Exp. Clin. Endocrinol. Vol. 97, No. 2/3, 1991, PP. 173-178

J. A. Barth, Leipzig

Antiidiotypic Antibodies against anti-TPO Antibodies in Sera of Patients with Autoimmune Thyroid Disorders BAiBARA Cz iiioci


With 3 Figures

Introduction Jeme (1974) postulated, that idiotypes (Id) and anti-idiotypes (anti-Id) constitute a complex network of interactions at the serum and lymphocyte levels. Anti-idiotypic antibodies that recognize and regulate the expression of Id determinants could theoretically play a key role in the induction of self-tolerance and the prevention of autoimmuni-

ty. Abnormalities of the Id-anti-Id network could, therefore, lead to expression or expansion of autoreactive cell clones and autoantibodies (Talai, 1978; Abdou, 1985). Anti-Id antibodies have been reported to appear spontaneously in humans in a variety of clinical conditions. Anti-Id were detected in myasthenia gravis (Dwyer et al., 1983), systemic lupus erythematosus (Zouali and Eyqem, 1983), reumathoid arthritis (Nasu et al., 1980), mixed cryoglobulinemia (Geltner et al., 1980), normal pregnancy (SuciuFoca et al., 1983), autoimmune thyroiditis (Sikorska, 1986). Because altered Id-anti-Id network might lead to the induction of autoimmune processes (Cooke et al., 1984), one could expect to find naturally occurring anti-Id antibodies to the variable regions of the anti-human thyroid peroxidase (anti-hTPO) autoantibodies in sera of patients with autoimmune thyroid disorders as a normal consequence of disease evolution. Anti-hTPO autoantibodies, by means of antibody dependent cell mediated cytotoxicity, might induce the pathogenic changes in the thyroid (Weetman and McGregor, 1984). Thus the mechanisms of regulation of this process could be very important to the management

of AITD. Materials and Methods Serum samples were obtained from 66 patients with AITD, randomly selected on the basis of anti-

hTPO autoantibodies titers; sex and age matched 40 healthy blood donors served as a control. All sera were inactivated by heating for 30 min at 56°C.

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Department of Biochemistry (Head: Prof. Dr. hab. Janusz Nauman), Medical Center of Postgraduate Education, Warsaw/Poland

Exp. Clin. Endocrinol. 97 (1991) 2/3


hTPO mAb) assisted column as previously described (Czarnocka et al., 1985). ELISA for anti-Id screening: Microtitration plates were coated with anti-hTPO mouse monoclonal antibodies at concentration 5 ng/ml overnight at 4°C. After washing and saturation remaining binding sites with BSA, the plates were incubated with either patient or normal control sera for 1.30 h at 37°C. HRP-labelled anti-human IgG was then added, and after 1.30 h incubation at 37°C, ABTS was added as substrate. The absorbance was read after one hour at 405 mn. Immunoglobulin fraction from human sera was purified by affinity chromatography on protein ASepharose. F(ab ') fragments were isolated from whole IgG molecules after digestion with pepsin coupled to Sepharose. Immunoadsorbents were prepared by coupling of irrelevant specificity mouse monoclonal antibody or normal BALB/c IgG to CNBr-activated Sepharose 4B. Anti-hTPO autoantibodies were adsorbed on hTPO coated nitrocellulose strips. Characteristics of mouse monoclonal anti-hTPO antibodies employed in this study were previously described (Ruf et al., 1989).


Antibody activities against mouse anti-hTPO mAbs in sera of patients and normal controls were measured by indirect ELISA procedure. Same patient sera with AITD had exhibited elevated (above upper limit of normal) binding to anti-hTPO mAbs (Fig. 1).


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n40 Fig. 1









Serum antibodies against mouse anti-hTPO monoclonal antibody in patients with AITD and

normal subjects assessed in ELISA. Results are expressed as absorbance at 405 nm. Sera were diluted 1/100.

