of Autoimmunity (1992) 5,557-570


Analysis of Anti-Idiotypic Antibodies Against Anti-Microsomal Antibodies in Patients with Thyroid Autoimmunity

N. Tandon, D. R. W. Jayne,* A. M. McGregor? and A. I?. Weetman Department of Medicine, University of Shefield Clinical Sciences Centre, Northern General Hospital, Shefield SS 7AU, *Department of Medicine, University of Cambridge

Clinical School, Addenbrooke’s


of Medicine,



King’s College Hospital Medical SE5 8RX,

(Received 13 March

CB2 2QQ and

School, London


1992 and accepted 22June


Anti-microsomal antibody (AMA) activity was inhibited in 14 of 16 sera and in all 12 IgG preparations from patients with postpartum thyroiditis following incubation with F(ab’), fragments from normal polyspecific immunoglobulin for therapeutic use (ivIg). Similar results were observed with sera from seven of seven patients with Graves’ disease and five of six patients with autoimmune hypothyroidism. Results of these competitive binding assays and affinity chromatography of AMA IgG on Sepharosebound F(ab’), fragments from ivIg indicated that AMA antibodies reacted with ivIg through idiotypic-anti-idiotypic interactions. Eight out of 10 IgG preparations from patients with autoimmune thyroid disease also showed inhibition of AMA activity when coincubated with autologous IgM at various 1gG:IgM molar ratios. These observations suggest that ivIg can inhibit anti-microsomal antibodies through idiotype-anti-idiotype interactions and that such interactions occur with IgM anti-idiotype antibodies in viva, providing evidence of a role for idiotypic network regulation in the control of thyroid autoimmunity.


Sera from patients suffering from Graves’ disease, Hashimoto’s thyroiditis, primary myxoedema and postpartum thyroiditis contain antibodies (predominantly IgG) Correspondence

to: A. P. Weetman at the above address. 557



14 $08.00/O

0 1992 Academic Press Limited


N. Tandon et al.

against the thyroid microsomal antigen now known to be thyroid peroxidase [ 11. Anti-microsomal antibody (AMA) activity varies with the fluctuations in clinical activity of these diseases, in postpartum thyroiditis, for example, being present in high titre during the acute phase and at a lower level or totally disappearing during remission [2]. The network theory of autoimmunity [3] proposes that antibody synthesis and lymphocytes involved in the immune response are regulated by a network of anti-idiotypic antibodies and lymphocytes. The presence of naturally occurring anti-idiotypic antibodies against autoantibodies has been described in many human autoimmune diseases [4], especially systemic lupus erythematosus [5] and myasthenia gravis [6]. The role of anti-idiotypic antibodies in the regulation of autoimmune disease processes has been examined in detail in animal models. For instance, in experimental autoimmune thyroiditis, heterologous anti-idiotypic antibodies to autoantibodies against rat thyroglobulin have been produced and shown to ameliorate thyroiditis in BUF rats [7]. Protective immunity against experimental autoimmune thyroiditis induced exclusively by a thyroglobulinspecific cytotoxic T cell clone has been demonstrated and shown to occur through the generation of anti-idiotypic antibodies recognizing the paratope of an antithyroglobulin monoclonal antibody specific to the pathogenic epitope of the thyroglobulin molecule [8]. Anti-idiotypic antibodies directed against human anti-thyroid stimulating hormone (TSH) receptor antibodies [9] and human anti-thyroglobulin antibodies [lo] have been generated and characterized. Anti-TSH antibodies have been found in patients with Graves’ disease, but whether they are anti-idiotypic to anti-TSH receptor antibodies is as yet unresolved [ll-131. In a recent report using the nujnu bioassay as an in vivo model for the study of human thyroid autoimmunity, more evidence has been presented suggesting a role for anti-idiotypic antibodies in the induction and modulation of remission in Graves’ disease [ 141. So far no attempt has been made to identify anti-idiotypic antibodies against microsomal antibodies, which recognize the third major thyroid autoantigen. In recent years, it has been shown that preparations of normal polyspecific immunoglobulin designed for intravenous therapy (ivIg) contain anti-idiotypic antibodies against antifactor VIII [15], anti-DNA, anti-thyroglobulin, anti-intrinsic factor [ 16, 171, anti-peripheral nerve [ 181, anti-neutrophil cytoplasmic antigen [ 191 and anti-acetylcholine receptor [20] autoantibodies from pathological sera. The kinetics of ivIg after infusion for treatment in autoimmune diseases have not been studied in depth. ivIg given to patients with myasthenia gravis resulted in an ivIg to serum IgG molar ratio of approximately 2:l during treatment [20-221. In another study evaluating the role of ivIg in treating patients with systemic vasculitis, a good correlation (r = 0.79; P-C 0.05) was found between in vitro inhibition of antineutrophil cytoplasmic antibodies and therapeutic response to ivIg [23]. This again suggests the relevance of in vitro observations to in vivo phenomena. The findings that F(ab’), fragments from ivIg neutralize F(ab’), fragments from autoantibody in a dose-dependent fashion, that ivIg coupled to an immunoaffinity matrix specifically retains autoantibodies, that ivIg does not contain detectable antibodies against the most commonly expressed allotypes in the F(ab’), region of human IgG and that ivIg recognizes idiotypes defined by heterologous anti-idiotypic


