0013-7227/90/1276-3160$02.00/0 Endocrinology Copyright© 1990 by The Endocrine Society
Vol. 127, No. 6 Printed in U.S.A.
Generation and Characterization of Monoclonal Antibodies Directed against Noniodinated and Iodinated Thyroglobulin, among which Are Antibodies against Hormonogenic Sites* M. T. DEN HARTOG, M. DE BOER, G. J. M. VEENBOER, AND J. J. M. DE VIJLDER Department of Experimental Pediatric Endocrinology, University of Amsterdam, Academic Medical Center (M. T.D.H., G.J.M. V., J.J.M.D. VJ, and the Laboratory of Biochemistry, Biotechnology Center, University of Amsterdam (M.D.B.), Amsterdam, The Netherlands
ABSTRACT. In this paper we describe the preparation and characterization of three different groups of monoclonal antibodies (Mabs) raised against iodinated and noniodinated human thyroglobulin (hTg). One group (A) of three Mabs is directed against hTg without discrimination between different iodine contents of Tg. The second group (B) of three Mabs has a higher affinity for iodinated Tg than for noniodinated Tg, and the Mabs are not species specific. The last group (C) of two Mabs generated against noniodinated hTg shows a higher affinity for
T
O STUDY the mechanism of thyroid hormone formation and mutations in this process, characteristics of thyroglobulin (Tg) are examined. Tg is a dimeric protein of 660,000 mol wt. The main function of Tg is the formation of thyroid hormones, T4 and T 3 (1). This hormone formation starts with the iodination of tyrosine residues in Tg (1). Coupling of two diiodotyrosine (DIT) residues synthesizes T4, and the coupling of a monoiodotyrosine (MIT) residue with a DIT residue gives T 3 or rT 3 (2-4). Each polypeptide chain of Tg contains at least four sites for hormone formation, termed hormonogenic sites (3, 5-8). One hormonogenic site is located at the Nterminal end of the Tg molecule, and the other three are located at the C-terminal end (9, 10). For rabbit Tg, Dunn et al. (11) described an extra T4-forming site at residue 1291. They did not detect the T4-forming site at residue 2569 described to be present in Tg from other species. For hormonogenesis Tg is iodinated in a prefer-
Received June 7,1990. Address requests for reprints to: Dr. M. T. Den Hartog, Department of Experimental Pediatric Endocrinology, University of Amsterdam, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. * This work was supported in part by the Netherlands Organization for Scientific Research (NWO) and the Ludgardine Bouwman Stichting.
noniodinated hTg than for iodinated hTg. From competition experiments with T4 we conclude that the second group of Mabs is directed against hormonogenic sites in the protein. From these Mabs, probably two are directed against the N-terminal hormonogenic site, and one against one of the C-terminal hormonogenic sites in Tg. Reactivity of the Mabs of group B with Tgs of various degrees of iodination indicates that the N-terminal T4 is formed first. (Endocrinology 127: 3160-3165,1990)
ential way. Early iodinated tyrosyl residues are preferentially involved in iodothyronine synthesis (4, 12-14). Even at a low degree of iodination, Tg forms thyroid hormone (12). At a higher degree of iodination, more tyrosines are iodinated, but the efficiency of thyroid hormone formation diminishes. In this paper we describe the generation and characterization of monoclonal antibodies (Mabs) against Tg, among which are Mabs against at least two hormonogenic sites. Our results indicate that the N-terminal T4 in Tg is formed first. Materials and Methods Tg Tg was isolated as described by Van Voorthuizen et al. (15). The iodine content of the Tg preparation was determined as described by De Vijlder et al. (16). We have isolated human Tg (hTg) with degrees of iodination of 0.00%, 0.20%, 0.53%, and 0.63% (wt/wt) from human thyroids obtained from patients with a total organification defect or larynx carcinoma and after obduction. With a RIA (17) we have determined the T4 content of the isolated hTg preparations after hydrolysis with pronase (10%, wt/wt; 48 h; 37 C) and leucine aminopeptidase (20%, wt/ wt; 24 h; 37 C). The T4 contents were, respectively, 0,1, 5, and 6 mol T4/mol Tg. We have also isolated Tg from dog, rat, mouse, goat, and bovine with degrees of iodination of 1.00%,
3160
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 04:29 For personal use only. No other uses without permission. . All rights reserved.
Mabs AGAINST hTg 0.82%, 0.22%, 0.59%, and 0.00%, respectively.1 We also had available 100,000 X g goiter supernatant from Dutch goats treated with T4 (18). These goats do not synthesize 330,000 mol wt Tg monomers, but only a glycosylated 40,000 mol wt Nterminal Tg fragment.
Antibodies The hybridomas producing the Mabs were generated by fusion with NS-1 myeloma cells of splenocytes from two groups of BALB/c mice immunized with hTg with an iodination degree of 0.00% or 0.63% (19). After limited dilution selection of these fusions, eight useful hybridomas were obtained. The experiments were performed with cell culture supernatants. Supernatants were obtained by culturing the hybridomas for 3 days without refreshing the medium. The supernatants were stored in 10-ml tubes at -20 C. Ascites fluid was obtained by injection of the hybridomas in the peritoneal cavity of BALB/c mice. The ascites fluid was heat inactivated at 56 C for 20 min. The glycoproteins were removed by dextran sulfate precipitation. After dialysis against PBS, aliquots were stored at -20 C. The monoclonal antibody affinity from the ascites fluid was measured using an enzyme-linked immunosorbant assay (ELISA) method. The 96-well polyvinyl plates (Falcon, Oxnard, CA) were coated with 1:1000 diluted goat [antimouse immunoglobulin G (IgG)] polyclonal antiserum from Sigma (St. Louis, MO). These plates were incubated with the ascites fluid in dilution. The relative affinity is defined as the dilution of the ascites fluid in the ELISA at which the Mab is not the limiting factor. A polyclonal antiserum against hTg (iodination degree, 0.20%, wt/wt) was raised in rabbits.
