WO1994025604A2

WO1994025604A2 - Steroid 21-hydroxylase fragments and assays using the same

Info

Publication number: WO1994025604A2
Application number: PCT/GB1994/000855
Authority: WO
Inventors: Jadwiga Maria Furmaniak-Wehr
Original assignee: R S R Ltd
Current assignee: R S R Ltd
Priority date: 1993-04-24
Filing date: 1994-04-22
Publication date: 1994-11-10
Anticipated expiration: 1995-10-24
Also published as: GB9308544D0; AU6661094A; JPH09500008A; WO1994025604A3

Abstract

Polypeptides comprising an amino acid sequence of steroid 21-hydroxylase containing an epitopic region for adrenal autoantibody are useful in assay for adrenal autoantibodies or the therapy of adrenal autoimmune disease. The polypeptides may be synthetic or expressed by novel transformants transfected with DNA encoding the polypeptides.

Description

Steroid 21-hvdroxylase fragments and assays using the same

The present invention relates to fragments of steroid 21 -hydro xylase and to immunoassays for adrenal autoantibodies involving such fragments.

Addison's disease is a disorder characterised by failure of the adrenal gland and is often an autoimmune disorder involving destruction of the adrenal cortex and the presence of adrenal autoantibodies in the patient's serum. The adrenal cortex is responsible for producing several steroid hormones including cortisol, aldosterone and testosterone. In autoimmune Addison's disease and other forms of the disease, levels of these hormones are reduced. This reduction in hormone levels is responsible for the clinical symptoms of the disease which include low blood pressure, muscle weakness, increased skin pigmentation and electrolyte imbalance.

An autoantigen against which Addison's disease autoantibodies are directed has been isolated and determined to be steroid 21-hydroxylase (p450c21)^1,2. It has been proposed to use steroid 21-hydroxylase or fragments thereof in assays for such antibodies

The present invention relates to steroid 21-hydroxylase fragments of most surprising identity which are useful in assays for adrenal autoantigen. The invention also relates to DNA encoding such fragments and to assays using them.

In one aspect the invention provides a polypeptide comprising an amino acid sequence which is shown in the amino acid sequence extending from amino acid residue No. 227 to amino acid residue No. 480 of sequence SEQ ID No 1 and which contains an epitopic region for adrenal autoantibody. In one embodiment such polypeptides also include an amino acid sequence which is shown in the amino acid sequence extending from amino acid residue No. 1 to amino acid residue No. 127 of sequence SEQ ID No. 1. Also provided by the invention is a polypeptide comprising an epitopic region for adrenal autoantibody and including an amino acid sequence shown in the amino acid sequence extending from amino acid residue No 434 to amino acid residue No 480 of sequence SEQ ID No. 1. Included in the invention is a polypeptide comprising an epitopic region for adrenal autoantibody and an amino acid sequence which is shown in the amino acid sequence extending from amino acid residue No. 227 to amino acid residue No. 480 of sequence SEQ ID No. 1 and which includes amino acid residue No. 434 of sequence SEQ ID No. 1.

The polypeptides of the invention may optionally be modified by one or more amino acid alterations (deletions, additions or substitutions) which do not cause loss of adrenal autoantibody binding properties.

The polypeptides of the invention may be of recombinant origin. The polypeptides may be glycosylated or unglycosylated.

The polypeptides of the invention may be synthetic peptides, synthesised by any known method, for example.

The polypeptides of the invention are useful in assays and may be labelled.

In a further aspect the invention provides DNA sequences encoding a polypeptide of the invention.

The invention in another aspect resides in DNA sequences v/hich encode an epitope to adrenal autoantibody and which comprise a nucleic acid sequence shown in the nucleic acid sequence extending from nucleic acid base No 733 to base No. 1494 of sequence SEQ ID No. 1. Such DNA sequences may also include a nucleic acid sequence shown in the nucleic acid sequence extending from nucleic acid base No 13 to nucleic acid base No 435 of sequence SEQ ID No. 1.

In a yet further aspect the invention provides DNA which encodes an epitope to adrenal autoantibody and which includes a nucleic acid sequence shown in the amino acid sequence extending from nucleic acid base No. 1354 to nucleic acid base No. 1494 of sequence SEQ ID No. 1.

Included in the invention is DNA which encodes an epitope to adrenal autoantibody and which comprises a nucleic acid sequence shown in the nucleic acid sequence extending from nucleic acid base 733 to base No. 1494 of sequence SEQ ID No. 1 and which includes bases Nos. 1354-1356 of sequence SEQ ID No. 1.

In another aspect, the invention comprises DNA sequences encoding an epitope to adrenal autoantibody and consisting of the nucleic acid sequence extending from nucleic acid base No. 733 to base No. 1494 of sequence SEQ ID No. 1 and sequences which have homology to at least part of the aforesaid sequence, encode an epitope to adrenal autoantibody and optionally include bases No. 1354-1356 of sequence SEQ ID No. 1.

The invention also includes DNA sequences which hybridise to any of the above- mentioned sequences and encode a polypeptide comprising an epitope to adrenal autoantigen. The hybridisation conditions used are a matter of individual choice.

Hybridisation conditions which may be used to find active sequences include, but are not limited to, 1 M NaCl/10 x Denhardt's solution/50 mM Tris-HCl (pH

7.4)/ 10 mM EDTA/0.1 % SDS/100 μg/ml denatured herring sperm DNA (Sigma) at 65°C for 16 h, with the following washing conditions, i.e. 2 x SSC/0.1 % SDS,

42° C— >0.5 x SSC/0.1 % SDS, 50°C— >0.1 x SSC/0.1 % SDS, 65°C— >0.1 x SSC/0.1 % SDS, 68°C.

The DNA provided by the invention includes DNA sequences degenerate to those described above having regard to the genetic code. Also included are allelic equivalents of the described sequences and their degenerate equivalents.

In other aspects, the invention provides an expression vector comprising DNA of the invention and a host transformed by such an expression vector. The invention also relates to a process for producing a transformant capable of expressing a polypeptide of the invention, comprising transforming a host with an expression vector of the invention.

In yet further aspects, the invention provides a method of preparing a polypeptide of the invention, comprising culruring a transformant capable of expressing such a polypeptide so as to permit expression of the DNA encoding the polypeptide, as well as a process for producing an expression vector of the invention, comprising inserting into a vector capable of transforming a host cell a DNA sequence of the invention in a position permitting expression of the DNA.