anti-idiotypic antibodies


antibodies on autoantibodies, suggest that ivIg contains anti-idiotypes against autoantibodies [15, 17, 241. Moreover, ivIg has been a useful tool for analysing whether anti-idiotypic antibodies can be detected in pathological sera [ 191. In this study, we have evaluated whether ivIg contains anti-idiotypic antibodies against microsomal antibodies and, using this material as a probe, whether pathological sera also contain such anti-idiotypes.

Materials and methods Patients

Sera from 30 unselected patients with anti-microsomal antibodies were collected and stored in aliquots at -70°C. The diagnoses were Graves’ disease in seven, autoimmune hypothyroidism (Hashimoto’s thyroiditis and primary myxoedema) in six and postpartum thyroiditis in 16 patients. IgG was prepared from 15 of these sera. Normal sera were obtained from healthy laboratory personnel without detectable thyroid antibodies.

Source of ivIg

ivIg used was Sandoglobulin@ (Sandoz Ltd, Basel, Switzerland), a preparation of pH 4 treated intact human polyspecific immunoglobulin for therapeutic use pooled from more than 3,000 normal blood donors.


of IgG, F( ab’) 2fragments from IgG and IgM

The IgG fraction from patients’ sera was prepared by chromatography on Protein G-Sepharose (Pharmacia, Uppsala, Sweden). F(ab’), fragments were prepared from ivIg and from patients’ IgG by pepsin (Sigma, Poole, Dorset) digestion (2% w/w) and chromatography on Protein A-Sepharose (Pharmacia). F(ab’), fragments were free of detectable Fc fragments as assessed by ELISA. The unbound fraction collected on running each serum sample down the Protein G-Sepharose column was used as a crude source of IgM. The IgG and IgM in the serum as well as this IgM preparation were measured by ELISA; the IgM preparation so produced contained less than 5% contaminating IgG.


of AMA


AMA activity in serum and IgG fractions were assessed by ELISA [25]. In brief, microsomal antigen, 10 pg/ml in carbonate-bicarbonate buffer pH 9.6, was coated overnight at 4°C onto polystyrene ELISA plates (Nunc, Kamstrup, Denmark). After washing, the plates were blocked for 1 h with 30/, bovine serum albumin in PBS-Tween and incubated with serum (diluted 1 :lOO in PBS-Tween) or IgG for 2 h (each in duplicate), both incubations at 37°C. The plates were then washed and incubated for 2 h at room temperature with a 1: 1,000 dilution of mouse anti-human


N. Tandon et al.

IgG alkaline phosphatase conjugate (Sigma). The substrate pNl?P (Sigma) was added after washing the plates and the absorbance, at 405 nm, of duplicate assays determined 30 min later. Dilutions of serum from a patient known to have a high titre of AMA served as a positive control and PBS-Tween was used as a negative control.

