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Experimental Studies |
FIRS Laboratories, RSR Ltd. (H.T., M.S.P., M.P., J.F.S., J.S., S.C., L.P., T.A., B.R.S., J.F.), Llanishen, Cardiff, United Kingdom CF4 5DU; the Institute of Semeiotica Medica, University of Padua (C.B., M.V.), Padua, Italy; and the Department of Medicine, University of Wales College of Medicine (H.T., M.S.P., J.S., S.C., T.A., B.R.S., J.F.), Cardiff, United Kingdom CF4 4XN
Address all correspondence and requests for reprints to: Dr. Bernard Rees Smith, FIRS Laboratories, RSR Ltd., Parc Ty Glas, Llanishen, Cardiff, United Kingdom CF4 5DU.
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Abstract |
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Overall, the 21-OH Ab assay based on 125I-labeled 21-OH showed good sensitivity, precision, and disease group specificity. This, combined with a simple assay protocol and the convenience of 125I handling and counting, make it attractive for routine use. Further investigations with the new assay should allow wider assessment of the prevalence and pattern of inheritance of adrenal autoimmunity. In addition, studies of the effect of treatment or possible preventative measures on 21-OH Ab levels in individuals without overt adrenal failure may suggest ways of delaying the onset of autoimmune Addison’s disease.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A convenient assay for 21-OH Abs would be of considerable value in the diagnosis and management of autoimmune adrenal disease, and we now describe such a method and its application to the analysis of different patient groups. In the assay, 125I-labeled recombinant human 21-OH is allowed to react with 21-OH Abs in test sera, and the immune complexes formed precipitate with solid phase protein A.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Sera were obtained from 60 patients with isolated Addison’s disease (37 women and 23 men; mean age, 31 yr; range, 6–57 yr), 12 patients with APS type I (8 women and 4 men; mean age, 26 yr; range, 13–45 yr), 27 patients with APS type II (23 women and 4 men; mean age, 44 yr; range, 16–83 yr), 9 patients with Addison’s disease due to tuberculosis (2 women and 7 men; mean age, 59 yr; range, 49–70 yr), and 30 patients positive for adrenal cortex Abs (ACA) by immunofluorescence without overt Addison’s disease (27 women and 3 men; mean age, 35 yr; range, 22–63 yr). Some of these sera have been described previously (4, 5, 6). Sera were also obtained from 77 patients with Graves’ disease (68 women and 9 men; mean age, 40 yr; range, 13–73 yr), 67 patients with Hashimoto’s thyroiditis (60 women and 7 men; mean age, 42 yr; range, 15–73 yr), 150 patients with insulin-dependent diabetes mellitus (IDDM; 87 women and 63 men; mean age, 20 yr; range, 1–67 yr), 32 patients with noninsulin-dependent diabetes mellitus (NIDDM; 7 women and 25 men; mean age, 55 yr; range, 23–73 yr), 17 patients with premature ovarian failure (POF; all women; mean age, 26 yr; range, 16–39 yr), and 35 patients with myasthenia gravis (27 women and 8 men; mean age, 45 yr; range, 18–68 yr). In addition, sera from 243 healthy blood donors were obtained. Sera from patients with Hashimoto’s thyroiditis were highly positive for thyroglobulin and/or thyroid peroxidase Abs (7), and sera from Graves’ patients were positive for TSH receptor Abs (8). All IDDM sera were positive for Abs to the 65-kDa isoform of glutamic acid decarboxylase (GAD65) (9), and all myasthenia gravis sera were positive for acetylcholine receptor Abs (10). The Abs in the above patient groups were measured with reagents available from RSR (Cardiff, UK). Disease diagnosis was based on clinical, immunological, and biochemical grounds.
