Autoimmunity, 1992, Vol. 11, pp. 141-149 Reprints available directly from the publisher Photocopying permitted by license only

0 1992 Harwood Academic Publishers GmbH

Printed in the United Kingdom

MAPPING EPITOPE SPECIFICITIES OF MONOCLONAL ANTIBODIES TO THYROID PEROXIDASE USING RECOMBINANT ANTIGEN PREPARATIONS D.L. EWINS, P.S. BARNETT, R.W.S. TOMLINSON, A.M. McGREGOR and J.P. BANGA Department of Medicine, Kings College School of Medicine and Dentistry, Bessemer Road, London SE5 9PJ

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(Received May 1 4 , 1991; in final form September 11,1991) Five separate monoclonal antibodies (MoAbs) to human thyroid peroxidase (hTPO) were raised by immunising Balb/c mice with hTPO purified from detergent solubilised thyroid microsomes by high performance liquid chromatography (HPLC). The epitope specificities of these MoAbs were determined by assessing their ability to bind to purified recombinant fusion protein fragments of human TPO (TPO(r)) generated in E . coli. A total of seven small overlapping fragments (averaging 104 amino acid residues) of hTPO, encompassing over 90% of the extracellular region of the molecule, were generated as glutathione S-transferase (GST) fusion proteins. The sequential epitopes on TPO(r) recognised by these MoAbs were analysed by both immunoblotting and enzyme linked immunosorbent assay (ELISA). Two different MoAbs (A4 and A5) recognised sequential epitopes within the TPO(r) preparation termed R la+b (residues 1-1 60) and more specifically, in the case of MoAb A4, within the subfragment R l b (residues 70-160). The inability of the other MoAbs (A I-A3) to recognise recombinant fragments, suggests they either recognise conformational determinants on the TPO molecule or epitopes that are present on the small regions of the TPO molecule which have not been expressed as recombinant proteins. KEY WORDS: Thyroid peroxidase, epitopes, monoclonal antibodies, recombinant antigens.

INTRODUCTION A common feature of patients with autoimmune thyroid disease (AITD), is the presence of high levels of circulating autoantibodies directed towards thyroid peroxidase (TPO)’. Thyroid peroxidase is an essential membrane bound enzyme involved in the generation of thyroid hormones from thyroglobulin. The autoimmune response to TPO is known to be directed towards a large number of autoantigenic epitopes on the molecule, including the enzymatic There remains some controversy as to whether the autoantigenic epitopes on TPO are purely conformational in nature’.‘ or a mixture of both conformational and sequential sites’. Purification of TPO from thyroid microsomes requires an initial solubilisation step utilising either detergent or trypsinisation to release a water soluble fragment. Solubilisation in non-ionic detergents such as Zwittergent allows purification by biochemical fractionation techniques using ion exchange and gel chromatography’.’, but has disadvantages associated with purity and potential toxicity if used in subsequent cell culture experiments (unpublished observations). Similarly trypsinisation results in the production of Please address correspondence to Dr D.L. Ewins, Department of Medicine. Stopford Building. Univcrsity of Manchester. Oxford Road. Manchester, MI3 9PT.

several fragments of TPO of various sizes”. Specific monoclonal antibodies have proved exceedingly useful in immunoaffinity purifying membrane antigens”. High purities and yields of detergent solubilised membrane antigens, including TPO, have been obtained, by affinity p ~ r i f i c a t i o n ’ ~ In . ’ ~this , report we describe our studies in generating monoclonal antibodies to hTPO purified by high performance liquid chromatography (HPLC) and their use in affinity purification of hTPO suitable for cellular experiments. We have recently described the preparation of recombinant fragments of TPO generated in E . coli, which were used to map the autoantigenic sites recognised by patients’ autoantibodies’. These recombinant fragments, encompassing a large proportion of the extracellular region of hTPO, were used to localise the antigenic sites recognised by murine monoclonal antibodies to hTPO.

MATERIALS AND METHODS 1 . Purification of human thyroid peroxidase

Thyroid microsomal membrane preparations, TPO(mic), were prepared from Graves’ thyroid tissue, removed during routine surgery, by differential centrifugationt4. Purified IiTPO was prepared by HPLC using a Mono-Q ion exchange column following solu141

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bilisation of the microsomes in Zwittergent 3-14x,9. The fractions eluting from the ion exchange column were examined for enzymatic activity by guaiacol oxidation’, for TPO(mic) binding by ELISA and by SDSpolyacrylamide gel electrophoresis (SDS-PAGE). The fractions containing the 110 kD polypeptide component under reducing conditions with the highest guaiacol activity were pooled and used as a source of purified hTPO for immunisation.

