Antibodies against nuclear antigens (IgG)

Test instructions for the ANA Profile 3 EUROLINE

ANTIBODIES IG-
ORDER NO. SUBSTRATE FORMAT
AGAINST CLASS
nRNP/Sm, Sm, SS-A, Ro-52, SS-B,
DL 1590-1601-3 G Scl-70, PM-Scl, Jo-1, CENP B, Ag-coated 16 x 01 (16)
IgG
DL 1590-6401-3 G PCNA, dsDNA, nucleosomes, immunoblot strips 64 x 01 (64)
Histones, rib. P-prot., AMA-M2

Indications: Sharp syndrome (MCTD), lupus erythematosus disseminatus (SLE), Sjögren’s syndrome,
progressive systemic sclerosis, poly-/dermatomyositis, overlap syndrome, limited form of progressive
systemic sclerosis (CREST syndrome), primary biliary liver cirrhosis.

Principles of the test: The EUROLINE test kit provides a qualitative in vitro assay for human
autoantibodies of the IgG class to 14 different antigens: nRNP, Sm, SS-A (SS-A native and Ro-52), SS-
B, Scl-70, PM-Scl, Jo-1, CENP B, PCNA, dsDNA, nucleosomes, histones, ribosomal P-protein and
AMA-M2 in serum or plasma. The test kit contains test strips coated with parallel lines of highly purified
antigens. In the first reaction step, diluted patient samples are incubated with the immunoblot strips. In
the case of positive samples, the specific IgG antibodies (also IgA and IgM) will bind to the
corresponding antigenic site. To detect the bound antibodies, a second incubation is carried out using an
enzyme-labelled anti-Human IgG (enzyme conjugate) catalysing a colour reaction.

Contents of the test kit:

Component Format Format Symbol
1. Test strips coated with the antigens:
nRNP/Sm, Sm, SS-A (SSA native and Ro-52),
SS-B, Scl-70, PM-Scl, Jo-1, CENP B, PCNA, 16 strips 4 x 16 strips .STRIPS.
dsDNA, Nucleosomes, Histones, rib. P-
protein, AMA-M2
2. Positive control
1 x 0.02 ml 4 x 0.02 ml .POS CONTROL 100x.
(IgG, human), 100x concentrate
3. Enzyme conjugate
Alkaline phosphatase-labelled anti-human IgG 1 x 3 ml 4 x 3 ml .CONJUGATE 10x.
(goat), 10x concentrate
4. Sample buffer
1 x 100 ml 3 x 100 ml .SAMPLE BUFFER.
ready for use
5. Wash buffer
1 x 50 ml 1 x 100 ml .WASH BUFFER 10x.
10x concentrate
6. Substrate solution
Nitrobluetetrazoliumchloride/5-Bromo-4-
1 x 30 ml 4 x 30 ml .SUBSTRATE.
chloro-3-indolylphosphate (NBT/BCIP), ready
for use
7. Incubation tray 2 x 8 channels ---
8. Test instruction 1 booklet 1 booklet
.LOT. Lot � Storage temperature
.IVD. In vitro determination �� � Unopened usable until

The following components are not provided in the test kits but can be ordered at EUROIMMUN under the
respective order numbers.
Performance of the test requires an incubation tray:
ZD 9899-0130 Incubation tray with 30 channels
ZD 9898-0130 Incubation tray with 30 channels (black, for EUROBlotCamera system)
ZD 9898-0148 Incubation tray with 48 channels (black, for EUROBlotCamera system)
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For the creation of work protocols and the evaluation of incubated test strips using EUROLineScan
green paper and adhesive plastic foil are required:
ZD 9880-0101 Green paper (1 sheet)
ZD 9885-0116 Adhesive foil for approx. 16 test strips
ZD 9885-0130 Adhesive foil for approx. 30 test strips
If you wish to perform a visual evaluation, you may order the required evaluation protocol under:
ZD 1590-0101-3 G Evaluation protocol visual ANA Profile 3 EUROLINE.

Storage and stability: The test kit must be stored at a temperature between +2°C to +8°C. Do not
freeze. Unopened, all test kit components are stable until the indicated expiry date.

Waste disposal: Patient samples, controls and incubated test strips should be handled as infectious
waste. Other reagents do not need to be collected separately, unless stated otherwise in official
regulations.

Preparation and stability of the reagents

Note: All reagents must be brought to room temperature (+18°C to +25°C) approx. 30 minutes before
use. Unopened, reagents are stable until the indicated expiry date when stored at +2°C to +8°C. After
initial opening, reagents are stable for 12 months or until the expiry date, if earlier, unless stated
otherwise in the instructions. Opened reagents must also be stored at +2°C to +8°C and protected from
contamination.

- Coated test strips: Ready for use. Open the package with the test strips only when the strips have
reached room temperature to prevent condensation on the strips. After removal of the strips the
package should be sealed tightly and stored at +2°C to +8°C.

- Positive control: The control is a 100x concentrate. For the preparation of the ready for use control
the amount required should be removed from the bottle using a clean pipette and diluted 1:101 with
sample buffer. Example: add 15 µl of control to 1.5 ml of sample buffer and mix thoroughly. The ready
for use diluted control should be used at the same working day.

- Enzyme conjugate: The enzyme conjugate is supplied as a 10x concentrate. For the preparation of
the ready for use enzyme conjugate the amount required should be removed from the bottle using a
clean pipette and diluted 1:10 with sample buffer. For one test strip, dilute 0.15 ml enzyme conjugate
with 1.35 ml sample buffer. The diluted enzyme conjugate should be used at the same working day.

