AESKULISA ANA HEp-2

REF 30-7115US
.
 Instruction manual

Contents

1. Intended Use................................................................................ 2

2. Clinical Applications and Principle of the Assay........................... 2

3. Kit Contents.................................................................................. 3

4. Storage and Shelf Life.................................................................. 3

5. Precautions of Use....................................................................... 4

6. Sample Collection, Handling and Storage.................................... 4

7. Assay Procedure.......................................................................... 5

8. Semiquantitative Interpretation.......................................................6

9. Technical Data.............................................................................. 6

10. Performance Data........................................................................ 7-8

11. Literature...................................................................................... 8

A : Pipetting scheme......................................................................... 18

B : Test Procedure.............................................................................. 19

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1. Intended Use

AESKULISA ANA-HEp2 is a solid phase enzyme immunoassay for the combined qualitative detection
of IgG antibodies against HEp2 cells in human serum. Each well is coated with lysed HEp2 cells and
specific antigens. The test collectively detects, in one well, total ANAs against double stranded DNA
(dsDNA), histones, SS-A (Ro), SS-B (La), Sm, snRNP/Sm, Scl-70, Jo-1 and centromeric antigens
along with sera positive for HEp2 immunofluorescence test (IFT).
The assay is a tool in the diagnosis of certain systemic rheumatic diseases and should be used in
conjunction with other serological tests and clinical findings.

2. Clinical Application and Principle of the Assay

Anti-nuclear antibodies (ANA) directed against a variety of nuclear and cytoplasmic antigens occur
in high frequency in systemic rheumatic diseases and thus are an important tool for the differential
diagnosis. For instance, SS-A (Ro) and SS-B (La) antibodies are associated with SLE and Sjögren’s
syndrome (SS), anti-dsDNA and anti-Sm antibodies with SLE, anti-histone antibodies with SLE and
drug-induced lupus, anti-RNP antibodies with mixed connective tissue disease (MCTD) and SLE, anti-
Scl-70 antibodies with scleroderma (progressive systemic sclerosis [PSS]), anti-Jo-1 antibodies with
polymyositis and dermatomyositis and anti-centromere antibodies with CREST syndrome.
Indirect immunofluorescence test (IFT) on eucaryotic cells like HeLa and HEp2 has been the
established method for the detection of ANAs. Although the IFT is a sensitive test, it is laborious when
testing large numbers of patient samples and is subject to errors from human interpretation and from
variability in fluorescent microscope. The ELISA test system is an excellent alternative to the IFT for
screening patient`s serum for the presence of ANAs of clinical significance. Single antibody specificities
have to be determined by more specific testing using ELISAs employing the specific target antigens for
a simple and reliable differentiation of ANAs.

Principle of the test

Serum samples diluted 1:101 are incubated in the microplates coated with the
specific antigen. Patient‘s antibodies, if present in the specimen, bind to the
antigen. The unbound fraction is washed off in the following step. Afterwards
anti-human immunoglobulins conjugated to horseradish peroxidase (conju-
gate) are incubated and react with the antigen-antibody complex of the samp-
les in the microplates. Unbound conjugate is washed off in the following step.
Addition of TMB-substrate generates an enzymatic colorimetric (blue) reaction,
which is stopped by diluted acid (color changes to yellow). The rate of color
formation from the chromogen is a function of the amount of conjugate bound
to the antigen-antibody complex and this is proportional to the initial concen-
tration of the respective antibodies in the patient sample.

