Arch Dermatol Res (1990) 282:76- 83

Archivea of

9 Springer-Verlag1990

Antikinetochore and antitopoisomerase I antibodies in systemic scleroderma: comparative study using immunoblotted recombinant antigens, immunofluorescence, and double immunodiffusion* M. Jarzabek-Chorzelska 1, M. Blaszczyk ~, Z. Kolacinska-Strasz 1, T. Chorzelski 1, S. Jablofiska 1, and G. G. Maul2 : Department of Dermatology, Warsaw School of Medicine, Koszykowa 82a, PL-02-008 Warszawa, Poland 2 Wistar Institute of Anatomy and Biology,Philadelphia, PA, USA Received July 22, 1989

Summary. In 135 patients with systemic scleroderma, we compared three different methods to determine antinuclear autoantibody (ANA) specificity: indirect immunofluorescence, double immunodiffnsion, and, employing recombinant antigens, immunoblotting using both marker autoantigens of this disease. A characteristic Sci-70 antibody pattern was found on HEp-2 cells in 83.8% of the patients, double immunodiffnsion was positive for the Scl70 antibodies in 81.9%, and immunoblot with the recombinant topoisomerase I (Topo I) was positive in 71% of the patients. For the centromere autoantibodies we found a high concordance between the anticentromere antibody (ACA) pattern on HEp-2 cells (27 patients positive) and the detection of recombinant kinetochore in immunoblotting (26 patients positive). The three testing techniques gave comparable results, except that the Topo I recombinant antigen used in immunoblotting reacted strongly with fewer than expected of the known Scl-70-positive sera. However, a method using recombinant antigens expressing all epitopes (rather than one of the epitopes of Topo I) will undoubtedly become the method of choice for detecting antibodies in systemic scleroderma. Using the immunoblotting technique with the recombinant antigens we detected in four patients antibodies against both Topo I and kinetochore. More severe symptoms of systemic scleroderma were found in patients who had both antibodies. The combined presence of both marker autoantibodies is therefore not as rare as previously reported and may predict severe disease. Key words: Anticentromere antibodies in scleroderma Antikinetochore antibodies in scleroderma - Topoisomerase I in scleroderma - Immunofluorescence Immunodiffusion - Immunoblotting

Almost all patients with systemic scleroderma (SSc) have been found to have antibodies against nuclear and/or nucleolar components [45] with a very high proportion of the sera recognizing either one of two marker antigens. Of these, the antibody against topoisomerase I (Topo I) [29, 40] is present in a higher number of patients with diffuse scleroderma, and the anticentromere antibody (ACA) is more common in patients with the relatively benign variant CREST (calcinosis, Raynaud, esophagus, sclerosis, telangiectasia), or limited acroscleroderma. The antigens have been cloned [14, 30] and are available as fi-galactosidase fusion proteins that can be used in Western blot or ELISA techniques. We have previously reported the detection of ACAs by indirect immunofluorescence in 25% of patients with acroscleroderma and in 52% of patients with minimal indurations confined to the digits (CREST syndrome or borderline cases of Raynaud's disease and incipient acroscleroderma) [8]. Similar results have been reported recently by Steen et al. [43]. Using double immunodiffusion against calf thymus extract antigens, anti-Scl-70 antibodies were identified in 77% of patients with diffuse scleroderma and 44% of acroscleroderma patients [20]. Additionally, these findings were confirmed by indirect immunofluorescence on HEp-2 cells, since anti-Scl-70 antibodies were found to have a characteristic immunofluorescence pattern. Steen et al. [43] found anti-Scl-70 antibodies in 33 % of diffuse scleroderma patients and in 18 % of patients with limited scleroderma. In the present study we compared the sensitivity of double immunodiffusion, indirect immunofluorescence, and immunoblotting with the use of recombinant antigens for the detection of Scl-70 and ACA antibodies characteristic for SSc.