Production of recombinant human 21-OH
Human 21-OH complementary DNA (cDNA) with STE2 leader sequence was placed under the control of the GAL1 promoter in pYES2 plasmid (pYES2/21-OH1) as described previously (5, 11). Saccharomyces cerevisiae strain c13ABYS86 (12) was transformed with the plasmid containing the 21-OH cDNA using the lithium chloride method (13). Yeast transformed with pYES2 plasmid not carrying 21-OH cDNA was used as the control. Transformants were grown, harvested, broken, and extracted as described previously (5, 14, 15). The extracts of 21-OH were purified by hydrophobic chromatography on octyl-Sepharose (Pharmacia, St. Albans, UK) followed by hydroxyapatite (Sigma Chemical Co., Dorset, UK) chromatography as described previously (5, 14, 15). The purity of 21-OH in the column fractions was assessed by electrophoresis on 9% polyacrylamide gels (SDS-PAGE) (16), and total protein content was measured by Bradford assay (17) using reagents from Bio-Rad (Hemel Hempstead, UK).
21-OH Ab assay based on 125I-labeled 21-OH expressed in yeast
Recombinant, purified human 21-OH was labeled with 125I to a specific activity of 500 kilobecquerels/µg protein using the chloramine-T method (18). After purification by gel filtration (Sephacryl S-300, Pharmacia), 50-µL aliquots of labeled material [30,000–40,000 cpm diluted in 150 mmol/L Tris-HCl (pH 8.3), 200 mmol/L NaCl, 10 mL/L Tween-20, 10 g/L BSA, and 0.5 g/L NaN3; hereafter called Tris buffer] were incubated at 4 C with duplicate 20-µL aliquots of undiluted test serum. After 18-h incubation, 50 µL solid phase protein A (RSR) were added, and incubation was continued for 1 h at 4 C. Tris buffer (1 mL) was then added, and after centrifugation (1500 x g for 30 min at 4 C), the supernatants were aspirated, and the pellets were counted for 125I.
Production of 35S-labeled human recombinant 21-OH and 35S immunoprecipitation assay
35S-Labeled human 21-OH was prepared using plasmid pYES2/21-OH1 in an in vitro transcription/translation system (Promega, Southampton, UK) as described previously (4, 6, 19) and stored in aliquots at -70 C (for up to 1 month). The 35S-labeled material was then used to measure 21-OH Abs in an immunoprecipitation assay as described in detail previously (6). Negative control and positive reference sera were included in each assay. The negative control consisted of sera pooled from 20 individual healthy blood donors. The positive reference was serum from a patient with Addison’s disease with high levels of 21-OH Abs. Results were expressed as a 21-OH Ab index calculated as 100 x [cpm (unknown) - (negative control)]/[cpm (positive reference) - cpm (negative control)]. An index value of 2.6 or greater (based on a mean ± 3 SD of 26 healthy blood donors) was considered to indicate the presence of 21-OH Abs.
Measurement of recombinant human 21-OH
The [35S]21-OH or [125I]21-OH immunoprecipitation assays were used to measure 21-OH concentrations by including a step in which duplicate aliquots of 21-OH test preparations were preincubated (1 h at room temperature) with 21-OH rabbit antibody before the addition of tracer. Serial dilutions of an in-house 21-OH reference preparation (recombinant material purified from yeast extracts) were used to produce a standard curve.
SDS-PAGE and Western blotting
Protein preparations were run (together with Sigma 6-H mol wt markers) on 9% SDS-PAGE under reducing conditions and either stained with Coomassie brilliant blue or blotted onto nitrocellulose membranes. Western blot analysis was carried out according to the method of Birk and Koepsell (20) using either mouse monoclonal antibody (RSR) or rabbit antibody to a glutathione-S-transferase-21-OH fusion protein (RSR) (21). The reactions were developed using antimouse or antirabbit horseradish peroxidase conjugate followed by ECL reagents (Amersham International, Little Chalfont, UK).