- Sample buffer: Ready for use.

- Wash buffer: The wash buffer is supplied as a 10x concentrate. For the preparation of the ready for
use wash buffer the amount required should be removed from the bottle using a clean pipette and
diluted 1:10 with distilled water. For one test strip, dilute 1 ml in 9 ml of distilled water. The ready-to-
use diluted wash buffer should be used at the same working day.

- Substrate solution: Ready for use. Close bottle immediately after use, as the contents are sensitive
to light.

Warning: The controls used have tested negative for HBsAg, and antibodies against HCV, HIV-1 and
HIV-2 using enzyme immunoassays or indirect immunofluorescence methods. Nonetheless all materials
should be treated as being a potential infection hazard and should be handled with care. Some of the
reagents are poisonous (buffer, substrate solution). Avoid contact with skin
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Preparation and stability of the patient samples

Sample material: Human serum or EDTA, heparin or citrate plasma.

Stability: Patient samples to be investigated can generally be stored at +2°C to +8°C for up to 14 days.
Diluted samples should be incubated within one working day.

Sample dilution: The patient samples for analysis are diluted 1:101 with sample buffer. For example,
add 15 µl of serum to 1.5 ml sample buffer and mix well by vortexing. Sample pipettes are not suitable
for mixing.

Incubation

Pretreat: Remove the required amount of test strips from the package and place them
each in an empty channel. The number on the test strip should be visible. Fill
the channels of the incubation tray according to the number of serum samples
that should be tested with 1.5 ml sample buffer each. Incubate for 5 minutes
at room temperature on a rocking shaker. Afterwards aspirate off all the liquid.

Incubate: Fill each channel with 1.5 ml of the diluted serum samples and incubate at
(1st step) room temperature (+18°C to +25°C) for 30 minutes on a rocking shaker.

Wash: Aspirate off the liquid from each channel and wash 3 x 5 minutes each with
1.5 ml working strength wash buffer on a rocking shaker.

Incubate: Pipette 1.5 ml diluted enzyme conjugate (alkaline phosphatase-labelled anti-

(2nd step) Human IgG) into each channel and incubate for 30 minutes at room tem-
perature (+18°C to +25°C) on a rocking shaker.

Wash: Aspirate off the liquid from each channel. Wash as described above.

Incubate: Pipette 1.5 ml substrate solution into the channels of the incubation tray.
(3rd step) Incubate for 10 minutes at room temperature (+18°C to +25°C) on a rocking
shaker.

Stop: Aspirate off the liquid from each channel and wash each strip 3 x 1 minute
with distilled water.

Evaluate: Place test strip on the evaluation protocol, air dry and evaluate.

For automated incubation with the EUROBlotMaster select the programme Euro01 AAb EL30.

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EUROIMMUN ANA Profile 3 EUROLINE (IgG)

Incubation protocol

Pretreat
Put the test strip into the incubation channel and fill each
channel with 1.5 ml sample buffer

5 min Shake

1. Step: Incubate
Aspirate off, pipette 1.5 ml of diluted serum sample (1:101) into
the incubation channel

30 min Shake

Wash
Aspirate off, wash 3 x 5 min with 1.5 ml working strength
wash buffer each

2. Step: Incubate
Aspirate off, pipette 1.5 ml enzyme conjugate into the
incubation channel

30 min Shake

Wash
Aspirate off, wash 3 x 5 min with 1.5 ml working strength
wash buffer each

3. Step: Incubate
Aspirate off, pipette 1.5 ml substrate into the incubation channel

10 min Shake

Stop
Aspirate off, rinse three times with 1.5 ml distilled water

Evaluation
EUROLineScan (digital)

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Interpretation of Results
Handling: For the evaluation of incubated test strips we generally recommend using the
EUROLineScan software. After stopping the reaction using deionised or distilled water, place the
incubated test strips onto the adhesive foil of the green work protocol using a pair of tweezers. The
position of the test strips can be corrected while they are wet. As soon as all test strips have been placed
onto the protocol, they should be pressed hard using filter paper and left to air-dry. After they have dried,
the test strips will be stuck to the adhesive foil. The dry test strips are then scanned using a flatbed
scanner (EUROIMMUN AG) and evaluated with EUROLineScan. For general information about the
EUROLineScan programme please refer to the EUROLineScan user manual (EUROIMMUN AG). The
code for entering the test into EUROLineScan is Ana3b.
If a visual evaluation must be performed, place the incubated test strips onto the respective work
protocol for visual evaluation. This protocol is available at EUROIMMUN under the order no.
ZD 1590-0101-3 G.
There is a control band on the strips. The incubation was performed correctly if a strong colour
reaction is visible on this control band. A white band at the position of an antigen has to be
interpreted as negative.
Antigens and their arrangement on the strips: The EUROLINE test strips have been coated with the
following antigens:
nRNP/Sm: Native U1-nRNP purified by affinity chromatography
from calf and rabbit thymus. nRNP/Sm
Sm: Native Sm antigen purified by affinity chromatography from
bovine spleen and thymus. The Sm antigen contains the core Sm
proteins of snRNP particles. D protein is the main component of
the Sm preparation. SS-A
SS-A: Native SS-A antigen purified by affinity chromatography
Ro-52
from bovine spleen and thymus.
Ro-52: Recombinant Ro-52 (52 kDa). The corresponding human SS-B
cDNA has been expressed with the baculovirus system in insect
cells.
SS-B: Native SS-B antigen purified by affinity chromatography Scl-70
from calf and rabbit thymus.
Scl-70: Native Scl-70 (DNA-Topoisomerase I) antigen purified by PM-Scl
affinity chromatography from bovine and rabbit thymus.
PM-Scl: Recombinant PM-Scl100. The corresponding human Jo-1
cDNA has been expressed with the baculovirus system in insect
cells.
Jo-1: Native Jo-1 (Histidyl-tRNA Synthetase) antigen purified by CENP B
affinity chromatography from calf and rabbit thymus.
CENP B: Recombinant centromere protein B. The corresponding PCNA
human cDNA has been expressed with the baculovirus system in
insect cells.
dsDNA
PCNA: Recombinant PCNA (36kDa). The corresponding human Nucleosomes
cDNA has been expressed with the baculovirus system in insect
cells.
Histones
dsDNA: Highly purified native, double-stranded DNA isolated
from salmon testes.
Rib. P-
Nucleosomes: Native nucleosomes purified from calf thymus. protein
Histones: A mixture of individually purified histone types isolated
from calf thymus. AMA-M2
Rib. P-protein: Native ribosomal P-proteins purified by affinity
chromatography from calf and rabbit thymus. Control
AMA-M2: Native M2 antigen (pyrutate-dehydrogenase complex)
purified from pork heart.