Material and methods * Supported by grants from the CPBP Nr. 06.01. of the Polish Academy of Sciences, AR grant 38907 from the National Institute of Health, and the Scleroderma Research Foundation Offprint requests to: Stefania Jabtofiska, MD (address see above)

Characterization of patients

The present series comprises 135 patients with SSc; 30 with minimal cutaneous indurations confined to the digits; 43 with

M. Jarzabek-Chorzelska et al.: Antikinetochore and antitopoisomerase I antibodies acroscleroderma; 41 with diffuse scleroderma; and 21 with transitional form acroscleroderma-diffuse scleroderma. Of these, 115 patients were positive either for ACA or for anti-Scl-70 antibodies; 20 were negative for both antibodies. As controls 100 patients with systemic lupus erythematosus (SLE) were included as were 100 normal Red Cross blood donors (females aged 1 7 - 4 5 years). Patients were classified according to the following criteria: a subset with minimal cutaneous involvement was recognized in patients with pronounced Raynaud's phenomenon, either with no indurations or indurations confined to the fingers, telangiectasia, and, in some patients, calcinosis. Acroscleroderma was diagnosed in patients with telangiectasia and limited skin changes confined to the hands, forearms, and face. Some cases showed calcium deposits and pronounced Raynaud's phenomenon that preceded indurations and visceral involvement by several years. The transitional form of acroscleroderma-diffuse scleroderma was diagnosed if the cutaneous lesions, primarily of acroscleroderma type, became widespread, while remaining more atrophic than indurative, with some involvement of the proximal extremities, and in some patients also of the thorax. In these patients the disease started as typical acroscleroderma, but the course became severe after more than 10 years of slow progression, and then at the time of testing the disease resembled diffuse scleroderma. Diffuse scleroderma was classified according to the preliminary ARA criteria of Masi et al. [28]. In these patients proximal cutaneous indurations involved the face, extremities, and trunk, with symmetrical hyper- and depigmentations. The course of the disease was, in general, rapid and severe, usually with a short period between the onset of Raynaud's phenomenon and that of indurations. SSc was considered to involve the lung if bilateral basilar interstitial pulmonary fibrosis was detected on chest X-ray, or if the patient showed 75% of predicted normal vital lung capacity or respiratory gas volume, or 75% of predicted normal for forced expiratory volume for first, second, or maximal expiratory flow. Patients with SSc were considered to have cardiac involvement if they had any one of the following findings on electrocardiography and echocardiography: arrhythmia, conduction disturbances, right heart failure secondary to pulmonary hypertension, or pericarditis. The disease was considered to involve the kidney in patients with abnormal clearance or abnormal urinalysis findings. The disease process was judged to extend to muscle in patients showing either primary muscle involvement on electromyography or the increased level of muscle enzyme (creatine phosphokinase, aldolase, and transaminases).

77

The l-ram wide nitrocellulose strips containing, respectively, recombinant Topo I, kinetochore, and La antigens were then reacted for 1 h with 1 ml of the patient's sera diluted 1 : 100 in Tris-buffered saline and Tween 20 (TBST). After the strips were washed for 15 rain with TBST they were incubated with 1 : 10 000 alkaline phosphataselabeled goat antihuman IgG (Promega, Madison, Wis.) for 1 h. After washing the strips again for 15 min in TBST, the reactions were visualized by incubating the strips with the chromogens nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphatase (BCIP) in alkaline phosphatase buffer (100raM Tris-HCl pH 9.5, 100 mM NaC1, 5 mM MgC1).

Results T h e n i t r o c e l l u l o s e test strips s h o w e d p o s i t i v e r e a c t i o n at the level o f the f u s i o n p r o t e i n (Fig. 1) if i n c u b a t e d with sera positive for the r e c o m b i n a n t T o p o I e p i t o p e , the k i n e t o c h o r e a n d L a epitopes. T h e d i s t i n c t p u r p l i s h c o l o r is u n m i s t a k a b l e a n d easily identified. C h a r a c t e r i s t i c i m m u n o f l u o r e s c e n c e p a t t e r n s o f A C A a n d anti-Scl-70 a n t i b o d i e s on H E p - 2 s u b s t r a t e are s h o w n on Figs. 2 a n d 3, respectively. T h e results o f the b l o t t i n g , d o u b l e i m m u n o d i f f u s i o n , a n d i m m u n o f l u o r e s c e n c e are p r e s e n t ed in Table l. E x c e p t for the case o f one p a t i e n t w i t h SSc w i t h m i n i m a l c u t a n e o u s i n v o l v e m e n t , all sera positive for A C A o n H E p - 2 cells were also p o s i t i v e with the recombinant kinetochore antigen on immunoblotting. I n two o f three p a t i e n t s n e g a t i v e for A C A o n H E p 2 cells a n d negative for a n t i k i n e t o c h o r e a n t i b o d i e s on i m m u n o b l o t s , a n d in one p a t i e n t p o s i t i v e for b o t h A C A a n d k i n e t o c h o r e , m i t o c h o n d r i a l a n t i b o d i e s were detected. I n o n e o f the three A C A - n e g a t i v e p a t i e n t s , p r i m a r y bilia r y cirrhosis was c o n f i r m e d , a n d in two o t h e r s there were s o m e signs o f the disease. T h e t h i r d p a t i e n t , n e g a t i v e for A C A o n H E p - 2 cells a n d for a n t i k i n e t o c h o r e a n t i b o d i e s in i m m u n o b l o t , h a d p r i m a r y p u l m o n a r y h y p e r t e n s i o n a n d s y m p t o m s o f S j 6 g r e n ' s s y n d r o m e . In this case L a a n d

