EUROLINE ANA Profile 3 plus DFS70 (IgG)

Test instruction
ORDER NO. ANTIBODIES AGAINST IG CLASS SUBSTRATE FORMAT
nRNP/Sm, Sm, SS-A, Ro-52, SS-B,
DL 1590-1601-30 G Ag-coated 16 x 01 (16)
Scl-70, PM-Scl, Jo-1, CENP B, PCNA,
DL 1590-6401-30 G IgG immunoblot 64 x 01 (64)
dsDNA, nucleosomes, histones,
DL 1590-5001-30 G strips 50 x 01 (50)
rib. P-prot., AMA M2, DFS70
Indications: The EUROLINE test kit provides qualitative in vitro determination of human autoantibodies
of the IgG class to 16 different antigens nRNP, Sm, SS-A, Ro-52, SS-B, Scl-70, PM-Scl, Jo-1, CENP B,
PCNA, dsDNA, nucleosomes, histones, ribosomal P-protein, AMA M2 and DFS70 in serum or
plasma to support the diagnosis of Sharp syndrome (MCTD), systemic lupus erythematosus (SLE),
Sjögren’s syndrome, progressive systemic sclerosis, poly-/dermatomyositis, overlap syndromes, limited
form of progressive systemic sclerosis (CREST syndrome) and primary biliary liver cholangitis.
Application: The EUROLINE “ANA Profile 3 plus DFS70 (IgG)” for the detection of antibodies against
16 different nuclear, cytoplasmic and mitochondrial antigens offers a multiplex approach for the deter-
mination of these antibodies in a single reaction, with optimal, fully automated processing and objective
evaluation of the test results using the EUROLineScan software. Moreover, this test enables the
simultaneous investigation of disease-relevant autoantibodies and antibodies against DFS70 which can
explain unclear immunofluorescence findings.
Principles of the test: The test kit contains test strips coated with parallel lines of highly purified anti-
gens. 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.
The format DL 1590-5001-30 G belongs to the Immunoblot-PreQ system. The test strips are already
placed into the incubation trays (EUROTray).
Contents of the test kit:
Component Format Format Format Symbol
1. Test strips coated with the antigens
nRNP/Sm, Sm, SS-A (native), Ro-52, 5 x 10
4 x 16
SS-B, Scl-70, PM-Scl, Jo-1, CENP B, 16 strips strips in .STRIPS.
strips
PCNA, dsDNA, nucleosomes, histones, EUROTrays
rib. P-protein, AMA M2 and DFS70
2. Positive control
1 x 0.02 ml 4 x 0.02 ml 5 x 0.1 ml .POS CONTROL 100x.
(IgG, human), 100x concentrate
3. Enzyme conjugate
Alkaline phosphatase-labelled anti- 1 x 3 ml 4 x 3 ml --- .CONJUGATE 10x.
human IgG (goat), 10x concentrate
4. Enzyme conjugate
Alkaline phosphatase-labelled anti-human --- --- 4 x 30 ml CONJUGATE
IgG (goat), ready for use
5. Sample buffer, ready for use 1 x 100 ml 3 x 100 ml 2 x 100 ml .SAMPLE BUFFER.

6. Wash buffer, 10x concentrate 1 x 50 ml 1 x 100 ml 1 x 100 ml .WASH BUFFER 10x.

7. Substrate solution
Nitroblue tetrazolium chloride/5-Bromo-4-
1 x 30 ml 4 x 30 ml 4 x 30 ml .SUBSTRATE.
chloro-3-indolylphosphate (NBT/BCIP),
ready for use
2x8
8. Incubation tray --- ---
channels
9. Test instruction 1 booklet 1 booklet 1 booklet
.LOT. Lot description Storage temperature
.IVD. In vitro diagnostic medical device Unopened usable until

Modifications to the former version are marked in grey.