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EUROIMMUN recommends interpreting results based on the signal intensity:

Signal Signal intensity
Result
Visual evaluation EUROLineScan
No signal 0-5 o Negative
Very weak band 6-10 (+) Borderline
Medium to strong band 11-25 or 26-50 +, ++ Positive
Very strong band with an intensity comparable Strong
>50 +++
to the control band positive

Results in the borderline range from 6 to 10 should be evaluated as increased but negative.

An indirect immunofluorescence test should always be performed in parallel with the determination of
cell nucleus antibodies by EUROLINE. On the one hand, this provides a check on plausibility as a
safeguard against false-positive results, on the other hand, by using EUROIMMUN HEp-2 cells, and in
particular in combination with frozen sections of primate liver, immunofluorescence permits the
detection of a wider range of cell nucleus antibodies, as not all cell nucleus antigens are presently
available in the EUROLINE.

Test characteristics
Calibration: The reactivity of each antigen is standardized by the human reference sera CDC-ANA #1 to
#11 of the “Center for Disease Control“ (Atlanta, USA). The reactivity of the CDC sera in the
EUROIMMUN ANA Profile EUROLINE is summarized in the following table:

CDC-1 CDC-2 CDC-3 CDC-4 CDC-5 CDC-6 CDC-7 CDC-8 CDC-9 CDC-10 CDC-11

Antigen Homoge Speckled/ Speckled RNP Sm Nucleolar SS-A Centro- Scl-70 Jo-1 PM-Scl
neous/ SS-B mere
rim
nRNP/Sm pos. neg. pos. pos. pos. neg. neg. neg. neg. neg. neg.
Sm pos. neg. pos. neg. pos. neg. neg. neg. neg. neg. neg.
SS-A neg. pos. pos. neg. neg. neg. pos. neg. neg. neg. neg.
Ro-52 neg. pos. pos. neg. neg. neg. pos. neg. neg. pos. neg.
SS-B neg. pos. pos. neg. neg. neg. neg. neg. neg. neg. neg.
Scl-70 neg. neg. neg. neg. neg. neg. neg. neg. pos. neg. neg.
PM-Scl neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. pos.
Jo-1 neg. neg. neg. neg. neg. neg. neg. neg. neg. pos. neg.
CENP B neg. neg. neg. neg. neg. neg. neg. pos. neg. neg. neg.
PCNA neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg.
dsDNA pos. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg.
Nucleosomes pos. neg. neg. neg. neg. neg. pos. neg. neg. neg. neg.
Histones pos. neg. neg. neg. neg. neg. pos. neg. neg. neg. neg.
Rib. P-protein neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg.
M2 neg. neg. neg. neg. neg. neg. neg. pos. neg. neg. neg.
The specificity of these sera was determined at the “Center for Disease Control“ by immunofluorescence
patterns (substrate: HEp-2 cells and primate liver), the results of double immunodiffusion or counter
immunoelectrophoresis (the sera are not in any case monospecific).

Measurement range: The EUROLINE is a qualitative method. No measurement range is provided. The
titre limit is given at a dilution of 1:101.

Cross reactions: The high analytical specificity of the test system is guaranteed by the quality of the
antigen substrates used (antigens and antigen sources). This EUROLINE specifically detects IgG class
antibodies to nRNP/Sm, Sm, SS-A, Ro-52, SS-B, Scl-70, PM-Scl, Jo-1, CENP B, PCNA, dsDNA,
Nucleosomes, Histones, ribosomal P-protein and AMA-M2. No cross reactions with other autoantibodies
have been found.

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Interference: Haemolytic, lipaemic and icteric sera up to a concentration of 5 mg/ml for haemoglobin, of
20 mg/ml for triglycerides and of 0.4 mg/ml bilirubin showed no effect on the analytical results of the
present EUROLINE.

Inter- and intra-assay variation: The inter-assay variation was determined by multiple analyses of
characterised samples over several days. The intra-assay variation was determined by multiple analyses
of characterised samples on one day. In every case, the intensity of the bands was within the specified
range. This EUROLINE displays excellent inter- and intra-assay reproducibility.

Sensitivity and specificity:

nRNP/Sm: For the detection of autoantibodies against RNP/Sm a sensitivity of 100% with reference to
the ELISA method was determined using 22 samples of patients with MCTD (mixed connective tissue
disease). The specificity was 100% for healthy blood donors (n = 50) and 96% in a panel of non-SLE
rheumatic diseases (Sjögren`s syndrome n = 14, scleroderma n = 18, polymyositis n = 25).