Inhibition of AMA


In preliminary experiments, sera were tested by ELISA at five dilutions between 1: 100 and 1: 10,000 so that a dilution curve could be constructed from each individual sample. Inhibition experiments were then performed with the concentration of each serum chosen to be in the linear portion of that individual’s AMA dilution curve and producing 50-75% of the maximal activity. Inhibition by ivIg F(ab’), fragments or autologous crude IgM preparations of AMA activity in patients’ sera or IgG preparations was assessed by incubating serial dilutions of inhibitor in PBS with a fixed dilution of sera (or a fixed amount of patients’ IgG) for 1 h at 37°C and 16 h at 4°C. Since the microsomal antigen used for the ELISA was not purified, the samples were also incubated with purified human thyroglobulin (25 pg/ml) for the same duration. The inhibition of AMA activity was expressed as the percentage reduction of optical density (O.D.) in the sample incubated in the presence of inhibitor relative to O.D. of the sample incubated with buffer alone. Percentage reduction = l-

OD sample + inhibitor OD sample

Results are expressed as the mean of a group of measurements



x 100 + SD.


F(ab’), fragments from ivIg were coupled to cyanogen-bromide-activated Sepharose (Pharmacia), as described previously [26], and 10 ml columns of the resulting packed gel were equilibrated with PBS. IgG preparations containing AMA activity were loaded on the columns and left to equilibrate for 18 h at 4°C. The columns were washed with PBS until no protein emerged in the run through and then eluted with glycine-HCl, 0.1 M pH 2.8. Protein (immunoglobulin) content and AMA activity were measured in the loaded material and in the eluted fractions. Specific autoantibody activity was expressed as the ratio between O.D. (measured at 405 nm) in the linear portion of the ELISA autoantibody titration curve and IgG concentration in l.tg (O.D./pg protein).

Results Inhibition of AMA

When sera were incubated activity was seen in all three with postpartum thyroiditis inhibition at the peak of the

activity by i.v.Zg

with F(ab’), fragments from ivIg, inhibition of AMA categories of patients studied. Sixteen sera from patients were selected from sequential samples and studied for AMA response (generally 5 to 6 months after delivery).



anti-idiotypic antibodies


0 00 0



0 0

80 0






0’ Postpartum thyroiditis

Graves’ disease

Autoimmune hypothyroidism

Figure 1. Inhibition of Ah4A activity in serum of patients with postpartum thyroiditis ( 0 ), Graves’ disease (A) and autoimmune hypothyroidism (0) by F(ab’), from ivIg. The data represent the maximal inhibition for each serum sample that was observed at a specific molar ratio between patient IgG and F(ab’), from ivIg for each serum.

Fourteen of these showed inhibition (a fall by 15% or more in AMA activity) (Figure 1, Table 1). IgG was prepared from eight of these sera and all of these preparations showed inhibition. In six patients, sera were also available 10 months after delivery when AMA levels had fallen and partial clinical remission had occurred. The mean ( t_ SD) absorbance of these sera in the AMA assay at 5 to 6 months was 1.26 + 0.25 and at 10 months 0.9lkO.19 (P~0.01 by paired t-test). In addition, IgG was prepared from four of these sera. Inhibition was observed with all six sera and all four IgG preparations. There was no consistent difference in the extent of inhibition between these pairs of sera (Table 2). Maximal inhibition of AMA activity ranged between 10 and 60%: inhibition of AMA activity in serum was 33.3 rfi16.7% and that in purified IgG preparations was 33.5 f 9.9%. Maximal inhibition was seen at a different molar ratio between patient’s IgG and F(ab’), from ivIg in each patient. Inhibition curves often showed a secondary peak in addition to the maximal inhibition peak (Figure 2). In the remaining curves, a steep fall in inhibition following the maximal inhibition was seen. The fall was followed by a flattening of the curve at higher IgG/ivIg F(ab’), ratios. The maximal inhibition was usually observed at an IgG/ivIg F(ab’), ratio of 0.05 to 0.1, but secondary peaks were often seen at ratios varying from 1 to 5. Similar results were observed with samples from patients with Graves’ disease and autoimmune hypothyroidism. All three sera and corresponding IgG preparations from patients with Graves’ disease showed inhibition. The maximal inhibition


