International Journal of Rheumatic Diseases 2009; 12: 100–106

ORIGINAL ARTICLE
Blackwell Publishing Asia

Anti-nucleosome antibodies in SLE

Anti-nucleosome antibodies as a disease activity marker in

patients with systemic lupus erythematosus
Suleiman SULEIMAN1, Daud KAMALIAH2, Afzal NADEEM1, Nyi Nyi NAING3 and
Che Hussin CHE MARAINA1
Departments of 1Immunology and 2Medicine, and 3Unit of Biostatistics and Research Methodology, School of Medical Sciences,
Universiti Sains Malaysia, Kubang Kerian, Kelantan, Malaysia

Abstract
Aim: To measure the level of anti-nucleosome antibodies in systemic lupus erythematosus (SLE) patients, to
determine the sensitivity and the specificity of these antibodies in the diagnosis of the disease and to evaluate
the relationship between the levels of anti-nucleosome antibodies, anti-dsDNA (double-stranded DNA) and
SLE disease activity.
Methods: A cross-sectional study was conducted. All patients attended either a medical specialist clinic or were
admitted to the medical wards of Hospital Universiti Sains Malaysia with the diagnosis of SLE (n = 90), other connective
tissue diseases (n = 45) or were normal controls (n = 90) within the period from July 2004 until September
2005. They were tested for anti-nucleosome antibodies by enzyme-linked immunosorbent assay and anti-DNA
antibodies by immunofluorescence. SLE disease activity was evaluated by SLE disease activity index (SLEDAI)
score.
Results: Out of 90 SLE patients, anti-nucleosome antibodies were positive in 47 (52.2%) patients, whereas
these antibodies were positive in three (6.7%) patients with other connective tissue diseases. Anti-dsDNA
antibodies were positive in 33 (36.7%) SLE patients, whereas these antibodies were positive in four (8.9%)
patients with other connective tissue diseases. Anti-nucleosome antibodies were positive in 40 (97.6%) patients
with active SLE, whereas these antibodies were positive in seven (14.3%) patients with inactive SLE. Anti-
nucleosome antibodies had a stronger correlation than anti-dsDNA antibodies with SLEDAI score. There was
a significant association between anti-nucleosome antibodies and disease activity.
Conclusion: Anti-nucleosome antibodies test is highly sensitive and specific for the diagnosis of SLE, espe-
cially when the anti-dsDNA antibodies are absent. They are additional disease activity markers in the assess-
ment of SLE disease activity.
Key words: anti-nucleosome antibodies, disease activity marker, SLE.

INTRODUCTION many different auto-antibodies in patient sera.1

Systemic lupus erythematosus (SLE) is an autoimmune
disorder of unknown aetiology. It can affect any organ
of the body and is characterized by the presence of
Correspondence: C. M. Che Hussin, Department of
Immunology, School Medical Sciences, Universiti Sains
Malaysia, Kubang Kerian, 16150 Kota Bharu, Kelantan,
Malaysia. Email: maraina@kck.usm.my
Anti-dsDNA (double-stranded DNA) antibodies are
considered the main diagnostic tool for SLE and
are useful markers of disease activity; however they are
only found in 40–60% of SLE patients and do not
always correlate with disease activity.2,3 Therefore, it is
important to find other auto-antibodies that may be
helpful in the diagnosis and assessment of disease
activity in SLE patients.