3161
well. After incubation (20 min; 20 C) the fluorescence was measured with a fluorscan Titertek. For determination of the isotypes of the Mabs, 96-well polyvinyl plates (Falcon) were used. The isotype-specific conjugates were goat antimouse IgG, goat antimouse IgM, goat antimouse IgGl, goat antimouse IgG2a, goat antimouse IgG2b, and rabbit antimouse Ig. Peroxidase was bound to all conjugates. They were obtained from Sigma (St. Louis, MO), except the last one which was from Dako-patts (Glostrup, Scotland). As substrate we used tetramethylbenzidine from Sigma. The bound peroxidase activity was revealed by the addition of 100 (A 0.25 mM tetramethylbenzidine in 0.1 M sodium phosphate buffer (pH 6.0) with 20 mM H2O2. The reaction was stopped after 15 min by the addition of 50 jtl 2 M H2SO4. The 450 nm absorbance was measured with a Titertek Flow Multiscan. ELISA for the study of monoclonal cross-reactivity with Tg from different species Nunc 96-well plates were coated with 20 ng Tg/well. The wells were filled with 100 /il of the appropriate Tg concentration in PBS and allowed to dry overnight at 37 C. The Tgs tested originated from human (two different iodine contents), dog, goat, cat, mouse, and bovine. After washing, undiluted supernatant of each monoclonal was added, and the plates were incubated (1 h; 37 C). Subsequently, the plates were, after another washing, incubated (1 h; 37 C) with the conjugate rabbit antimouse Ig conjugated to alkaline phosphatase, as described in the protocol for the sandwich ELISA. Bound phosphatase activity was revealed by the addition of 4-methylumbeffiferyl phosphate (Sigma). Competition experiment with T4
Sandwich ELISA The reactivity of the Mabs with hTg preparations of various iodination degrees and antibody isotypes was determined using a solid phase sandwich enzyme immunoassay. A 1:1000 dilution in PBS of an IgG fraction (obtained by using protein-A-Sepharose; Pharmacia, Uppsala, Sweden) of the polyclonal antiserum against hTg was adsorbed to the wells of 96-well microtiter plates (Nunc, Roskilde, Denmark; 2 h; 37 C). Unbound antibody was removed by washing the plates with PBS containing 0.05% Tween (wt/wt; 3 times) and demineralized water (once). Subsequently, hTg of known iodine content was added in various dilutions and incubated (16 h; 4 C). After washing, 100 n\ of the Mab to be tested (undiluted supernatant) were added to each well and incubated (2 h; 37 C). After another washing the plates were incubated (1 h; 37 C) with rabbit antimouse Ig conjugated to alkaline phosphatase (Dako-Immunoglobulines, Copenhagen, Denmark). This conjugate was used in a 1:1000 dilution in PBS-Tween with 1% (wt/wt) BSA to prevent aspecific binding. After washing thoroughly with PBS-Tween (5 times) and demineralized water (twice), 100 /*1 4 mM 4-methylumbeffiferyl phosphate in 0.1 M diethanolamine buffer, pH 10.3, containing 1 mM MgCl2 were added to each 1 Bovine thyroglobulin with an iodination degree of 0.00% was taken from an animal that had presumably been treated with antithyroid drugs.
Microtiter plates (96-well; Nunc) were coated with the polyclonal antiserum in a 1:1000 dilution in PBS, as described for the sandwich ELISA. After incubation and washing the plates were coated with 40 ng hTg/well, with a iodine content of 0.63%. After incubation (16 h; 4 C), the plates were washed, and blocking was performed by incubation (1 h; 37 C) with a 1% (wt/vol) BSA solution in PBS. After washing, the plates were incubated with 50-fil T4 (Sigma) solutions, ranging from 500-0 ng T4/well, together with 50 n\ of the supernatant of each Mab. After incubation (2 h; 37 C), the plates were washed and incubated with the conjugate, as described for the sandwich ELISA, using 4-methylumbeffiferyl phosphate as substrate. Electrophoresis and immunoblotting hTg (iodine content, 0.20%) was treated with 0.8% sodium dodecyl sulfate (SDS) and 2% 2-mercapthoethanol (5 min; 100 C). This (2 ng/lane) was run on a 7.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE; 2 h; 100 V). After electrophoresis the gels were electroblotted to a nitrocellulose filter in 25 mM Tris-HCl, 192 mM glycine, and 20% methanol, pH 8.3 (1 h; 100 V; 4 C). The mol wt standards for SDS-PAGE were obtained from Sigma. After blotting, the nitrocellulose filters were blocked with 1% BSA in PBS-Tween (1 h; room temperature). Incubation with the Mabs (ascites fluid) was performed overnight in PBS-Tween with 0.1% BSA also at room temper-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 04:29 For personal use only. No other uses without permission. . All rights reserved.
Mabs AGAINST hTg
3162
ature. After washing with PBS-Tween (five times) coupling of 1:3000 diluted horseradish peroxidase-conjugated rabbit antimouse total Ig (Dako-Patts) in PBS-Tween was performed for 1 h at 20 C. The filters were washed with PBS-Tween (five times) and PBS (once) and 10 min in 50 mM Tris-HCl, pH 7.6. They were stained with 1.4 mM diaminobenzidine tetrahydrochloride (Sigma) in 50 mM Tris-HCl, pH 7.6, with 3 mM H2O2. The reaction was stopped after 15 min by the addition of 1% (vol/vol) HC1 and directly washed with water. Filters were air dried.
Endo • 1990 Voll27«No6
D co
o iz
Results All of the Mabs, coded Al, A2, and A3 (group A); Bl and B2 (group B); and Cl and C2 (group C), are of the IgGl isotype, except B3 (group B) which is a IgG2a, as determinated with specific polyclonal antisera as described in Materials and Methods. The reactivity of some of the Mabs against hTgs of various iodination degrees tested in a sandwich ELISA is shown in Fig. 1. The reactivity of Mab A3 is independent of the iodine and thyroid hormone content. For the Mabs Al and A2, similar curves were found (results not shown). The Mabs Bl, B2, and B3 have a higher affinity for iodinated than for low iodinated Tg, as can be seen for Mab B2 in Fig. 1. The Mabs Cl and C2 show a higher affinity for low iodinated than for high iodinated Tg, as shown in Fig. 1 for Mab C2. To study whether the epitopes were also present in Tg of other species than man, we have tested the reactivity of the Mabs against Tg of various animals. A direct ELISA was performed, because polyclonal antisera against Tg of the various species were not available. In Table 1 the results are shown. The Mabs that have a higher affinity for Tg with a higher iodine content also recognize dog, mouse, rat, and goat Tg. All of these Tgs had a high iodine content, bovine Tg did not react, since it was not iodinated. The other Mabs do not react with Tg of the various species tested. It is remarkable that Mabs Al and A2 and Cl and C2 did not recognize hTg as they did in the sandwich ELISA (Table 1, last two columns). To study whether Mabs Bl, B2, and B3 recognize hormonogenic sites in Tg we performed competition experiments with T4. From Fig. 2 it is clear that Mabs Bl, B2, and B3, which recognize higher iodinated Tgs, are the Mabs that are inhibited by T4. hTg with an iodine content of 0.20% (wt/wt) was blotted on nitrocellulose filters after SDS-PAGE under reduced conditions. The filters were incubated with the various Mabs. Mabs Al, A2, Cl, and C2 show little or no reaction with hTg. Mabs A3, Bl, B2, and B3 give various bands (Fig. 3). The patterns were different, indicating that the Mabs recognize different epitopes on Tg. All Mabs recognize bands with mol wt of 240,000,
20
5 1.25 0.31 ng Tg / well
0.07
o
to T3
(0
CO
D O
20
5
1.25
0.31
0.07
0
0.07
0
ng Tg / well
T5
ro
CO
ZJ
o
1.25
0.31
ng Tg / well
FIG. 1. Sandwich ELISA. Plates are coated with polyclonal anti-Tg serum incubated with hTg [iodine content, 0.00% (A-A), 0.20% (OO), 0.53% (+-+), or 0.63% (D-d)] in dilution from 0-80 ng Tg/well and cell culture supernatants from Mabs A3, B2, and C2.