Immunofluorescence studies
ACA were detected by a classical indirect immunofluorescence technique, using thin cryostatic sections of normal bovine adrenal gland as the source of antigen and fluorescein isothiocyanate-conjugated goat antihuman IgG, as previously described (22). Sera were tested undiluted, and if positive, ACA titers determined by retesting sera in serial 2-fold dilutions until reaching the end point.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, a and b. On SDS gels
stained with Coomassie blue, a 55-kDa band representing 21-OH was
clearly evident after one run on hydrophobic chromatography (Fig. 1a, lane 3). Finally, purified material ran essentially as a single 55-kDa
band (Fig. 1a, lane 4). When the same samples were analyzed by Western
blotting using a monoclonal antibody to 21-OH, a 55-kDa band
representing 21-OH was clearly identified in the various preparations
from yeast transformed with pYES2/21-OH1 plasmid (Fig. 1b, lanes 2–4).
There was no reactivity of the 21-OH monoclonal antibody with
preparations of yeast transformed with control plasmid (i.e.
not containing the 21-OH cDNA; lane 1 in Fig. 1, a and b). Similar
results were obtained when a rabbit antibody to 21-OH was used in
Western blotting analysis (data not shown).
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shows a dilution curve obtained with a 21-OH
Ab-positive Addison’s serum. [125I]21-OH binding ranged
from 34% of the total counts added for the undiluted serum to 6% for
serum diluted 1:64. Normal pooled sera showed binding of about 2.5% at
each dilution.
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The [125I]21-OH Ab assay interassay coefficient of variation was 1.5% (n = 6) for serum with higher levels of 21-OH Abs (mean, 13.9 U/mL), 2.2% (n = 6) for serum with medium levels of 21-OH antibodies (mean, 9.8 U/mL), and 2.6% (n = 6) for serum with lower levels of 21-OH Abs (mean, 3.1 U/mL). The intraassay coefficient of variation was 1.3% (n = 6) for serum with higher levels of 21-OH Abs (mean, 13.9 U/mL), 2.1% (n = 6) for serum with medium levels of 21-OH Abs (mean, 9.9 U/mL), and 2.2% (n = 6) for serum with lower levels of 21-OH Abs (mean, 3.2 U/mL).
The results of 21-OH Ab measurements using the
[125I]21-OH Ab assay in sera from healthy blood donors,
patients with autoimmune adrenal disease, and patients with other
autoimmune and nonautoimmune diseases are shown in Fig. 3 and Table 1. In the case of sera from
healthy blood donors, 6 of 243 showed 21-OH Ab levels greater than 1
U/mL (1.1, 1.1, 1.2, 1.5, 3.4, and 3.7 U/mL). The 21-OH Ab activity in
all six of the sera could be readily neutralized by the addition of
unlabeled 21-OH (for examples, see Table 2).
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).
There was good agreement between 21-OH Abs measured by
[125I]21-OH- and [35S]21-OH-based assays in
a study of 129 sera from patients with autoimmune adrenal disease
(Table 1) with a Pearson correlation coefficient of r = 0.86
(n = 129; P < 0.001; Fig. 4).
However, there were a few discrepancies (Fig. 4 and Table 1). One
Addison’s serum that was low positive in the 35S assay
(21-OH Ab index of 3.0) was negative in the 125I assay. In
contrast, 6 sera that were negative in the 35S assay were
low positive in the 125I assay (from 1.1–3.1 U/mL).
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. Although there
was good overall agreement between these assays, 9 sera (4 Addison, 2
APS type I, and 3 APS type II) that were negative for ACA by
immunofluorescence were positive for 21-OH Abs by 125I
assay (range, 1.1–8.7 U/mL). Also, 7 sera that were ACA positive by
immunofluorescence (6 ACA-positive patients without overt Addison’s
disease and 1 ACA-positive Addison’s patient) were negative for 21-OH
Abs by 125I assay. In the group of patients with adrenal
failure (isolated Addison’s disease, APS type I, and APS type II;
Table 1), 16 sera had levels of 21-OH Ab between 1–10 U/mL
(i.e. lower levels) as measured by 125I assay.