Sm: For the detection of autoantibodies against Sm a sensitivity of 100% with reference to the ELISA
method was determined using 45 samples of patients with SLE. The specificity was 100% for healthy
blood donors (n = 50) and 100% in a panel of non-SLE rheumatic diseases (Sjögren`s syndrome n = 14,
scleroderma n = 18, polymyositis n = 25).

SS-A: For the detection of autoantibodies against SS-A a sensitivity of 100% with reference to the ELISA
method was determined using 14 samples of patients with Sjögren`s syndrome. The specificity was
100% for healthy blood donors (n = 50) and 95% in a panel of non-SLE rheumatic diseases
(scleroderma n = 18, MCTD n = 22).

Ro-52: For the detection of autoantibodies against Ro-52 a sensitivity of 100% with reference to the
westernblot method was determined using 103 samples of patients with SLE and Sjögren`s syndrome
(SLE n = 23, Sjögren`s syndrome n = 77 and neonatal lupus erythematosus n = 3). The specificity was
100% for healthy blood donors (n = 65). Antibodies against Ro-52 are not disease specific and can be
detected in samples from patients suffering from myositis, scleroderma and other rheumatic diseases
(11-16), i.e. in 7 of 20 samples of scleroderma patients autoantibodies against Ro-52 were detected.

SS-B: For the detection of autoantibodies against SS-B a sensitivity of 100% with reference to the ELISA
method was determined using 14 samples of patients with Sjögren`s syndrome. The specificity was
100% for healthy blood donors (n = 50) and 97% in a panel of non-SLE rheumatic diseases
(scleroderma n = 18, MCTD n = 22).

Scl-70: For the detection of autoantibodies against Scl-70 a sensitivity of 100% with reference to the
ELISA method was determined using 18 samples of patients with scleroderma. The specificity was 100%
for healthy blood donors (n = 50) and for a panel of non-SLE rheumatic diseases (MCTD n = 22,
Sjögren`s syndrome n = 14, myositis n = 25).

PM-Scl: In 14 of 20 sera of patients with polymyositis, having a nucleolar-positive pattern in the indirect
immunofluorescence (HEp-2-cells/primate liver), autoantibodies against PM-Scl were detected. The
specificity was 100% for healthy blood donors (n = 50) and 99% in a panel of non-SLE rheumatic
diseases (MCTD n = 22, Sjögren`s syndrome n = 14, scleroderma n = 18).

Jo-1: For the detection of autoantibodies against Jo-1 a sensitivity of 100% with reference to the ELISA
method was determined using 5 samples of patients with myositis. The specificity was 100% for healthy
blood donors (n = 50) and 99% in a panel of non-SLE rheumatic diseases (scleroderma n = 18, MCTD
n = 22, Sjögren`s syndrome n = 14).

CENP B: In 19 of 20 sera of patients with scleroderma, having a centromer-positive pattern in the

indirect immunofluorescence (HEp-2-cells/primate liver), autoantibodies against CENP B (sensitivity
95%) were detected. The specificity was 100% for healthy blood donors (n = 50) and 97% in a panel of
non-SLE rheumatic diseases (MCTD n = 22, Sjögren`s syndrome n = 14, myositis n = 25).

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PCNA: In 13 of 20 patient sera, having a cyclin I-positive pattern in the indirect immunofluorescence
(HEp-2-cells/primate liver), autoantibodies against PCNA were detected. The specificity was 100% for
healthy blood donors (n = 50) and 99% in cyclin I-negative sera of patients with SLE (n = 83).
dsDNA: For the detection of autoantibodies against dsDNA a sensitivity of 94% with reference to the
ELISA method was determined using 36 samples of patients with SLE. The specificity was 100% for
healthy blood donors (n = 50) and for a panel of non-SLE rheumatic diseases (Sjögren`s syndrome
n = 14, scleroderma n = 18).
Nucleosomes: For the detection of autoantibodies against nucleosomes a sensitivity of 97% with
reference to the ELISA method was determined using 34 samples of patients with SLE. The specificity
was 100% for healthy blood donors (n = 50) and in a panel of non-SLE rheumatic diseases (Sjögren`s
syndrome n = 14, scleroderma n = 18).
Histones: For the detection of autoantibodies against histones a sensitivity of 78% with reference to the
ELISA method was determined using 41 samples of patients with SLE. The specificity was 100% for
healthy blood donors (n = 50) and 97% in a panel of non-SLE rheumatic diseases (Sjögren`s syndrome
n = 14, scleroderma n = 18).
Ribosomal P-protein: For the detection of autoantibodies against ribosomal P-protein a sensitivity of
82% with reference to the ELISA method was determined using 49 samples of patients with SLE. The
specificity was 100% for healthy blood donors (n = 50) and in a panel of non-SLE rheumatic diseases
(Sjögren`s syndrome n = 14, scleroderma n = 18).
AMA-M2: For the detection of autoantibodies against AMA-M2 a sensitivity of 100% with reference to
the ELISA method was determined using 36 samples of patients with primary biliary liver cirrhosis. The
specificity was 100% for healthy blood donors (n = 50) and 99% in a panel of other liver diseases
(autoimmune hepatitis n = 28, toxic liver damage n = 38, viral hepatitis B/C n = 69).
Reference range: The reference range was determined using a cohort of healthy blood donors (n = 50).
All blood donors were negative.
Antigens: nRNP and Sm belong to a group of small ribonucleoproteins (snRNP, small nuclear ribo-
nucleoproteins) which consist of low molecular weight RNA with a high uridine content (U-RNA)
complexed with various proteins (molecular weights 9 - 70 kDa). The RNA component is termed U1 to
U6, depending on its behaviour in chromatography. Besides the particular RNA, the particles of U-nRNP
contain six different core proteins (B, B', D, E, F, G), U1-nRNP additionally contains particle-specific
proteins (70K, A, C). Antibodies to U1-nRNP are directed against one or more of the particle-specific
proteins 70K, A or C. In contrast, antibodies to Sm can also react with one or more core proteins. The U-
nRNP particles are involved in splicing of the pre-mRNA (pre-messenger RNA) - they split off the non-
coding mRNA sequences (introns) and insert the coding mRNA sequences (exons) to recreate the
messenger RNA.
The native SS-A antigen is a small ribonucleoprotein composed of one of five RNA molecules (Y1, Y2,
Y3, Y4 or Y5 RNA; 80-112 bases) and a 60 kDa protein. The SS-A band in the EUROLINE consists of
the native SS-A antigen. A 52 kDa protein (52 kDa) is also associated with the SS-A/Ro complex, but
whether this protein is a component of the SS-A/Ro complex is controversially discussed in the literature.
Isolated antibody reactions with Ro-52 should not be evaluated as anti-SS-A positive or specific
for SLE or Sjögren’s syndrome, since they can occur in many different autoimmune diseases.
We recommend interpreting the EUROLINE with reference to the ANA screening test (HEp-2
cells/primate liver) as follows:
IIFT EUROLINE
Result
HEp-2 cells Ro-52 (52 kDa) SS-A (60 kDa)
ANA negative positive negative Anti-SS-A negative
ANA positive positive negative Anti-SS-A negative
positive or
ANA positive positive Anti-SS-A positive
negative