200,000, and 180,000. It is remarkable that Bl (lane 3) and B2 (lane 4) recognize bands at 20,000 and 78,000; B3 (lane 5) does not show a Tg fragment of low mol wt. To study whether the Mabs of group B could react with an N-terminal Tg fragment we coated wells with
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 04:29 For personal use only. No other uses without permission. . All rights reserved.
Mabs AGAINST hTg
3163
TABLE 1. Cross-reactivity of Mabs with Tgs obtained from various species with various iodination degrees sandwich ELISA human human dog goat rat mouse bovine human human 0.00% 0.63% 0.00% 0.63% _ _ _ _ _ _ + + direct ELISA
Al A3 Bl B2 B3 Cl C2
+ -
+ + + + _ -
+ + + _ -
_ _ + + + + + + _ _ -
_ + + + / _ -
_ - _ -
+ + +
+ + + +
A direct ELISA was performed with plates coated with 20 ng Tg/ well human (iodine contents 0.00% and 0.63%), dog, goat, rat, mouse, and bovine origin (iodine contents as mentioned in Materials and Methods). + means that the detected signal was at least two times the background. On plates coated with BSA [1% (wt/vol)] or with the polyclonal antiserum against Tg (1/1000 diluted) alone, there was no signal detected with any of the Mabs. As reference the results of a sandwich ELISA with human Tg (iodine contents 0.00% and 0.63%) are given.
5OO
125
31.25
7.5
1.8
0.45 O
ng T4/well
B
normal goat Tg and the goitrous proteins (18). The results are shown in Fig. 4. Mabs Bl, B2, and B3 reacted with normal goat Tg, and Bl and B2, in contrast to B3, reacted with goitrous Tg.
Discussion Eight Mabs against Tg are described. These Mabs can be classified in three groups. The Mabs of groups A and B were obtained from mice that had been injected with Tg containing 0.63% (wt/wt) iodine. The Mabs of group C were from mice that had been immunized with noniodinated Tg. The Mabs Al, A2, and A3 (group A) recognize on each Tg an epitope that is not influenced by the iodine and thyroid hormone content and as a consequence do not distinguish between Tgs of various iodination degrees. Binding of Tg to polyvinyl and nitrocellulose diminished the reactivity of Mabs Al and A2 for Tg. This indicates that the reacting epitopes depend on the Tg configuration, which alters on binding to surfaces. Another possibility is that these epitopes are hidden by binding to a surface. The reactivity of the Mabs is not influenced when Tg is bound to precoated polyclonal antibody against Tg. From control experiments it was clear that the Mabs do not react with the polyclonal antiserum alone. The same effect is observed with Mabs Cl and C2. These Mabs have a higher affinity for low or noniodinated Tg than for iodinated Tg. When the conformation is changed by iodination (12) or denaturation, the recognition of these Mabs for Tg decreased strongly. Mabs Bl, B2, and B3 have higher affinities for iodinated Tg then for low iodinated Tg. Competition experiments with T4 indicate that these Mabs are most likely
125
31.25
7.5
1.8
0.45 O
ng T4/well FIG. 2. Competition experiment with T4. Sandwich ELISA performed with the polyclonal antiserum and hTg (40 ng/well) with a iodine content of 0.63%. Competition was performed by incubation of T4 together with supernatant from the Mabs Al ( • - • ) , A2 (D-D), A3 ( 0 0), and Bl (Y-V) in A and B2 (+-+), B3 (A-A), Cl (O-O), and C2
(•-•) in B. directed against epitopes near or in the thyroid hormonecontaining sites of the Tg molecule. From further competition studies we have found that MIT does not inhibit the Tg-Mabs interaction, while DIT, compared with T4, causes only a very slight inhibition (results not shown). As expected, we can conclude that the iodination degree of Tg also influences what kind of immune response will be induced. The hormonogenic peptides of Tg from various species are homologous, while other parts of the molecule show less homology (9, 10). In agreement with these facts we show that the three Mabs of group B all show cross-reactivity with Tgs from other species if this Tg is iodinated. Incubating a Western blot of hTg after SDS-PAGE with Mabs Bl and B2 showed several bands, among which was a band at about 20,000 mol wt. This is
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 04:29 For personal use only. No other uses without permission. . All rights reserved.
Mabs AGAINST hTg
3164
Endo • 1990 Vol 127 • No 6
10
Mr x 1000
1 2
3
4 5
180116 • 844836•*•
moles T 4 / mol Tg
26-
FIG. 5. Reactivity of Mabs B2 (+-+), Bl (D-D), and B3 (•-•) with
FIG. 3. Immunoblotting of hTg (iodine content, 0.20%) with ascites
fluid from Mabs A3 (lane 2), Bl (lane 3), B2 (lane 4), and B3 (lane 5). Lane 1, molecular weight markers (Sigma). Mabs Al, A2, C2, and Cl in the same experiment showed no reaction (results not shown). The relative affinity of the ascites fluid dilution used was the same for each Mab. Mr, Mol wt.