N. Tandon et al.

Table 1. Inhibition

of AMA activity in sera of patients with postpartum thyroiditis, Graves’ disease or autoimmune hypothyroidism by F(ab’), fragments from ivlg

Patient PTl PT2 PT3 PT4 PT5 PT6 PT7 PTS PT9 PTlO PTll PT12 PT13 PT14 PT15 PT16 GDl GD2 GD4 AHI AH2 AH3 AH4 AH5 AH6

O.D. (basal)

O.D. (at maximal inhibition)

0.924 0.426 0.689 1.205 0.751 0.873 0.456 0.331 0.425 0.431 0.469 0.582 0.411 0.947 0.562 0.873 1.172 0.319 0.778 1.062 0.838 0.823 0.922 0.969 0.840

0.718 0.361 0.433 0.487 0.326 0.553 0.223 0.130 0.342 0.238 0.203 0.365 0.336 0.643 0.486 0.553 0.611 0.173 0.620 0.829 0.674 0.646 0.813 0.761 0.611

IgG/ivIg ratio causing maximal inhibition 10.0 0.1 0.05

0.05 0.05 0.05 0.1 0.1 0.1 0.1 0.1 0.1 5.0 0.1 0.1 0.05 0.1 0.1 0.1 0.1 5.0 0.5 0.1 0.1 0.1

*Diluted sera were incubated with serial dilutions of F(ab’), fragments of ivIg in PBS, and binding was evaluated in an AMA ELISA. The optical density in the presence of either PBS (basal) or ivIg F(ab’), fragments (at a concentration which caused maximal inhibition of AMA activity) is shown. PT= Postpartum thyroiditis, GD = Graves’ disease, AH = autoimmune hypothyroidism.

observed was 35.7 f 12.5% in the case of serum AMA activity and 35.13 k 13.0% in the case of AMA activity in purified IgG preparations. IgG from an additional four patients whose sera had not been studied in inhibition experiments showed inhibition varying from 1%32% with a mean of 22.3+6.7%. Five of six sera of patients with autoimmune hypothyroidism and three of the four IgG preparations from these patients also showed inhibition: inhibition of serum AMA activity was 2 1.8 + 7.3% whereas the inhibition of AMA activity in purified IgG preparations was 24.1 f 8.9%. To ascertain that F(ab’), fragments from ivIg were not causing inhibition by directly binding to the antigen, an ELISA was carried out to detect AMA activity in ivIg. The ivIg F(ab’), fragments were added to the ELISA over the range of concentrations (0.02-2.0 mg/ml) in which they were used for the inhibition assays. In two separate experiments, the absorbance of the wells loaded with ivIg F(ab’), fragments never exceeded that of control wells loaded with PBS, demonstrating the absence of AMA activity in the ivIg used in our experiments.


anti-idiotypic antibodies


Table 2. Maximal inhibition of AMA activity by F(ab’), fragments from ivIg in patients with postpartum thyroiditis during the acute phase and partial clinical remission* “/bInhibition

Patient PTl PT2 PT3 PT4 PT5 PT6


acute phase

y0 Inhibition

15.8 11.0 31.8 29.7 56.6 22.8




22.6 15.3 37.2 59.6 22.8 36.7

*I: 100 dilution of patient serum (collected during the acute phase of illness and also during partial clinical remission) was incubated with varying concentrations of F(ab’), fragments from ivIg and then analysed for inhibition of AMA activity by ELISA. PT = Postpartum thyroiditis.



40 ‘;j + .g .c 2


5 20


0 I



0. I

I 1.0

IgG/i.v.lg F(ab),




Figure 2. Inhibition of AMA activity in serum of individual patients with postpartum thyroiditis ( l ), Graves’ disease (A) and autoimmune hypothyroidism (0) by F(ab’), fragments from ivIg.


of serum and IgG AMA


The AMA levels in many patients were higher in the isolated IgG preparation than in the corresponding whole serum (Figure 3). This difference in AMA activity was expressed as a percentage change as follows:


N. Tandon et al.



A 100 80 s.