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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 9895-0130 Incubation tray with 30 channels
ZD 9898-0144 Incubation tray with 44 channels (black, for the EUROBlotOne and EUROBlotCamera
system)
If using Immunoblot-PreQ (DL 1590-5001-30 G), no additional incubation tray is needed.
For the creation of work protocols and the evaluation of incubated test strips using EUROLineScan
green paper and adhesive 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 a visual evaluation is to be performed in individual cases, the required evaluation protocol can be
ordered under:
ZD 1590-0101-30 G Visual evaluation protocol EUROLINE ANA Profile 3 plus DFS70 (IgG).
If using Immunoblot-PreQ (DL 1590-5001-30 G), the strips should stay in the EUROTray during
evaluation. For the evaluation we generally recommend using a EUROIMMUN camera system
connected to EUROLineScan software. Strips need to be dry before starting the evaluation.
Preparation and stability of the reagents
Note: This test kit may only be used by trained personnel. Test strips and incubation trays are intended
for single use . 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, 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 (+18°C to +25°C) to prevent condensation on the strips. After removal of
the strips/Immunoblot-PreQ 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 tip 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 tip and diluted 1:10 with sample buffer. For one test strip, dilute 0.15 ml enzyme
conjugate with 1.35 ml sample buffer. The ready for use diluted enzyme conjugate should be used at
the same working day.
- Enzyme conjugate: Ready for use
Note: Only for DL 1590-5001-30 G!
- 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 tip and
diluted 1:10 with distilled water. For one test strip, dilute 1 ml in 9 ml of distilled water. The ready for
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 .
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.
Warning: The controls of human origin have tested negative for HBsAg, anti-HCV, anti-HIV-1 and
anti-HIV-2. Nonetheless all materials should be treated as being a potential infection hazard and should
be handled with care. Some of the reagents contain sodium azide in a non-declarable con-centration.
Avoid skin contact.

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Preparation and stability of the patient samples

Samples: 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 using a clean
pipette tip. For example, add 15 µl of sample to 1.5 ml sample buffer and mix well by vortexing. Sample
pipettes are not suitable for mixing.

Incubation
If using Immunoblot-PreQ (DL 1590-5001-30 G), manual incubation is not possible. Please see below for
options of automated incubation.

Pretreat: Remove the required amount of test strips from the package and place them
each in an empty channel. (Make sure that the surface of the test strips is not
damaged!).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.
Use of Immunoblot-PreQ: Set up the required antigen profiles according to
the work protocol and insert into the incubation device.
Incubate for 5 minutes at room temperature (+18°C to +25°C) on a rocking
shaker. Afterwards aspirate off all the liquid.

Incubate: Fill each channel with 1.5 ml of the diluted serum samples using a clean
(1st step) pipette tip.
Incubate for 30 minutes at room temperature (+18°C to +25°C) 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

(2nd step) anti-human IgG) into each channel.
Incubate for 30 minutes at room temperature (+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.
Immunoblot-PreQ: The evaluation of the test strips is realised exclusively via
the EUROIMMUN camera systems.

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

For automated incubation with the EUROBlotOne select the program EURO 01/02.

For automated incubation of Immunoblot-PreQ with the EUROBlotOne see instruction manual
EUROBlotOne (YG_0153_A_UK_CXX).

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EUROLINE ANA Profile 3 plus DFS70 (IgG)