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2. Production of monoclonal antibodies Female Balb/c mice (6-8 weeks old) were immunised with 50 pg purified hTPO in complete Freund’s adjuvant into several sites subcutaneously and intramuscularly. Three booster injections of 25 jig of hTPO in incomplete Freund’s adjuvant were given at three week intervals. Animals containing the highest titre of antibodies to TPO(mic) by ELISA were boosted with 20 p g of purified hTPO in saline solution intraperitoneally. Monoclonal antibodies were generated using methods already d e ~ c r i b e d ’The ~ . class and subclass of each monoclonal antibody was ascertained using a commercial kit (Serotec, UK). The monoclonal antibodies were purified from ascitic fluid by ion exchange chromatography on a TSK.DEAE 5PW column, using a sodium acetate gradientI6. The eluted fractions were examined for anti-TPO activity by ELISA and purity ascertained by SDS-PAGE under reducing conditions. The ability of each MoAb to inhibit TPO enzymatic activity was assessed by determining their ability to inhibit TPO-induced oxidation of guaiacol2.”.

protein/ml gel) to remove non-specific binding components before passage down the monoclonal A5Sepharose 4B column ( 2 m l packed beads) at a flow rate of 200 pI/min. The column was washed with three bed volumes of Tris/saline buffer containing 0.5% sodium cholate followed by 50 mM diethylamine pH 8.5 to remove non-specific binding components. The bound material was eluted with 50 mM diethylamine pH 1 1.5 in Tris/saline buffer containing 0.5% sodium cholate and was collected in solid glycine to immediately neutralise the eluate prior to dialysis against phosphate buffered saline containing 0.5% sodium deoxycholate at 4°C. The purity and molecular weight of the resulting affinity-purified TPO (APTPO) was determined by SDS-PAGE run under reducing conditions. The enhanced enzymatic activity was demonstrated by calculating the specific enzymatic activity (guaiacol units/mg protein).

4. Recombinant preparations of thyroid peroxidase The cloning and expression of thyroid peroxidase as a

3. Monoclonal antibody affinity purification of thyroid peroxidase Ascitic fluid containing monoclonal antibody A5 was precipitated with 40% ammonium sulphate, dialysed against phosphate buffererd saline and used to couple to CNBr-activated Sepharose 4B according to the manufacturer’s instructions (Pharmacia-LKB, Milton Keynes, UK), at 10 mg antibody/ml gel. Human TPO was purified from solubilised crude thyroid microsomes by affinity chromatography using a modification of the method described previouslylx. Twenty mg thyroid microsomes were solubilised in 2% w/v sodium cholate (4 ml), in 10 mM Tris HCI, 0.15 M NaCl pH 8.1 (Tris/saline buffer) by glass to glass hornogenisation and left on ice for 60 min. Following centrifugation at 45,000 rpm for 60 min, the supernatant containing solubilised thyroid membrane proteins was diluted in Tris/saline buffer to give a final protein concentration of 1.25 mg/ml in 0.5% detergent. Ten ml solubilised microsomes were passed down an ovalbumin-Sepharose column (5 mg

m

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Figure 1 Protein staining of SDS-PAGE gel to show purified recombinant fragments. Lane I, non-recombinant GST: Lanes 2, 3. 3 . 5 and 6 refer to Rla+b. R l u . R2a. K2b and R3 recombinant polypeptides respectively. in refers to molecular weight markers (top to bottom. on right) 116, 97. 66, 45 and 29 kD: (on left) 66. 45, 36. 20. 20 and 14 kD.

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recombinant glutathione S-transferase (GST) fusion protein using the expression plasmid vector pGEX-2T has been described’. Briefly, overlapping recombinant fragments encompassing over 90% of the extracellular region of the TPO molecule were purified from inclusion bodies and used in this study. These recombinant fragments represented the following amino

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Table 1 Immunochemical properties and anti-TPO ELISA titres of purified monoclonal antibodies to human TPO. Monoclonal antibody

lsotype

Anti-TPO titre (at I mglml)

Guaiarol inhibition

KCHM A 1 KCHM A2 KCHM A3 KCHM A4 KCHM AS KCHM A6

IgG I IgG I IgG2a IgG I IgGl lgG I

1:100,000 1 : 10,000 1 50,000 I :so,000 1 :s,o00

30% 0% 0% 0% 0% 0%

-

acid residues on the hTPO molecule; R l a (residues 1-85), R l b (residues 70-160), R l c (residues 145-250), R2a (residues 320-456), R2b (residues 457-589) and R3 (residues 577-845). In addition a preparation R 1a+b (residues 1-1 60), encompassing regions R l a and R l b was also expressed. The recombinant TPO fragments were purified by preparative polyacrylamide gel electrophoresis using an electroendosmosis preparative gel electrophoresis unit (Genofit, Switzerland). The purity of the recombinant fusion proteins purified by this method was assessed by SDS-PAGE as shown in Figure 1.