The invention includes a method of producing a yeast expression vector comprising a DNA sequence of the invention, comprising (a) replacing the bases coding for the first 13 amino acids of the steroid 21-hydroxylase gene sequence with bases coding for a yeast leader sequence, (b) before or after said replacement digesting the steroid 21-hydroxylase gene sequence with one or more restriction enzymes, such that steps (a) and (b) together result in a DNA fragment with the yeast leader sequence and encoding a polypeptide comprising an epitope to adrenal autoantibody or result in plurality of DNA fragments which may be ligated together to form a DNA sequence encoding such a polypeptide, if necessary providing a stop codon at the 3 ' end of the fragments or of that one of the plurality of fragments which is to form the 3' end of the coding sequence, and (c) ligating d e DNA fragment or fragments into a yeast expression vector.

In one preferred embodiment of step (a), the bases coding for the first 13 amino acids of steroid 21-hydroxylase are replaced with a DNA sequence comprising the first 42 base pairs of coding sequence from the STE2 gene.

Preferably, in step (a), the first 41 base pairs of the steroid 21-hydroxylase gene sequence (21 -OH) are removed therefrom and the resultant fragment is ligated into yeast expression vector pYES2, as cut with BamHI and SphI, using a BamHI-Narl linker comprising 11 base pairs of non-coding and 42 base pairs of coding STE2 gene sequence, to prepare a pYES2/21-OH construct. Preferably, in step (b), the construct is digested with BamHI, PvuII and SphI to obtain BamHI-PvuII and PvuII-SphI fragments, which in step (c) are ligated into a yeast expression vector.

Preferably the expression vector is constructed by cloning into the EcoRI and SphI sites of pYES2 a linker including one or more stop codons and a restriction site for a restriction enzyme to be used in transforming a host, for example E. Coli.

The invention further provides a yeast expression vector comprising a DNA coding sequence for a polypeptide of the invention ligated at its 5' end to a yeast leader sequence, especially the STE2 leader sequence, and controlled by the GALI promoter. Also included are host cells, e.g. E. Coli or Saccharomyces cerevisiae, transformed by such an expression vector or an expression vector obtainable by a method described herein.

In another aspect there is provided a method of producing a yeast expression vector comprising a DNA sequence of the invention, the method comprising ligating into a yeast expression vector a DNA sequence comprismg a yeast leader sequence, control sequences and a DNA sequence of the invention in proper reading frame.

The invention relates to assays for adrenal autoantibody which use a polypeptide of the invention, and to assay kits comprising such a peptide. Methods of using the polypeptides of the invention to detect for adrenal autoantibody and kits comprising such polypeptides are set forth in the accompanying claims.

In a yet further aspect there is provided a pharmaceutical formulation comprising a polypeptide of the invention formulated for pharmaceutical use.

The invention is further described by way of example only with reference to accompanying sequence listing and drawings. In the sequence listing:

SEQ ID No. 1 shows the amino acid sequence and nucleic acid coding sequence of steroid 21-hydroxylase and the cleavage sites of restriction enzymes used in experiments described hereinafter. The sequence was obtained from a publically available source and is derived from the work of White et al.²³

SEQ ID No. 2 shows the nucleic acid sequence of a novel linker molecule useful in preparing a polypeptide of the invention and itself the subject of one aspect of the invention.

In the drawings:

Figure 1 is a schematic restriction enzyme map of the human steroid 21- hydroxylase gene;

Figure 2 is a schematic diagram of full-length, truncated and internally deleted steroid 21-hydroxylase in vectors pYES2 and pYES;

Figure 3 shows a western blot of polypeptides produced in vitro and reacted with rabbit antibody to steroid 21-hydroxylase;

Figure 4 shows a western blot of polypeptides produced in vitro and reacted with adrenal autoantibody; and

Figure 5 shows an SDS-PAGE analysis followed by autoradiography of ³⁵S- methionine labelled steroid 21-hydroxylase and polypeptides produced in vitro.

Polypeptides

The invention relates to the preparation of polypeptides antigenic to adrenal autoantibody and the use of such polypeptides in assays. In accordance with the invention, such polypeptides comprise an amino acid sequence of a region of steroid 21-hydroxylase towards its carboxyl terminal end. The polypeptides may be modified by one or more amino acid alterations (additions, deletions or substitutions) so long as they retain the property of binding to adrenal autoantibody.

The fragment between amino acid residues 227 and 480 of the accompanying sequence listing SEQ ID No. 1 binds effectively to adrenal autoantibody. It is most surprising that the binding sites is in a region at the carboxyl terminal end. The polypeptides comprise an epitope to adrenal autoantibody and preferably comprise an amino acid sequence which is shown in the amino acid sequence extending from amino acid residue 227 to amino acid residue 480 of SEQ ID No. 1. Amino acid and nucleic acid sequences defined herein by reference to the terminals residues of the sequence are inclusive of the terminal residues. Residue 480 is the final amino acid at the carboxyl terminal of steroid 21-hydroxylase. In preferred embodiments, the polypeptides comprise an amino acid sequence which is shown in the amino acid sequence extending from amino acid residue 494 to amino acid residue 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390 or 400.

The region between amino acid residues 434 and 480 has been shown to be important for autoantibody binding. The invention therefore includes polypeptides which include a sequence comprising residue 434 (e.g. the sequence extending from residue 434 to residue 480) and an epitope for adrenal autoantibody. Also included are those polypeptides which include a sequence comprised in the region between residues 433 and 480, for example polypeptides including at least the sequence between residues 400 and 480.

The polypeptides may be recombinant or synthetic for example. DNA

DNA sequences coding for the polypeptides of the invention are therefore included in the invention. Also included is DNA comprising a coding sequence shown in SEQ ID No. 1 and encoding an epitope to adrenal autoantibody. Further included are DNA sequences coding for polypeptides which bind to adrenal autoantibody and which hybridise to DNA comprising a sequence shown in SEQ ID No. 1 and encoding a polypeptide of the invention. Thus, the invention provides DNA sequences which encode an epitope to adrenal autoantibody and which comprise a nucleic acid sequence shown in the nucleic acid sequence extending from nucleic acid base No 733 to base No. 1494 of sequence SEQ ID No. 1. Such DNA sequences may also include, for example, a nucleic acid sequence shown in the nucleic acid sequence extending from nucleic acid base No 13 to nucleic acid base No 435 of sequence SEQ ID No. 1.