Incubation protocol

Pretreat
(Manual incubation)
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 working-strength 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 in-
cubated 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) and evaluated with EUROLineScan. Alternatively, imaging and evaluation is
possible directly from the incubation trays (EUROBlotCamera and EUROBlotOne). For general
information about the EUROLineScan program please refer to the EUROLineScan user manual
(YG_0006_A_UK_CXX, EUROIMMUN). The code for entering the test in the EUROLineScan is
Ana_DFS.
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 1590-0101-30 G.
If using Immunoblot-PreQ (DL 1590-5001-30 G), the strips should stay in the EUROTray during
evaluation. For the evaluation we generally recommend using a EUROIMMUN camera system
connected to EUROLineScan software. Strips need to be dry before starting the evaluation.
Note: Correct performance of the incubation is indicated by an intense staining of the 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: U1-nRNP purified by affinity chromatography from calf and nRNP/Sm
rabbit thymus.
Sm: Sm antigen purified by affinity chromatography from bovine spleen Sm
and thymus. The Sm antigen contains the core proteins of snRNP
particles. D protein is the main component of the Sm pre-paration. SS-A
SS-A: SS-A (60 kDa) antigen purified by affinity chromatography from Ro-52
bovine spleen and thymus.
SS-B
Ro-52: Recombinant Ro-52 (52 kDa). The corresponding human cDNA
has been expressed with the baculovirus system in insect cells.
SS-B: SS-B antigen purified by affinity chromatography from calf and Scl-70
rabbit thymus.
PM-Scl
Scl-70: Scl-70 (DNA topoisomerase I) antigen purified by affinity
chromatography from bovine and rabbit thymus. Jo-1
PM-Scl: Recombinant PM-Scl100. The corresponding human cDNA has
been expressed with the baculovirus system in insect cells.
CENP B
Jo-1: Jo-1 (histidyl-tRNA synthetase) antigen purified by affinity
chromatography from calf and rabbit thymus.
CENP B: Recombinant centromere protein B. The corresponding human PCNA
cDNA has been expressed with the baculovirus system in insect cells.
PCNA: Recombinant PCNA (36kDa). The corresponding human cDNA dsDNA
has been expressed with the baculovirus system in insect cells. Nucleosomes
dsDNA: Highly purified native, double-stranded DNA isolated from salmon Histones
testes.
Nucleosomes: Native nucleosomes purified from calf thymus. Rib. P-
Histones: A mixture of individually purified histone types isolated from calf
protein
thymus. AMA M2
Rib. P-protein: Ribosomal P-proteins purified by affinity chroma-tography.
DFS70
AMA M2: Purified M2 antigen (pyruvate dehydrogenase complex).
DFS70: Recombinant DFS70 (full length). The human cDNA was Control
expressed in mammalian cells.

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EUROIMMUN recommends interpreting results based on the signal intensity:

Signal intensity
Signal
EUROLineScan Result
Visual evaluation
Flatbed scanner
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 to
>50 +++ Strong positive
the control band

Results in the borderline range (+) should be evaluated as increased but negative. The table above
contains values for the evaluation using a flatbed scanner. The values for other instruments supported
by EUROLineScan can be found in the EUROLineScan program. To do so, mark the corresponding
assay in the test list (main menu “Help” → “Test”) and click on details and select the corresponding
instrument in “image source”.
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 safe-
guard 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 de-
tection of a wider range of cell nucleus antibodies, as not all cell nucleus antigens are presently available
in the EUROLINE.
For the medical diagnosis, the clinical symptoms of the patient and, if available, further findings should
always be taken into account alongside the serological result. A negative serological result does not
exclude the presence of a disease.

Test characteristics
Calibration: The reactivity of each antigen is standardised by the human reference sera CDC-ANA #1 to
#11 of the “Center for Disease Control and Prevention” (CDC, Atlanta, USA). The reactivity of the CDC
sera in the EUROIMMUN ANA Profile 3 plus DFS70 EUROLINE is summarised 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.
AMA-M2 neg. neg. neg. neg. neg. neg. neg. pos. neg. neg. neg.

The specificity of these sera was determined at the CDC 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.

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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, AMA M2 and DFS70. No cross reactions with other auto-
antibodies have been found.

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 98% in a panel of non-SLE
rheumatic diseases (Sjögren`s syndrome n = 14, systemic sclerosis 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,
systemic sclerosis 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 97.4% in a panel of non-SLE rheumatic diseases (systemic
sclerosis 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, systemic sclerosis and other rheumatic
diseases. As an example, the prevalence of autoantibodies against Ro-52 in sera from patients with
systemic sclerosis (n = 20) was determined to be 31.6%.

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 100% in a panel of non-SLE rheumatic diseases (systemic
sclerosis = 18, MCTD n = 22).