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3. Kit Contents

To be reconstituted:

5x Sample Buffer 1 vial, 20 ml - 5x concentrated (capped white: yellow solution)

Containing: Tris, NaCl, BSA, sodium azide < 0.1% (preservative)

50x Wash Buffer 1 vial, 20 ml - 50x concentrated (capped white: green solution)
Containing: Tris, NaCl, Tween, sodium azide < 0.1% (preservative)
Ready to use:
Negative Control 1 vial, 1.5 ml (capped green: clear solution)
Containing: Human serum (diluted), sodium azide < 0,1% (preservative)

Positive Control 1 vial, 1.5 ml (capped red: yellow solution)

Containing: Human serum (diluted), sodium azide < 0,1% (preservative)

Cut-off Control 1 vial, 1.5 ml (capped blue: yellow solution)

Containing: Human serum (diluted), sodium Azide < 0,1% (preservative)

Conjugate 1 vial,15 ml IgG (capped blue: blue solution)

Containing: goat polyclonal anti-human immunoglobulins conjugated to horseradish peroxidase

TMB Substrate 1 vial, 15 ml (capped black)

Containing: Stabilized TMB/H2O2

Stop Solution 1 vial, 15 ml (capped white: colorless solution)

Containing: 1M Hydrochloric Acid

Microtiterplate 12x8 well strips with breakaway microwells

Coating see paragraph 1

Material required but not provided:

Microtiter plate reader 450 nm reading filter and optional 620 nm reference filter (600-690 nm). Glass
ware, test tubes for dilutions. Vortex mixer, precision pipettes (10, 100, 200, 500, 1000 µl) or multipipette.
Microplate washing device (multichannel pipette or automated system), adsorbent paper.
Our tests are designed to be used with purified water according to the definition of the United States
Pharmacopeia (USP 26 - NF 21) and the European Pharmacopeia (Eur.Ph. 4th ed.).

4. Storage and Shelf Life

Store all reagents and the microplate at 2-8°C/35-46°F, in their original containers. Once prepared,
reconstituted solutions are stable for 1 month at 4°C/39°F, at least. Reagents and the microplate
shall be used within the expiry date indicated on each component, only. Avoid intense exposure
of TMB solution to light. Store microplates in designated foil, including the desiccant, and seal
tightly.

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5. Precautions of Use

5.1 Health hazard data

THIS PRODUCT IS FOR IN VITRO DIAGNOSTIC USE ONLY. Thus, only staff trained and specially advised in
methods of in vitro diagnostics may perform the kit. Although this product is not considered particularly
toxic or dangerous in conditions of normal use, refer to the following for maximum safety :

Recommendations and precautions

This kit contains potentially hazardous components. Though kit reagents are not classified being irritant
to eyes and skin we recommend to avoid contact with eyes and skin and wear disposable gloves.
WARNING! Calibrators, Controls and Buffers contain sodium azide (NaN3) as a preservative . NaN3
may be toxic if ingested or adsorbed by skin or eyes. NaN3 may react with lead and copper plumbing
to form highly explosive metal azides. On disposal, flush with a large volume of water to prevent azide
build-up. Please refer to decontamination procedures as outlined by CDC or other local/national gui-
delines.
Do not smoke, eat or drink when manipulating the kit.
Do not pipette by mouth.
All human source material used for some reagents of this kit (controls, standards e.g.) has been tested
by approved methods and found negative for HbsAg, Hepatitis C and HIV 1. However, no test can
guarantee the absence of viral agents in such material completely. Thus handle kit controls, standards
and patient samples as if capable of transmitting infectious diseases and according to national require-
ments.

5.2 General directions for use

Do not mix or substitute reagents or microplates from different lot numbers. This may lead to variations
in the results.
Allow all components to reach room temperature (20-26°C/64-78.8°F) before use, mix well and follow
the recommended incubation scheme for an optimum performance of the test.
Never expose components to higher temperature than 37°C/ 98,6 °F.
Always pipette substrate solution with brand new tips only. Protect this reagent from light. Never pipette
conjugate with tips used with other reagents prior.

A definite clinical diagnosis should not be based on the results of the performed test only, but
should be made by the physician after all clinical and laboratory findings have been evaluated.
The diagnosis is to be verified using different diagnostic and medicinal methods if the patient
has got infectious diseases accompanied by medication.

6. Sample Collection, Handling and Storage

Use preferentially freshly collected serum samples. Blood withdrawal must follow national require-
ments.