The region between bases 1354 and 1494 has been shown to encode an amino acid sequence important for autoantibody binding. The invention therefore includes DNA sequences which include a sequence comprising base 1354 (e.g. the sequence extending from base 1354 to base 1356 or from 1354 to base 1394 and encoding an epitope to adrenal autoantibody.

The invention includes DNA sequences which hybridise to those described above and encode an epitope to adrenal autoantibody. Sequences degenerate to those previously described, allelic equivalents and their degenerate equivalents are included.

The DNA is expressed by being incorporated into an expression vector which in turn is used to transform a host, e.g. E. Coli or S. Cerevisiae, which expresses the polypeptide.

DNA encoding a polypeptide of the invention may be prepared using the pYES2/21-OH construct previously described^1*2. Preparation of a DNA sequence and Polypeptide

In an exemplary experiment, the pYES2/human 21 -OH construct under control of the GAL1 promotor including the yeast STE2 leader sequence was modified by cloning a unique linker designed to include useful restriction sites and introduce translation stop codons (TGA). A series of truncations and deletions 3' from the leader sequence have been made, expressed in yeast and tested for binding to rabbit antibody to 21 -OH and adrenal autoantibody. The truncations and deletion were made using PvuII, Saul, PmaCI and Stul.

In addition, protein expression and reaction with antibodies have been analysed using an in vitro translation system.

A deletion between the two PvuII sites had no effect on antibody and autoantibody binding to recombinant protein. The truncations at the Saul and PmaCI sites resulted in loss of the ability of recombinant protein to bind rabbit antibody and autoantibody. Recombinant 21 -OH after truncation at the Stul site reacted with rabbit antibody but not with adrenal autoantibody. Addison pool serum and four individual sera from patients with high levels of adrenal autoantibody were used in these experiments. The full-length construct and all modified constructs were expressed in Saccharomyces Cerevisiae and using an in vitro coupled reticulocyte lysate translation system (sold under the trade mark TNT by Promega Corp. , 2800 Woods Hollow Road, Madison, Wl 53711-5399, USA). Expressed proteins were analysed by western blotting using rabbit antibody and Addison sera. In addition, the recombinant protein in this system labelled with 35-S-methionine and analysed on SDS-PAGE followed by autoradiography.

Assays

The polypeptides of the invention, for example protein obtained using the steroid 21-hydroxylase gene with the PvuII-PvuII deletion, may be used in assays for adrenal autoantibody. The assays may be competitive or non-competitive and examples are radioimmunoassays (RIA), enzyme immunoassays (El A) and enzyme- linked immunosorbent assays (ELISA). Further details of suitable assays can be found in the literature³ and especially in GB 2256046, which describes two illustrative assay techniques for adrenal autoantibodies.

The assays may be performed using a suitable kit. Assays involving the interaction of a polypeptide of the invention and a sample may be performed using a kit comprising antigenic protein. The reader is referred to GB 2256046 for further information. The polypeptides of the present invention may be used in the methods and kits of GB 2256046 in place of the proteins and protein fragments described in GB 2256046.

The invention also includes the use of antibody against a polypeptide of the invention to detect steroid 21-hydroxylase. Such antibodies may be prepared by any known method, for example, and may be polyclonal or, preferably, monoclonal. Further description of suitable antibodies and their preparation may be found in GB 2256046.

Therapy of Adrenal Autoimmune Disease

It is thought that autoimmune diseases are caused at least in part by autoantigens persistently activating T cells. The autoantigen in the case of adrenal autoimmune disease can be any epitope of the antigenic molecule which is recognised by a T cell receptor capable of helping a B cell make antibody to the antigenic molecule, or a T cell otherwise involved in the autoimmune disorder.

Adrenal autoimmune disorders should therefore be treatable by disrupting the action of T cells involved in the disorder. T cells such as, for example, those infiltrating the adrenal gland, in lymphoid organs or in the circulation can act as

T helper (Th) cells in responding to adrenal autoantigen epitopes. Such Th cells help B cells make specific anti-autoantigen antibodies. T cells can also act in mediating cell-mediated immune responses and act on adrenal cortex cells by the release of cytokines or directly. The destruction of adrenal cortex cells can result, when cytotoxic T cells specific for the adrenal autoantigen are activated, or by an inflammatory response mediated by a different T cell class.

If such T cells were interfered with, the production of anti-autoantigen antibodies and of T cells destructive of adrenal cortex cells would be suppressed. The T cells could be disrupted by a polypeptide of the invention which is capable of binding to the T cell receptor (TCR) of an adrenal autoantigen specific T cell, i.e. containing a T cell epitope of the autoantigen.

Such a polypeptide acts as a competitive antagonist for the autoantigen in the adrenal cortex and thereby inhibits antigen specific cellular interactions which result in the production of adrenal autoantibody or adrenal autoantigen specific cell-mediated immunity. Exemplary references describing the use of peptides to treat autoimmune disease are included in the attached bibliography^4"10.

Another therapeutic approach is to suppress the autoimmune response to adrenal autoantigen by administering T cells¹¹"¹⁷ specific to a polypeptide of the invention or a protein or protein fragment (e.g. peptide) mimicking the TCR of such cells^{16 17}. Administration of such a "vaccine" induces "counter-autoimmunity" which is thought to be mediated by T cells specific to the TCR of the autoimmune T cells¹¹-¹⁶'¹⁸-¹⁹.

The autoimmune response may also be suppressed by T suppressor (Ts) cells capable of specifically recognising an anti-adrenal autoantigen B cell or T cell^20,21. Such therapy may be effected by administering to the patient a polypeptide of the invention capable of inducing Ts cells or by administering Ts cells specific to a polypeptide of the invention and capable of suppressing an anti-adrenal autoantigen response.

The autoimmune TCR specific to the adrenal autoantigen may be characterised using methods described in the literature²². Disruption of interaction of the autoimmune T cells with the adrenal autoantigen may alleviate or prevent adrenal autoimmune disease. T cells which will disrupt such interaction and which are specific for the autoimmune TCR for adrenal autoantigen may be prepared²³.

