Avidity of thyroglobulin antibody in sera from patients with Hashimoto s thyroiditis with different thyroid functional statuscei_

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1 Clinical and Experimental Immunology ORIGINAL ARTICLE doi:1.1111/j x Avidity of thyroglobulin antibody in sera from patients with Hashimoto s thyroiditis with different thyroid functional statuscei_ Y. Zhang, Y. Gao, M. Li, L. Xie, Y. Huang, Y. Gao and X. Guo Department of Endocrinology, University First Hospital, Beijing, China Accepted for publication 15 February 21 Correspondence: Y. Gao, Department of Endocrinology, Peking University First Hospital, Beijing 134, China. bjgaoying@yahoo.com [Correction added after online publication 3 June 21: Errors in Figure 2 and Figure 3 were corrected.] Summary The mechanism of disease progression in Hashimoto s thyroiditis (HT) is still unclear. Thyroglobulin antibody (TgAb) is a diagnostic hallmark of HT. The aim of our study was to evaluate the avidity of TgAb in sera from HT patients with different thyroid functional status. Sera from 5 patients with newly diagnosed HT were collected and divided into three groups according to thyroid function: patients with hypothyroidism (H, n = 18), subclinical hypothyroidism (sh, n = 18) and euthyroidism (Eu, n = 14). Titres and avidity of TgAb were determined by enzyme-linked immunosorbent assays (ELISAs). Avidity constant (ak) was determined as the reciprocal value of the thyroglobulin molar concentration in the liquid phase resulting in 5% inhibition of TgAb binding to thyroglobulin in solid-phase ELISAs. The titres and ak of TgAb were performed using log-transformation, and expressed as lgt and lgak, respectively. Mean lgt of TgAb in sera was in H, in sh, and in Eu, respectively. The median avidity of TgAb was in H, in sh, in Eu, respectively. lgt and lgak of TgAb were at significantly lower levels in Eu than in sh and H (P < 5). Correlation was found between lgt and lgak (r = 594, P < 5). lgak was also related to TSH (r= 38, P < 5). Our study indicated that patients with high-avidity TgAb might be at high risk of developing subclinical, even to overt, hypothyroidism. Keywords: avidity, Hashimoto s thyroiditis, thyroglobulin antibody Introduction Autoimmune thyroid diseases (AITD) are among the most frequent organ-specific autoimmune diseases, including Hashimoto s thyroiditis (HT), Graves disease, atrophic thyroiditis, primary myxoedema and post-partum thyroiditis [1]. Thyroglobulin antibody (TgAb) in Hashimoto s disease was first described by Doniach and Roitt in 1956 [2]. Approximately 82 1% of patients with HT are TgAbpositive [3] and it is an important sera marker of HT. In clinical practice, HT patients with high titres of sera TgAb manifested differently in thyroid functional status; some were euthyroid, some subclinical hypothyroid and some even overtly hypothyroid. The exact mechanism of disease progression in HT is not clear. Our previous work revealed that the predominant TgAb immunoglobulin (Ig)G subclasses in sera from HT patients were IgG1, IgG2 and IgG4, and patients with high titres of IgG1 and IgG2 might be at high risk of developing overt hypothyroidism, indicating that the distribution of IgG subclasses of TgAb might be involved in the disease progression of HT [4]. Avidity is another immunological autoantibody characteristic. We speculated that studies on TgAb avidity might also provide some useful information on the pathogenesis of HT and might help us to identify the HT patients at high risk of progressing to thyroid function failure. The aim of our study was to evaluate TgAb avidity in sera from patients with HT of different thyroid functional status. Patients and methods Study population A total of 5 patients with newly diagnosed HT in Peking University First Hospital during August 25 to November 26 were collected on the basis of high levels of TgAb ( 65% of a known strong positive control) in the current study. Diagnosis was made based on the presence of a firm, symmetrically enlarged thyroid. None of the patients had other autoimmune diseases, such as pernicious anaemia, 21 British Society for Immunology, Clinical and Experimental Immunology, 161:

2 Y. Zhang et al. systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), type 1 diabetes mellitus or any evidence of co-existent pregnancy or tumour. Patients receiving anti-thyroid drugs or l-thyroxine therapy were also excluded. There was no evidence of hereditary and acquired variations in the concentrations of thyroxine-binding globulin in sera from the patients. This study complied with the Helsinki Declaration and was approved by the Ethics Committee of Peking University First Hospital. All patients gave written informed consent. According to thyroid function, all 5 patients were divided into three groups: patients with hypothyroidism (H) (n = 18, three males, 15 females), subclinical hypothyroidism (sh) (n = 18, four males, 14 females) and euthyroidism (Eu) (n = 14, two males, 12 females). Detection of thyroid function Serum samples were collected at diagnosis and kept frozen at -2 C until use. Chemiluminescence immunoassays were used to detect total triiodothyronine (TT3), total tetraiodothyronine (TT4) and thyroid-stimulating hormone (TSH) [ADVIA Centaur (Bayer Healthcare Diagnostics, Tarrytown, NY, USA)]. Detection of TgAb titres TgAb was measured by enzyme-linked immunosorbent assay (ELISA). Costar microtitre plates (Data Packaging Corporation, Spencer, MA, USA) were coated with 2 mg/ml human thyroid thyroglobulin (Tg) (Calbiochem Merck KGaA, Darmstadt, Germany) in 5 mol/l carbonate/ bicarbonate buffer, ph 9 6 at 37 C for 1 h. The volume in each well was 1 ml in all steps and every sample was added in duplicate. Every plate contained a positive control, a negative control and blank controls. The plates were washed three times with phosphate-buffered saline (PBS) containing 1% Tween 2 (PBST) between stages. The wells were blocked with 3% bovine serum albumin (BSA, Sigma-A73; Sigma, St Louis, MO, USA) for 1 h at 37 C, then sera from patients were diluted to 1 : 4 with PBST and incubated for 3 min at 37 C; the binding was revealed with a peroxidaseconjugated goat anti-human IgG (H+L) (Zhongshan Golden Bridge Biotechnology Co. Ltd., Beijing, China) diluted at 1 : 5, followed by the addition of a mixture of diaminobenzidine, citric acid and 3% hydrogen peroxide; the reaction was stopped with 1 mol/l hydrochloric acid after 1 min. The results were recorded as optical density at 49 nm (Bio-Rad model 68, Hercules, CA, USA). Samples were considered positive if they exceeded mean 3 standard deviations from 1 euthyroid sera in normal blood donors (no clinical, autoantibody evidence of thyroid disease). The TgAb titre was defined as the maximum serum dilution from 1 : 4 to 1 : 49 6 giving a positive binding, and was expressed as logarithm value (lgt). Avidity constant determination The avidity constant (ak) was determined by antigeninhibition ELISAs. Briefly, as shown in Fig. 1a, the serum concentration required for competitive assay was first (a) 12 Percentage of OD of positive control (b) 7 Percentage of OD of positive control c Serum dilution of thyroglobulin antibody b Tg, μg/ml Fig. 1. (a) Determination of the patient s serum dilution giving about 65% at the maximum binding of thyroglobulin antibody (TgAb) in the standard thyroglobulin (Tg) enzyme-linked immunosorbent assay (ELISA). The titres of sera 1, 2 and 3 were 1 : 12 4, 1 : 24 8 and 1 : 49 6, respectively. We chose 65% as the starting-point. (b) Inhibition profiles of TgAb. Inhibition of TgAb binding to Tg in the solid phase by increasing concentration of fluid-phase Tg. The apparent affinity constant (ak) was determined as the reciprocal value of Tg molar concentration resulting in 5% inhibition of TgAb binding to Tg in the fluid-phase ELISA. ak was, respectively, , and l/mol for sera a, b and c. a British Society for Immunology, Clinical and Experimental Immunology, 161: 65 7