8 5OOO
1250
o
313
78
ng Tg/well FIG. 4. ELISA. Plates incubated with goat Tg (0, D, and O) and with Tg antigens from the goitrous Dutch goats (18) (•, • , and • ) in dilution from 0-5000 ng Tg/well and tested with ascites fluid from Mabs Bl (O and • ) , B2 (0 and • ) , and B3 ( • and • ) . The relative affinity of the ascites fluid dilution used was the same for each Mab.
probably the N-terminal Tg fragment containing T4 in the hormonogenic site (6, 7, 20). The other Mab of this group, B3, did not give a reaction with this Tg fragment. Together with the results that only the Mabs Bl and B2 reacted in the ELISA with goiter supernatant of Dutch goats (18), which contains an N-terminal Tg fragment (mol wt, 40,000), we suggest that Mabs Bl and B2 are directed against the N-terminal hormonogenic site, and B3 against one of the C-terminal hormonogenic sites. De Baets et al. (20) also described Mabs directed against the hormonogenic sites containing T4. However, these antibodies were sensitive to surface binding and could not be
Tgs with different iodination degrees (20 ng Tg/well) plotted against
the T4 content of the Tgs measured in a sandwich ELISA.
used in blotting experiments. In contrast, Mabs A3, Bl, B2, and B3 are useful for this purpose. When the reactivities of Mabs Bl, B2, and B3 are plotted against the T4 content of the various hTgs, as is shown in Fig. 5, Mabs B2 and Bl react with Tg with a T4 content of 1 mol/mol Tg, in contrast to Mab B3 that reacts only with Tg containing more T4. This may indicate that thyroid hormone is formed first at the N-terminal hormonogenic site of Tg and only at higher iodination degrees does hormone formation occur at one of the C-terminal sites. This suggestion is in agreement with the finding that residue 5 (the N-terminal hormonogenic site) is first iodinated (21). For more precise characterization of the Mabs described further epitope localization will be required.
Acknowledgments We wish to thank Janine de Randamie for the iodine determinations and the laboratory for endocrinology (head, Dr. E. Endert) for performing the RIAs.
References 1. Taurog A 1986 Hormone synthesis: thyroid iodine metabolism. In: Ingbar SH, Braverman LE (eds) Werner's The Thyroid, ed 5. Lippincott, Philadelphia, pp 53-97 2. Deme D, Gavaret JM, Pommier J, Nunez J 1976 Maximal number of hormonogenic iodotyrosine residues in thyroglobulin iodinated by thyroid peroxidase. Eur J Biochem 70:7-13 3. Chernoff SB, Rawitch AB 1981 Thyroglobulin structure-function. J Biol Chem 256:9425-9430 4. Maurizis JC, Marriq C, Rolland M, Lissitzky S 1981 Thyroid hormone synthesis and reactivity of hormone-forming tyrosine residues of thyroglobulin. FEBS Lett 132:29-32 5. Marriq C, Arnaud C, Rolland M, Lissitzky S 1980 An approach to the structure of thyroglobulin. Eur J Biochem 111:33-47 6. Dunn JT, Dunn AD, Heppner DG, Kim PS 1981 A discrete thyroxine-rich iodopeptide of 20,000 daltons from rabbit thyroglobulin. J Biol Chem 256:942-947 7. Dunn JT, Kim PS, Dunn AD 1982 Favored sites for thyroid
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 04:29 For personal use only. No other uses without permission. . All rights reserved.
Mabs AGAINST hTg
8.
9. 10. 11. 12. 13. 14.
hormone formation on the peptide chains of human thyroglobulin. J Biol Chem 257:88-94 Marriq C, Rolland M, Lissitzky S 1982 Structure-function relationship in thyroglobulin: amino acid sequence of two different thyroxine-containing peptides from porcine thyroglobulin. EMBO J 4:397-401 Mercken L, Simons MJ, Swillens S, Massaer M, Vassart G 1985 Primary structure of bovine thyroglobulin deduced from the sequence of its 8,431 base complementary DNA. Nature 316:647-651 Malthiery Y, Lissitzky S 1987 Primary structure of human thyroglobulin deduced from the sequence of its 8,448 base complementary DNA. Eur J Biochem 165:491-498 Dunn JT, Anderson PC, Fox JW, Fassler CA, Dunn AD, Hite LA, Moore RC 1987 The sites of thyroid hormone formation in rabbit thyroglobulin. J Biol Chem 262:16948-16952 Lamas L, Taurog A, Salvatore G, Edelhoch H 1974 Preferential synthesis of thyroxine from early iodinated tyrosyl residues in thyroglobulin. J Biol Chem 249:2732-2737 Haraguchi K, Endo T, Onaya T, Sho K, Ohmiya Y, Kondo Y 1988 Evidence for a preferential iodination site within the thyroglobulin molecule. Mol Cell Endocrinol 59:111-115 Sho K, Hayashi H, Ohmiya Y, Kondo Y 1988 Preferential formation of triiodothyronine residues in newly synthesized [14C]tyrosine-labeled thyroglobulin molecules in follicles reconstructed in a
15.
16. 17. 18.
19. 20.
21.
3165
suspension culture of hog thyroid cells. Mol Cell Endocrinol 59:117-124 Van Voorthuizen WF, De Vijlder JJM, Van Dijk JE, Tegelaers WHH 1978 Euthyroidism via iodide supplementation in hereditary congenital goiter with thyroglobulin deficiency. Endocrinology 103:2105-2111 De Vijlder JJM, Van Voorthuizen WF, Van Dijk JE, Rijnberk A, Tegelaers WHH 1978 Hereditary congenital goiter with thyroglobulin deficiency in a breed of goats. Endocrinology 102:1214-1222 Chopra IJ 1972 A radioimmunoassay for measurement of thyroxine in unextracted serum. J Clin Endocrinol Metab 34:938-947 Sterk A, Van Dijk JE, Veenboer GJM, Moorman AFM, De Vijlder JJM 1989 Normal-sized thyroglobulin messenger ribonucleic acid in Dutch goats with a thyroglobulin synthesis defect is translated into a 35,000 molecular weight N-terminal fragment. Endocrinology 124:477-483 Kohler G, Milstein C 1975 Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256:495-497 De Baets MH, Theunissen R, Kok K, De Vijlder JJM, Van BredaVriesman PJC 1987 Monoclonal antibodies to human thyroglobulin as probes for thyroglobulin structure. Endocrinology 120:11041111 Palumbo G, Gentile F, Condorelli GL, Salvatore G 1990 The earliest site of iodination in thyroglobulin is residue number 5. J Biol Chem 265:8887-8892
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 04:29 For personal use only. No other uses without permission. . All rights reserved.