©Asia Pacific League of Associations for Rheumatology


Recently attention has been attracted to the nucleo-
some, the fundamental unit of chromatin. It consists of
the core particle composed of an octamer of histones
(H2A-H2B-H3-H4)2 around which the helical DNA is
wrapped and H1 is located at the point where DNA
enters and exits the nucleosome.4
Anti-nucleosome antibodies, which only target the
native nucleosome particle, but not the individual
components (DNA and histone), occur early in the
disease and precede the formation of anti-dsDNA and
anti-histone antibodies.5,6 The nucleosome is generated
during cell apoptosis and it has been identified to be
the most important immunogen in SLE pathogenesis.2,7
The serum reactivity to nucleosome was demonstrated
in 70–80% of SLE patients, which is more than the
positivity of anti-dsDNA antibodies and can be demon-
strated in the early phase of the disease,7 and was
found to correlate better than anti-dsDNA antibodies
with SLE disease activity.8
The aims of this study were to measure the level of
anti-nucleosome as well as anti-dsDNA antibodies in
SLE patients and to evaluate the level of these auto-
antibodies with SLE disease activity.
MATERIALS AND METHODS
Patients
A cross-sectional study was conducted at Hospital
Universiti Sains Malaysia in Kelantan, Malaysia among
90 SLE patients. Those who fulfilled at least four
criteria of SLE according to the American College of
Rheumatology1 were included. Clinical and biologic
information was obtained at each patient’s visit and
from the medical records. This information was used to
determine the SLE Disease Activity Index (SLEDAI)
score, which was used because it has been validated
as a clinical index for measurement of disease act-
ivity in SLE9 and it has been routinely used in our
institution.
The control group comprised of 90 healthy blood
donors. Forty-five patients were diagnosed as hav-
ing other connective tissue diseases (CTDs), which in-
cluded 39 patients with rheumatoid arthritis (86.7%),
two with mixed CTD (4.4%), three with scleroderma
(6.7%) and one with Sjögren’s syndrome (2.2%). In
Malaysia, rheumatoid arthritis is commonly seen com-
pared to other CTDs. This explains the predominance
of rheumatoid arthritis in the other group of CTDs.
Sera were obtained after blood coagulation by centri-
fugation at 50 g for 5 min, and then the sera were
separated and stored at –20°C until they were used.
International Journal of Rheumatic Diseases 2009; 12: 100–106
Anti-nucleosome antibodies in SLE
The Universiti Sains Malaysia’s Research and Ethics
Committee approved the study. The study protocol was
explained to all patients and their informed consents
were taken.
Laboratory methods
Detection of anti-nucleosome antibodies
Anti-nucleosome antibodies were detected by using
a commercial enzyme-linked immunosorbent assay
(AESKULISA Nucleo-h: Aeskulab, Wendelsheim, Germany).
It is a solid phase enzyme immunoassay with human
native nucleosomes isolated from the eukaryotic cell
line Henrietta Lacks (HeLa) for the quantitative and
qualitative detection of antibodies against nucleosomes
and their components (dsDNA and histones) in
human serum.
Assay procedure
Reagents and sample preparation
– The sample buffer was diluted with distilled water at
a ratio of 1 : 5.
– The wash buffer was diluted with distilled water at a
ratio of 1 : 50.
– The serum sample was diluted with sample buffer at
a ratio of 1 : 100.
Procedure
A total of 100 μL of each patient’s diluted serum was
pipetted into designated microwells; 100 μL calibrators,
negative and positive controls were then pipetted into
the designated microwells. All samples were run in
duplicate. The microwells were then incubated for
30 min at room temperature. The wells were then
washed three times with 300 μL washing buffer
(diluted 1 : 50). One hundred microliters conjugate
(anti-human immunoglobulins conjugated to horse-
radish peroxidase and thimerosal 0.01%) was then
pipetted into each well. The mixture was incubated for
15 min at room temperature. The wells were again
washed three times with 300 μL washing buffer. Then,
100 μL of tetramethylbenzidine (TMB) substrate was
pipetted into each well, followed by incubation of the
wells for 15 min at room temperature, in the dark;
100 μL of stop solution was then pipetted into each
well.
The wells were again incubated for a minimum of
5 min. This was followed by careful agitation of the
plate for 5 sec. Finally, the absorbance was read at
450 nm (optionally 450/620 nm) within 30 min of the
101
S. Suleiman et al.