3 TgAb avidity in sera from HT patients determined for each patient as the serum dilution giving approximately 65% of the maximum binding in the standard Tg ELISA. The competitive binding assay was performed by incubating the diluted patients sera with increasing amounts of Tg (from 1 mg/ml to 1 mg/ml) in PBST for 2 h at 37 C. The diluted sera giving approximately 65% of the maximum binding with and without Tg inhibition were then both transferred to Tg-coated plates for the standard ELISA procedure. The TgAb binding was expressed as the percentage of control binding determined in the absence of fluidphase Tg. ak was determined as the reciprocal value of molar concentration of Tg in the liquid phase resulting in 5% inhibition of TgAb binding (Fig. 1b), and was calculated using the following formula: ak (l/mol) = 66 KDa [1/Tg (mg/ml)] 1 6. Titres of thyroglobulin antibody Eu P < 5 P < 5 sh Group H Statistical analysis The titre and ak of sera TgAb were performed using log transformation and expressed as lgt and lgak, respectively. The comparison was performed using the independent Student s t-test, one-way analysis of variance (anova) and Pearson s test to compare quantitative parameters. The spss version 11 5 statistical analysis program (SPSS Inc., Chicago, IL, USA) was used. A P-value less than 5 was considered to have significant difference. Result Demographic data Nine of the 5 HT patients with high titres of TgAb were male and 41 were female, with an average age of (14 82) years. No significant difference could be found in age and gender distribution between the three groups. Table 1 summarizes the levels of TT3, TT4 and TSH, TgAb titre and avidity in the three groups. TSH was of significantly higher levels in H than in sh and Eu (P < 5, respectively); TT3 and TT4 levels in the H group were significantly lower than those in the other two groups (P < 5, respectively). Fig. 2. Thyroglobulin antibody (TgAb) titres in sera of different groups. The transverse lines represent median and quartiles, respectively. Eu, euthyroidism; H, hypothyroidism; sh, subclinical hypothyroidism. Titres and avidity of TgAb Titres of TgAb in all three groups are shown in Fig. 2. As shown in Table 1, mean lgt of TgAb was in H, in sh, and in Eu, respectively. lgt was of significantly lower level in Eu than in H and sh (P < 5, respectively), and lgt in sh was significantly lower than that in H (P < 5.). The median avidity of TgAb was in H, in sh and in Eu, respectively (Fig. 3). lgak of TgAb was of significantly lower level in Eu than in H and sh (P < 5, respectively), and there was no significant difference between H and sh (P > 5). Correlation was found between lgak and lgt (r = 594, P < 5). In the H and sh groups, lgak was related to lgt (r = 563, P < 5; r = 771, P < 5, respectively); however, no correlation was found in the Eu group (r = 81, P > 5). Figure 4 shows the change of lgak/lgt according to lgt. Although no significant difference of lgak/ lgt was found in these three groups, there was a trend towards patients with thyroid dysfunction having a higher avidity than euthyroid patients in the case of the similar Table 1. The levels of total triiodothyronine (TT3), total tetraiodothyronine (TT4), thyroid stimulating hormone (TSH), and thyroglobulin antibody (TgAb) levels and avidity in sera from patients with H, sh and Eu. ak ( 1 7 l/mol) Group n TSH (miu/l) TT3 (nmol/l) TT4 (nmol/l) lgt Median Range lgak H *, ** *, ** *, ** *, ** ** sh ** ** Eu *P < 5 compared with sh; **P < 5 compared with Eu. Reference value range: TT3: nmol/l; TT4: nmol/l; TSH: miu/l. ak, avidity constant of TgAb; Eu, euthyroidism; H, hypothyroidism; lgak, logarithm value of avidity constant of TgAb; lgt, logarithm value of TgAb titre; sh, subclinical hypothyroidism. 21 British Society for Immunology, Clinical and Experimental Immunology, 161:

4 Y. Zhang et al. ak of thyroglobulin antibody (1 7 mol/l 1 ) Eu P < 5 P < 5 sh Group Fig. 3. Avidity constant of thyroglobulin antibody (TgAb) in different groups. The transverse lines represent median and quartiles, respectively. Eu, euthyroidism; H, hypothyroidism; sh, subclinical hypothyroidism. titres of TgAb. lgak was also correlated with the levels of serum TSH (r = 38, P < 5). We divided sh group into two groups according to the TSH levels; the cut-off value was 1 miu/l and no significant difference was found in lgt and lgak in the group with 5 miu/l < TSH < 1 miu/l and the group with TSH > 1 miu/l. H IgG subclasses distribution of TgAb and TPOAb might indicate the different progression of HT, which suggested that the immunological characteristics of the serological marker antibody might be involved in HT pathogenesis [4,13]. Avidity is another immunological character of autoantibody; this is the strength with which a multivalent antibody binds a multivalent antigen. Antibody avidity indicates the overall strength of interaction between antibody and antigen [14]. The precise mechanism by which the avidity contributes to pathogenesis is debated. Gharavi et al. proposed that in non-organ-specific, immune-complex-mediated disorders such as SLE and RA, low-avidity autoantibodies might be equally pathogenic as high-avidity autoantibodies, whereas in organ-specific autoantibody-associated diseases such as Goodpasture s syndrome and myasthenia gravis, autoantibody avidity might play a more critical role [15]. AITD is a type of organ-specific autoimmune disease, therefore we speculated that TgAb avidity might be involved in the pathogenesis of HT. In the literature, Hjort assessed the thyroglobulinneutralizing capacity of TgAb by the reversed haemagglutination-inhibition technique and proved that TgAb from HT patients displayed higher avidity than those with non-toxic goitre, which may correspond to a significant 3 5 Discussion It is well known that TgAb is not only a diagnostic marker for HT; but it also plays an important role in HT pathogenesis. Although, traditionally, it is believed that AITDs are T cellmediated diseases, there is growing evidence to suggest that TgAb may be involved in the initiation of autoimmune thyroiditis. For example, experimental autoimmune thyroiditis can be transferred passively by TgAb [5]. Furthermore, in vitro some investigators have indicated that TgAb had some effect on antibody-dependent cellular cytotoxicity (ADCC), which might be involved in thyroid damage [6]. In epidemic investigation, patients with positive TgAb rather than those without might be more likely to experience thyroid failure during follow-up [7,8]. However, it is difficult to determine the disease progression based only on TgAb titres, as HT patients with high levels of TgAb have different thyroid functional status: some remain euthyroid for a long time, others progress quickly to hypothyroidism. The progression rate of euthyroidism to thyroid dysfunction is variable. Cambridge et al. suggested that whether the autoantibody response was pathogenic would depend upon many factors, including the immunological characteristics of the autoantibodies, such as epitope specificity, avidity, subclass and idiotype [9]. Caturegli and co-workers have reported that the IgG subclasses and epitope heterogeneity of TgAb might be utilized to predict full-blown thyroid disorders [1 12]. Our previous studies have also shown that the lgak/lgt lgt Fig. 4. lgak/lgt to lgt ratio in different groups. lgak: logarithm value of avidity constant of thyroglobulin antibody (TgAb); lgt, logarithm valueoftitresoftgab; : hypothyroid group; : subclinical hypothyroid group; : euthyroid group. lgak/lgt to lgt ratios in subclinical and overt hypothyroid groups almost lie in the right upper side of this figure, which indicates with the same titres of TgAb that HT patients with thyroid dysfunction might have a trend towards higher TgAb avidity than euthyroid patients British Society for Immunology, Clinical and Experimental Immunology, 161: 65 7

