Immunology Letters, 32 (1992) 259 - 264 Elsevier IMLET 01786

New antigenic clusters on human thyroglobulin defined by an expanded panel of monoclonal antibodies Majida Bouanani, V6ronique Hanin, Madeleine Bastide and Bernard Pau Laboratoire d'lmmunologie et de Parasitologie, Facult~ de Pharmacie, CNRS, Montpellier, France (Received 20 February 1992; accepted 24 March 1992)

I. Summary Twenty-seven hybridomas secreting monoclonal antibodies (mAb) directed against new antigenic clusters on human thyroglobulin (hTg) were obtained by fusion of the mouse myeloma P3-X63Ag8 653 with spleen cells from BALB/c mice immunized with a mixture of hTg and six anti-hTg mAb with the aim of masking the corresponding antigenic clusters previously reported. Fourteen mAb were selected, produced in ascitic fluid and characterized. All these mAb were of the IgG1 subclass. Five new antigenic clusters on the hTg molecule were defined by the 14 mAb, extending the initial antigenic map of hTg to 11 clusters. These mAb were used in an attempt to probe the interaction between hTg and the autoantibodies from patients with Hashimoto's thyroiditis who do not recognize antigenic cluster II, a cluster whose recognition by anti-hTg autoantibodies is significantly associated with thyroid disorders. 2. Introduction Thyroglobulin (Tg), a major glycoprotein synthesized by the thyroid gland, is one of the autoantigen targets during thyroid autoimmune disease [1]. Among the various thyroid proteins, Tg is also Key words: Human thyroglobulin; Monoclonal antibody; Epitopic specificity Correspondence to: Dr. Majida Bouanani, Laboratoire d'Immunologie et de Parasitologie - CNRS UMR 9921, Facult6 de Pharmacie, 15 Av. Charles Flahault, 34060 Montpellier Cedex 01, France. Tel.: 67 63 52 02; Fax" 67 54 75 33.

the autoantigen responsible for experimental autoimmune thyroiditis [2]. Several investigators have studied the epitope mapping of human Tg (hTg) to understand the relationship between this molecule and the immune response [3 - 9]. Although 40 antigenic determinants were recognized by polyclonal rabbit antiserum [10] in a heterogeneous system, the use of monoclonal antibodies (mAb) has led to a better characterization of antigenic determinants of the hTg molecule. We have reported an antigenic map of hTg defined by 15 mAb directed against 6 antigenic clusters in hTg [11]. Other investigators have also used panels of mAb to characterize the antigenic sites on hTg molecule [3, 8, 9]. Furthermore, mAb to hTg are excellent probes to study the epitopic specificity of anti-hTg autoantibodies (aAb) present in thyroid disorders [3, 8, 9, 12, 13]. In previous studies using our panel of mAb, we demonstrated that the epitopic specificity patterns of anti-hTg aAb from patients with thyroid disease were different from those of healthy individuals [12]. Indeed, the anti-hTg aAb of these patients showed a restricted epiptopic specificity against a particular antigenic region, namely cluster II, whereas the natural anti-hTg aAb in the sera of healthy individuals were polyreactive, recognizing antigenic clusters III, IV and in particular cluster V [13]. Recently, we demonstrated that the sera from patients with both a primary SjSgren's syndrome and an associated thyroid disease strongly interact with region II as do the sera from patients with Hashimoto's thyroiditis [14]. These findings emphasize the critical importance of cluster II in autoimmune thyroid disease. However, 40°70 of the sera from patients with thyroid disease do not interact

0165-2478 / 92 / $ 5.00 © 1992 Elsevier Science Publishers B.V. All rights reserved

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with any of the antigenic clusters defined by our 15 mAb. These results suggest that there are other antigenic clusters on the hTg molecule. The aim of our present study was to produce new mAb to enlarge the initial antigenic map of hTg in an attempt to identify other antigenic regions that might be recognized by those patients with Hashimoto's thyroiditis whose sera do not interact with cluster II. To this end, two populations of Hashimoto's thyroiditis patients, with or without epitope II specificity, were used to determine antigenic regions complementary to cluster II. This study describes a new and expanded panel of mAb to hTg. A novel immunization protocol was used to obtain this panel. We obtained fourteen mAb directed against five additional antigenic clusters on the hTg molecule, extending the initial antigenic map of hTg to eleven clusters.

in an attempt to mask the antigenic regions previously determined. The first immunization was performed using complete Freund's adjuvant (CFA), it was followed by another immunization in physiological saline three weeks later.