2 2



u .c 0,

2 8 E



g at


: 0.0


A’ L____-000 0 A

-20 Figure 3. Percentage change in AMA activity in the IgG preparation compared with corresponding serum AMA activity. Values greater than 0 reflect enhanced activity in IgG compared with whole serum. 0 Hashimoto’s thyroiditis; A Graves’ disease; 0 postpartum thyroidiris.

o/ochange in AMA activity = O.D. of AMA activity in IgG-

O.D. of AMA activity in serum

O.D. of AMA activity in whole serum

x 100

and therefore values greater than 0 represent an increase in activity in the IgG fraction. There was a significant difference (P~0.05, paired t-test) in favour of enhanced activity in the IgG fraction compared with whole serum. AJinity


Purified IgG from the sera of two patients with postpartum thyroiditis, one patient with autoimmune hypothyroidism and five patients with Graves’ disease were chromatographed on Sepharose-bound F(ab’), fragments from ivIg. Acid elution of the columns yielded between 0.75% and 12.5% of loaded IgG (mean 6.5 54.5%). Specific AMA activity in the eluate varied from values below that of the loaded material to a three-fold enrichment (Table 3). Inhibition of AMA

activity in IgG preparations

by autologous IgM

In the following inhibition experiments, if the concentrations of the IgG preparation and the IgM preparation being used were in the same relative proportion as their concentrations in serum, the 1gM:IgG ratio was considered to be 1. Since whole IgG


anti-idiotypic antibodies


Table 3. Ajj%zitychromatography of IgG containing AMA activity on Sepharose bound

F(ab’), fragments from ivIg* Eluted


Specific activity

Specific activity Patient


Amount 1,200 1,280 2,280 2,200 1,900 160 160 280


(O.D./W 0.317 0.280 0.07 0.08 0.2 0.308 6.05 6.6


104 96 208 56 190 20 1.5 2


(O.D./@ 0.072 0.021 0.06 0.003 0.007 0.007 3.75 20.7

*IgG was chromatographed on a column of F(ah’), fragments from i.v.Ig coupled to cyanogenbromide-activated Sepharose. The IgG eluted from this column was analysed for AMA activity and this was compared with AMA activity in whole IgG (loaded) from these patients. GD = Graves’ disease; AH = autoimmune hypothroidism; PT = postpartum thyroiditis.

and not IgG F(ab’), was used in the inhibition studies, it was necessary to rule out anti-Fc activity in the IgM fractions as a cause of inhibition. All IgM preparations were tested for rheumatoid factor (RF) activity by latex agglutination and no preparation was RF positive. Eight of the 10 IgG preparations (five from patients with postpartum thyroiditis, four from patients with Graves’ disease and one from a patient with autoimmune hypothyroidism) showed inhibition when autologous IgM (at varying 1gM:IgG ratios) was used to inhibit AMA activity (Table 4). The maximal inhibition varied from 12 to 42% with a mean of 24.3 + 9.4%. Considering the patient groups separately, the inhibition observed in IgG from patients with postpartum thyroiditis was 22.56 + 10.1 o/oand 28.4 + 9.1 o/0in the case of IgG from patients with Graves’ disease. In three instances, paired IgG preparations from patients with postpartum thyroiditis in the acute phase and partial remission were available. Inhibition observed in the acute phase IgG preparations was 25.6-t 11.4%, whereas it was 19.8 & 7.1 y0 in IgG preparations from the latter group. Four of the IgM preparations had AMA activity. To exclude occupation of antigenic sites by IgM as a cause of apparent inhibition of IgG activity by these particular preparations, a modified ELISA was performed. The plates were coated and blocked in the usual fashion. While the IgG preparations were coincubated with autologous IgM, PBS was added to one set of wells of the ELISA plate and to a matching set of wells in the same plate, autologous IgM (at the maximal concentration used for inhibition) was added. After the coincubation, the plates were washed. IgG alone was added to the wells which had been treated with IgM, and IgG which had coincubated with IgM was added to wells treated with PBS. Routine ELISA was then performed. AMA activity in IgG preparations was reduced when they were coincubated with IgM, compared with the activity, in these preparations