However, only 8 (50%) of these sera had detectable ACA by
immunofluorescence.
To confirm the specificity of 21-OH Ab measurements, neutralization
studies were carried out on some sera using unlabeled 21-OH, as shown
in Table 2. Addition of unlabeled 21-OH neutralized 21-OH Abs in all
sera shown, including low positive sera from healthy blood donors,
IDDM, and Graves’ patients. A similar effect was observed with 21-OH
Abs in Addison’s sera (Table 2). Addition of unlabeled control
proteins produced in yeast; recombinant GAD65 and YP50 (a
50-kDa protein isolated from yeast transformed with control plasmid
pYES2) had no effect. Further studies were carried out on the healthy
blood donor serum that had a 21-OH Ab level of 3.7 U/mL. In particular,
this serum was serially diluted in 21-OH Ab-negative human serum and
reassayed. This showed that the serum 21-OH Ab had a similar dilution
profile to 21-OH Ab in Addison’s sera (data not shown). Furthermore,
increasing concentrations (0.15–60 µg/mL serum) of unlabeled 21-OH
had a similar effect on 125I-labeled 21-OH binding to 21-OH
Ab in this healthy blood donor serum and 21-OH Ab in Addison’s sera
(data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The amount of extractable recombinant 21-OH in yeast cells was about 150 µg/g cells, and this can be compared to about 200 µg native 21-OH/g human adrenal tissue (data not shown). However, human adrenal tissue is not readily available in large amounts, and this limitation explains in part why preparations of native human 21-OH have not been used for 125I labeling. In contrast, yeast can be grown easily and safely on a relatively large scale. In addition to being a useful expression system for producing 21-OH, yeast can be used to produce other important human autoantigens, including glutamic acid decarboxylase (9).
Using our assay based on 125I-labeled 21-OH, we could detect 21-OH Abs in 43 of 60 (72%) Addison’s sera. This can be compared with prevalences of 64–86% for 21-OH Abs in Addison’s patients reported by different laboratories using 35S-labeled 21-OH based assays or immunoblotting analysis (4, 5, 6, 23, 24, 25). A high prevalence of 21-OH Abs was also observed in APS type I and APS type II sera (11 of 12 and 27 of 27, respectively) using 125I-labeled 21-OH. The one 21-OH Ab-negative APS type I patient had hypoparathyroidism, candidiasis, Addison’s disease, vitiligo, alopecia, and keratopathy. His brother also had APS type I with the same clinical symptoms, but was in the 21-OH Ab-positive group. Our previous data (4, 6) and reports by Song et al. (24) and Uibo et al. (26) have shown similar prevalences of 21-OH Ab in APS type I and type II. These results strongly support the concept that 21-OH is a major autoantigen in autoimmune adrenal disease.
There was good agreement between 21-OH Ab results obtained by 125I and 35S assays (r = 0.86; n = 129). Only one serum (ACA negative) that was low positive in the 35S assay was negative by 125I assay, whereas six sera that were negative by 35S assay were positive in the 125I assay. These six sera were from patients with a clinical diagnosis of Addison’s disease (two isolated Addison, three APS type I, and one APS type II), although all but one were negative for ACA. In contrast to the 35S assay, undiluted serum was used with 125I-labeled 21-OH, and this should allow detection of Abs present at lower concentrations. In addition, the precision of the 125I assay was good, with inter- and intraassay coefficients of variation of about 2%. Overall, therefore, the 125I assay appears to be superior to the 35S assay in terms of sensitivity. Also, the production of 35S-labeled 21-OH is expensive and time consuming, whereas 125I-labeled 21-OH can be prepared easily, and handling and counting of this isotope are much more convenient than 35S.