Vol. 127, No. 6 Printed in U.S.A.
Generation and Characterization of Monoclonal Antibodies Directed against Noniodinated and Iodinated Thyroglobulin, among which Are Antibodies against Hormonogenic Sites* M. T. DEN HARTOG, M. DE BOER, G. J. M. VEENBOER, AND J. J. M. DE VIJLDER Department of Experimental Pediatric Endocrinology, University of Amsterdam, Academic Medical Center (M. T.D.H., G.J.M. V., J.J.M.D. VJ, and the Laboratory of Biochemistry, Biotechnology Center, University of Amsterdam (M.D.B.), Amsterdam, The Netherlands
ABSTRACT. In this paper we describe the preparation and characterization of three different groups of monoclonal antibodies (Mabs) raised against iodinated and noniodinated human thyroglobulin (hTg). One group (A) of three Mabs is directed against hTg without discrimination between different iodine contents of Tg. The second group (B) of three Mabs has a higher affinity for iodinated Tg than for noniodinated Tg, and the Mabs are not species specific. The last group (C) of two Mabs generated against noniodinated hTg shows a higher affinity for
T
O STUDY the mechanism of thyroid hormone formation and mutations in this process, characteristics of thyroglobulin (Tg) are examined. Tg is a dimeric protein of 660,000 mol wt. The main function of Tg is the formation of thyroid hormones, T4 and T 3 (1). This hormone formation starts with the iodination of tyrosine residues in Tg (1). Coupling of two diiodotyrosine (DIT) residues synthesizes T4, and the coupling of a monoiodotyrosine (MIT) residue with a DIT residue gives T 3 or rT 3 (2-4). Each polypeptide chain of Tg contains at least four sites for hormone formation, termed hormonogenic sites (3, 5-8). One hormonogenic site is located at the Nterminal end of the Tg molecule, and the other three are located at the C-terminal end (9, 10). For rabbit Tg, Dunn et al. (11) described an extra T4-forming site at residue 1291. They did not detect the T4-forming site at residue 2569 described to be present in Tg from other species. For hormonogenesis Tg is iodinated in a prefer-
Received June 7,1990. Address requests for reprints to: Dr. M. T. Den Hartog, Department of Experimental Pediatric Endocrinology, University of Amsterdam, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. * This work was supported in part by the Netherlands Organization for Scientific Research (NWO) and the Ludgardine Bouwman Stichting.
noniodinated hTg than for iodinated hTg. From competition experiments with T4 we conclude that the second group of Mabs is directed against hormonogenic sites in the protein. From these Mabs, probably two are directed against the N-terminal hormonogenic site, and one against one of the C-terminal hormonogenic sites in Tg. Reactivity of the Mabs of group B with Tgs of various degrees of iodination indicates that the N-terminal T4 is formed first. (Endocrinology 127: 3160-3165,1990)
ential way. Early iodinated tyrosyl residues are preferentially involved in iodothyronine synthesis (4, 12-14). Even at a low degree of iodination, Tg forms thyroid hormone (12). At a higher degree of iodination, more tyrosines are iodinated, but the efficiency of thyroid hormone formation diminishes. In this paper we describe the generation and characterization of monoclonal antibodies (Mabs) against Tg, among which are Mabs against at least two hormonogenic sites. Our results indicate that the N-terminal T4 in Tg is formed first. Materials and Methods Tg Tg was isolated as described by Van Voorthuizen et al. (15). The iodine content of the Tg preparation was determined as described by De Vijlder et al. (16). We have isolated human Tg (hTg) with degrees of iodination of 0.00%, 0.20%, 0.53%, and 0.63% (wt/wt) from human thyroids obtained from patients with a total organification defect or larynx carcinoma and after obduction. With a RIA (17) we have determined the T4 content of the isolated hTg preparations after hydrolysis with pronase (10%, wt/wt; 48 h; 37 C) and leucine aminopeptidase (20%, wt/ wt; 24 h; 37 C). The T4 contents were, respectively, 0,1, 5, and 6 mol T4/mol Tg. We have also isolated Tg from dog, rat, mouse, goat, and bovine with degrees of iodination of 1.00%,
3160
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 04:29 For personal use only. No other uses without permission. . All rights reserved.
Mabs AGAINST hTg 0.82%, 0.22%, 0.59%, and 0.00%, respectively.1 We also had available 100,000 X g goiter supernatant from Dutch goats treated with T4 (18). These goats do not synthesize 330,000 mol wt Tg monomers, but only a glycosylated 40,000 mol wt Nterminal Tg fragment.
Antibodies The hybridomas producing the Mabs were generated by fusion with NS-1 myeloma cells of splenocytes from two groups of BALB/c mice immunized with hTg with an iodination degree of 0.00% or 0.63% (19). After limited dilution selection of these fusions, eight useful hybridomas were obtained. The experiments were performed with cell culture supernatants. Supernatants were obtained by culturing the hybridomas for 3 days without refreshing the medium. The supernatants were stored in 10-ml tubes at -20 C. Ascites fluid was obtained by injection of the hybridomas in the peritoneal cavity of BALB/c mice. The ascites fluid was heat inactivated at 56 C for 20 min. The glycoproteins were removed by dextran sulfate precipitation. After dialysis against PBS, aliquots were stored at -20 C. The monoclonal antibody affinity from the ascites fluid was measured using an enzyme-linked immunosorbant assay (ELISA) method. The 96-well polyvinyl plates (Falcon, Oxnard, CA) were coated with 1:1000 diluted goat [antimouse immunoglobulin G (IgG)] polyclonal antiserum from Sigma (St. Louis, MO). These plates were incubated with the ascites fluid in dilution. The relative affinity is defined as the dilution of the ascites fluid in the ELISA at which the Mab is not the limiting factor. A polyclonal antiserum against hTg (iodination degree, 0.20%, wt/wt) was raised in rabbits.
