Diagnosis of autoimmune adrenal disease

Details Diagnosis of autoimmune adrenal disease Diagnosis of autoimmune adrenal disease

1999-11-08

2002-01-29

Nolan, Patrick J. (Department: 1644)

Chemistry: analytical and immunological testing

For preexisting immune complex or auto-immune disease

C436S507000, C436S536000, C436S542000

Reexamination Certificate

active

06342394

ABSTRACT:

The present invention relates to diagnosis of overt, subclinical or potential autoimmune adrenal disease. Examples of autoimmune adrenal diseases include Addison's disease (AD) and autoimmune polyglandular syndrome (APS) type I and type II.
More especially, the present invention relates to use of the major autoantigen in autoimmune adrenal disease, human steroid 21-hydroxylase (21-OH) and to the epitopes on the 21-OH molecule reactive with 21-OH autoantibodies (Ab) in a patient's serum. The present invention concerns particular amino acid sequences of the 21-OH peptide, important for 21-OH Ab binding, and methods for using such sequences for diagnosis and prediction of the development of autoimmune adrenal disease.
The adrenal cortex of supra renal glands produce steroid hormones, such as cortisol and aldosterone. In addition, androgens (such as testosterone) are also secreted by the adrenal cortex.
Signs and symptoms of Addison's disease are related to the insufficiency of the adrenal cortex and include low blood pressure, muscle weakness, increased skin pigmentation and electrolyte imbalance. The severity of the symptoms depend on the extent of adrenal failure and if not treated, this disorder will inevitably lead to adrenocortical crisis and subsequent death.
Among the various causes of Addison's disease, the autoimmune origin is currently the most common. Autoantibodies to the adrenal cortex were first described in 1957 using the complement fixation technique. In 1988, an adrenal specific 55 kDa protein was immunoprecipitated from adrenal microsomes using Addison sera. This protein was subsequently shown to be human steroid 21-hydroxylase and identified to be the major adrenal autoantigen in different forms of autoimmune adrenal disease. Measurement of 21-OH Ab is a useful diagnostic tool in cases of suspected or overt adrenal failure and 21-OH Abs can be helpful markers of progression towards autoinmmune Addison's disease in patients with other organ-specific autoimmune diseases, such as type I diabetes, especially in children.
Previous studies have shown that autoantibody binding sites on 21-hydroxylase are in the main part conformational and are formed by the central and the C-terminal parts of the protein. Three fragments of 21-OH amino acid (AA) sequence (AA 164-356; 272-356 and 338-360) have been reported in U.S. Pat. No. 5,376,533 to be reactive with 21-OH Abs and potentially useful in specific diagnostic assays to identify the autoantibody associated with Addison's disease.
According to the present invention, we have determined that other parts of the 21-hydroxylase sequence are important for autoantibody binding in patients with different forms of autoimmune adrenal disease.
According to the present invention there is provided a method to aid in the diagnosis of overt, subclinical or potential autoimmune adrenal disease, said method comprising:
(a) providing a sample of body fluid from a subject;
(b) contacting the sample with
(i) at least one epitope region of 21-hydroxylase, which epitope region is either present in said 21-hydroxylase or in a fragment thereof and which epitope region is essential for binding monoclonal or polyclonal antibodies to 21-hydroxylase and/or autoantibodies to 21-hydroxylase; and
(ii) monoclonal or polyclonal antibodies to 21-hydroxylase; and
(c) monitoring the degree of binding of said autoantibodies present in said sample to said epitope region.
Depending on the assay system used, a substantial change in the amount of binding (a substantial increase or substantial decrease) of the autoantibodies to the relevant epitope region indicates that the subject may have autoimmune adrenal disease or be at risk of developing said disease.
In one embodiment of the present invention, the monoclonal or polyclonal antibodies may be immobilised to a solid phase; alternatively, at least one epitope region of 21-hydroxylase may be immobilised to a solid phase. The solid phase may be a magnetic or a non-magnetic material.
The monoclonal or polyclonal antibodies typically comprise any of human antibodies, rodent antibodies, rabbit antibodies or recombinant antibodies or parts or fragments thereof.
The relevant epitope region(s) may be directly labelled; alternatively, in some embodiments, the epitope region(s) may be indirectly labelled (for example, by binding the epitope region(s) to labelled monoclonal antibodies).
According to the present invention, 21-hydroxylase labeled with, for example,
125
I is incubated with none, one, or more monoclonal or polyclonal antibodies to 21-hydroxylase. The antibodies are directed to epitope regions on 21-hydroxylase which preferably include amino acids 391 to 405 (SEQ ID No: 1 & 2), 406 to 411 (SEQ ID No: 3 & 4) and/or amino acids 335 to 339 (SEQ ID No: 5 & 6). These monoclonal or polyclonal antibodies bind to the epitope regions on the labeled 21-hydroxylase, thus preventing the subsequent binding of patient serum autoantibodies to the epitope regions. According to the invention, a mixture of labeled 21-hydroxylase (which therefore comprises labeled epitope regions of 21-hydroxylase) and monoclonal or polyclonal antibodies is contacted with the sample of body fluid from a patient (typically with autoantibodies to 21-hydroxylase). The ability of a particular monoclonal or polyclonal antibody to 21-hydroxylase to lower the binding of 21-hydroxylase autoantibodies to 21-hydroxylase indicates the presence of autoantibodies to the same epitope region as the particular monoclonal or polyclonal antibody.
The monoclonal or polyclonal antibody, which itself may be labeled, is selected for reactivity with epitope regions along the amino acid sequence of 21-hydroxylase, the relevant epitope regions preferably comprising amino acid sequence (AA) 391 to 405 (SEQ ID Nos: 1 & 2), (AA) 406 to 411 (SEQ ID No: 3 & 4) and/or (AA) 335 to 339 (SEQ ID No: 5 & 6).
The method according to the present invention preferably comprises monitoring the reactivity of autoantibodies to 21-hydroxylase in a subject's body fluid (such as serum, whole blood or amniotic fluid) with one or more of the three different epitope regions mentioned above. The reactivity with the three different epitope regions may be monitored individually or in combination.
According to the present invention, the components of the reaction, namely 21-hydroxylase or its relevant epitope region(s), and monoclonal or polyclonal antibodies to 21-hydroxylase, may be labelled with suitable radioisotopic or non-isotopic labels. Suitable isotopic labels include
125
I,
35
S,
14
C and
3
H; suitable non-isotopic labels include enzymes, dyes, gold, chemiluminescent substances and fluorescent substances.
The reactivity between the various components may be monitored using a variety of methods, such as an enzyme linked immunosorbent assay (ELISA), solid or liquid phase radio-immunoassays (RIA), immunochromatographic assays, immunoprecipitation assays, or the like.


REFERENCES:
patent: 4198389 (1980-04-01), Wadsworth
patent: 5376533 (1994-12-01), Maclaren et al.
patent: 2256046 (1992-11-01), None
patent: WO9425604 (1994-11-01), None

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