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It has been shown in various studies that anti-SS-A positive sera always contain antibodies against
native SS-A (60 kDa protein) and may additionally exhibit antibodies against Ro-52. For example, in a
Japanese study (EUROIMMUN) sera from 103 patients with SLE and Sjögren’s syndrome (SLE n = 26,
Sjögren’s syndrome n = 77), which were characterized as anti-SS-A positive by double immunodiffusion,
were investigated. 102 sera reacted with native SS-A, and 90 sera reacted additionally with the Ro-52
band. But no serum showed only a reaction with the Ro-52 band. This study demonstrates that
antibodies against native SS-A can be reliably detected using the native SS-A. In rare cases and in
suspected cases of neonatal lupus syndrome, the Ro-52 band may provide important supplementary
information.

The SS-B antigen is a phosphoprotein with a molecular weigth of 48 kDa. It functions in the cell nucleus
as a helper protein for RNA polymerase III.

The Scl-70 antigen has been identified as the enzyme DNA Topoisomerase-I. The molecular weight of
the native antigen is 100 kDa. Originally, only a metabolic product of molecular weight 70 kDa was found
in the western blot. The DNA Topoisomerase-I is situated in the nucleoplasm and, in a particularly high
concentration, in the nucleolus. The enzyme participates in the replication and transcription of the DNA
double helix.

The PM-Scl antigen is a complex of 11-16 polypeptides with molecular weights of between 20 and 110
kDa. The main antigens are two polypeptides of 75 and 100 kDa, which are known as PM-Scl75 and
PM-Scl100. 90-98% of PM-Scl autoantibody positive sera react with PM-Scl100 and 50-63% with PM-
Scl75. The two antigens are independent of one another and do not show any cross reactivity. PM-Scl is
mainly localized in the nucleoli, but also occurs in the nucleoplasm. The function of the polypeptide
complex has not yet been fully explained. It is suspected that PM-Scl plays a role in splicing of the 5.85
rRNA and some U-snRNAs.

The Jo-1 antigen is identical to Histidyl-tRNA synthetase, a cytoplasmic phosphoprotein with a molecular
weight of 50 kDa. It joins the amino acid histidine in the cytoplasm to its corresponding tRNA.

Four different proteins were identified as centromere autoantigens: centromere protein-A (17 kDa),
centromere protein-B (80 kDa), centromere protein-C (140 kDa) and centromere protein-D (50 kDa). All
sera containing anti-centromere antibodies pre-characterized in indirect immunofluorescence tests are at
least reactive with centromere protein B.

PCNA – proliferating cell nuclear antigen – with a molecular weight of 36 kDa is expressed cell cycle
dependent. The active, trimeric form is a cofactor of DNA polymerases and takes part in the regulation of
DNA repair. In indirect immunofluorescence on HEp-2 cells autoantibodies against PCNA produce a
pattern called cyclin I. Half of the nuclei of all interphase cells display a bright, fine-granular basic
fluorescence, whereby the nucleoli are excluded. The same fluorescence patterns can be seen in the
other half, but the intensity is less by a factor of about 10.

Antibodies against DNA are distinguished into two different types: Antibodies against native, double-
stranded DNA (dsDNA) and antibodies against denatured, single-stranded DNA. Antibodies defined as
reactive with dsDNA recognize mainly epitopes in the deoxyribose phosphate backbone of the double
helix and are therefore reactive with both double- and single-stranded DNA. On the other hand, anti-
bodies defined as reactive with ssDNA recognize polymers of purine and pyrimidine bases which are not
accessible in the double-stranded form.

Nucleosomes are highly organized functional subunits of chromosomes consisting of histones (types
H1, H2A, H2B, H3 and H4) and dsDNA. Their centre consists of a H3-H3-H4-H4 tetramer which is
flanked on two sites by a H2A-H2B dimer each. The histone core particle is surrounded by two coils of
the DNA double helix (146 base pairs in total). The nucleosomes are joined in a row in a string-of-pearls
fashion, the DNA (linker DNA) is associated with the histone H1 in the region of the bond.