A n t i b o d y testing Scl-70 antigen was prepared from calf thymus using the method of Tan et al. [46], as modified by Kumar et al. [23]. Double immunodiffusion was performed according to the method of Tan et al. [46], as standardized for the detection of antibodies to RNP and Sm by Kumar et al. [23]. All sera were also tested on HEp-2 cells for ACA and other antinuclear antibodies, using the method of Beutner et al. [4]. The conjugate used was fluorescein-labeled goat antihuman IgG (Heintel, Vienna, Austria), with a fluorescein to protein ratio of 2.2, 16 U/ml. This was diluted 1:128 to contain 1/8 U per ml. Derived as previously described [15, 30], fl-galactosidase fusion proteins were isolated from lysogens. The Topo I fusion protein contained the carboxy terminal 109 amino acids of Topo I. A kinetochore fusion protein, part of the 140 kDa kinetochore antigen, contained the carboxy terminal amino acids. Its primary sequence showed that it is not the antigen described by Earnshaw et al. [14]. The La fusion protein contained a 30 kDa fragment and reacted with all available La-positive sera (n = 50; unpublished). All recombinant clones were obtained from BioDiagnostics, Wynnewood, Pa. Predetermined amounts of the respective antigen were separated according to the method of Laemmli [24] and electroblotted [47] onto nitrocellulose.

Fig. 1. Immunoblot of three different SSc sera with recombinent antigens. I: 1, positive Topo I; 2, negative kinetochore; 3:, negative La. H: 1, negative Topo I; 2, positive kinetochore; 3, negative La. IlI: 1, negative Topo I; 2, negative kinetochore; 3, positive La

78

M. Jarzabek-Chorzelska et al. : Antikinetochore and antitopoisomerase I antibodies

Fig. 2. Indirect immunofluorescence on HEp-2 cells. Staining of the centromeres in the interphase cells and a characteristic chromosomal pattern in the metaphase cells in the center and lower right

Fig. 3. Indirect immunofluorescence on HEp-2 cells. Diffusely grainly staining of the nucleoplasm with dotted nucleoli, characteristic for Scl-70 antibody

Table 1. Results of indirect immunofluorescence (IIF), double immunodiffusion (DID), and immunoblot (IB) studies of antibodies against Scl-70, ACA, Topo I, kinetochore, and others Diagnosis

IIF pattern on HEp-2 ceils

DID

Scl-70 Subset of SSc with minimal cutaneous involvement (n = 30) 0 Acroscleroderma and diffuse scleroderma (n = 105) 88 SLE (n = 108) 0 Normal controls (n = 100) 0

IB

ACA

Other

Scl-70

Other

Topo I

Kinetochore

27 0 0 0

3 17 108 7

0 86" 0 0

1 7 73 5

0 71 0 0

26 4b 0 0

" In two cases negative for Scl-70 in DID, the IIF pattern on HEp-2 cells was of the type o f Scl-70, and Topo I was positive in IB b In all four cases Topo I coexisted (see Table 2)

M. Jarzabek-Chorzelska et al.: Antikinetochore and antitopoisomerase I antibodies

79

Table 2. Patients with concomitant antibodies Yopo I and kinetochore

Duration in years Raynaud's phenomenon (R) Scleroderma (S) Period in years between R and S

1. D.G. F 32 years old Diffuse scleroderma

2. G.T. F 37 years old Diffuse scleroderma

3. G.S. F 32 years old Acroscleroderma

4. C.M. F 39 years old Transitional form (acro diffuse)

11 7

2 0.5

5 1

10 5

4

1.5

4

Indurative edema of the hands

+

+

+

+

5

Sclerodactyly grip

+

+

-

-

Acrolysis

+

+

+

+

Central involvement

+

+

-

+

Calcinosis

+

+

+

+

Telangiectasiae

+

+

+

+

Arthralgia

+

+

+

+

Visceral involvement Esophagus Heart Lungs Kidney Muscle

+ + -

+ + + + +

+ -

+ + +

Capillaroscopy

ND

R< S

R=S

R> S

IIF on HEp-2 cells

2 560 f. sp. nucl.