3161
well. After incubation (20 min; 20 C) the fluorescence was measured with a fluorscan Titertek. For determination of the isotypes of the Mabs, 96-well polyvinyl plates (Falcon) were used. The isotype-specific conjugates were goat antimouse IgG, goat antimouse IgM, goat antimouse IgGl, goat antimouse IgG2a, goat antimouse IgG2b, and rabbit antimouse Ig. Peroxidase was bound to all conjugates. They were obtained from Sigma (St. Louis, MO), except the last one which was from Dako-patts (Glostrup, Scotland). As substrate we used tetramethylbenzidine from Sigma. The bound peroxidase activity was revealed by the addition of 100 (A 0.25 mM tetramethylbenzidine in 0.1 M sodium phosphate buffer (pH 6.0) with 20 mM H2O2. The reaction was stopped after 15 min by the addition of 50 jtl 2 M H2SO4. The 450 nm absorbance was measured with a Titertek Flow Multiscan. ELISA for the study of monoclonal cross-reactivity with Tg from different species Nunc 96-well plates were coated with 20 ng Tg/well. The wells were filled with 100 /il of the appropriate Tg concentration in PBS and allowed to dry overnight at 37 C. The Tgs tested originated from human (two different iodine contents), dog, goat, cat, mouse, and bovine. After washing, undiluted supernatant of each monoclonal was added, and the plates were incubated (1 h; 37 C). Subsequently, the plates were, after another washing, incubated (1 h; 37 C) with the conjugate rabbit antimouse Ig conjugated to alkaline phosphatase, as described in the protocol for the sandwich ELISA. Bound phosphatase activity was revealed by the addition of 4-methylumbeffiferyl phosphate (Sigma). Competition experiment with T4
Sandwich ELISA The reactivity of the Mabs with hTg preparations of various iodination degrees and antibody isotypes was determined using a solid phase sandwich enzyme immunoassay. A 1:1000 dilution in PBS of an IgG fraction (obtained by using protein-A-Sepharose; Pharmacia, Uppsala, Sweden) of the polyclonal antiserum against hTg was adsorbed to the wells of 96-well microtiter plates (Nunc, Roskilde, Denmark; 2 h; 37 C). Unbound antibody was removed by washing the plates with PBS containing 0.05% Tween (wt/wt; 3 times) and demineralized water (once). Subsequently, hTg of known iodine content was added in various dilutions and incubated (16 h; 4 C). After washing, 100 n\ of the Mab to be tested (undiluted supernatant) were added to each well and incubated (2 h; 37 C). After another washing the plates were incubated (1 h; 37 C) with rabbit antimouse Ig conjugated to alkaline phosphatase (Dako-Immunoglobulines, Copenhagen, Denmark). This conjugate was used in a 1:1000 dilution in PBS-Tween with 1% (wt/wt) BSA to prevent aspecific binding. After washing thoroughly with PBS-Tween (5 times) and demineralized water (twice), 100 /*1 4 mM 4-methylumbeffiferyl phosphate in 0.1 M diethanolamine buffer, pH 10.3, containing 1 mM MgCl2 were added to each 1 Bovine thyroglobulin with an iodination degree of 0.00% was taken from an animal that had presumably been treated with antithyroid drugs.
Microtiter plates (96-well; Nunc) were coated with the polyclonal antiserum in a 1:1000 dilution in PBS, as described for the sandwich ELISA. After incubation and washing the plates were coated with 40 ng hTg/well, with a iodine content of 0.63%. After incubation (16 h; 4 C), the plates were washed, and blocking was performed by incubation (1 h; 37 C) with a 1% (wt/vol) BSA solution in PBS. After washing, the plates were incubated with 50-fil T4 (Sigma) solutions, ranging from 500-0 ng T4/well, together with 50 n\ of the supernatant of each Mab. After incubation (2 h; 37 C), the plates were washed and incubated with the conjugate, as described for the sandwich ELISA, using 4-methylumbeffiferyl phosphate as substrate. Electrophoresis and immunoblotting hTg (iodine content, 0.20%) was treated with 0.8% sodium dodecyl sulfate (SDS) and 2% 2-mercapthoethanol (5 min; 100 C). This (2 ng/lane) was run on a 7.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE; 2 h; 100 V). After electrophoresis the gels were electroblotted to a nitrocellulose filter in 25 mM Tris-HCl, 192 mM glycine, and 20% methanol, pH 8.3 (1 h; 100 V; 4 C). The mol wt standards for SDS-PAGE were obtained from Sigma. After blotting, the nitrocellulose filters were blocked with 1% BSA in PBS-Tween (1 h; room temperature). Incubation with the Mabs (ascites fluid) was performed overnight in PBS-Tween with 0.1% BSA also at room temper-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 04:29 For personal use only. No other uses without permission. . All rights reserved.
Mabs AGAINST hTg
3162
ature. After washing with PBS-Tween (five times) coupling of 1:3000 diluted horseradish peroxidase-conjugated rabbit antimouse total Ig (Dako-Patts) in PBS-Tween was performed for 1 h at 20 C. The filters were washed with PBS-Tween (five times) and PBS (once) and 10 min in 50 mM Tris-HCl, pH 7.6. They were stained with 1.4 mM diaminobenzidine tetrahydrochloride (Sigma) in 50 mM Tris-HCl, pH 7.6, with 3 mM H2O2. The reaction was stopped after 15 min by the addition of 1% (vol/vol) HC1 and directly washed with water. Filters were air dried.
Endo • 1990 Voll27«No6
D co
o iz
Results All of the Mabs, coded Al, A2, and A3 (group A); Bl and B2 (group B); and Cl and C2 (group C), are of the IgGl isotype, except B3 (group B) which is a IgG2a, as determinated with specific polyclonal antisera as described in Materials and Methods. The reactivity of some of the Mabs against hTgs of various iodination degrees tested in a sandwich ELISA is shown in Fig. 1. The reactivity of Mab A3 is independent of the iodine and thyroid hormone content. For the Mabs Al and A2, similar curves were found (results not shown). The Mabs Bl, B2, and B3 have a higher affinity for iodinated than for low iodinated Tg, as can be seen for Mab B2 in Fig. 1. The Mabs Cl and C2 show a higher affinity for low iodinated than for high iodinated Tg, as shown in Fig. 1 for Mab C2. To study whether the epitopes were also present in Tg of other species than man, we have tested the reactivity of the Mabs against Tg of various animals. A direct ELISA was performed, because polyclonal antisera against Tg of the various species were not available. In Table 1 the results are shown. The Mabs that have a higher affinity for Tg with a higher iodine content also recognize dog, mouse, rat, and goat Tg. All of these Tgs had a high iodine content, bovine Tg did not react, since it was not iodinated. The other Mabs do not react with Tg of the various species tested. It is remarkable that Mabs Al and A2 and Cl and C2 did not recognize hTg as they did in the sandwich ELISA (Table 1, last two columns). To study whether Mabs Bl, B2, and B3 recognize hormonogenic sites in Tg we performed competition experiments with T4. From Fig. 2 it is clear that Mabs Bl, B2, and B3, which recognize higher iodinated Tgs, are the Mabs that are inhibited by T4. hTg with an iodine content of 0.20% (wt/wt) was blotted on nitrocellulose filters after SDS-PAGE under reduced conditions. The filters were incubated with the various Mabs. Mabs Al, A2, Cl, and C2 show little or no reaction with hTg. Mabs A3, Bl, B2, and B3 give various bands (Fig. 3). The patterns were different, indicating that the Mabs recognize different epitopes on Tg. All Mabs recognize bands with mol wt of 240,000,
20
5 1.25 0.31 ng Tg / well
0.07
o
to T3
(0
CO
D O
20
5
1.25
0.31
0.07
0
0.07
0
ng Tg / well
T5
ro
CO
ZJ
o
1.25
0.31
ng Tg / well
FIG. 1. Sandwich ELISA. Plates are coated with polyclonal anti-Tg serum incubated with hTg [iodine content, 0.00% (A-A), 0.20% (OO), 0.53% (+-+), or 0.63% (D-d)] in dilution from 0-80 ng Tg/well and cell culture supernatants from Mabs A3, B2, and C2.