Scl-70: For the detection of autoantibodies against Scl-70 a sensitivity of 94% with reference to the
ELISA method was determined using 18 samples of patients with systemic sclerosis. 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 100% in a panel of non-SLE rheumatic
diseases (MCTD n = 22, Sjögren`s syndrome n = 14, systemic sclerosis 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 100% in a panel of non-SLE rheumatic diseases (systemic sclerosis n = 18,
MCTD n = 22, Sjögren`s syndrome n = 14).

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CENP B: In 19 of 20 sera of patients with systemic sclerosis, 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 100% in a panel of
non-SLE rheumatic diseases (MCTD n = 22, Sjögren`s syndrome n = 14, myositis n = 25).

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 100% in cyclin I-negative sera of patients with SLE (n = 83).

dsDNA: For the detection of autoantibodies against dsDNA a sensitivity of 93% 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, systemic sclerosis n = 18).

Nucleosomes: For the detection of autoantibodies against nucleosomes a sensitivity of 97% with
reference to the EUROIMMUN Anti-Nucleosomes ELISA (IgG) method was determined using
34 samples of patients with SLE. The clinical prevalence determined by the ELISA (CE-notified test,
coated with native mononucleosomes free from histone H1 and non-histone proteins, Patent
EP1476750B1/US7566545 (B2)) amounts to 53%. The specificity was 100% for healthy blood donors
(n = 50) and in a panel of non-SLE rheumatic diseases (Sjögren`s syndrome n = 14, systemic sclerosis
n = 18).

Histones: For the detection of autoantibodies against histones a sensitivity of 75% with reference to the
ELISA method was determined using 40 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, systemic sclerosis 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 46 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, systemic sclerosis 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 100% in a panel of other liver diseases
(autoimmune hepatitis n = 28, toxic liver damage n = 38, viral hepatitis B/C n = 69).

DFS70: The investigation of sera from 198 healthy blood donors showed a prevalence of 3.5% (n = 7)
for autoantibodies against DFS70. All positive samples showed a granular ANA pattern and a granular
colouring of the chromosomes (typical of anti-DFS70) in the indirect immunofluorescence test with
HEp-2 cells. The investigation of sera from 50 samples with positive, partly unclear ANA pattern showed,
with respect to the reference method ELISA, a sensitivity of 92.3% at a specificity of 91.7% for the
detection of autoantibodies against DFS70. With respect to the reference method Westernblot (whole
cell lysate from HEp-2 cells with specific detection of the DFS70 band), the investigation showed a
sensitivity of 100% at a specificity of 85.7% in the same samples (n = 49).

Reference range: The reference range was determined using a cohort of healthy blood donors (n = 50).
All blood donors were negative (exception see DFS70).