N. Tandon et al. Table


4. Inhibition

of AMA activity in purified autologous IgM*

Max inhibition




IgG preparations


22.1 41.3 21.8 28.5 16.4 24.1 18.2 20.0 38.7 11.8


at max inhibition

50 10 2.5 0.5 50 20 10 5 10 5

*Appropriate dilutions of IgG preparations from patient sera were incubated with a range of dilutions of autologous IgM and then analysed for inhibition of AMA activity in the ELISA. The 1gM:IgG ratio was considered to be 1 if the concentrations used were in the same relative proportion as their concentrations in serum. disease; AH = autoimmune GD = Graves’ hypothyroidism; PT = postpartum thyroiditis.

Table 5. Comparison either pretreatment

of AMA activity in four IgG preparations after of wells with IgM or preincubated with IgM* Absorbance

Wells pretreated with IgM IgG preincubation with IgM

(405 nm)





0.530 0.455

1.000 0.490

0.700 0.645

0.923 0.578

*IgG preparations were incubated with autologous IgM for 2 h. Simultaneously, either PBS or autologous IgM was added to wells of an ELISA plate coated with microsomal antigen. After 2 h, the plate was washed. IgG alone was added to wells pretreated with IgM and IgG which had been coincubated with IgM added to wells treated with PBS. Then the ELISA was performed fo assess AMA activity in the two IgG preparations.

when added to wells (without prior preincubation) pretreated with IgM (Table 5). These data indicate that the inhibition of AMA activity observed was not due simply to saturation of antigenic sites by IgM but by IgG-IgM interactions. To substantiate that the interaction between IgG and IgM was idiotypic-antiidiotypic in nature, a direct interaction between autologous IgG and IgM was evaluated. Whole IgG from three of the patients was used to coat an ELISA plate at two different concentrations (10 and 100 pg/ml). The wells were then loaded with either PBS or autologous IgM and the binding of IgM assessed by adding antihuman IgM alkaline phosphatase conjugate followed by substrate. In all three

Anti-microsomal anti-idiotypic antibodies 567 Table 6. IgM binding

to autologous IgG in patients with autoimmune thyroid disease* o/0 Increase


Concentration of IgG used to coat wells @g/ml)


IgM dilution

in optical density lt

50 20 52 34 46 82

10 100 10 100 10 100

IgM dilution 2

31 26 18 10 24 20

Whole IgG from patients was used at two concentrations (10 and 100 pg/ml) to coat an ELISA plate. The wells were then loaded with either PBS or autologous IgM and binding was assessed by adding anti-human IgM alkaline phosphatase conjugate followed by substrate. Percentage excess of IgM binding compared to controls was calculated as follows: Percentage increase =

OD of IgM binding to IgG-

OD of PBS binding to IgG

OD of PBS binding to IgG

x 100

tIgM dilution 1= dilution of IgM causing maximal inhibition in AMA ELISA (see Table 4); IgM dilution 2 = 1: 10 dilution of dilution 1. PT = Postpartum thyroiditis; GD = Graves’

disease; AH = autoimmune hypothyroidism.

patients, IgM binding to the IgG could be demonstrated (Table 6). The observation that none of the IgM preparations contained rheumatoid factor activity supports the presence of an idiotype-anti-idiotypic interaction.

Discussion AMA are present in the sera of patients with autoimmune thyroid disease, and changes in AMA titres are seen with variations in clinical activity of the disease [ 1,2]. We have demonstrated that anti-idiotypic antibodies against AMA are present in pooled normal immunoglobulin for therapeutic use (ivIg) and also in IgM derived from patients’ sera. Incubation of serum containing AMA with F(ab’), fragments from ivIg resulted in inhibition of AMA activity in 88% of sera from 25 patients with clinical autoimmune thyroid disease. A similar proportion of IgG preparations from these patients also showed inhibition of AMA activity for ivIg F(ab’),. The preincubation studies permitted detection of anti-idiotypic activity directed against the antigen binding site on the F(ab’), region, since they revealed inhibition of autoantibody binding to autoantigen. Inhibition of AMA activity by ivIg F(ab’), was dose-dependent with maximal inhibition occurring at a molar ratio between patients’ IgG and ivIg, which varied between 0.05 and 5.0. In addition to the peak of maximal inhibition, a second peak of inhibition was often seen. Other studies have also shown a wide range of maximal inhibitory ratios varying from 0.4 to 23.2 for