agitation. The patients were considered to have anti-
nucleosome antibodies if the level = 15 U/mL.
Detection of anti-dsDNA antibodies
Anti-dsDNA antibodies were detected by using a
commercial indirect immunofluorescence assay (anti-
dsDNA antibodies system: Scimedx Corp., Denville, NJ,
US), a useful test for detection of antibodies to native
dsDNA by indirect immunofluorescence and it utilizes
the giant mitochondrion kinetoplast of the non-
pathogenic hemoflagellate Crithidia luciliae as a
substrate for pure DNA.
Assay procedure
Reagents and sample preparation
Preparation of phosphate-buffered saline (PBS): The
contents of the powder buffer pack were poured into a
container where deionized or distilled water was added
to bring it to a final volume of 1.0 L. The sample diluent
was diluted with PBS at a ratio of 1 : 2. The sample was
diluted with PBS at a ratio of 1 : 10.
Procedure
One drop (25–50 μL) of diluted serum samples and
controls was added on the substrate fixed on the slide.
Then the slide was placed in a moist chamber and
incubated for 30 min at room temperature. The slide
was rinsed in a light stream of PBS and washed thoroughly
in a staining dish containing fresh PBS for 5 min.
Special blotting paper was used for blotting the slide.
One drop of fluorescein isothiocynate (FITC) conjugate
was then put into each well and incubated for 30 min
in the dark. The washing was repeated and the slide
blotted. Several drops of mounting medium were
positioned and a coverslip was gently applied to avoid
air bubble formation. The slide was examined under
fluorescence microscope (×40) (Olympus, Tokyo,
Japan). Serial dilutions were performed for positive
samples and a titre of 1 : 10 or higher was regarded as
positive.
Another way of measuring anti-dsDNA antibodies is
by ELISA method, which is more sensitive. We were
using the immunoflourescence method in our study
since it is already used in our laboratory set-up and due
to budget limitations.
Statistical analysis
The SPSS version 11.0 (SPSS Inc., Chicago, IL, US) was
used for data entry and statistical analysis. Descriptive
statistics was expressed by mean and standard deviation
102
for continuous variables and frequency and percentage
for categorical variables. For non-normally distributed
continuous variables, median and interquartile ranges
(IQR) were applied. Histograms and box and whisker
plots were used to test the normality of continuous
variables. Non-parametric tests were used because of
non-normal distribution of the variables in this study.
The Mann-Whitney test was used to compare the median
differences between the two groups. Non-parametric
Spearman rank correlation coefficient was assessed to
find the correlation between two continuous variables.
Pearson chi-square test was applied to investigate the
association between categorical variables. A correlation
between related categorical variables was analysed
using McNemar’s test. The level of significance (α) was
set at 0.05 and P-value < 0.05 was accepted as significant.
Epi-info version 6.0 was used to determine sensitivity
and specificity.
RESULTS
Clinical characteristics
There were 82 (91.1%) female and 8 (8.9%) male SLE
patients. Ages ranged from 15 to 62 years with mean
(± SD) age of 31.4 (± 11.25). Forty-one (45.6%)
patients were found to have active SLE (SLEDAI score
≥ 5) and 49 (54.4%) had inactive SLE (SLEDAI score
< 5). There were 80 (88.9%) female and 10 (11.1%)
male healthy blood donors. Ages ranged from 20 to 50
years with a mean of 23.8 years (SD = 4.59). There
were 42 (93.3%) female and three (6.7%) male ‘other
CTD’ patients. Ages ranged from 19 to 63 years with a
mean of 45.4 years (SD = 12.39).
Organ/system involvement in SLE patients were as
follows: musculoskeletal (n = 62, 68.8%), renal (n = 56,
62.2%), skin and mucous membrane (n = 48, 53.3%),
hematological (n = 45, 50%), cerebral (n = 19, 21.1%),
ocular (n = 7, 7.8%), cardiac (n = 6, 6.7%), gastrointest-
inal (n = 3, 3.3%) and pulmonary (n = 2, 2.2%).
Levels of anti-nucleosome and anti-dsDNA
antibodies
Anti-nucleosome antibodies were found to be positive
in 47 (52.2%) SLE patients with a median (IQR) of
17.5 U/mL (67.5), whereas it was found in three
(6.7%) patients with other CTDs with a median of
4.6 U/mL (3.2). The median concentration of anti-
nucleosome antibodies was significantly different between
SLE patients and patients with other CTDs (P < 0.001)
(Table 1). Anti-dsDNA antibodies were found in 33
(36.7%) SLE patients and in four (8.9%) patients with
International Journal of Rheumatic Diseases 2009; 12: 100–106
 Anti-nucleosome antibodies in SLE