The present invention provides pharmaceutical (including veterinary pharmaceutical) formulations comprising a polypeptide of the invention.

The invention also includes pharmaceutical formulations comprising a polypeptide of the invention capable of binding to the TCR of an adrenal autoantigen specific T cell or B cell.

In other aspects, the invention provides T cells specific for a polypeptide of the invention and proteins or protein fragments (e.g. peptides) which mimick the receptor of such T cells. Included in the invention are counter-autoimmune T cells, i.e. T cells induced by such adrenal autoantibody specific T cell or TCR mimickig protein or protein fragment. Pharmaceutical preparations including any of the aforesaid agents are further provided by the invention.

Ts cells capable of interacting specifically with an anti-adrenal autoantigen B cell or T cell may be formulated into pharmaceutical formulations. The invention therefore includes the use of a polypeptide of the invention to generate such anti- autoimmune T cells.

Other Uses of the Inventive Polypeptides

The inventive polypeptides may be used to purify adrenal autoantibody, for example by adsorbing such a polypeptide on a substrate, passing a sample containing the antibody over the substrate and then washing the substrate to release the antibody. Example

The 21 -OH cDNA sequence (obtainable from plasmid pCD//pC21/3c - ATCC 57421) has previously been modified by the replacement of the natural signal sequence with the STE2 signal sequence and cloned into yeast expression vector pYES2, where the 21-OH sequence is controlled by the GAL1 promotor.

The full length human steroid 21-hydroxylase has been successfully expressed in yeast (Saccharomyces cerevisiae) transformed using the pYES2/21-OH construct. The recombinant protein reacts with rabbit antibody to 21 -OH and human adrenal autoantibodies¹'².

This example describes the preparation of a polypeptide containing an epitope to adrenal autoantibody using the pYES2/human 21 -OH construct with 21 -OH controlled by the GAL1 promotor and including the yeast STE2 leader sequence. A series of truncations and deletions 3' from the leader sequence were made, expressed in yeast and tested for binding to rabbit antibody to 21 -OH and adrenal autoantibody. In addition, protein expression and reaction with antibodies were analysed using the in vitro coupled reticulocyte lysate translation system.

To isolate modified 21 -OH gene fragments, restriction sites within the gene sequence were used. The strategy involved isolation of suitable fragments from pYES2/21-OH which contains the full coding sequence of 21 -OH (Fig. 1). The construction of pYES2/21-OH full length human 21-hydroxylase gene placed downstream of the GAL1 promotor in pYES2, (Invitrogen, distributed by British Biotechnology Products Ltd, Abingdon, UK) with the native signal peptide replaced by the leader sequence from the yeast STE2, gene has of course been described previously^1,2. In addition to the 21-hydroxylase coding sequence, pYES2/21-OH has 650 bp of non-coding sequence following the TGA stop codon which includes the polyadenylation signal and site.

The 21 -OH full-length sequence with the STE2 leader sequence and the 650 bp non-coding sequence was cloned into the BamHI and SphI sites of the pYES2 multicloning site. The 5' end of the insert sequence was cloned into BamHI site and the 3' end into SphI site.

The vector pYES3 is a novel derivative of pYES2 constructed by cloning a specially-designed, unique linker into the EcoRI and SphI sites of the multiple cloning site of pYES2. The linker is shown in SEQ ID No. 2 of the accompanying sequence listing. The linker, prepared by annealing two chemically synthesised oligonucleotides, was designed to include PmaCI and Saul restriction sites for cloning and a series of TGA stop codons which ensured translation termination of all the truncated 21-hydroxylase genes.

21 -OH gene fragments comprising a constant 5' end, but differing length of 21 -OH coding sequence were isolated from pYES2/21-OH. This was accomplished by digestion with BamHI and either PmaCI, Saul or Stul, whose cleavage sites are indicated in sequence SEQ ID No. 1 of the sequence listing. A series of constructs was made by ligating these fragments into pYES3 (digested with BamHI and either PmaCI or Saul).

The two restriction sites included in the linker consisted of one "sticky end" site (Saul) and one "blunt end" site (PmaCI). These sites were used for cloning in the following way:- the 21 -OH full-length sequence gene (containing BamHI site at the 5' end) after truncation at Saul site was cloned into the compatible sites in pYES3 (BamHI and Saul; the Saul site was provided by the incorporated linker).

Similarly, the version of 21-OH which had been truncated at the PmaCI site was cloned into the BamHI and PmaCI sites of pYES3 (a compatible PmaCI site in the vector was provided by the linker sequence). Digestion of the 21 -OH gene with Stul yielded the "blunt ended" gene sequence which could then be ligated into the "blunt end" site (PmaCI) of pYES3.

A construct with an internal PvuII deletion was prepared by cloning the 21 -OH BamHI-PvuII and PvuII-SphI fragments excised from pYES2/21-OH into pYES3 cut with BamHI and SphI.

A schematic diagram of full-length, truncated and internally deleted 21 -OH constructs is shown in Fig. 2.

The cloning procedure involved a restriction digest of the pYES2/21-OH construct with the appropriate restriction endonucleases followed by analysis on 1 % agarose gel. The 21 -OH insert of the appropriate size was cut out of the agarose gel and purified using the Gene-clean reagents (Bio 101, supplied by Stratech Scientific). . This insert was ligated into pYES3 cut with appropriate enzymes and transformed into E.Coli. In the case of PvuII deletion, the fragments BamHI-PvuII and PvuII- Sphl were cut out from pYES2/21-OH, gene cleaned and ligated into PYES3 prepared by digestion with BamHI and SphI in a three-way ligation reaction.

Plasmid DNA was isolated from the transformed E.Coli and analysed by digestion with restriction endonucleases and electrophoresis on agarose gels. When the construct appeared to be correct, larger scale plasmid preparation was performed using Qiagen™- 100 kits (Qiagen, supplied by Hybaid Ltd). Pure preparations of plasmid DNA were checked by digestion with restriction enzymes in different combinations and when correct, used to transform the yeast, Saccharomyces cerevisiae, or used in an in vitro transcription/translation system (TnT coupled reticulocyte lysate system, Promega Corp., 2800 Woods Hollow Road, Madison, WI 53711-5399, USA). The yeast transformants were grown under inducing conditions (YEP-galactose) as described before¹*² and analysed on SDS-PAGE followed by Western blot.