5. Enzyme-linked immunosorhent assay (ELZSA) The ability of HPLC-purified MoAbs to bind to APTPO, trypsinised porcine TPO and recombinant TPO preparations was assessed by ELISA. Microtitre plates (Nunc-Immunoplate Maxisorp F96, Nunc,

Figure 2 Prolein staining of SDSPAGE gel. Lanes 1 and 2 demonstratc thyroid microsome preparations before and after passage down MoAb A5 affinity column. Lane 3 demonstrates affinity purified TPO eluted off the affinity column. M refers to molecular weight markera (arrowcd) ai (from top to bottom) 205, 116, 97, 66. 45 and 29 kD. Note the decrease in protein staining at I10 kD. corresponding to TPO, in the microsome preparation after passage down the affinity column.

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Denmark) were coated with 200 p l of APTPO, porcine TPO or purified recombinant TPO fragments (Rla+b, R l c , R2a, R2b and R3) at the optimally determined concentration of 0.1 pg/ml in carbonate-bicarbonate buffer (pH 9.6), incubated overnight at 4OC and then washed with PBS-Tween (pH 7.4). Subfragments R l a and R l b were not used to coat the plates due to limited antigen availability. Each plate was blocked with 0.5% bovine serum albumin (BSA) in PBS Tween for 1 hr before adding serial dilutions (1:lOO to 1:10x106) of HPLC-purified MoAb preparations in PBS Tween. The coated plates were incubated at room temperature for 2 hr before washing and adding alkaline phosphatase-conjugated anti-mouse IgG (Sigma, Poole) and subsequently paranitrophenyl phosphate (Sigma, Poole) at 1 mg/ml in diethanolamine buffer (pH 9.8)14.The optical density (OD) was recorded at 405 nm after 1 hr using a microELISA plate reader (Titertek Multiscan MCC/340, Flow Laboratories) and the titre determined. 6. Immunoblotting The binding of the monoclonal antibodies to thyroid microsomes, APTPO and recombinant TPO fragments under reducing conditions was assessed by immunoblotting’’. Briefly, the antigen preparations were run on SDS-PAGE gels and then immunoblotted onto

nitrocellulose membranes (Hybond C, Amersham International) prior to incubation with HPLC-purified MoAbs (at 0.5 pglml). Bound MoAbs were detected by the addition of ‘251-labelled,affinity purified rabbit anti-mouse IgG (approximately 4 pCi/pg) followed by autoradiography. A rabbit polyclonal antiserum to GST, made using GST purified from transfected E. coli by glutathione affinity chromatography, was also used in immunoblotting experiments with the recombinant fusion proteins.

RESULTS 1. Production of monoclonal antibodies A total of six MoAbs were generated from the Balb/c mice (Table 1). The HPLC-purified MoAbs were all of IgG class and their purity was confirmed’ by SDSPAGE which demonstrated two single protein bands corresponding to IgG light chains (26 kD) and heavy chains (53 kD) (not shown). Although all the six MoAbs bound to crude thyroid microsome preparations by ELISA, only five MoAbs (Al-A5) showed reactivity towards APTPO by ELISA (Table l), suggesting that MoAb A6 is directed towards some component present in the

a) APTPO 2.4 -

2.0 1.6; OD

1.2 -

b) Rla+b 0

MoAbAl MoAb A2

MoAb A3 MoAb A4

2.0 -

MoAb A5

1.6 OD

1.2 -

0.8 -

0.8 -

0.4 -

0.4 -

0-

0, I

I

1

I

I

Titre Figure 3 Titration curves showing the effect of increasing dilutions of MoAbs AI-A5 on their ability to bind to (a) affinity-purified thyroid peroxidase (APTPO), and to (b) recombinant TPO fragment R la+b. Titres were determined from the dilutions giving half maximal absorbancc (OD) reading.

TPO EPITOPES

HPLC-purified preparation used to immunise the mice other than TPO. One of the MoAbs (Al) decreased TPO enzymatic activity as evidenced by a 30% reduction in guaiacol oxidation (Table 1).