5 TgAb avidity in sera from HT patients fraction of euthyroid HT detected currently by cytological and ultrasound criteria [16]. Our current study shows that TgAb avidity was of a significantly lower level in Eu than in H and sh. Nielsen indicated that TgAb facilitated the formation of complement-activating Tg/TgAb complexes, the binding of immune complexes to B cells and the subsequent proliferation of B and T cell subsets [17]. Antibodies with high binding capacity (strong avidity) could react with antigens more quickly and strongly, therefore we speculated that high-avidity TgAb in HT sera might be involved more strongly in the immune process series and patients with strong avidity antibody might be at high risk of developing overt hypothyroidism. Furthermore, we tried to determine whether better discrimination between euthyroidism and thyroid dysfunction could be obtained based on TgAb titres and avidity. There was no significant difference in lgak/lgt in these three groups, which might be due to the limited number of patients. However, as shown in Fig. 4, there was a trend towards higher lgak/lgt variation according to lgt in the sh and H groups than in the Eu group, which indicated that in the case of high levels of TgAb, HT patients with strong avidity antibody might be at high risk of developing thyroid function failure. As immunological characteristics of TgAb, it would be of interest to study whether there is any relationship between IgG subclass distribution and avidity. Data from 45 patients enrolled in both studies were analysed further. Titres of IgG subclasses were also expressed as logarithm values. Because IgG1/IgG4 was used by several investigators as an evaluation parameter in the inflammation process [18], the lgak to lg(igg1)/lg(igg4) ratio was compared; however, no significant difference was found between these three groups. Further study on the TgAb IgG subclass avidity might provide more information. It is well known that the antigen-specific antibody undergoes affinity improvement after the primary response. To achieve affinity maturation, molecular modifications of the immunoglobulin genes triggered by antigen are pivotal [19]. Selective expansion of antibody clones on the basis of antigen affinity produces mature antibodies that are high in both affinity and specificity [2]. Studies on myeloperoxidase anti-neutrophil cytoplasmic antibody (MPO ANCA) in ANCA-associated vasculitis (AAV) and propylthiouracil-induced AAV have shown that antigen plays a key role in avidity maturation [21,22]. In our study, from euthyroidism to thyroid function failure, TgAb might experience a shift from low to high affinity (affinity maturation) assisted by antigen stimulation. We suppose that the process of affinity maturity of TgAb might be associated, at least in part, with HT progression. In the current study, TgAb avidity was measured by ELISAs, which have been proved to have a higher detection rate than haemagglutination techniques [23,24]. As Bruderer et al. have noted, the ability of antigen inhibition ELISAs to measure the ak of a heterogeneous mixture of antibodies was based on the fact that ak measurement of lower-affinity antibodies is not influenced by the presence of higher-affinity subpopulations [25]. The more accurate determination of antibody affinity would rely upon the direct measurement of affinity-purified antibodies; however, this could not be applied to a large number of samples [26]. In conclusion, our study has shown that the immunological characteristics of TgAb might be related to HT progression, and patients with high TgAb avidity might be at high risk of progressing to thyroid function failure. Further studies on the role and the relationships between these immunological characteristics such as titres, avidity and IgG subclasses might provide more useful information on the pathogenesis of HT. Acknowledgements This work was supported by the National Natural Science Foundation (no. 3853) and Beijing Natural Science Foundation (no.78296). Disclosure There are no potential conflicts of interest, including all relevant financial interests in any company or institution that might benefit from the publication. References 1 Ajjan RA, Weetman AP. Pathogenesis of autoimmune thyroid disease. In: Paul S, ed. Contemporary immunology: autoimmune reactions. Totowa, NJ: Humana Press Inc, 1999: Roitt IM, Doniach D, Campbell PN, Hudson RV. Autoantibodies in Hashimoto s disease (lymphadenoid goitre). Lancet 1956; ii: Toyoda N, Nishikawa M, Iwasaka T. Anti-thyroglobulin antibodies. Nippon Rinsho 1999; 57: (Review) (in Japanese). 4 Li MR, Guo XH, Gao Y et al. 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