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The anti-hTPO autoantibodies titer was measured by ELISA with highly purified hTPO coated microtitration plates. Human TPO was immunopurified on anti-hTPO monoclonal antibody (anti-


B. CZARNOCKA and J. NAUMAN, Anti-hTPO-anti-idiotype

The observed high absorbance values in ELISA were due to cross-reactivity between HRP-labelled anti-human 1gO second antibody and mouse IgG, but also to naturally occurring anti-mouse allotype anti-Id antibodies in sera of normal individuals and autoimmune patients. The majority of sera showed, to various degree, binding to both pooled normal BALB/c IgG and irrelevant specificity mouse monoclonal antibody. It was also found by others (Schroff et al., 1985; Schwartz et al., 1978) that significant level of anti-

mouse IgG was detected by ELISA in sera of normal individuals. It is possible, that

K antibody. Anti-hTPO autoantibodies were adsorbed on the hTPO coated 1.0-






E q,)


Ls o-Q E

mAb 47



Binding of F(ab ')2 fragment of the anti-Id positive patient serum to anti-hTPO mAB 64 in an ELISA. Plates were coated with idiotype or BALB/c IgG as a control at concentration 5 tg/ml and incubated with Fc and F(ab ')2 fragments of the anti-Id active serum of Fc and F(ab ')2 fragments of IgG of the normal individual. Fig. 2

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nonspecific interaction between the human and mouse antibodies occur via Fc receptors. The binding of IgG fragments prepared from positive patient sera to anti-hTPO mAb is demonstrated in Fig. 2. The whole sera were preadsorbed on the affinity column with coupled to CNBrSepharose 4B, an irrelevant specificity monoclonal BALB/c origin,


Exp. Clin. Endocrinol. 97 (1991) 2/3

nitrocellulose, and 1gO fraction purified on protein A-Sepharose column. F(ab ')2

fragments were isolated by pepsin digestion of IgG molecules. The specific binding was observed between F(ab' )2 fragments isolated from positive patient sera. Fc fragments of positive sera as well as F(ab' )2 and Fc fragments of normal controls did not react specifically with anti-hTPO mAbs. These results exclude the possibility that the binding assay was detecting classical reumathoid factor activity.











Titration curves of the AITD patient sera exhibiting the anti-Id activity. Microtitration plates were coated with anti-hTPO mAb 47 at a concentration 5 tg/ml. The data are expressed as A405nm, after subtraction of the background.

The binding of anti-Id positive sera to anti-hTPO mAbs was dose dependent. The typical titration curves of several positive patients sera are shown in Fig. 3. The amount of anti-idiotypic antibodies varied from serum to serum. The estimated titers were not high in a majority of positive sera, and ranged from 1/250 to 1/10000, however the relatively elevated titers were found in few sera, which were used for analysis.

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The isolated F(áb' )2 and Fc fragments were tested for the reaction with normal BALB/c immunoglobulins or isolated F(ab ')2. No significant binding was observed (Fig. 2). These results indicate that observed binding between anti-hTPO mAb and positive sera of patients with AITD was not due to isotypic or allotypic determinants of mouse immunoglobulins, therefore have the characteristics of anti-idiotypic antibodies.

B. Cz iuocic and J. NAUMAN, Anti-hTPO-anti-idiotype



This paper describes the incidence of anti-hTPO anti-idiotypic auto-antibodies in pa-

tients with autoimmune thyroid diseases that recognize Id on anti-hTPO mouse monoclonal antibodies. The antibody activities of 1gO from positive patient sera were not

reacted with Id on anti-hTPO monoclonal antibodies but not on monoclonal antibody of irrelevant specificity or normal BALB/c 1gO. It is unlikely that detected by ELISA binding was due to either free or complexed hTPO. The SDS-PAGE and blotting study (data not shown) indicated that positive sera did not contain hTPO. Moreover, removal of IgG fractions from sera with anti-idiotypic activity resulted in complete lost of binding to anti-hTPO mAbs. Fc fragments did not

tract with idiotypic antibodies. Demonstrated data suggest that anti-anti-hTPO antibodies may occur naturally during the course of AITD. The frequency of occurrence of

anti-idiotypic antibodies was rather high; 22 out of 66 tested patient sera had antiidiotypic autoantibodies. Shared idiotypy between mouse and human antibodies of the same specificity has also been reported by others (Dwyer et al., 1983; Schwartz et al., 1978). The broad cross-reactivity among anti-hTPO monoclonal antibodies suggests that similar idiotypic determinants exist in the anti-hTPO antibodies tested. The expression of common Id on antibodies and autoantibodies from different species implies structural similarities between the binding site of these antibodies and could suggest that hypervariable region genes responsible for the particular Id expression have been evolutionary highly conserved. Acknowledgements. This work was supported by MZ-X Grant from the Ministry of Health and Social Welfare. We thank to Drs. J. Ruf and P. Carayon for their generous gifts of monoclonal anti-hTPO antibodies.