3. Materials and Methods

The mouse sera and the mAb were tested for their binding capacity to hTg by the indirect ELISA method previously reported [13]. The reactivity of mouse sera and mAb against the initial six antigenic clusters was measured by an inhibition assay [14]. Inhibition was evaluated as follows: a decrease in absorbance of at least 80% compared with the absorbance in the absence of antibodies, was considered as total inhitibion, and a decrease of 30 - 80°70 as partial inhibition. A normal mouse serum without anti-hTg activity was used as the negative control for 0% inhibition. Maximum inhibition (100°70) for each AP-mAb was obtained with an excess of the same unlabeled mAb.

3.1. Patients Twenty sera from patients with Hashimoto's thyroiditis (HT) were provided by Dr. P. Montagne (School of Medicine, Nancy, France). These sera were divided into two pools on the basis of their epitope II specificity as determined by an inhibition assay (see below). Pool 1 was composed of 11 sera that had positive epitope II specificity (II +) and pool 2 was composed of 9 sera with negative epitope II specificity ( I I - ) .

3.4. Fusion and cell production Spleen cells were fused with P3-X63-Ag8 653 myeloma cells using a method adapted from Galen et al. [15]. Antibody-secreting hybridomas were subcloned by the limiting dilution technique [16] and then frozen in liquid nitrogen. Ascitic fluids were produced follwing intraperitoneal injection of cloned hybridoma cells into pristane-treated BALB/c mice. 3.5. Screening methods

3.2. Preparation of the thyroglobulin-monoclonal

antibody complexes HTg (10/zg) in phosphate buffered saline (PBS) pH 7.2 was preincubated for 18 h at 4 °C with either 10/zg (complex 1) or 5/zg (complex 2) of each of six anti-hTg mAb (1, 2, 4, 7, 10 and 12) representing the six antigenic clusters previously defined [7].

3.6. Purification and labeling of mAb MAb were purified from the ascitic fluids by affinity chromatography on protein A-Sepharose according to the instructions of the manufacturer. The purified mAb, labeled with alkaline phosphatase as described previously [11], were used for epitope mapping.

3.3. Immunization procedure 3.7. Determination of mAb subclass Two BALB/c mice (S1 and $2) were immunized subcutaneously with complex 1 (70 #g of total protein), two others ($3 and $4) were immunized with complex 2 (40/~g of protein) and two mice ($5 and $6) with 10/zg of hTg alone. This strategy was used 260

Isotypes of mAb were determined by ELISA using anti-mouse Ig subclass-rabbit biotinylated antibodies detected by a horseradish peroxidase-streptavidin conjungate (Amersham).

3.8. Affinity determination The association constant (Ka) of five mAb representative of each of the five new antigenic clusters was determined by a technique previously described [11].

sera from two of the mice immunized with the hTgmAb complexes compared with the sera from mice immunized with hTg alone ($5 and $6). Mice S1 and $4 were chosen for fusion because of their very low antibody activity against the initial six antigenic clusters and in particular region II.

3.9. Epitope mapping

4.2. Selection and characterization o f mAb

The antigenic clusters recognized by the new mAb were studied by competitive ELISA [11]. The ability of each unlabeled mAb to inhibit the binding of the same or other AP-mAb was determined by incubation with various amounts of unlabeled mAb followed by a constant amount of the same or other AP-mAb.

Based on the results of the indirect ELISA, 40 antibody-secreting hybridomas were selected for their binding to absorbed hTg. The inhibition assay was used to determine whether the epitopes recognized by these 40 antibody-secreting hybridomas were different from those previously defined. Among the 40 hybridomas, 27 secreted mAb that did not crossreact with any of the 15 mAb produced previously. Fourteen of these 27 mAb (mAb 21, 23 - 28, 30 - 35) were produced in ascitic fluids and purified. All these anti-hTg mAb were of the IgG1 isotype.

4. Results

4.1. Serum reactivity o f immunized mice The antibody activity and epitopic specificity of the sera from mice immunized with the hTg-mAb complexes or by hTg alone are shown in Figs. 1 and 2. The binding of all sera to hTg in the indirect ELISA was very high. No significant difference in antibody activity was observed between the sera of mice immunized with hTg alone and those of mice immunized with hTg-mAb complexes. Nevertheless, screening by the inhibition assay showed differences in the epitopic specificity towards the six antigenic regions (I to VI) previously defined. Indeed, the activity against cluster II decreased in the 2,0 ' ~ l ' ~ - - - , 4 a ~

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The hTg antigenic determinants recognized by the purified mAb were determined by competitive ELISA. The capacity of one mAb to inhibit another one was considered positive when the inhibition of the binding AP-mAb to adsorbed hTg was greater than 30%. In this way, five new antigenic clusters on the hTg molecule could be defined by the 14 mAb, extending the initial antigenic map of hTg to

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New antigenic clusters on human thyroglobulin defined by an expanded panel of monoclonal antibodies.

Twenty-seven hybridomas secreting monoclonal antibodies (mAb) directed against new antigenic clusters on human thyroglobulin (hTg) were obtained by fu...
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