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2. Antibody affinity purification of human TPO from thyroid microsomes Preliminary affinity purification experiments with MoAbs A4 and A5, shown to bind to hTPO under reducing conditions, demonstrated a better recovery of hTPO with MoAb A5, despite its lower anti-TPO titre by ELISA, hence this MoAb was used for subsequent purifications. Affinity purification from sodium deoxycholate solubilised thyroid microsomes using MoAb A5 coupled to Sepharose beads resulted in the production of a pure preparation of TPO with specific enzymatic activity enhanced by a factor of 53-fold. SDS-PAGE demonstrated that the APTPO preparation comprised a polypeptide of molecular weight 110 kD (Figure 2, Lane 3), whereas the microsome preparation before passage down the affinity column demonstrated multiple protein bands (Figure 2, Lane l). 3. ELISA

MoAbs A1-A5 showed reactivity towards APTPO by ELISA with titres of up to 1:100,000 (Table 1 and Figure 3). Using the TPO(r) fragments the epitope specificities of MoAbs A4 and A5 were both shown to reside within fragment Rla+b (residues 1-160), with titres of 1:50,000 and l:S,OOO respectively. None of the other MoAbs, including MoAb A6, bound to any of the other recombinant fragments by ELISA. Interestingly, none of the MoAbs recognised trypsinised preparations of purified porcine TPO although this antigen was still recognised by a polyclonal rabbit antiserum to porcine TPO by ELISA (not shown). 4. Immunoblotting The specificity of the monoclonal antibodies was assessed by immunoblotting using thyroid microsomal membrane preparations and APTPO separated by SDS-PAGE. Monoclonal antibody A4 showed binding to a 110 kD polypeptide in thyroid microsome preparations under reducing conditions which co-migrated with APTPO (Figure 4, lanes 1 and 3 respectively). Similar reactivity with both microsomes and APTPO was demonstrated with MoAb AS (results not shown). Interestingly, MoAb A6 only showed binding to an 80 kD component in the microsome preparation, and not APTPO, providing further evidence that this MoAb is recognising a contaminating protein in the

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Figure 4 Autoradiograph demonstrating binding of MoAb A4 to thyroid microsomes and APTPO. Lane I , thyroid microsomes; lane 2, thyroid microsomes after passage down affinity column; lane 3, affinity purified TPO. m refers to molecular weight markers (from top to bottom) 116, 97, 66, 45 and 29 kD. The autoradiograph was exposed for 3 days.

immunising preparation. The MoAbs A 1, A2 and A3 failed to show any specific binding to thyroid microsomes or APTPO under the reducing conditions of the immunoblot (despite binding to both by ELISA). The purified recombinant preparations of hTPO were used in immunoblotting experiments to ascertain the epitope specificity of all the MoAbs, but in par-

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Figure 5 Autoradiographs demonstrating binding of (a) MoAb A4, (h) MoAb AS and (c) a polyclonal anti-GST antiserum to the recombinant fusion proteins. Lanes 2-6 repreaent recombinant fusion proteins Rla+b, R l c . R2a, R2b and R3 respectively. Lane I represents recombinant GST alone. The weaker higher molecular weight band seen in lane I represents aggregated GST. The arrows indicate molecular weight markers (from top to bottom) 66,45,36,29 and 20 kD respectively.

ticular the two MoAbs A4 and AS which had shown reactivity to thyroid microsomes and APTPO. Both these antibodies showed reactivity to the amino terminal recombinant fragment of hTPO (Rla+b) in agreement with the ELISA data (Figure 5, Panel a and b). In addition MoAb A4 also bound to the subfragment R I b (residues 70-160) being well visualised after 5 days of exposure (Figure 6, Lane 1). All the purified recombinant fusion fragments of hTPO denionstrated binding to a polyclonal anti-GST antiserum by immunoblotting (Figure 5 , Panel c). DISCUSSION

Monoclonal antibodies to TPO were first described where crude thyroid microsomes were used as a source of antigen". Although the specificity of the antibodies was confirmed by imniunofluorescence'". the antibodics were principally of IgM class and hence unsuitable for imiiiunoblotting or purification of TPO by antibody affinity chromatography. The purification of porcine or human TPO preparations by biochemical techniques employing trypsinisation or gel purification of detergent solubilised TPO allowed the generation of monoclonal antibodies specific for TPO I ?. I \.?o.? I