200,000, and 180,000. It is remarkable that Bl (lane 3) and B2 (lane 4) recognize bands at 20,000 and 78,000; B3 (lane 5) does not show a Tg fragment of low mol wt. To study whether the Mabs of group B could react with an N-terminal Tg fragment we coated wells with
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 04:29 For personal use only. No other uses without permission. . All rights reserved.
Mabs AGAINST hTg
3163
TABLE 1. Cross-reactivity of Mabs with Tgs obtained from various species with various iodination degrees sandwich ELISA human human dog goat rat mouse bovine human human 0.00% 0.63% 0.00% 0.63% _ _ _ _ _ _ + + direct ELISA
Al A3 Bl B2 B3 Cl C2
+ -
+ + + + _ -
+ + + _ -
_ _ + + + + + + _ _ -
_ + + + / _ -
_ - _ -
+ + +
+ + + +
A direct ELISA was performed with plates coated with 20 ng Tg/ well human (iodine contents 0.00% and 0.63%), dog, goat, rat, mouse, and bovine origin (iodine contents as mentioned in Materials and Methods). + means that the detected signal was at least two times the background. On plates coated with BSA [1% (wt/vol)] or with the polyclonal antiserum against Tg (1/1000 diluted) alone, there was no signal detected with any of the Mabs. As reference the results of a sandwich ELISA with human Tg (iodine contents 0.00% and 0.63%) are given.
5OO
125
31.25
7.5
1.8
0.45 O
ng T4/well
B
normal goat Tg and the goitrous proteins (18). The results are shown in Fig. 4. Mabs Bl, B2, and B3 reacted with normal goat Tg, and Bl and B2, in contrast to B3, reacted with goitrous Tg.
Discussion Eight Mabs against Tg are described. These Mabs can be classified in three groups. The Mabs of groups A and B were obtained from mice that had been injected with Tg containing 0.63% (wt/wt) iodine. The Mabs of group C were from mice that had been immunized with noniodinated Tg. The Mabs Al, A2, and A3 (group A) recognize on each Tg an epitope that is not influenced by the iodine and thyroid hormone content and as a consequence do not distinguish between Tgs of various iodination degrees. Binding of Tg to polyvinyl and nitrocellulose diminished the reactivity of Mabs Al and A2 for Tg. This indicates that the reacting epitopes depend on the Tg configuration, which alters on binding to surfaces. Another possibility is that these epitopes are hidden by binding to a surface. The reactivity of the Mabs is not influenced when Tg is bound to precoated polyclonal antibody against Tg. From control experiments it was clear that the Mabs do not react with the polyclonal antiserum alone. The same effect is observed with Mabs Cl and C2. These Mabs have a higher affinity for low or noniodinated Tg than for iodinated Tg. When the conformation is changed by iodination (12) or denaturation, the recognition of these Mabs for Tg decreased strongly. Mabs Bl, B2, and B3 have higher affinities for iodinated Tg then for low iodinated Tg. Competition experiments with T4 indicate that these Mabs are most likely
125
31.25
7.5
1.8
0.45 O
ng T4/well FIG. 2. Competition experiment with T4. Sandwich ELISA performed with the polyclonal antiserum and hTg (40 ng/well) with a iodine content of 0.63%. Competition was performed by incubation of T4 together with supernatant from the Mabs Al ( • - • ) , A2 (D-D), A3 ( 0 0), and Bl (Y-V) in A and B2 (+-+), B3 (A-A), Cl (O-O), and C2
(•-•) in B. directed against epitopes near or in the thyroid hormonecontaining sites of the Tg molecule. From further competition studies we have found that MIT does not inhibit the Tg-Mabs interaction, while DIT, compared with T4, causes only a very slight inhibition (results not shown). As expected, we can conclude that the iodination degree of Tg also influences what kind of immune response will be induced. The hormonogenic peptides of Tg from various species are homologous, while other parts of the molecule show less homology (9, 10). In agreement with these facts we show that the three Mabs of group B all show cross-reactivity with Tgs from other species if this Tg is iodinated. Incubating a Western blot of hTg after SDS-PAGE with Mabs Bl and B2 showed several bands, among which was a band at about 20,000 mol wt. This is
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 04:29 For personal use only. No other uses without permission. . All rights reserved.
Mabs AGAINST hTg
3164
Endo • 1990 Vol 127 • No 6
10
Mr x 1000
1 2
3
4 5
180116 • 844836•*•
moles T 4 / mol Tg
26-
FIG. 5. Reactivity of Mabs B2 (+-+), Bl (D-D), and B3 (•-•) with
FIG. 3. Immunoblotting of hTg (iodine content, 0.20%) with ascites
fluid from Mabs A3 (lane 2), Bl (lane 3), B2 (lane 4), and B3 (lane 5). Lane 1, molecular weight markers (Sigma). Mabs Al, A2, C2, and Cl in the same experiment showed no reaction (results not shown). The relative affinity of the ascites fluid dilution used was the same for each Mab. Mr, Mol wt.