160 f. sp. nucl.

20 f. sp.

640 horn. f. sp.

Scl-70 pattern

+

+

+

+

ACA pattern"

+

+

-

-

Immunodiffusion

Scl-70

Scl-70

Scl-70

Scl-70

Immunoblot b

Topo I kinetochore

Topo I kinetochore La

Topo I kinetochore

Topo I kinetochore La c

Remarks on patients

Pronounced vascular lesions with extensive deep ulcerations of legs and dorsal feet

Pronounced vascular involvement, gangrenous ulcerations of the fingers and toes

Arterial hypertension

" In some cells, in addition to the typical fine speckled Scl-70 pattern, there were evenly distributed grains suggestive of centromere staining b Prevailing antibody in italics c All three antibodies weak

R o a n t i b o d i e s were d e t e c t e d in d o u b l e i m m u n o d i f f u s i o n , w i t h L a a n t i b o d y c o n f i r m e d also b y i m m u n o b l o t using the r e c o m b i n a n t L a antigen. O f the 105 cases o f a c r o s c l e r o d e r m a a n d diffuse s c l e r o d e r m a , the anti-Scl-70 a n t i b o d y p a t t e r n was f o u n d on H E p - 2 cells in 88 ( 8 3 . 8 % ) o f the p a t i e n t s , 86 were positive for Scl-70 in d o u b l e i m m u n o d i f f u s i o n , a n d 71 were p o s i t i v e f o r the r e c o m b i n a n t T o p o I e p i t o p e b y immunoblot. Various other antibodies (RNP, Ro, La, a n d u n i d e n t i f i e d lines) were d e t e c t e d in seven p a t i e n t s using double immunodiffusion. O f two p a t i e n t s f o u n d to be p o s i t i v e f o r a n t i b o d i e s a g a i n s t the r e c o m b i n a n t e p i t o p e T o p o I a n d n e g a t i v e in d o u b l e i m m u n o d i f f u s i o n , one h a d clinical f e a t u r e s o f a c r o s c l e r o d e r m a a n d the o t h e r o f diffuse s c l e r o d e r m a . B o t h diseases were slowly p r o g r e s s i v e . It is i m p o r t a n t to n o t e t h a t these sera were r e p e a t e d l y n e g a t i v e b y d o u b l e immunodiffusion.

I n f o u r p a t i e n t s , there was c o n c o m i t a n t p r e s e n c e o f T o p o I a n t i b o d y a n d k i n e t o c h o r e a n t i b o d y w h e n tested w i t h the respective r e c o m b i n a n t antigens. A n t i - S c l - 7 0 was detected by double immunodiffusion and imrrmnofluorescence, w h e r e a s A C A was n o t d e t e c t e d o n H E p - 2 cells due to m a s k i n g p h e n o m e n a . N e i t h e r A C A n o r anti-Scl-70 a n t i b o d i e s were f o u n d in 100 S L E patients. H o w e v e r , 9 % o f the S L E p a t i e n t s h a d a n t i b o d i e s to the r e c o m b i n a n t L a , results t h a t matched those obtained by immunodiffusion. T h e c o m p a r i s o n o f Scl-70 p o s i t i v e b u t T o p o I - a n t i body-positive and Topo I-antibody-negative patients did n o t s h o w a n y significant difference in disease e x p r e s s i o n in all t h r e e subsets. O f f o u r p a t i e n t s (Table 2) p o s i t i v e f o r b o t h T o p o I a n d k i n e t o c h o r e a n t i b o d i e s , two were o f diffuse s c l e r o d e r m a t y p e w i t h very extensive v a s c u l a r i n v o l v e m e n t a n d g a n g r e n o u s u l c e r a t i o n . I n p a t i e n t 2, k i n e t o c h o r e anti-