In this study, six monoclonal antibodies were produced by immunising mice with HPLC-purified hTPO (a method that yields purities of TPO in excess of 60%1'). Experimental data provide strong evidence that five of these MoAbs (AI-AS) are specific for TPO. Firstly, they all bind to APTPO by ELISA and two of them (A4 and AS) maintain this reactivity under the reducing and denaturing conditions of Western blotting. The fact that one of the MoAbs (AS) was used to produce the APTPO which was subsequently used to confirm its specificity could engender a circular argument. However, there is compelling evidence that the APTPO preparation is indeed hTPO; it runs at the predicted molecular weight of approximately 110 kD on SDS-PAGE and has enhanced peroxidase enzymatic activity. The relatively small enhancement o f specific enzyme activity (S3-fold) may well be related to partial denaturation of the enzyme by the high pH conditions used for elution off the affinity column. Secondly, two of the MoAbs ( A 4 and AS) also react with purified recombinant human TPO preparations. Finally, a further MoAb ( A l ) decreases TPO enzymatic activity suggesting it binds to TPO at, or near to, the enzymatic site. Upon immunoblotting, three MoAbs (A 1-A3) failed to show any binding to microsomal antigens, TPO(r) preparations or APTPO despite binding to

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TPO EPITOPES

1

2

Figure 6 Autoradiograph demonstrating binding of MoAb A4 to recombinant TPO preparations R l b (lane I ) and Rla+b (lane 2). None of the other recombinant proteins demonstrated binding to MoAb A4. M refers to molecular weight markers (from top to bottom) 116, 97, 66, 45 and 29 kD.The autoradiograph was exposed for S days.

APTPO by ELISA. It can be postulated that these MoAbs recognise conformational determinants which are labile in the presence of SDS and/or reducing conditions. MoAbs A4 and A5 maintained their reactivity to microsomal antigens and APTPO by immunoblotting and hence recognise determinants that are likely to be sequential. This is further supported by the fact that they recognise the recombinant protein fragments. Similarly, the failure of MoAbs Al-A3 to bind to TPO(r) preparations by ELISA, despite binding to APTPO, provides further evidence that these MoAbs recognise conformational epitopes that are preserved on the APTPO molecule but are absent in the TPO(r)

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preparations. It is probable that the comparatively small recombinant protein fails to take up the appropriate 3-dimensional structure or the lack of an important post-translational modification such as glycosylation or phosphorylation may affect antibody binding to the prokaryotically-produced recombinant protein . Using purified overlapping recombinant TPO preparations for immunoblotting, the epitope specificity of monoclonal antibodies A4 and A5 was shown to reside within recombinant fragment Rla+b (residues 1-160), and in the case of MoAb A4 this has been further localised to fragment R1 b (residues 70-160). The relatively weak binding to R l b is likely to be related to the small amounts of the recombinant protein loaded onto the gel due to its poor expression in E . coli (unpublished data). Due to the overlapping nature of the recombinant fragments it is likely that the sequential epitope recognised is encompassed within amino acid residues 85-145. This epitope mapping was confirmed by the ELISA data showing MoAbs A4 and A5 binding specifically to recombinant fragment Ral+b ( R l b was not tested due to limited supply of the antigen). The fact that all the recombinant GST-fusion proteins (and GST alone) demonstrated binding to a polyclonal anti-GST antiserum by immunoblotting indicates that the lack of binding of these recombinant proteins to other MoAbs is due to an inability of such MoAbs to recognise sequential determinants within these fragments rather than any failure of the immunoblotting process per- se. It is of interest that none of the MoAbs bound to porcine TPO by ELISA despite all binding to human TPO. Preparation of the porcine TPO involved an initial solubilisation step utilising trypsin which results in the removal of the first 109 amino acids from the amino terminus of the native moleculezz. It can be postulated that MoAbs A4 and A5 fail to bind to the trypsinised porcine TPO for one of two reasons; either the epitope recognised by them (shown to reside within amino acid residues 85-145) has been removed by trypsinisation or there is significant species differences in the amino acid sequence of porcine and human TPO in the region of the epitope, although amino acid sequence analysis within this region demonstrates 69% identity. If the former explanation is correct this implies that the epitope recognised is located between amino acid residues 85 and 109. Although the number of monoclonal antibodies mapped by this procedure is small, it is interesting that antibodies to the amino termina1 of hTPO have been readily generated by immunisation of mice, whereas anti-TPO autoantibodies in humans with AITD have been shown to be directed predominantly to the carboxyl-terminal of the TPO r n o l e c ~ l e ~ ~ ” ~ ’ ~ . Interestingly by recombinant DNA expression studies,

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monoclonal antibodies to hTPO generated in other l a b ~ r a t o r i e s ”have ~ ~ been mapped to recognise epitopes within residues 266-281 and 698-728 respectively6

Mapping epitope specificities of monoclonal antibodies to thyroid peroxidase using recombinant antigen preparations.

Five separate monoclonal antibodies (MoAbs) to human thyroid peroxidase (hTPO) were raised by immunising Balb/c mice with hTPO purified from detergent...
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