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Histones are basic DNA-associated proteins with molecular weights from 11.2 kDa to 21.5 kDa. Their
function is to stabilise the DNA double helix and also they might play a role in gene regulation
mechanisms. Five distinct histone types exist: H1, H2A, H2B, H3, and H4. Histones are associated with
DNA forming highly organized nucleosomal structures.

The ribosomal P-protein is composed of 3 proteins of the 60S-subunit of the ribosomes. These proteins
are called P0 (molecular weight 38 kDa), P1 (19 kDa) and P2 (17 kDa). The main antigenic epitope is
localized at the carboxy-terminus, which contains an identical sequence of 17 amino acids within all
three proteins.

The M2 antigen system has been shown to comprise three biochemically related multi-enzyme
complexes of the inner mitochondrial membrane which catalyze the oxidative decarboxylation of
pyruvate, 2-oxoglutarate and branched-chain 2-oxoacids. Six proteins have been identified as M2
antigens: E2 (74 kDa) of the pyruvate dehydrogenase complex, Protein X (55 kDa) of the pyruvate
dehydrogenase complex, E1 alpha subunit (45 kDa) of the pyruvate dehydrogenase complex and E1
beta subunit (36 kDa) of the pyruvate dehydrogenase complex; furthermore E2 (51 kDa) of the
branched-chain 2-oxoacid dehydrogenase complex and E2 (51 kDa) of the 2-oxoglutarate
dehydrogenase complex. The E2-enzymes are responsible for the transfer of acetyl groups to coenzyme
A, protein X is a subunit of the pyruvate dehydrogenase complex with an unknown function.

Clinical significance
Antibodies against nuclear antigens are directed against various cell nuclear components (biochemical
substances in the cell nucleus) [1, 2]. These encompass nucleic acids, cell nucleus proteins and
ribonucleoproteins. They are a characteristic finding in many diseases, in particular rheumatic diseases
[3, 4, 5]. The frequency (prevalence) of anti-nuclear antibodies in inflammatory rheumatic diseases is
between 20% and 100%, the lowest occurring in rheumatoid arthritis at between 20% and 40%.
Therefore, differential ANA diagnostics is indispensable in the identification of individual rheumatic
diseases as well as useful in the diagnosis of further autoimmune diseases [3, 6, 7, 8, 9, 10].
Rheumatic diseases:
- Sharp syndrome (mixed connective tissue disease = MCTD),
- systemic lupus erythematosus (SLE) [11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21]
- Sjögren’s syndrome (primary Sjögren’s syndrome) [5],
- systemic sclerosis (systemic scleroderma, SSc) [1, 22],
- limited form of systemic sclerosis (CREST syndrome) [27],
- poly-/dermatomyositis [11, 14],
- rheumatoid arthritis [5].
U1-nRNP
(Uridine 1- low-molecular-weight ribonuclear protein)
Antigen Disease Prevalence
Sharp syndrome 95 % - 100 %
Systemic lupus erythematosus (SLE) 15 % - 40 %
U1-nRNP
Systemic sclerosis 2 % - 12 %
Poly-/dermatomyositis 12 % - 16 %
The antibodies are directed exclusively against the core proteins A, C and 70kD of U1-nRNP [7, 17, 24].
High antibody titers against U1-nRNP with a sensitivity of 95% to 100% for the determination of
autoantibodies against nRNP/Sm are characteristic markers for Sharp syndrome, a multi-symptomatic
and multiform mixed connective tissue disease combining characteristics of rheumatoid arthritis, SLE,
systemic sclerosis and polymyositis. It has not yet been clarified if it is an independent disease. The
antibody titer correlates with the disease activity. Antibodies against U1-nRNP can also be found in
patients with SLE (15%-40%), systemic sclerosis (2%-12%) and polymyositis (12%-16%) [13].
Sm
(Sm, Smith antigen – name of the index patient; group of small ribonuclear proteins, which are involved
in splicing pre-mRNA. They consist of RNA with high uridine content, U-RNA, and various proteins;
molecular weight 9-70kDa)
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Antigen Disease Prevalence

Sm Systemic lupus erythematosus (SLE) 5 % - 40 %
Antibodies against Sm are highly specific (over 90%) for SLE and can be found in 5%-40% of patients
[11, 12, 13]. Together with antibodies against dsDNA, they can be considered pathognomonic for this
condition. The prevalence in Caucasians amounts to 5%-10%. It is much higher in other ethnic groups,
e.g. in Arabs (20 %-42 %), Chinese (around 30 %) or Black Africans (around 30 %) [15, 16, 25, 26, 27,
28]. For this reason, the prevalence given in many American studies is 20%-40%.
SS-A (Ro)
(Soluble substance A or Sjögren’s syndrome A or Robert antigen – name of the index patient;
cytoplasmic protein complex consisting of one Y1-, Y2-, Y3-, Y4- or Y5-RNA molecule and a 60-kDa
protein subunit)
Antigen Disease Prevalence
Sjögren’s syndrome 40 % - 95 %
SS-A (Ro) Systemic lupus erythematosus (SLE) 20 % - 60 %
Neonatal lupus erythematosus 95 % -100 %
Antibodies to SS-A are directed against the 60 kDa subunit and are associated with various autoimmune
diseases [7, 8]. They most commonly occur in patients with Sjögren's syndrome (40% - 95% of cases),
but also in SLE (20% - 60%) and primary biliary cirrhosis (20%), and occasionally also in autoimmune
hepatitis and viral hepatitis [2, 12, 16]. Apart from this, antibodies to SS-A can be found in practically
100% of cases of neonatal lupus erythematosus (neonatal LE syndrome) [29]. They are transmitted
diaplacentally to the fetus and cause inflammatory reactions as well as a congenital AV block when the
mother is anti-SS-A positive, in particular when anti-SS-B (Anti-La) are also present [11, 30, 31, 32].
Differentiation of anti-SS-A antibodies from those against the so-called Ro52 antigen (52 kDa protein,
RING dependent E3 ligase) is of decisive diagnostic importance, since antibodies against Ro-52 are not
disease-specific, but are also detected in myositis, systemic sclerosis, other collagenoses, neonatal
lupus erythematosus, primary biliary cirrhosis, autoimmune hepatitis and viral hepatitis [29, 32, 33, 34,
35, 36, 37].
SS-B (La)
(Soluble substance B or Sjögren’s syndrome B or Lane antigen – name of the index patient; transcription
termination factor in the nucleus for RNA polymerase III)