8 5OOO
1250
o
313
78
ng Tg/well FIG. 4. ELISA. Plates incubated with goat Tg (0, D, and O) and with Tg antigens from the goitrous Dutch goats (18) (•, • , and • ) in dilution from 0-5000 ng Tg/well and tested with ascites fluid from Mabs Bl (O and • ) , B2 (0 and • ) , and B3 ( • and • ) . The relative affinity of the ascites fluid dilution used was the same for each Mab.
probably the N-terminal Tg fragment containing T4 in the hormonogenic site (6, 7, 20). The other Mab of this group, B3, did not give a reaction with this Tg fragment. Together with the results that only the Mabs Bl and B2 reacted in the ELISA with goiter supernatant of Dutch goats (18), which contains an N-terminal Tg fragment (mol wt, 40,000), we suggest that Mabs Bl and B2 are directed against the N-terminal hormonogenic site, and B3 against one of the C-terminal hormonogenic sites. De Baets et al. (20) also described Mabs directed against the hormonogenic sites containing T4. However, these antibodies were sensitive to surface binding and could not be
Tgs with different iodination degrees (20 ng Tg/well) plotted against
the T4 content of the Tgs measured in a sandwich ELISA.
used in blotting experiments. In contrast, Mabs A3, Bl, B2, and B3 are useful for this purpose. When the reactivities of Mabs Bl, B2, and B3 are plotted against the T4 content of the various hTgs, as is shown in Fig. 5, Mabs B2 and Bl react with Tg with a T4 content of 1 mol/mol Tg, in contrast to Mab B3 that reacts only with Tg containing more T4. This may indicate that thyroid hormone is formed first at the N-terminal hormonogenic site of Tg and only at higher iodination degrees does hormone formation occur at one of the C-terminal sites. This suggestion is in agreement with the finding that residue 5 (the N-terminal hormonogenic site) is first iodinated (21). For more precise characterization of the Mabs described further epitope localization will be required.
Acknowledgments We wish to thank Janine de Randamie for the iodine determinations and the laboratory for endocrinology (head, Dr. E. Endert) for performing the RIAs.
References 1. Taurog A 1986 Hormone synthesis: thyroid iodine metabolism. In: Ingbar SH, Braverman LE (eds) Werner's The Thyroid, ed 5. Lippincott, Philadelphia, pp 53-97 2. Deme D, Gavaret JM, Pommier J, Nunez J 1976 Maximal number of hormonogenic iodotyrosine residues in thyroglobulin iodinated by thyroid peroxidase. Eur J Biochem 70:7-13 3. Chernoff SB, Rawitch AB 1981 Thyroglobulin structure-function. J Biol Chem 256:9425-9430 4. Maurizis JC, Marriq C, Rolland M, Lissitzky S 1981 Thyroid hormone synthesis and reactivity of hormone-forming tyrosine residues of thyroglobulin. FEBS Lett 132:29-32 5. Marriq C, Arnaud C, Rolland M, Lissitzky S 1980 An approach to the structure of thyroglobulin. Eur J Biochem 111:33-47 6. Dunn JT, Dunn AD, Heppner DG, Kim PS 1981 A discrete thyroxine-rich iodopeptide of 20,000 daltons from rabbit thyroglobulin. J Biol Chem 256:942-947 7. Dunn JT, Kim PS, Dunn AD 1982 Favored sites for thyroid
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 04:29 For personal use only. No other uses without permission. . All rights reserved.
Mabs AGAINST hTg
8.
9. 10. 11. 12. 13. 14.
hormone formation on the peptide chains of human thyroglobulin. J Biol Chem 257:88-94 Marriq C, Rolland M, Lissitzky S 1982 Structure-function relationship in thyroglobulin: amino acid sequence of two different thyroxine-containing peptides from porcine thyroglobulin. EMBO J 4:397-401 Mercken L, Simons MJ, Swillens S, Massaer M, Vassart G 1985 Primary structure of bovine thyroglobulin deduced from the sequence of its 8,431 base complementary DNA. Nature 316:647-651 Malthiery Y, Lissitzky S 1987 Primary structure of human thyroglobulin deduced from the sequence of its 8,448 base complementary DNA. Eur J Biochem 165:491-498 Dunn JT, Anderson PC, Fox JW, Fassler CA, Dunn AD, Hite LA, Moore RC 1987 The sites of thyroid hormone formation in rabbit thyroglobulin. J Biol Chem 262:16948-16952 Lamas L, Taurog A, Salvatore G, Edelhoch H 1974 Preferential synthesis of thyroxine from early iodinated tyrosyl residues in thyroglobulin. J Biol Chem 249:2732-2737 Haraguchi K, Endo T, Onaya T, Sho K, Ohmiya Y, Kondo Y 1988 Evidence for a preferential iodination site within the thyroglobulin molecule. Mol Cell Endocrinol 59:111-115 Sho K, Hayashi H, Ohmiya Y, Kondo Y 1988 Preferential formation of triiodothyronine residues in newly synthesized [14C]tyrosine-labeled thyroglobulin molecules in follicles reconstructed in a
15.
16. 17. 18.
19. 20.
21.
3165
suspension culture of hog thyroid cells. Mol Cell Endocrinol 59:117-124 Van Voorthuizen WF, De Vijlder JJM, Van Dijk JE, Tegelaers WHH 1978 Euthyroidism via iodide supplementation in hereditary congenital goiter with thyroglobulin deficiency. Endocrinology 103:2105-2111 De Vijlder JJM, Van Voorthuizen WF, Van Dijk JE, Rijnberk A, Tegelaers WHH 1978 Hereditary congenital goiter with thyroglobulin deficiency in a breed of goats. Endocrinology 102:1214-1222 Chopra IJ 1972 A radioimmunoassay for measurement of thyroxine in unextracted serum. J Clin Endocrinol Metab 34:938-947 Sterk A, Van Dijk JE, Veenboer GJM, Moorman AFM, De Vijlder JJM 1989 Normal-sized thyroglobulin messenger ribonucleic acid in Dutch goats with a thyroglobulin synthesis defect is translated into a 35,000 molecular weight N-terminal fragment. Endocrinology 124:477-483 Kohler G, Milstein C 1975 Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256:495-497 De Baets MH, Theunissen R, Kok K, De Vijlder JJM, Van BredaVriesman PJC 1987 Monoclonal antibodies to human thyroglobulin as probes for thyroglobulin structure. Endocrinology 120:11041111 Palumbo G, Gentile F, Condorelli GL, Salvatore G 1990 The earliest site of iodination in thyroglobulin is residue number 5. J Biol Chem 265:8887-8892
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 04:29 For personal use only. No other uses without permission. . All rights reserved.