Sera from 6 of 30 patients who were positive for ACA by immunofluorescence but had no clinical signs of adrenal failure were negative for 21-OH Abs by 125I assay. The reason for this discrepancy is not clear at present, but may be related to the specificity of the ACA immunofluorescence test. In the case of patients with overt adrenal failure (Addison’s, APS type I and APS type II), all ACA positive sera in this group were also positive for 21-OH Abs by 125I assay except 1. Furthermore, in this overt disease group, measurements with the 125I assay seemed to be more sensitive, as 16 patient sera with lower levels (1–10 U/mL) of 21-OH Abs detectable by 125I assay only showed positive for ACA by immunofluorescence in 8 cases (50%).
In our studies with the assay based on 125I-labeled 21-OH, low levels of 21-OH Abs were detected in 6 of 243 (2.5%) healthy blood donors, 1 of 77 (1.3%) Graves’ sera, 4 of 150 (2.7%) IDDM sera, and 1 of 67 (1.5%) sera from patients with Hashimoto’s thyroiditis. 21-OH Abs were not detected in any of the myasthenia gravis, NIDDM, or POF sera studied. Unlabeled 21-OH (but not unlabeled GAD65 or the yeast protein YP50) neutralized the 21-OH Abs in the healthy and control group sera, thus confirming the presence of specific 21-OH Abs. Furthermore, dilution studies and experiments with different concentrations of unlabeled 21-OH indicated that the 21-OH binding characteristics of the antibodies present in one of the healthy blood donor sera were similar to those of 21-OH Abs in Addison’s sera.
To date, the prevalence of 21-OH Abs in healthy blood donors and in autoimmune control groups has not been studied extensively. 21-OH Abs were not detected in healthy blood donors sera or in autoimmune control sera in studies by Colls (6), Chen (4), and Uibo (26). However, Falorni (25) found 21-OH Abs in 1 of 70 (1.4%) healthy blood donors using the 35S assay. These results can be compared with the observation that low levels of GAD65 Abs are also found in a small proportion of healthy blood donors or in patients with autoimmune diseases other than IDDM (9, 27, 28).
Prevalences of ACA measured by immunofluorescence in large groups of sera from patients with autoimmune diseases (IDDM, autoimmune thyroid disease, myasthenia gravis, atrophic gastritis, and systemic lupus erythematosus) have been reported to range from below 1% to 5–8%, with the highest prevalence (from 2.5–20%) for patients with idiopathic hypoparathyroidism (29, 30, 31, 32, 33). ACA measured by immunofluorescence were not usually found in sera from healthy blood donors, except for 1 of 1127 sera in the study by Nerup (29) and 1 of 338 sera in the study by Betterle et al. (31, 32, 33, 34, 35). The prevalence of 21-OH Abs measured by 125I assay seems higher than that of ACA measured by immunofluorescence in the autoimmune control groups and healthy blood donors, and this probably reflects the greater sensitivity of the 125I assay.
Consequently, an assay is now available that will allow easy, reliable, routine assessment of adrenal autoimmunity. This type of assessment is particularly important in young patients who present with other autoimmune disorders, as the presence of adrenal Ab in this group of individuals indicates that adrenal insufficiency will develop soon (36). Assessment of adrenal autoimmunity is also important in all patients who are suspected of having reduced adrenal reserve or adrenal failure (36, 37, 38, 39, 40, 41). Further investigations with the new assay on the prevalence of 21-OH Abs in the general population and in different disease groups and on their pattern of inheritance should follow. In addition, studies of the effect of treatment or possible preventive measures on 21-OH Ab levels in individuals without overt adrenal failure may point to ways of delaying the onset of Addison’s disease.
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Footnotes |
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2 Recipient of a fellowship from RSR.
Received October 21, 1996.
Revised January 21, 1997.
Accepted February 5, 1997.
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References |
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and
P-450C-21. Isolation, partial characterization and
comparison to P-450scc. J Biol Chem. 257:12696–12704.This article has been cited by other articles:
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). Sera were diluted in Tris buffer. 0 on
horizontal axis indicates undiluted serum. See text for experimental
details.
).