80

M. Jarzabek-Chorzelska et al.: Antikinetochore and antitopoisomerase I antibodies

body response was even stronger than the response to Topo I and there was, additionally, a weak band of La (Fig. 4). The course of the disease was in this patient very severe, with fatal outcome. In patient 3 with acroscleroderma type disease, the antibody response to kinetochore was weaker than that to Topo I (Fig. 4); and in patient 4 who had transitional type acroscleroderma-diffuse scleroderma, both antibody responses were weak; La antibody was present in addition. This patient had arterial hypertension and kidney involvement, and the course of the disease was severe. The indirect immunofluorescence pattern on HEp-2 cells in patients 1 and 2 was, in general, characteristic of anti-Scl-70 antibodies; some interphase cells displayed features suggestive of centromere fluorescence (Fig. 5). Discussion

The frequency of detection of the antibodies characteristic of SSc varies considerably, and has been reported to be from 25% to 96% for ACA [ 6 - 8 , 16, 22, 25, 3 1 -

Fig. 4. Immunoblot with

recombinant antigens of sera, with coexistent Topo I and kinetochore antibodies. 1, Topo I; 2, kinetochore; 3, La. In case first (DG) Topo I is stronger that kinetochore, while in the second (GT) kinetochore is stronger than Topo I

33, 35, 42, 43, 46, 48] and from 18% to 67% for anti-Scl70 [2, 5, 6, 17, 20, 25, 26, 31, 32, 43, 44]. This wide variation appears to be partly due to the differences in the classification of the subsets of the disease, and partly to different, not well-standardized techniques. The high detectability of anti-Scl-70 antibodies in our patients, as compared with other reports, might be due to the difference in the source of the antigen and its method of preparation [23], and, to some extent, to the classification of SSc patients. The variety, classified by us as a subset of SSc with minimal cutaneous involvement, corresponds to type 1, i.e., sclerodactyly of the Barnett classification [7], to SSc without or with limited indurations by Giordano et al. [17], and to type 1 of the German classification [25]. We have avoided the term CREST syndrome, previously used by us for the subset of acroscleroderma with minimal cutaneous indurations [8], since the term has not been defined precisely and is regarded by some authors as synonymous with acroscleroderma [38]. The present results confirm our previous findings and those of others [6, 8, 42, 43, 46] that ACA and anti-Scl70 antibodies are highly characteristic of SSc. In our series, these antibodies were not disclosed in control sera and/or SLE. ACA as a single antibody was found exclusively in the subset of SSc with minimal cutaneous indurations confined to the digits or in patients with longlasting Raynaud's disease with some features of incipient SSc. In contrast to the almost total absence of cutaneous changes in those patients, there was visceral involvement in the majority of them, as found in our previous series [8], and as also noted by others [25, 37, 42, 43]. The ACA detected by indirect immunofluorescence on HEp-2 cells were almost invariably confirmed by immunoblot with the use of human recombinant kinetochore antigen. Thus, there was a very high concordance between the detection of ACA on HEp-2 cells and the kinetochore antibodies in immunoblot. In the only case negative for kinetochore antibodies, the ACA titer (320) was the lowest of the whole series. Improvement in

Fig. 5. Patient no 2 in Table 2 (GT). Indirect immunofluorescenceon HEp-2 cells. In the center mixed pattern, with centromere staining clearly visible in some cells. To the right a typical Scl-70 pattern