Full length or modified proteins expressed in yeast or in TnT were analysed by western blotting using rabbit antibody and Addison sera. Addison pool serum and four individual sera from patients with high levels of adrenal autoantibody were used in these experiments. In addition, the recombinant proteins in TnT were labelled with ³⁵S-methionine and analysed on SDS-PAGE followed by autoradiography. The analyses are shown in Figures 3 and 4. Figure 3 is an analysis by SDS-PAGE and western blotting of polypeptides produced in vitro and reacted with rabbit antibody (Lane 1 reaction with in vitro transcription/ translation of vector only; Lane 2 : vector with 21 -OH sequence truncated at Stul site; Lane 3 : vector with 21 -OH sequence truncated at Saul site; Lane 4 : vector with 21 -OH sequence truncated at PmaCI site; Lane 5 : vector with 21 -OH internal sequence deletion between PvuII and PvuII sites; Lane 6 : vector with full length 21 -OH sequence; Lane 7 : full length 21 -OH expressed in yeast). Figure 4 is an analysis by SDS-PAGE and western blotting of proteins produced by in vitro transcription/translation and reacted with adrenal autoantibody (Lanes as Fig. 3).

The deletion between two PvuII sites had no effect on antibody and autoantibody binding to recombinant protein (Figs. 3 and 4). The truncations at the Saul and PmaCI sites resulted in loss of the ability of the recombinant protein to bind rabbit antibody and autoantibody (Figs. 3 and 4). Recombinant 21 -OH after truncation at the Stul site reacted with rabbit antibody (Fig. 3), but not with adrenal autoantibody (Fig. 4).

These results are summarised in Table 1:-

Modification Length^* Mol. wt. Rabbit Auto¬ (SDS- antibody antibody PAGE) binding binding

Y V Y V

None AA 1-480 55K + -f- + +

Truncation AA 1-433 46K + + (-) (-) at Stul site

Truncation AA 1-364 43K (-) (-) (-) (-) at Saul site

Truncation AA 1-266 32K (-) (") (-) (-) at PmaCI site

Deletion AA 1-127 45K + - + + PvuII-PvuII and

227-480

The length is expressed in terms of the imtial and final amino acids as numbered in SEQ ID No. 1.

Key: AA = amino acids, Y = expressed in yeast, V = expressed in vitro, + = reaction with antibody, (-) = no reaction with antibody.

The introduced truncations and deletions did not affect the expression of the protein as shown in the in vitro translation experiments, the results of which appear in Figure 5, which is an SDS-PAGE analysis followed by autoradiography of ³⁵S-methionine labelled 21 -OH and 21 -OH fragments produced by the TnT in vitro transcription/translation system (Lanes as Figure 3). There was a good agreement between the results obtained on western blot analysis of proteins expressed in the rabbit reticulocyte system and in yeast.

Analysis of autoantibody binding to recombinant 21 -OH with mid-sequence deletions and 3 ' truncations indicated that the autoantibody binding site was located in a region at the carboxyl terminal end of the protein comprised in the region between residues 434 and 480. The deletion of large middle portions of the sequence did not affect autoantibody binding. This would suggest that the autoantibody epitope(s) on 21 -OH are most probably linear which is consistent with previous observations. The number of autoantigenic epitopes situated on the fragment involved in autoantibody binding is almost certainly restricted.

The data indicate that the short fragment of 21 -OH between amino acids 434 and 480 is important for adrenal autoantibody binding. Furthermore, derivatives

(fragments) of 21-OH consisting of midsequence deletions such as the 396 AA preparations shown in Table 1 are suitable for use in adrenal autoantibody assays.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: RSR Limited

(B) STREET: Avenue Park

(C) CITY: Cardiff

(E) COUNTRY: UK

(F) POSTAL CODE (ZIP) : CF2 7HE

(ii) TITLE OF INVENTION: Assay for Adrenal Autoantigen (iii) NUMBER OF SEQUENCES: 2

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1509 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iii) ANTI-SENSE: NO fvi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(D) DEVELOPMENTAL STAGE: roetus

(F) TISSUE TYPE: adrenal gland

(ix) FEATURE:

(A) NAME/KEY: sig_peptide

(B) LOCATION: 13..54 fix) FEATURE:

(A) NAME/KEY: mat_peptide

(B) LOCATION: 55..1494

(D) OTHER INFORMATION: product= "steroid 21- ydroxylase" i ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 13..1494 ( i x ) FEATURE :

( A ) NAME/ KEY : rn ι sc_ f eature

( B ) LOCATION : ( 4 35 ' 4 36 )

(D) OTHER INFORMATION: /standard_name= "PvuII cleavage site"

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: (732-733)

(D) OTHER INFORMATION: /standard_name= "PvuII cleavage site"

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: (852-853)

(D) OTHER INFORMATION: /standard_name= "PmaCI cleavage site"

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: (1148-1149)

(D) OTHER INFORMATION: /standard_name= "'Saul cleavage site"

(IX) FEATURE:

(A) NAME/KEY: mιsc_feature

(B) LOCATION: (1354"1355)

(D) OTHER INFORMATION: /standard_name= "Stul cleavage site"

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: (53^Λ54)

(D) OTHER INFORMATION: /standard_name= "Narl cleavage site"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

GGGCGTCTCG CC ATG CTG CTC CTG GGC CTG CTG CTG CTG CCC CTG CTG 48

Met Leu Leu Leu Gly Leu Leu Leu Leu Pro Leu Leu -14 -10 -5

GCT GGC GCC CGC CTG CTG TGG AAC TGG TGG AAG CTC CGG AGC CTC CAC 96 Ala Gly Ala Arg Leu Leu Trp Asn Trp Trp Lys Leu Arg Ser Leu His 1 5 10

CTC CCG CCT CTT GCC CCG GGC TTC TTG CAC CTG CTG CAG CCC GAC CTC 144 Leu Pro Pro Leu Ala Pro Gly Phe Leu His Leu Leu Gin Pro Asp Leu 15 2C 25 ^" 30

