:770-7.

6. Strakosch CR, Joyner D, Wall JR. Thyroid-stimulating antibod.ies in patients with autoimmune disorders. J Clin Endocrino]Metab 1978;47:361-5.

7. Bliddal H, Kirkegaard C, Siersbaek-Nielsen, et al. Prognosticvalue of thyrotropin binding inhibiting immunoglobulins (Thil) inlong-term antithyroid treatment. ‘�‘i therapy given in combinationwith carbimazole and in euthyroid ophthalmopathy. Acts Endo.crinol (Copenhagen) 1981;98:364-9.

8. Brown RB, Kertiles 12, Rosenfield C, et al. Thyrotropin-receptomautoantibodies in children and young adults with Graves’ disease,Am J Dis Child 1986;140:238-41.

9. Zakarija M, McKenzie JM, Banovac K. Clinical significance olassay of thyroid-stimulating antibody in Graves’ disease. AnnIntern Med 1980;93:28-32.

10. Kleinmann RE, Braverman LE, Vagenakis AG, et al. A ne�method for measurement of human thyroid-stimulating immuno-globulins. J Lab Clin Med 1980;95:581-9.

11. Hinds WE, Takai N, Rapoport B, et al. Thyroid-stimulatingbioassay using cultured human thyroid cells. J Clin EndocrinolMetab 1981;52:1204-10.

12. Beall GN, Chopra IJ, Solomon DH, Kruger SR. Serum proteininhibition of thyrotropin binding to human thyroid tissue. J CliiiEndocrinol Metab 1978;47:967-73.

13. Smith BR, Mum’o DS, Dorrington KJ. The distribution of thelong-acting thyroid stimulator among G-immunoglobuluns. Biochin,Biophys Acts 1969;188:89-100.

14. Kuzuya N, Chiu SC, Ikeda H, et al. Correlation betweenthyroid stimulators and 3,5,3’-triiodothyronine suppressibility inpatients during treatment for hyperthyroidism with thionainidedrugs: comparison of assays by thyroid-stimulating and thyroid.displacing activities. J Clin Endocrinol Metab 1979;48:706-11.

15. Wall JR, Strakosch CR, Bandy P, Bayly R. Nature of thyrotro-pin displacement activity in subacute thyroiditis. J Clin EndocrinolMetab 1982;54:349-53.

16. Baumstark JS, Laffin RJ, Bardawil WA. A preparative method 21. Van Herle Al. Serum thyroglobulin measurement in thefor the separation of 7S gamma globulin from human serum. Arch diagnosis and management of thyroid disease. Thyroid TodayBiochem Biophys 1964;108:514-22. 1981;4:1-5.

17. Moore WV, Wolff J. Thyroid-stimulating hormone binding � 22. de Bruin TWA, Van der Heide P, Querido A. Thyrotropin-beef thyroid membranes: relation to adenylate cyclase activity. J binding inhibition by anti-thyrotrophin receptor antibodies inBiol Chem 1974;249:6255-63. Graves’ disease which is not reflected by 1.6 M ammonium sulfate

precipitates. Clin Endocrinol 1982;17:77-84.18. Brown RB, Kertiles 12, Reichlin S. Partial purification ofthyrotropin binding inhibitory immunoglobulins from normal hu- 23. Hashizuine K, Fenzi G, DeGroot U. Thyroglobulin inhibitionman plasma. J Clin Endocrinol Metab 1983;56:156-62. of thyrotropin binding to thyroid plasma membrane. J Clin Endo-crinol Metab 1978;46:679-85.19. Beau GN, Chopra LI, Solomon DH, et al. Studies of the TSH 24. Wood LC, Burger A, Peterson M, et al. Induction of a LATS-likeradioreceptor assay. Acta Endocrinol 1979;90:217-26. response by thyroglobulin in the McKenzie assay system. Endocri-

20. Fahey JL, Terry EW. Ion exchange chromatography and gel nology 1973;92:1538-43.ifitration. In: DM Weir, ed. Handbook of experimental immunology, 25. Valenta LI. Thyroid peroxidase, thyroglobulin, cAMP and2nd ed. Oxford: Blackwell Scientific Publications, 1973:7.1-7.16. DNA in human thyroid. J Clin Endocrinol Metab 1976;43:466-9.

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