N. Tandon et al.

anti-thyroglobulin, anti-intrinsic factor and anti-DNA autoantibodies [16]. The higher ratios were mainly observed in the case of anti DNA autoantibodies, which may reflect the presence of a higher frequency of anti-idiotypic specificities against anti-DNA antibodies in ivIg. Affinity chromatography experiments using Sepharose-bound F(ab’), fragments from ivIg showed retention of AMA activity in the column. However, only one of the eight patients studied showed an autoantibody enrichment (3-fold) in the acid eluate from the affinity column. This is unlike the more consistent enrichment observed with autoantibodies to factor VIII [15], neutrophil cytoplasm [19], DNA, intrinsic factor and thyroglobulin [16]. However, similar results have been observed in the anti-acetylcholine receptor autoantibody system [20]. Affinity chromatography selects predominantly high affinity interactions between variable regions of autoantibodies and ivIg. If the AMA network has a preponderance of low affinity idiotype anti-idiotype interactions between ivIg and autoantibodies, these will not be detected in the acid eluate of the column. Hence, the absence of enrichment observed in most cases may actually underestimate the true extent of the idiotype anti-idiotype interaction. ivIg has been shown to contain no anti-idiotypic antibody and no antibodies against the commonest allotypes expressed by the F(ab’), region of human IgG [15]. The role of anti-idiotypic IgM in the control of expression of natural IgG autoantibodies has been well studied in mice [27], but less so in man. In patients with systemic vasculitis it was shown that remission sera contained IgM capable of specifically interacting with IgG idiotypes of anti-neutrophil cytoplasmic antibodies from acute phase sera [ 191. We studied the ability of autologous IgM to inhibit the binding of AMA to antigen. Eight of 10 IgG preparations were inhibited on being coincubated with IgM before testing for AMA activity by ELISA. The maximal inhibition observed with IgM was more than that seen with ivIg F(ab’), in four of these patients. Four IgM preparations used had low, but detectable AMA activity and ancillary experiments were needed to demonstrate the mechanism for IgM-mediated inhibition of IgG AMA activity. Furthermore, IgG AMA activity was significantly higher than in serum, suggesting that removal of IgM with anti-idiotypic activity increased IgG binding to the microsomal antigen. Other experiments showed that the ELI SA plates were coated with an excess of antigen and so any inhibition of AMA activity seen was unlikely to be due to reduced availability of antigenic sites in the system. These data and the absence of any rheumatoid factor activity tn the IgM preparations suggest that the inhibition of IgG AMA activity by IgM was mediated by IgM interacting with the F(ab’), portion of IgG. In conclusion, our study demonstrates that antibodies able to bind and inhibit anti-microsomal autoantibodies by idiotype-anti-idiotype interactions are present in ivIg derived from normal individuals, and that IgM anti-idiotype antibodies to AMA are present in certain patients’ sera. These AMA anti-idiotypic antibodies only incompletely inhibit AMA in some patients’ sera and in others appear to be absent. Therefore, they are likely to provide only a partial regulatory effect in autoimmune thyroid disease, suggesting that treatment with ivIg would have limited success. This, in turn, would explain our ability to detect AMA activity in sera from patients, despite coexisting IgM anti-idiotypic antibodies.

Anti-microsomal anti-idiotypicantibodies


Acknowledgements This work was supported by the Wellcome Trust and Medical Research Council. N.T. is in receipt of a Cambridge Nehru Scholarship and Overseas Research Studentship. D.R.W. J. has a Clinical Research Fellowship from Gonville and Caius College, Cambridge. References 1. Banga, J. P., P. S. Bamett, and A. M. McGregor. 1991. Immunological and molecular characteristics of the thyroid peroxidase autoantigen. Autoimmunity 8: 335-343 2. Jansson, R., P. A. Dahlberg, and F. A. Karlsson. 1988. Postpartum thyroiditis. Ballidre’s Clin. Endocrinol.