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Clinical significance
Antibodies against nuclear antigens (ANA) are directed against various cell nuclear components
(biochemical substances in the cell nucleus). These encompass nucleic acids, cell nuclear proteins and
ribonucleoproteins. The serological detection of autoantibodies against individual or several cell nuclear
autoantigens is an essential element in the diagnosis of autoimmune diseases, particularly rheumatic
diseases. The frequency (prevalence) of anti-nuclear antibodies in inflammatory rheumatic diseases is
between 20% and 100% (in rheumatoid arthritis between 20% and 40%). Therefore, differential ANA
diagnostics to detect autoantibodies against different nuclear antigens is indispensable for the
identification of individual rheumatic diseases. ANA analysis is also helpful in the diagnosis of other
autoimmune diseases, such as primary biliary cholangitis (PBC) or autoimmune hepatitis (AIH).
The ANA profiles offer innovative test combinations based on the lineblot technology (EUROLINE).
Positive test results provide important serodiagnostic information for the diagnosis of the rheumatic
diseases below, as well as further autoimmune diseases such as PBC.
1. Systemic lupus erythematosus (SLE)
SLE is a chronic inflammatory autoimmune disease which occurs in phases and mainly affects the
connective tissue and various organic systems. Worldwide, women are ten times more frequently
affected by collagenosis than men, whereby there are regional differences, e.g. 12.5 in 100,000
women in central Europe and up to 100 in 100,000 women in the US have SLE. The predilection age
is between 15 and 30 years. The clinical symptoms vary greatly and can include butterfly erythema,
discoid hyperkeratotic skin changes, purpura, arthralgia, myalgia, kidney insufficiency,
neuropsychiatric abnormalities, polyneuropathy, pericarditis, cardiomyopathy, pleuritis, lung fibrosis,
anaemia, hepatomegaly and splenomegaly. An SLE attack is often accompanied by fever.
In drug-induced lupus around 50 to 75% of patients treated with procainamide and 25 to 30% of those
treated with hydralazine develop ANA without symptoms of SLE during long-term therapy. A third of
these patients demonstrate autoantibodies against histones and after varied duration of therapy show
polyarthralgia, pleuritis and pericarditis. These ANA persist for years after the drugs have been
discontinued and the symptoms have abated.
2. Sharp syndrome (mixed connective tissue disease = MCTD)
Sharp syndrome is a multi-symptomatic and multiform MCTD combining symptoms of rheumatoid
arthritis (RA), SLE, systemic sclerosis (SSc) and polymyositis. It has not yet been clarified if it is an
independent disease.
3. Sjögren’s syndrome (primary Sjögren’s syndrome, SS)
SS is a chronic inflammatory autoimmune disease of the exocrine glands which can be found in one
to four million people in the US alone. Nine out of ten patients are women. The main clinical feature of
primary SS is ocular and oral dryness as a result of the destruction of lachrymal and salivary glands
by lymphocytic infiltration. The pancreatic glands, the mucous-secreting glands of the intestine,
bronchia or vagina and the sudoriferous glands may also be affected. Around 5% of SS patients
develop malignant lymphoma. In secondary SS the disease signs of primary SS occur as
accompanying symptoms of RA, SSc, SLE, polymyositis/dermatomyositis, PBC and AIH.
4. Systemic sclerosis (systemic scleroderma, SSc)
SSc is an autoimmune connective tissue disease, which affects the skin and the inner organs. It
affects around 2 to 50 in 100,000 persons worldwide (USA: 25 in 100,000), and is around three to four
times more common in women than in men.
Shortening of the lingual frenum and Raynaud’s syndrome are early symptoms of SSc. In the
following phase oedema of the hands and feet develop. The skin becomes stiff and in later stages
atrophic, waxy and thin. Finally, deformation of the hands occurs. The fingers become fixed in a bent
position (claw hand) and are highly tapered at the ends (Madonna fingers). Furthermore, the
characteristic masklike face with rigid mimic develops. Finally, callosity of the inner organs,
particularly of the digestive tract, lungs, heart and kidneys occurs. At present, lung involvement is the
most frequent cause of death from SSc. Manifest SSc is the collagenosis with the highest vital risk for
the patient. The 10-year survival rate is 55%.