CCC ATC TAT CTG CTT GGC CTG ACT CAG AAA TTC GGG CCC ATC TAC AGG 192 Pro lie Tyr Leu Leu Gly Leu Thr Gin Lys Phe Gly Pro lie Tyr Arg 35 40 45 CTC CAC CTT GGG CTG CAA GAT GTG GTG GTG CTG AAC TCC AAG AGG ACC 240 Leu His Leu Gly Leu Gin Asp Val Val Val Leu Asn Ser Lys Arg Thr 50 55 60

ATT GAG GAA GCC ATG GTC AAA AAG TGG GCA GAC TTT GCT GGC AGA CCT 288 lie Glu Glu Ala Met Val Lys Lys Trp Ala Asp Phe Ala Gly Arg Pro 65 70 75

GAG CCA CTT ACC TAC AAG CTG GTG TCT AAG AAC TAC CCG GAC TTG TCC 336 Glu Pro Leu Thr Tyr Lys Leu Val Ser Lys Asn Tyr Pro Asp Leu Ser 80 85 90

TTG GGA GAC TAC TCC CTG CTC TGG AAA GCC CAC AAG AAG CTC ACC CGC 384 Leu Gly Asp Tyr Ser Leu Leu Trp Lys Ala His Lys Lys Leu Thr Arg 95 100 105 110

TCA GCC CTG CTG CTG GGC ATC CGT GAC TCC ATG GAG CCA GTG GTG GAG 432 Ser Ala Leu Leu Leu Gly lie Arg Asp Ser Met Glu Pro Val Val Glu 115 120 125

CAG CTG ACC CAG GAG TTC TGT GAG CGC ATG AGA GCC CAG CCC GGC ACC 480 Gin Leu Thr Gin Glu Phe Cys Glu Arg Met Arg Ala Gin Pro Gly Thr 130 135 140

CCT GTG GCC ATT GAG GAG GAA TTC TCT CTC CTC ACC TGC AGC ATC ATC 528 Pro Val Ala lie Glu Glu Glu Phe Ser Leu Leu Thr Cys Ser lie lie 145 150 155

TGT TAC CTC ACC TTC GGA GAC AAG ATC AAG GAC GAC AAC TTA ATG CCT 576 Cys Tyr Leu Thr Phe Gly Asp Lys lie Lys Asp Asp Asn Leu Met Pro 160 165 170

GCC TAT TAC AAA TGT ATC CAG GAG GTG TTA AAA ACC TGG AGC CAC TGG 624 Ala Tyr Tyr Lys Cys lie Gin Glu Val Leu Lys Thr Trp Ser His Trp 175 180 185 190

TCC ATC CAA ATT GTG GAC GTG ATT CCC TTT CTC AGG TTC TTC CCC AAT 672 Ser lie Gin lie Val Asp Val lie Pro Phe Leu Arg Phe Phe Pro Asn 195 200 205

CCA GGT CTC CGG AGG CTG AAG CAG GCC ATA GAG AAG AGG GAT CAC ATC 720 Pro Gly Leu Arg Arg Leu Lys Gin Ala lie Glu Lys Arg Asp His lie

210 ^* 215 220

GTG GAG ATG CAG CTG AGG CAG CAC AAG GAG AGC CTC GTG GCA GGC CAG 768 Val Glu Met Gin Leu Arg Gin His Lys Glu Ser Leu Val Ala Gly Gin 225 230 235

TGG AGG GAC ATG ATG GAC TAC ATG CTC CAA GGG GTG GCG CAG CCG AGC 816 Trp Arg Asp Met Met Asp Tyr Met Leu Gin Gly Val Ala Gin Pro Ser 240 245 250

ATG GAA GAG GGC TCT GGA CAG CTC CTG GAA GGG CAC GTG CAC ATG GCT 864 Met Glu Glu Gly Ser Gly Gin Leu Leu Glu Gly His Val His Met Ala 255 260 265 270 GCA GTG GAC CTC CTG ATC GGT GGC ACT GAG ACC ACA GCA AAC ACC CTC 912 Ala Val Asp Leu Leu lie Gly Gly Thr Glu Thr Thr Ala Asn Thr Leu

275 280 285

TCC TGG GCC GTG GTT TTT TTG CTT CAC CAC CCT GAG ATT CAG CAG CGA 960 Ser Trp Ala Val Val Phe Leu Leu His His Pro Glu lie Gin Gin Arg

290 295 300

CTG CAG GAG GAG CTA GAC CAC GAA CTG GGC CCT GGT GCC TCC AGC TCC 1008 Leu Gin Glu Glu Leu Asp His Glu Leu Gly Pro Gly Ala Ser Ser Ser

305 310 315

CGG GTC CCC TAC AAG GAC CGT GCA CGG CTG CCC TTG CTC AAT GCC ACC 1056 Arg Val Pro Tyr Lys Asp Arg Ala Arg Leu Pro Leu Leu Asn Ala Thr

320 325 330

ATC GCC GAG GTG CTG CGC CTG CGG CCC GTT GTG CCC TTA GCC TTG CCC 1104 lie Ala Glu Val Leu Arg Leu Arg Pro Val Val Pro Leu Ala Leu Pro