Antigen Disease Prevalence

Sjögren’s syndrome 40 % - 95 %
SS-B (La) Systemic lupus erythematosus (SLE) 10 % - 20 %
Neonatal lupus erythematosus 75 %
Antibodies against SS-B are found almost exclusively in females (29:1), in cases of Sjögren’s syndrome
(40%-95%), SLE (10%-20%) and neonatal lupus erythematosus (75%) [12, 30, 31, 32]. In Sjögren's
syndrome, combined SS-A and SS-B antibodies mainly occur [2].
Scl-70
(Enzyme DNA topoisomerase I, in the nucleoplasm and, in a particularly high concentration, in the
nucleolus, plays a role in the replication and transcription of the DNA double helix)
Antigen Disease Prevalence
Systemic sclerosis 25 % - 75 %
Scl-70 - diffuse form 40 % - 65 %
- limited form 5 % - 15 %
Systemic sclerosis (SSc) can manifest itself in two forms, which cannot always be clearly differentiated:
the diffuse cutaneous and the limited cutaneous form [36]. Antibodies against Scl-70 are detected in
25%-75% of patients with SSc, whereby the prevalence is 40%-65% in the diffuse form and 5%-15% in
the limited form [39, 40].

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PM-Scl
(Antigen complex of 11-16 polypeptides with molecular weights of between 20 and 110 kDa. These are
located predominantly in the nucleoli and are involved in the formation of ribosomal RNA. The main
antigens are PM-Scl100 and PM-Scl75. These two antigens are independent of one another and do not
show any cross reactions. It is assumed that PM-Scl is involved in splicing of the 5.85 rRNA and some
U-snRNA)
Antigen Disease Prevalence
Systemic sclerosis including overlap syndrome 10 % - 20 %
Polymyositis/systemic sclerosis overlap syndrome 18 %
PM-Scl
Systemic sclerosis (anti-PM-Scl75 positive) 10 %
Systemic sclerosis (anti-PM-Scl100 positive) 7%
90% - 98% of PM-Scl autoantibody positive samples in systemic sclerosis including overlap syndrome
react with PM-Scl100 and 50% - 63% with PM-Scl75. The specificity amounts to 99% (PM-Scl100) or
98% (PM-Scl75), and the sensitivity to 6.6% or 11.8%, respectively. PM-Scl antibodies are detected in
18% of patients with polymyositis/systemic sclerosis overlap syndrome. Here the autoantibodies are
generally directed against both main antigens, PM-Scl75 and PM-Scl100. If progressive systemic
sclerosis is present, antibodies against PM-Scl75 show a prevalence of almost 10% and those against
PM-Scl100 a prevalence of 7% [5, 6, 9, 10, 15, 40].
Jo-1
(Cytoplasmic histidyl-tRNA synthetase)
Antigen Disease Prevalence
Jo-1 Polymyositis / dermatomyositis 25 % - 35 %
Antibodies to Jo-1 are found in polymyositis and dermatomyositis with a prevalence of 25% - 35% [11,
34, 35]. They are often associated with a concurrent interstitial fibrosis of the lung/fibrous alveolitis.
Centromeres
(Four different proteins, CENP A, B, C, D: centromere protein A with a molecular weight of 17 kDa,
centromere B with a molecular weight of 80 kDa, centromere C with a molecular weight of 140 kDa and
centromere D with a molecular weight 50 kDa)
Antigen Disease Prevalence
Systemic sclerosis, limited form 80 % - 95 %
Centromeres Systemic sclerosis, diffuse form 8%
Primary biliary cirrhosis 10 % - 30 %
Autoantibodies against centromeres (anti-centromere antibodies = ACA) are associated with the limited
from of systemic sclerosis and can be found in 80%-95% of patients [33]. They are detected in only 8 %
of patients with the diffuse form, but also occur in 10%-30% of patients with primary biliary cirrhosis [23,
39, 41, 42, 43].
PCNA
(Proliferating cell nuclear antigen, Cyclin I, helper protein for DNA polymerase delta with molecular
weight of 36 kDa. Key role in regulating the cell cycle: when it appears the S-phase begins. The protein
is broken down by the middle of the G2 phase)
Antigen Disease Prevalence
PCNA Systemic lupus erythematosus (SLE) 3%
A specificity of 99% has been determined for the detection of autoantibodies against PCNA. However
the prevalence is only 3% [5, 6, 9].
dsDNA
(Double-stranded DNA, dsDNA, native DNA)
Antigen Disease Prevalence
Double-stranded
Systemic lupus erythematosus (SLE) 40 % - 90 %
DNA