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SSc is divided into limited and diffuse forms, depending on the cutaneous distribution. In the limited
form, skin involvement is limited to the distal extremities. In the diffuse form (also proximal systemic
sclerosis) the symptoms are diffusely distributed over the trunk, the proximal and distal extremities
and the face.
5. Myositis (poly-/dermatomyositis)
The autoimmunogenic myositides (idiopathic inflammatory myopathies) are systemic autoimmune
diseases with inflammation of the skeletal musculature, symmetric and proximal accentuated pain and
muscle weakness. They occur with an incidence of 0.1-1 per 100,000 per year, a prevalence of
1-6 per 100,000 and ratio of men to women of 1 to 2. They can be divided into polymyositis of adults
(around 30%), dermatomyositis of adults (around 30%), paraneoplastic polymyositis of the lungs,
ovaries, mammary glands, gastrointestinal tract and in myeloproliferative diseases (around 8%),
infantile myositis/dermatomyositis with accompanying vasculitis (around 7%), as well as myositides in
association with autoimmune diseases such as RA, lupus erythematosus, MCTD and rare forms such
as granulomatosis, eosinophile, focal and inclusion body myositis (around 20%). It should be noted
that dermato-/polymyositis is often of paraneoplastic origin, particularly in elderly patients.
Dermatomyositis symptoms can occur before the tumour is even diagnostically detectable.
Polymyositis (PM) is a systemic inflammatory disease of the skeletal muscles of unknown aetiology
with perivascular lymphocytic infiltration. When the skin is involved, the disease is known as
dermatomyositis (DM). Clinical symptoms of PM are recurring bouts of fever, muscle weakness,
arthralgia, possibly Raynaud’s syndrome, trouble with swallowing and involvement of the inner
organs. In DM, skin symptoms appear as purple-coloured exanthema on the eye lids, nose bridge and
cheeks, periorbital oedema, local erythema and scaly eczema dermatitis.
6. Rheumatoid arthritis (RA)
RA is both one of the most common autoimmune disorders and the most common chronic
inflammatory joint disease. The disease affects around 1% of the world population, whereby 75% of
patients are female. It is characterised by inflammation of the synovial membrane, which spreads
symmetrically from the small to large joints leading to the destruction of the joints in the late phase
accompanied by a systemic involvement of the soft tissue. Initial symptoms include painful swelling of
basic finger joints with morning stiffness in the joints. Reliable and earliest possible diagnosis is
indispensable to keep the disease under control with suitable therapy and to avoid irreversible joint
damage.
7. Primary biliary cholangitis (PBC)
PBC is a chronic non-suppurative destructive cholangitis with progressive inflammatory destruction of
the small biliary ducts and liver cirrhosis in the final stage. In 80 to 90% of cases the patients are
female, mainly between 20 and 60 years of age. In rare cases, the disease also affects children. In
Germany the prevalence is around 3 to 4 cases per 100,000 inhabitants. Demographic differences
(Caucasians, Africans, etc.) are minimal.
PBC can be subdivided into various stages using liver biopsy. In around 6% of cases there is an
increased risk of hepatocellular carcinoma. In the final stage of PBC (decompensated cirrhosis) only
liver transplantation will save the patient's life. In around 75% of cases the transplant patients recover
fully from PBC. Some patients, however, suffer a PBC relapse after transplantation, but only with a
very slow disease course.
In addition to the typical PBC histological characteristics, specific serodiagnostic parameters are
important for confirming suspected cases of PBC: 1. Biochemical markers of cholestasis, such as
increased levels of alkaline phosphatase (AP) and gamma-glutamyl transferase (γGT) in serum, 2.
Presence of PBC-specific autoantibodies, in particular autoantibodies against mitochondria (AMA)
which are directed against the component M2 (family of oxo-acid dehydrogenases), and 3. Additional
determination of ANA, in particular against nuclear granules (nuclear dots, sp100 and PML) and
against nuclear membrane (gp210), which are also pathognomonically relevant. Autoantibodies
against centromere proteins are found regularly in a proportion of patients with overlap syndrome with
SSc.

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Overview

Autoantibodies
Autoimmune disease Prevalence
against
nRNP/Sm MCTD 95%
Sm SLE 5% - 40%
SS-A SS or SLE 40% - 95% or 20% - 60%
Neonatal lupus erythematosus 95% - 100%
Ro-52 SS or SLE 70% - 90% or 40% - 60%
SSc or idiopathic inflammatory myopathy 20% or 20% - 40%
SS-B SS or SLE 40% - 95% or 10% - 20%
Neonatal lupus erythematosus 75%
Scl-70 SSc 25% - 75%
Diffuse or limited form of SSc 40% - 65% or 5% - 15%
PM-Scl SSc including overlap syndrome 10% - 20% or 5% - 20%
PM/SSc overlap syndrome 18%
SSc (anti-PM-Scl75 positive) 24% - 50%
SSc (anti-PM-Scl100 positive) 7%
Jo-1 Myositis (polymyositis/dermatomyositis) 25% - 35%
CENP A SSc - limited form or SSc - diffuse form 80% - 95% or 5% - 10%
CENP B SSc - limited form or SSc - diffuse form 80% - 95% or 8%
PBC 10% - 30%
PCNA SLE 3%
dsDNA SLE 40% - 90%
Nucleosomes SLE 40% - 70%
Histones Drug-induced SLE 95% - 100%
SLE or RA 50% or 15% - 50%
Ribosomal P- SLE 10%
protein
AMA M2: PBC or other chronic liver diseases up to 96% or 30%
SSc 7% - 25%
DFS70 Atopic dermatitis 4% - 10%
Rheumatic diseases 5% - 10%
Mi-2α DM approx. 20%
Mi-2β DM, associated with neoplasia approx. 10%
(e.g. colon or breast carcinoma)
Ku SLE/myositis/SSc up to 10% / 40% / 5%
RP11 SSc 5%
RP155 SSc 7%
Sp100 PBC 21%
PML PBC 13%
gp210 PBC 26%