335 340 345 350

CAC CGC ACC ACA CGG CCC AGC AGC ATC TCT GGC TAC GAC ATC CCT GAG 1152

His Arg Thr Thr Arg Pro Ser Ser lie Ser Gly Tyr Asp lie Pro Glu

355 360 365

GGC ACA GTC ATC ATT CCG AAC CTC CAA GGC GCC CAC CTG GAT GAG ACG 1200

Gly Thr Val lie lie Pro Asn Leu Gin Gly Ala His Leu Asp Glu Thr

370 375 380

GTC TGG GAG AGG CCA CAT GAG TTC TGG CCT GAT CGC TTC CTG GAG CCA 1248

Val Trp Glu Arg Pro His Glu Phe Trp Pro Asp Arg Phe Leu Glu Pro

385 390 J95

GGC AAG AAC TCC AGA GCT CTG GCC TTC GGC TGC GGT GCC CGC GTG TGC 1296

Gly Lys Asn Ser Arg Ala Leu Ala Phe Gly Cys Gly Ala Arg Val Cys

400 405 410

CTG GGC GAG CCG TTG GCG CGC CTG GAG CTC TTC GTG GTG CTG ACC CGA 1344

Leu Gly Glu Pro Leu Ala Arg Leu Glu Leu Phe Val Val Leu Thr Arg

415 420 425 430

CTG CTG CAG GCC TTC ACG CTG CTG CCC TCC GGG GAC GCC CTG CCC TCC 1392

Leu Leu Gin Ala Phe Thr Leu Leu Pro Ser Gly Asp Ala Leu Pro Ser

435 440 445

CTG CAG CCC CTG CCC CAC TGC AGT GTC ATC CTC AAG ATG CAG CCT TTC 1440

Leu Gin Pro Leu Pro His Cys Ser Val lie Leu Lys Met Gin Pro Phe

450 455 460

CAA GTG CGG CTG CAG CCC CGG GGG ATG GGG GCC CAC AGC CCG GGC CAG 1488

Gin Val Arg Leu Gin Pro Arg Gly Met Gly Ala His Ser Pro Gly Gin

465 470 475

AAC CAG TGATGGGGCA GGACC 1509 Asn Gin 430 INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..4

(D) OTHER INFORMATION: /standard_name= "EcoRI restriction end"

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: (7~8)

(D) OTHER INFORMATION: /standard_name= "Saul cleavage site"

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: (16^Λ17)

(D) OTHER INFORMATION: /standard_name= "PmaCI cleavage site"

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 31..33

(D) OTHER INFORMATION: /standard_name= "SphI restriction end"

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 8..10

(D) OTHER INFORMATION: /standard__name= "Stop codon"

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 18..20

(D) OTHER INFORMATION: /standard_name= "Stop codon"

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 22..24

(D) OTHER INFORMATION: /standard_name= "Stop codon"

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 26..28

(D) OTHER INFORMATION: /standard name-- "Stop codon" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

AATTCCCTGA GGCCACGTGA CTGACTGAGC ATG ^• 33

GGGACTC CGGTGCACTG ACTGACTC

Bibliography

1. Bednarek J et al (1992) FEBS Lett 309: 51-55

2. UK Patent Application No. 2256046, R S R Limited

3. Vunakis H V, Langone J L (editors), Methods in Enzymology, Academic Press, Vols. 70, 73, 74, 84 and 92 [Immunochemical Techniques parts A (1980), B (1981), C (1981), D (1982), EQ983)].

4. Janeway C (1989) Nature 341:482.

5. Acha-Orbea H et al. (1989) Ann. Rev. Immunol 7:371-405.

6. Kumar V er al. (1989) Ann. Rev. Immunol 7:657-682

7. Urban J L et al. (1989) Cell 54:577-592.

8. Wraith D C et al. (1989) Cell 57:709-715.

9. Wraith D C et al. (1989) Cell 59:247-255.

10. Urban J L et al. (1989) Cell 59:257-271.

11. Cohen I R (1986) Immunol. Revd. 94:5-21.

12. (1986) Prog. Immunol. V 491-499.

13. (1988) Scientific Amer. 258:52-60.

14. (Feb 15, 1989) Hosp. Prac. 57-64. 15. Cohen I R et al. (1988) Immunol. Today 9:332-335.

16. Vandenbark AA et al . ( 1989) Nature 341 : 541 -544.

17. Howell M D et al. (1989) Science 246:668-671.

18. Sun D et al. (1988) Nature 332:843-845.

19. Sun D et al. (1988) Eur. J. Immunol. 18: 1993-1999.

20. Green D et al. (1983) Ann. Rev. Immunol. 1:439.

21. Benacerraf B in The Biology of Immunologic Disease, H P Publishing Co Inc, New York 1983 (49-62).

22. Burns F J et al. (1989) J. Exp. Med. 169:27.

23. White P C et al. (1986) Proc. Natl. Acad. Sci. USA 83:5111-5115.

Claims

1. A polypeptide comprising an amino acid sequence which is shown in the amino acid sequence extending from amino acid residue No. 227 to amino acid residue No. 480 of sequence SEQ ID No. 1 and which contains an epitopic region for adrenal autoantibody.

2. A polypeptide of claim 1 which further comprises an amino acid sequence which is selected from the amino acid sequence extending from amino acid residue

No. 1 to amino acid residue No. 127 of sequence SEQ ID No. 1.

3. A polypeptide comprising an epitopic region for adrenal autoantibody and including an amino acid sequence shown in the amino acid sequence extending from amino acid residue No. 434 to amino acid residue No. 480 of sequence SEQ

ID No. 1.

4. A polypeptide comprising an epitopic region for adrenal autoantibody and an amino acid sequence which is shown in the amino acid sequence extending from amino acid residue No. 227 to amino acid residue No. 480 of sequence SEQ ID No. 1 and which includes amino acid residue No. 434 of sequence SEQ ID No. 1.

5. A polypeptide of any of claims 1 to 4 modified by one or more amino acid alterations (deletions, additions or substitutions) which do not cause loss of adrenal autoantibody binding properties.

6. A polypeptide of any of claims 1 to 5 which is of recombinant origin.

7. A polypeptide of any of claims 1 to 5 which is synthetic.

8. A polypeptide of any of claims 1 to 7 which is labelled.

9. A DNA sequence encoding a polypeptide of any of claims 1 to 5.

10. A DNA sequence which encodes an epitope to adrenal autoantibody and which comprises a nucleic acid sequence shown in the nucleic acid sequence extending from nucleic acid base No. 733 to base No. 1494 of sequence SEQ ID

No. 1.

11. A DNA sequence of claim 10 which further comprises a nucleic acid sequence shown in the nucleic acid sequence extending from nucleic acid base No.

13 to nucleic acid base No. 435 of sequence SEQ ID No. 1.

12. A DNA sequence which encodes an epitope to adrenal autoantibody and which includes a nucleic acid sequence shown in the amino acid sequence extending from nucleic acid base No. 1355 to nucleic acid base No. 1494 of sequence SEQ ID No. 1.

13. A DNA sequence which encodes an epitope to adrenal autoantibody and which comprises a nucleic acid sequence shown in the nucleic acid sequence extending from nucleic acid base No 744 to base No. 1494 of sequence SEQ ID

No. 1 and which includes bases Nos. 1354-1356 of sequence SEQ ID No. 1.

14. A DNA sequence which hybridises to a DNA of any of claims 9 to 13 encodes a polypeptide comprising an epitope to adrenal autoantigen.