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The detection of autoantibodies against deoxyribonucleic acid (DNA) is essential in the diagnosis of SLE
[5, 6, 9]. Autoantibodies against DNA are divided into two different types: antibodies against double-
stranded, native DNA (dsDNA) and antibodies against single-stranded, denatured DNA. Antibodies
defined as reactive with dsDNA recognise mainly epitopes in the (outer) deoxyribose phosphate
backbone of the double helix and are therefore reactive with both double- and single-stranded DNA. On
the other hand, antibodies defined as reactive with ssDNA recognize epitopes of purine and pyrimidine
bases. They may also react with epitopes of the deoxyribose phosphate backbone.
Anti-dsDNA antibodies are found almost exclusively in SLE [44]. The prevalence of antibodies against
dsDNA amounts to 20% to 90% depending on the detection method and disease activity. Antibodies
against dsDNA are also occasionally detected in patients with other autoimmune diseases and infections
and, in rare cases, in clinically healthy people [45]. 85% of people in the latter group develop SLE within
5 years of initial detection of anti-dsDNA. However, SLE cannot be entirely excluded if anti-dsDNA
antibodies are not detected [45].
Nucleosomes
(Functional subunits of chromosomes in the cell nucleus consisting of histones and dsDNA)
Antigen Disease Prevalence
Nucleosomes Systemic lupus erythematosus (SLE) 40 % - 70 %
Antibodies against nucleosomes have been found in the serum of patients with systemic lupus
erythematosus [45, 46]. Until recently, their relevance as a characteristic marker for SLE was limited
since up to 70% of sera from systemic sclerosis patients reacted with conventionally prepared
nucleosomes. Antibodies against nucleosomes detected using the new, highly purified nucleosome
preparation by EUROIMMUN as antigen have a specificity of almost 100% for SLE: with this test no
reactions have been found with sera from blood donors or systemic sclerosis, Sjögren’s syndrome or
polymyositis patients [50, 51, 52, 53, 54].
Histones
(Nuclear proteins, types H1, H2A, H2B, H3, H4, which form nucleosomes together with dsDNA,
functional subunits of chromosomes in the cell nucleus)
Antigen Disease Prevalence
Drug-induced lupus erythematosus 95 % - 100 %
Histones Systemic lupus erythematosus (SLE) 50 %
Rheumatoid arthritis 15 % - 50 %
Autoantibodies can form against all five histone types. Most frequent are antibodies against H1 and H2B
[5, 9, 55]. They are a constant find in drug-induced (procainamide, hydralazine, isoniazide and other)
lupus erythematosus (95%). Around 50%-75% of patients treated with procainamide and 25%-30% of
those treated with hydralazine develop anti-nuclear antibodies without symptoms of SLE during long-
term therapy. A third of these patients demonstrate antibodies against histones and after varied duration
of therapy show clinical signs of drug-induced lupus erythematosus: polyarthralgia, pleuritis, pericarditis.
The anti-nuclear antibodies persist for years after the drugs have been discontinued and the symptoms
have abated [20, 21, 56]. Antibodies against histones also occur in around 50% of patients with non-
drug-induced lupus erythematosus and in 5% - 50% of patients with rheumatoid arthritis [55].
Ribosomal P-proteins
(3 proteins of the 60S ribosomal subunit, referred to as P0 with a molecular weight of 38 kDa, P1 with a
molecular weight of 19 kDa and P2 with a molecular weight of 17 kDa; the main immuoreactive epitope
is localised at the carboxy terminal, and in all 3 proteins consists of an identical sequence of 17 amino
acids)
Antigen Disease Prevalence
Ribosomal P- Systemic lupus erythematosus (SLE) 10 %
proteins
Antibodies against ribosomal P-proteins (ARPA) are specific for SLE [7, 12, 21]. In a multicentre study
performed by EUROIMMUN serum samples from 360 SLE patients, 79 patients suffering from other
collagenoses (systemic sclerosis, Sjögren’s syndrome, dermatomyositis/polymyositis, Sharp syndrome)
and 206 healthy blood donors were investigated for ARPA. ARPA were detected in 34 (9.4%) of the 360
SLE patients and 3 (12.5%) of the 24 patients with Sharp syndrome. In 2 of these 3 patients antibodies

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against dsDNA were detected, indicating an overlap with SLE. ARPA were not found in any of the
patients with systemic sclerosis, Sjögren’s syndrome or dermatomyositis/polymyositis or in any of the
healthy blood donors. In SLE the titer level of antibodies against ribosomal P-proteins did not correlate
with the disease activity. The prevalence of ARPA was identical in SLE patients with or without CNS
involvement, nephritis or hepatitis. ARPA may be detected more frequently in patients with other
phenomena accompanying SLE, e.g. psychosis, although this is not statistically significant [57].
AMA-M2
(E2 enzyme and protein X of the pyruvate dehydrogenase complex are the main anti-mitochondrial M2
antigens)
Antigen Disease Prevalence
AMA-M2 Primary biliary liver cirrhosis >90 %
High titres of antibodies against M2 (AMA-M2) are characteristic of primary biliary liver cirrhosis (PBC),
whereby the E2 enzyme and protein X of the pyruvate dehydrogenase complex are the preferred
antigens. PBC is an immune-mediated chronic inflammatory cholestatic liver disease of unknown
aetiology. The disease is characterised by female predominance (>90 %) with most cases
observed between the ages of 40 and 60. PBC incidence in different parts of the world is estimated
to be 4 to 31 cases/million per year [10, 23, 39, 43, 58, 59].

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