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Literature references
1. Alba P, Bento L, Cuadrado MJ, Karim Y, Tungekar MF, Abbs I, Khamashta MA, D'Cruz D, Hughes
GR. Anti-dsDNA, anti-Sm antibodies, and the lupus anticoagulant: significant factors
associated with lupus nephritis. Ann Rheum Dis 62 (2003) 556-560.
2. Bogdanos DP, Komorowski* L. (*EUROIMMUN AG). Disease-specific autoantibodies in primary
biliary cirrhosis. Clin Chim Acta 412 (2011) 502-512.
3. Brouwer R, Hengstman GJ, Vree Egberts W, Ehrfeld H, Bozic B, Ghirardello A, Grondal G, Hietarinta
M, Isenberg D, Kalden JR, Lundberg I, Moutsopoulos H, Roux-Lombard P, Vencovsky J, Wikman A,
Seelig HP, van Engelen BG, van Venrooij WJ. Autoantibody profiles in the sera of European
patients with myositis. Ann Rheum Dis 60 (2001) 116-123.
4. Chan EKL, Daimoiseaux J, Carballo OG, Conrad K, de Melo Cruvinel W, Francescantonio PLC,
Fritzler MJ, Garcia-De La Torre I, Herold M, Mimori T, Satoh M, von Mühlen CA, Andrade LEC.
Report of the first international consensus on standardized nomenclature of antinuclear
antibody HEp-2 cell patterns 2014-2015. Front Immunol 6 (2015) 412.
5. Ganapathy V, Casiano CA. Autoimmunity to the nuclear autoantigen DFS70 (LEDGF): what
exactly are the autoantibodies trying to tell us? Arthritis Rheum 50 (2004) 684-688.
6. Ghirardello A, Bassi N, Palma L, Borella E, Domeneghetti M, Punzi L, Doria A. Autoantibodies in
polymyositis and dermatomyositis. Curr Rheumatol Rep 15 (2013) 335.
7. Hanke K, Brückner CS, Becker M, Meyer* W, Schlumberger* W, Riemekasten G. (*EUROIMMUN
AG). Anti-CENP-A and anti-CENP-B antibodies show high concordance and similar clinical
associations in patients with systemic sclerosis despite completely different underlying
protein sequences. In: Conrad K et al. (Hrsg.). From Pathogenesis to Therapy of Autoimmune
diseases: Autoantigens, Autoantibodies, Autoimmunity. Pabst Science Publishers (2009) 392-393.
8. Hanke K, Brückner CS, Dähnrich* C, Huscher D, Komorowski* L, Meyer* W, Janssen* A, Backhaus
M, Becker M, Kill A, Egerer K, Burmester G, Hiepe F, Schlumberger* W, Riemekasten G.
(*EUROIMMUN AG). Antibodies against PM/Scl-75 and PM/Scl-100 are independent markers
for different subsets of systemic sclerosis patients. Arthritis Research & Therapy (2009) 11:R22.
9. Hanke K, Uibel S, Brückner CS, Dähnrich* C, Egerer K, Hiepe F, Schlumberger* W, Riemekasten G.
(*EUROIMMUN AG). Antibodies to CENP-B antigen identify a subgroup of systemic sclerosis
patients presenting more frequently sicca syndrome and less frequently lung fibrosis, cardiac
and vascular involvement – analysis of the Charité SSc cohort. In: Conrad K et al. (Hrsg.). From
Etiopathogenesis to the Prediction of Autoimmune Diseases: Relevance of Autoantibodies. Pabst
Science Publishers 5 (2007) 477-478.
10. Hartung K, Seelig HP. Laboratory diagnostics of systemic autoimmune diseases. Part 1.
Collagenoses. [Article in German] Z Rheumatol 65 (2006) 709-724.
11. Haugbro K, Nossent JC, Winkler T, Figenschau Y, Rekvig OP. Anti-dsDNA antibodies and disease
classification in antinuclear antibody positive patients: the role of analytical diversity. Ann
Rheum Dis 63 (2004) 386-394.
12. Li J, Shen Y, He J, Jia R, Wang X, Chen X, Wang D, Han L, Zhu L, Chi X, Saschenbrecker* S,
Dähnrich* C, Stöcker* W, Schlumberger* W, Li ZG. (*EUROIMMUN AG). Significance of
antibodies against the native ribosomal P protein complex and recombinant P0, P1, and P2
proteins in the diagnosis of Chinese patients with systemic lupus erythematosus. J Clin Lab
Anal 27 (2013) 87-95.
13. Mahler M, Parker T, Peebles CL, Andrade LE, Swart A, Carbone Y, Ferguson DJ, Villalta D, Bizzaro
N, Hanly JG, Fritzler MJ. Anti-DFS70/LEDGF antibodies are more prevalent in healthy
individuals compared to patients with systemic autoimmune rheumatic diseases. J Rheumatol
39 (2012) 2104-2110.
14. EUROIMMUN AG. Meyer W, Scheper T, Janssen A, Torkler S, Schlumberger W, Stöcker W.
EUROLINE Myositis Profile: A newly developed line immunoassay for the detection of
myositis specific antibodies. In: Conrad K et al. (Hrsg.). From Etiopathogenesis to the Prediction of
Autoimmune Diseases: Relevance of Autoantibodies. Pabst Science Publishers 5 (2007) 612-613.