M. Jarzabek-Chorzelska et al.: Antikinetochore and antitopoisomerase I antibodies

the signal amplification may make this low titered serum positive. Negative findings in immunoblot in this case might also be due to the presence of antibodies directed against an epitope not present in the recombinant kinetochore antigen. In the SSc subset of patients with minimal cutaneous involvement three patients had neither anti-Scl-70 antibodies nor ACA but other antibodies; two patients had mitochondrial and one had Ro and La antibodies. Three patients with mitochondrial antibodies, with or without ACA, had symptoms of primary biliary cirrhosis known to be associated, in certain cases, with acroscleroderma with minimal cutaneous indurations [3, 27, 48]. Other cases negative for ACA were also negative for the recombinant kinetochore antigen. ACA, as detected by indirect immunofluorescence, and kinetochore antibodies, detected by immunoblot with the use of nonrecombinant antigens, were reported by others to show a very high correlation [11-13, 32]. The molecular weight of antigens varied considerably. The kDa of 18 apparent antigens ranged between 14 and 140 with a cluster of 8 reported values between 19.5 and 26 kDa [9, 11, 12, 18, 19, 32, 34]. Earnshaw et al. [13] found that the principal antigens were kinetochore proteins of 17, 80, and 140 kDa. All ACA-positive sera studied by these authors recognized protein 80 kDa regarded as diagnostic for ACA, and most sera also recognized two other antigens at lower titers. In the study by McNeilage et al. [32], all 42 ACA-positive sera were reactive with the 72 kDa polypeptide, and 35 of these sera also reacted with the 19.5 kDa polypeptide. Antibodies eluted from both the 72 kDa and the 19.5 kDa polypeptides reacted with kinetochore when retested on HEp-2 cells. Thus, both polypeptides were shown to be antigenic components of kinetochore. In our present study anti-Scl-70 antibody was detected by double immunodiffusion in 81.9% of the patients. Topo I antibodies were shown by immunoblot to be present in 71% of the patients, and the indirect immunofluorescence pattern characteristic of anti-Scl-70 antibodies was present in 83.8% of the patients. Two patients positive for Topo I antibodies with the characteristic indirect immunofluorescence pattern for Scl-70 antibodies, were negative in double immunodiffusion. Scl-70 antigen has been characterized as an abundant nuclear D N A enzyme, Topo I [19, 29, 44]. The molecular mass of the antigen has been found variously to be 70 [10], 86 [49], 89 [32], 9 5 - 1 0 0 [1], 95 [19], and 1 0 0 105 kDa [29]. The lower molecular weight antigens could be regarded as degradation products [19, 21, 29, 49]. A good agreement between the results of double immunodiffusion or counter immunoelectrophoresis and immunoblot, with slightly greater accuracy for immunoblot, has been reported [1, 32, 49]. In our study, sensitivity to Scl-70 was higher in double immunodiffusion than in immunoblot. This might be due to the use of the recombinant antigen. It is assumed that some epitopes are lacking here, as the Topo I fusion protein contains only the 109 amino acids at the carboxyl end of the 105 kDa Topo I [29]. A detailed evaluation of clinical and laboratory data did not reveal any significant

81

differences between patients positive for Scl-70, both positive and negative for the recombinant Topo I epitope. The diagnostic significance of a recombinant La antigen was documented using both the ELISA and immunoblot methods. La was found in 94% (47/50) of patients with primary Sj6gren's syndrome, and in none of 50 patients with SLE [50], whereas positive findings should be expected in a proportion of cases when nonrecombinant antigens are used [36, 41, 45]. Our recombinant La antigen reacted with 9% of all SLE sera (n = 100) and some of the SSc sera. It must therefore contain those epitopes missing in the clone described by Whittingham et al. [50]. The use of the immunoblot technique made it possible to detect in sera of four patients the coexistence of Topo I and kinetochore antibodies, regarded as mutually exclusive [20, 43]. All of these patients had severe disease. Three of them had central cutaneous involvement and concomitantly very extensive vascular changes. It should be stressed that in the most severe case of diffuse scleroderma with fatal outcome there was a surprisingly strong antibody reaction with kinetochore, and a weak one with Topo I. On HEp-2 cells overlapping patterns of anti-Scl70 antibodies and ACA appeared in some cells, although anti-Scl-70 antibodies predominated. Some mitotic cells showed features of ACA. The ACA pattern was primarily overlooked and eventually found, after we had positive results from immunoblot for kinetochore antibodies. The simultaneous occurrence of ACA and anti-Scl-70 antibodies, as detected also by indirect immunofluorescence on chromosomal spreads and double immunodiffusion [39] or by indirect immunofluorescence, immunoblot, and counterimmunoelectrophoresis [32] appeared to be a very rare event, and its clinical significance is as yet unclear. The finding of four such cases out of 135 patients with confirmed scleroderma indicates that the combined presence of both marker autoantibodies is not as rare as has been assumed. With the use of immunoblot it was also possible to confirm kinetochore antibodies in a patient with concomitant presence of mitochondrial and ACA and La antibodies. The identification of ACA on HEp-2 might be difficult in such cases. Eventually the immunoblot method with the use of recombinant antigens will undoubtedly be the best and most reliable technique for detecting disease-specific antibodies in SSc. However, until three-dimensional Topo I epitopes can be obtained, the use of all three methods, indirect immunofluorescence on HEp-2 cells, double immunodiffusion, and immunoblot allows the highest detectability of ACA and anti-Scl-70 antibodies, Acknowledgements. We would like to thank Dr. E.H. Beutner and Dr. V. Kumar of the NY State University at Buffalo for the prescription for preparation of the calf thymus extract for double immunodiffusion and Mrs. R. Hochenberg and P. White for technical assistance.

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