15. An expression vector comprising a DNA sequence of any of claims 9 to 14.

16. A host transformed by an expression vector of claim 15.

17. A process for producing a transformant capable of expressing a polypeptide of any of claims 1 to 5 comprising transforming a host with an expression vector of claim 15.

18. A method of preparing a polypeptide of any of claims 1 to 5, comprising culturing a transformant capable of expressing such a polypeptide so as to permit expression of the DNA encoding the polypeptide.

19. A process for producing an expression vector of claim 15, comprising inserting into a vector capable of transforming a host cell a DNA sequence of any of claims 9 to 14 in a position permitting expression of the DNA sequence.

20. A method of producing a yeast expression vector comprising a DNA sequence of the invention, comprising (a) replacing the bases coding for the first 13 amino acids of the steroid 21-hydroxylase genes sequence with bases coding for a yeast leader sequence, (b) before or after said replacement digesting the steroid 21-hydroxylase gene sequence with one or more restriction enzymes, such that steps (a) and (b) together result in a DNA fragment with the yeast leader sequence and encoding a polypeptide comprising an epitope to adrenal autoantibody or result in plurality of DNA fragments which may be ligated together to form a DNA sequence encoding such a polypeptide, if necessary providing a stop codon at the 3' end of the fragments or of that one of the plurality of fragments which is to from the 3' end of the coding sequence, and (c) ligating the DNA fragment or fragments into a yeast expression vector.

21. A method of claim 20 wherein in step (a), the bases coding for the first 13 amino acids of steroid 21-hydroxylase are replaced with a DNA sequence comprising the first 42 base pairs of coding sequence from the STE2 gene.

22. A method of claim 21, wherein the first 41 base pairs of the steroid 21- hydroxylase gene sequence are removed therefrom and the resultant fragment is ligated into yeast expression vector pYES2 as cut with BamHI and SphI, using a BamHI-Narl linker comprising 11 base pairs of non-coding and 42 base pairs of coding STE2 gene sequence, to prepare a pYES2/21-OH construct.

23. A method of any of claims 20 to 22, wherein in step (b), the construct is digested with BamHI, PvuII and SphI to obtain BamHI-PvuII and PvuII-SphI fragments, which in step (c) are ligated into a yeast expression vector.

24. A method of claim 23, wherein the expression vector is constructed by cloning into the EcoRI and SphI sites of pYES2 a linker including one or more stop codons and a restriction site for a restriction enzyme to be used in transforming a host, for example E. Coli.

25. A yeast expression vector comprising a DNA coding sequence for a polypeptide of any of claims 1 to 5 ligated at its 5' end to the STE2 leader sequence or another yeast leader sequence and controlled by the GALI promotor.

26. A host cell, e.g. E. Coli or Saccharomyces cerevisiae, transformed by such an expression vector of claim 25 or an expression vector obtainable by a method of any of claims 20 to 24.

27. A method for detecting adrenal autoantibodies in a sample, comprising contacting with the sample a polypeptide of any of claims 1 to 8.

28. A method of claim 27, wherein the polypeptide is labelled and the method further comprises detecting for labelled polypeptide/autoantibody complex or unbound labelled polypeptide.

29. A method of claim 27 which further comprises contacting the polypeptide and the sample with labelled antibody to the polypeptide and then detecting labelled antibody bound to the polypeptide.

30. A method of claim 29, wherein the polypeptide is immobilised on a substrate.

31. A method of claim 27 wherein the polypeptide is immobilised on a substrate and the method further comprises, after contacting the polypeptide with the sample, contacting the polypeptide with labelled ligand for adrenal autoantibody and detecting bound labelled ligand.

32. A method of claim 31 , wherein the ligand is a polypeptide of any of claims

1 to 7, an antibody to adrenal autoantibody, an anti-immunoglobulin, protein A or protein G.

33. A method of any of claims 28 to 32, wherein the label comprises a radioisotope, enzyme, fluorescent compound, chemiluminescent compound, bioluminescent compound or metal chelate.

34. A method for detecting adrenal autoantibodies in a sample, comprising contacting with the sample immobilised antibody against a polypeptide of any of claims 1 to 7, which antibody has bound thereto a labelled ligand comprising a polypeptide of any of claims 1 to 7, and detecting labelled ligand bound to immobilised antibody or labelled ligand bound to antibody in the sample.

35. A kit for detecting adrenal autoantibodies in a sample, comprising a polypeptide of any of claims 1 to 7.

36. A kit of claim 35, wherein the protein or protein fragment is labelled.

37. A kit of claim 35, which further comprises a labelled antibody to the polypeptide and wherein the polypeptide is optionally immobilised on a substrate.

38. A kit of claim 36, wherein the polypeptide is immobilised on a substrate and the kit further comprises a labelled ligand for adrenal autoantibody.

39. A kit as claimed in claim 38, wherein the ligand is a polypeptide of any of claims 1 to 7, an antibody to adrenal autoantibody, an anti-immunoglobulin, protein A or protein G.

40. A kit as claimed in any one of claims 35 to 39, wherein the label comprises a radioisotope, enzyme, fluorescent compound, chemiluminescent compound, bioluminescent compound or metal chelate.

41. A kit for detecting adrenal autoantibodies in a sample, comprising immobilised antibody against a polypeptide of any of claims 1 to 7 having bound thereto a ligand comprising a polypeptide of any of claims 1 to 7.

42. The use in an assay for adrenal autoantibodies of a polypeptide of any of claims 1 to 8 or of an antibody to a polypeptide of any of claims 1 to 7.

43. A pharmaceutical formulation comprising as active ingredient a polypeptide of any of claims 1 to 7 formulated for pharmaceutical use.

44. A pharmaceutical composition comprising as active ingredient a polypeptide of any of claims 1 to 7 and a pharmaceutically acceptable diluent, excipient or carrier.

45. A polypeptide of any of claims 1 to 7 for use as a pharmaceutical, the protein fragment optionally being a peptide.

46. The use of a polypeptide of any of claims 1 to 7 to generate a T cell specific for adrenal autoantigen.

47. An oligonucleotide having the sequence of sequence SEQ ID No.2.

48. The oligonucleotide of claim 47 when inserted between the EcoRI and SphI restriction sites of the multiple cloning site of plasmid pYES2.