Ñeference [1] ABoUDU, N. I.: The idiotype-anti-idiotype network in human autoimmunity. J. Clin. Immunol. 5 (1985) 365-369. [21 CooI, A.; LYDYARD, P. M.; R0ITT, M. I.: Autoimmunity and idiotypes. Lancet 2(1984)723-727. CZARNOCKA, B.; Rus, J.; FERRAND, M.; CARAYON, R; LISSITZKY, S.: Purification of the human

thyroid peroxidase and its identification as the microsomal antigen involved in autoimmune thyroid diseases. FEBS Lett. 190 (1985) 147-152. DWYER, D. S.; BRADLEY, R. J.; URGUHATR, K. C.; KERNEY, J. E: Naturally occurring anti-idiotypic

antibodies in myasthenia gravis patients. Nature 301 (1983) 611-614. GELTNER, D.; FRIu.IN, C.; FRANGIONE, B.: Antiidiotypic activity in the 1gM fractions of mixed cyroglobulins. J. Immunol. 125 (1980) 1530-1535.

JERNE, N. K.: Towards theory of the immune system. Ann. Immunol. (Paris) 125C (1974) 373-389. NASU, H.; CHIA, S.; KNUTSON, D. W.; BARNETT, E. V.: Naturally occurring human antibodies to the

F(ab ')2 portion of 1gO. Clin. Exp. Immunol. 42 (1980) 378-386. Ruiz, J.; 1bUBERT, E.-M.; CzARJ.Iocic&, B.; DURAND-GORDE, M.-J.; Faiu&i.m, M.; CARAYON, P.:

Relationship between immunological structure and biochemical properties of human thyroid peroxidase. Endocrinology 125 (1989) 1211-1218. SIKORSKA, M. H.: Anti-thyroglobulin anti-idiotypic antibodies in sera of patients with Hashimoto's thvroiditis and Graves' disease. J. Immunol. 137 (1986) 3786-3795.

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directed toward mouse allotype or isotype, since positive sera or F(ab')2 fragments


Exp. Clin. Endocrinol. 97 (1991) 2/3 Scisaopp, R. W.; FooN, K. A.; BEATTY, S. M.; OLDEAM, R. K.; MoROAI, A. C.: Human antimurine immunoglobulic responses in patients receiving monoclonal antibody therapy. Cancer Res.

45 (1985) 879-885. SCHWARTZ, M.; NOVICK, D.; GIVOL, D.; FUSCH, S.: Induction of anti-idiotypic antibodies by im-

munisation with syngeneic spleen cells educated with acetyicholine receptor. Nature 273 (1978)

543-545. HLA receptors induced by pregnancy. Proc. Natl. Acad. Sci. USA 80 (1983) 830-834. TAi..aj., N.: Autoimmunity and the immunologic network. Arth. Rheum. 21 (1978) 853-861. WEETMAN, P. A.; McG1GoR, M. A.: Autoimmune thyroid disease; developments in our understanding. Endoc. Rev. 5 (1984) 309-355. ZOUALI, M.; E'YQUEM, A.: Idiotypic-anti-idiotypic interactions in SLE: demonstration of oscillary levels of anti-DNA autoantibodies and reciprocal anti-idiotypic activity in a single patient. Ann. Immunol. (Paris) 134C (1983) 377-391.

Author's address: Dr. B. CzAnocc, Department of Biochemistry, Medical Center of Postgraduate Education, Marymoncka 99, PL-01-813 Warsaw

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[121 SCIU-FOCA, N.; REED, E.; ROHOWSKY, C.; KUNO, P.; KINo, D. W.: Anti-idiotypic antibodies to anti-

Antiidiotypic antibodies against anti-TPO antibodies in sera of patients with autoimmune thyroid disorders.

Exp. Clin. Endocrinol. Vol. 97, No. 2/3, 1991, PP. 173-178 J. A. Barth, Leipzig Antiidiotypic Antibodies against anti-TPO Antibodies in Sera of Pati...
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