12
 Medizinische
EUROIMMUN Labordiagnostika
AG

15. EUROIMMUN AG. Meyer W, Scheper T, Janssen A et al. A comprehensive line immunoassay for
the detection of autoantibodies in primary biliary cirrhosis. In: Conrad K, Chan EK, Fritzler MJ,
Sack U, Shoenfeld Y, Wiik A, editors. From Etiopathogenesis to the Prediction of Autoimmune
Diseases: Relevance of Autoantibodies. Report on the 8th Dresden Symposium on Autoantibodies,
5 ed. Dresden: Pabst Science Publishers (2007) 323-325.
16. EUROIMMUN AG. Schlumberger W, Dähnrich C, Frahm S, Siegemund M, Meyer W, Suer W,
Stöcker W. Diagnostic relevance of autoantibodies against nucleosomes. Autoimmunity
Reviews 1 (2002) 32.
17. EUROIMMUN AG. Stöcker W, Schlumberger W, Krüger C. Alle Beiträge zum Thema
Autoimmundiagnostik und Labordiagnostik der Infektionskrankheiten. In: Gressner A, Arndt T (Hrsg.)
Lexikon der Medizinischen Laboratoriumsdiagnostik. 2. Auflage. Springer Medizin Verlag,
Heidelberg (2012).
18. EUROIMMUN AG. Suer W, Dähnrich C, Schlumberger W, Stöcker W. Autoantibodies in SLE but
not in scleroderma react with protein-stripped nucleosomes. J Autoimmun 22 (2004) 325-334.
19. van Boekel MA, Vossenaar ER, van den Hoogen FH, van Venrooij WJ. Autoantibody systems in
rheumatoid arthritis: specificity, sensitivity and diagnostic value. Arthritis Res 4 (2002) 87-93.
20. Varga J. Systemic sclerosis: an update. Bull NYU Hosp Jt Dis 66 (2008) 198-202.

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Version: 20/03/2018