2: 619-636

3. Jerne, N. K. 1974. Towards a network theory of the immune system. Ann. Immunol. Pasteur.)




4. Burdette, S. and R. S. Schwartz. 1987. Current concepts: immunology. Idiotypes and idiotypic networks. N. Eng1.J.


317: 219-224

5. Abdou, N. I., H. Wall, H. B. Lindsley, J. F. Halsey, and T. Suzuki. 1981. Network theory in autoimmunity: in vitro suppression of serum anti-DNA antibody binding to DNA by anti-idiotypic antibody in systemic lupus erythematosus. J. Clin. Invest. 67: 1297-1304

6. Dwyer, D. S., R. J. Bradley, C. K. Urquhart, 7. 8. 9. 10.



and J. F. Kearney. 1983. Naturally occurring anti-idiotypic antibodies in myasthenia gravis patients. Nature 301: 611-614 Zanetti, M. and P. E. Bigazzi. 1981. Anti-idiotypic immunity and autoimmunity. I In vitro and in vivo effects of anti-idiotypic antibodies to spontaneously occurring autoantibodies to rat thyroglobulin. Eur. J. Zmmunol. 11: 187-195 Roubaty, C., C. Bedin, and J. Charreire. 1990. Prevention of experimental autoimmune thyroiditis through the anti-idiotypic network. J. Immunol. 144: 2167-2172 Baker, J. R., Y. G. Lukes, and K. D. Burman. 1984. Production, isolation and characterization of rabbit anti-idiotypic antibodies directed against human antithyrotropin receptor antibodies. J. Clin. Invest. 74: 488-495 Kojima, K., T. Matsuyama, and H. Tanaka. 1986. Suppression of in vitro human antithyroglobulin antibody secretion by private and cross-reactive anti-idiotypic antibodies. Clin. Immunol. Immunopathol. 39: 337-344 Ochi, Y., T. Nagamune, Y. Nakajima, M. Ishida, Y. Kajita, T. Hachiya, and H. Ogura. 1989. Anti-TSH antibodies in Graves’ disease and their failure to interact with TSH receptor antibodies. Acta Endocrinol. (Copenh.) 120: 773-777 Noh, J., N. Hamada, H. Saito, H. Oyanagi, N. Ishikawa, N. Momotani, K. Ito, and H. Morii. 1989. Evidence against the importance in the disease process of antibodies to bovine thyroid-stimulating hormone found in some patients with Graves’ disease. J. Clin. Endocrinol.


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13. Raines, K. B., J. R. Baker, Y. G. Lukes, L. Wartofsky, and K. D. Burman. 1985. Antithyrotropin antibodies in the sera of Graves’ disease patients. J. Clin. Endocrinol. Metabol.

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14. Paschke, R., J. Teuber, I. Enger, R. Schmeidl, U. Schwedes, and K. H. Usadel. 1990. Evidence for a role of anti-idiotypic antibodies in the induction of remission in Graves’ disease. J. Autoimmunity 3: 441-448 15. Rossi, F., Y. Sultan, and M. D. Kazatchkine. 1988. Anti-idiotypes against autoantibodies and alloantibodies to VIII:C (anti-hemophilic factor) are present in therapeutic polyspecific normal immunoglobulins. Clin. Exp. Immunol. 74: 311-316 16. Rossi, F. and M. D. Kazatchkine. 1989. Anti-idiotypes against autoantibodies in pooled normal human polyspecific normal immunoglobulins. J. Zmmunol. 143: 4104-4109 17. Dietrich, G. and M. D. Kazatchkine. 1990. Normal IgG for therapeutic use (IVIg) contain anti-idiotypic specificities against an immunodominant, disease associated, cross-reactive idiotype of human antithyroglobulin autoantibodies. J. Clin. Invest. 85: 620-625


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Analysis of anti-idiotypic antibodies against anti-microsomal antibodies in patients with thyroid autoimmunity.

Anti-microsomal antibody (AMA) activity was inhibited in 14 of 16 sera and in all 12 IgG preparations from patients with postpartum thyroiditis follow...
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