Table 1 Positivity and level of anti-nucleosome antibodies in SLE and other CTDs
Variable SLE (n = 90) median (IQR) Other CTDs (n = 45) median (IQR) Z statistic† P-value*
Anti-nucleosome (U/mL) 52% 7%
17.5 (67.5) 4.6 (3.2) –5.64 < 0.001
*Level of significance set at 0.05.
SLE, systemic lupus erythematosus; CTDs, connective tissue diseases; IQR, interquartile range.
†Mann-Whitney test applied.

Table 2 Sensitivity and specificity of autoantibodies in diagnosis of systemic lupus erythematosus

Autoantibodies Sensitivity (%) (95% CI) Specificity (%) (95% CI) PPV (%) (95% CI) NPV (%) (95% CI)
Anti-nucleosome 52 (41.5–62.8) 98 (93.1–99.4) 94 (82.5–98.4) 75 (68.2–81.5)
Anti-dsDNA 37 (26.9–47.5) 97 (92.1–99.0) 89 (73.6–96.5) 70 (62.5–76.0)
PPV, positive predictive value; NPV, negative predictive value.

Table 3 Level of anti-nucleosome antibodies in active and inactive SLE

Variable Active SLE (n = 41) Median (IQR) Inactive SLE (n = 49) Median (IQR) Z statistic† P-value*
Anti-nucleosome (U/mL) 74.6 (121.5) 7.6 (9.2) –7.32 < 0.001
*Level of significance set at 0.05.
†Mann-Whitney test.
SLE, systemic lupus erythematosus; IQR, interquartile range.

Table 4 Sensitivity and specificity of anti-nucleosome and anti-dsDNA antibodies in the diagnosis of active systemic lupus
erythematosus
Autoantibodies Sensitivity (%) (95% CI) Specificity (%) (95% CI) PPV (%) (95% CI) NPV (%) (95% CI)
Anti-nucleosome 98 (85.6–99.9) 86 (72.1–93.6) 85 (71.1–93.3) 98 (86.2–99.9)
Anti-dsDNA 61 (44.5–75.4) 84 (69.8–92.2) 76 (57.4–88.3) 72 (58.3–82.6)
PPV, positive predictive value; NPV, negative predictive value.

other CTDs. None of these two auto-antibodies were
tested positive in healthy blood donors.
Anti-nucleosome antibodies had a sensitivity of 52%
and a specificity of 98%, whereas anti-dsDNA antibodies
had a sensitivity of 37% and a specificity of 97% for
the diagnosis of SLE (Table 2).
(60.9%) active-SLE patients and in eight (16.3%)
inactive-SLE patients.
Anti-nucleosome antibodies had a sensitivity of 98%
and a specificity of 86%, whereas anti-dsDNA antibodies
had a sensitivity of 61% and a specificity of 84% for the
diagnosis of active SLE (Table 4).

Anti-nucleosome antibodies and SLE Relationship between anti-nucleosome,

disease activity
Anti-nucleosome antibodies were found to be positive
in 40 (97.6%) active-SLE patients with a median (IQR)
of 74.6 U/mL (121.5), while they were found in seven
(14.3%) inactive-SLE patients with a median (IQR)
of 7.6 U/mL (9.2). The median concentration of
anti-nucleosome antibodies was significantly different
between active- and inactive-SLE patients (P < 0.001)
(Table 3). Anti-dsDNA antibodies were found in 25
anti-dsDNA antibodies and SLEDAI score
Spearman’s rank correlation coefficient showed an ex-
cellent correlation between anti-nucleosome antibod-
ies and SLEDAI (ρ = 0.77, P < 0.001) (Fig. 1). A good
correlation was also observed between anti-dsDNA
antibodies and SLEDAI score (ρ = 0.51, P < 0.001).
There was a significant correlation between anti-
nucleosome antibodies and anti-dsDNA antibodies
(P = 0.047).

International Journal of Rheumatic Diseases 2009; 12: 100–106 103

S. Suleiman et al.
Figure 1 A scatter plot showing an excellent positive cor-
relation between anti-nucleosome antibodies and Systemic
Lupus Erythematosus Disease Activity Index (SLEDAI) score.
Relationship between anti-nucleosome and
anti-dsDNA antibodies in active SLE
In active SLE patients, a positive level of both anti-
nucleosome and anti-dsDNA antibodies was found in
23 (56.1%) patients, but 17 (41.5%) patients had a
positive level of anti-nucleosomes without anti-dsDNA
antibodies. Only one (2.4%) patient had a positive
level of anti-dsDNA without anti-nucleosome antibodies.
Additionally, a significant correlation was found
between the level of anti-nucleosomes and those of
anti-dsDNA antibodies (ñ = 0.37, P < 0.001). There was
a significant association between anti-nucleosome
antibodies and disease activity (P < 0.001).
DISCUSSION
Systemic lupus erythematosus has no single diagnostic
marker, which makes it difficult to detect unless
clinicians identify it through a combination of clinical
manifestations and some laboratory findings.10 Accurate
diagnosis of SLE is important because treatment can
reduce morbidity and mortality of patients, particularly
from lupus nephritis.11,12 Therefore, much effort has
been spent to find a useful and early marker of this
complication.
In this study we found that the prevalence of anti-
nucleosome antibodies was higher than the prevalence
of anti-dsDNA antibodies in SLE patients. This may
indicate a role of anti-nucleosome antibodies in the
disease process, as well as their usefulness in obtaining
104
more accurate diagnosis of SLE. Similar findings were
also reported in previous studies conducted on the
prevalence of anti-nucleosome antibodies.7,13–15 Most
studies have found that anti-nucleosome antibodies are
quite sensitive and specific for SLE.7,13,16
In the current study, anti-nucleosome antibodies
showed a sensitivity of 52% in SLE patients and it was
higher than the sensitivity of anti-dsDNA (37%),
whereas the specificity was comparable. These results
agreed with a group of studies showing both high
sensitivity and specificity of anti-nucleosome antibodies
in the diagnosis of SLE.14,17 The sensitivity of anti-
dsDNA antibodies was lower in this study compared
with other previous studies. This could be due to the
differences among commercial test kits and assay tech-
niques used, such as substrate differences, the isotype
of antibodies detected, antibody affinity, assay-specific
parameters, as well as problems with standardization
and calibration.8,18–20 Because of these difficulties in the
detection of anti-dsDNA antibodies, our study, which
was also supported by almost all the previous studies,
suggested that the anti-nucleosome antibodies test
might be a good alternative test for the diagnosis of SLE
rather than anti-dsDNA antibodies test.
Our study showed that anti-nucleosome antibodies
were positive in almost all active-SLE patients (98%),
and at the same time the serum levels of these antibodies
was significantly higher in active than in inactive SLE.
On the other hand, anti-dsDNA antibodies were found
to be positive only in 61% of active-SLE patients, and
these auto-antibodies were found to be positive in a
small percentage of inactive-SLE patients. Anti-nucleosome
antibodies showed a higher sensitivity than anti-dsDNA
to detect active SLE. This could be due to anti-dsDNA
antibodies that can be detected as negative early in a
disease course or after treatment. Some flares occur
with no change in the level of anti-dsDNA antibodies.
Moreover, the decrease in anti-dsDNA antibodies at the
time of flare is consistent with the hypothesis that
anti-dsDNA antibodies are deposited in the tissue.21,22
Longitudinal monitoring of anti-nucleosome anti-
bodies in correlation with changes of clinical disease
activity in SLE patients would be another convincing
way of demonstrating its usefulness.
In most published work, the level of anti-nucleosome
antibodies has been reported to be higher in active
SLE.7,23,24 The current data also showed that anti-
nucleosome and anti-dsDNA antibodies were found to
have a significant correlation with SLEDAI score, but
the correlation of anti-nucleosome antibodies with
SLEDAI score was found to be better than anti-dsDNA
International Journal of Rheumatic Diseases 2009; 12: 100–106

antibodies, especially among patients who were tested
negative for anti-dsDNA antibodies.
Numerous studies have been conducted to evaluate
the relationship between anti-nucleosome antibod-
ies and SLE disease activity by using various indices.
Their data showed that anti-nucleosome antibodies
have significant correlation with SLE disease activity.
Our data is consistent with those reported by some
authors.13,23–27 However, there are controversial data
from some previous studies showing that there is no
correlation between anti-nucleosome antibodies and
SLE disease activity.15,17,28 This can be explained by the
clinical characteristics of the patients included in the
study; evaluation of the disease activity is done by
using different disease activity indices or following
therapeutic management.24 In these matters, our results
indicate that both anti-dsDNA and anti-nucleosome
antibodies are suitable and useful parameters to evaluate
disease activity, and anti-nucleosome antibodies are
helpful when anti-dsDNA antibodies are absent.
The anti-nucleosome antibodies ELISA test that was
used in this study is a straightforward laboratory test
that can be performed on a large number of samples in
a relatively short period of time. The level of anti-
nucleosome antibodies appears to be a useful parameter
in the differential diagnosis of CTD. Anti-nucleosome
antibodies are sensitive and specific markers for the
diagnosis of SLE. Therefore, the determination of
circulating anti-nucleosome antibodies could be a
useful parameter in addition to the laboratory tests that
can help in the diagnosis and monitoring effect in the
treatment of SLE. We support other groups of researchers
who have suggested that anti-nucleosome antibodies
should be introduced into routine work-up in the
diagnosis and management of SLE.13,23–27
ACKNOWLEDGMENTS
We thank University Sains Malaysia for providing the
short-term grant (304/PPSP/6131272) that funded our
research project. Our profound gratitude goes to
Associate Professor Dr Mustaffa Musa for his invaluable
advice and overall comments. Without their cooperation,
this study would not have been possible. Last but not
least, our sincere thanks to all patients whose valuable
data have been used to fulfil the objective of this study.
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