Do not use icteric, lipemic, hemolysed or bacterially contaminated samples. Sera with particles should
be cleared by low speed centrifugation (<1000 x g). Blood samples should be collected in clean, dry
and empty tubes. After separation, the serum samples should be used immediately, respectively stored
tightly closed at 2-8°C/35-46°F up to three days, or frozen at -20°C/-4°F for longer periods.

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7. Assay Procedure

7.1 Preparations prior to pipetting

Dilute concentrated reagents:
Dilute the concentrated sample buffer 1:5 with distilled water (e.g. 20 ml plus 80 ml).
Dilute the concentrated wash buffer 1:50 with distilled water (e.g. 20 ml plus 980 ml).
Samples
Dilute serum samples 1:101 with sample buffer (1x)
e.g. 1000 µl sample buffer (1x) + 10 µl serum. Mix well !
Washing
Prepare 20 ml of diluted wash buffer (1x) per 8 wells or 200 ml for 96 wells
e.g. 4 ml concentrate plus 196 ml distilled water.
Automated washing:
Consider excess volumes required for setting up the instrument and dead volume of robot
pipette.
Manual washing:
Discard liquid from wells by inverting the plate. Knock the microwell frame with wells downside
vigorously on clean adsorbent paper. Pipette 300 µl of diluted wash buffer into each well, wait
for 20 seconds. Repeat the whole procedure twice again.
Microplates
Calculate the number of wells required for the test. Remove unused wells from the frame,
replace and store in the provided plastic bag, together with desiccant, seal tightly (2-8°C/35-46°F).

7.2 Work flow

For pipetting scheme see Annex A, for the test procedure see Annex B

● Pipette 100 µl of each patient‘s diluted serum into the designated microwells.
● Pipette 100 µl calibrators OR cut-off control and negative and positive controls into
the designated wells.
● Incubate for 30 minutes at room temperature (20-26°C/64-78.8°F).
● Wash 3x with 300 µl washing buffer (diluted 1:50).
● Pipette 100 µl conjugate into each well.
● Incubate for 30 minutes at room temperature (20-26°C/64-78.8°F).
● Wash 3x with 300 µl washing buffer (diluted 1:50).
● Pipette 100 µl TMB substrate into each well.
● Incubate for 30 minutes at room temperature (20-26°C/64-78.8°F), in the dark.
● Pipette 100 µl stop solution into each well, using the same order as pipetting the
substrate.
● Incubate 5 minutes minimum.
● Agitate plate carefully for 5 sec.
● Read absorbance at 450 nm (optionally 450/620 nm) within 30 minutes.

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8. Semiquantitative Interpretation

Read the optical density of the cut-off control and the patient samples. Compare patient ODs with the
OD of the cut-off control. All samples which are higher than cut-off are considered positive. A positive
result may indicate the presence of one or more autoantibodies. Confirmatory testing for individual
autoantibody should be performed.
Negative: OD Patient < OD cut-off
Positive: OD Patient > OD cut-off

Calibrators O.D. 450/620 nm CV % (Variation)

Negative Control 0.081 2.6
Cut-off Control 0.52 1.8
Positive Control 1.259 0.7

Example of interpretation
We recommend pipetting cut-off control in parallel for each run.

Cut-off control Patient sample Interpretation

0.52 OD 0.25 OD Negative
0.52 OD 0.52 OD Borderline
0.52 OD 0.67 OD Positive
0.52 OD 1.75 OD Positive

Do not use this example for interpreting patients results!

We recommend to retest samples, that are borderline. For lot specific data, see enclosed quality
control leaflet. Medical laboratories might perform an in-house Quality Control by using own controls
and/or internal pooled sera, as foreseen by EU regulations.

For semi-quantification of the results, each patient-OD value can be expressed by the Index-Value.
The Index-Value is calculated by dividing the patient-OD by the cut-off OD:

OD (patient sample)
Index Value =
OD (cut-off control)

Negative: Index Value < 1.0

Positive: Index Value > 1.0

9. Technical Data

Sample material: serum

Sample volume: 10 µl of sample diluted 1:101 with 1x sample buffer
Total incubation time: 90 minutes at room temperature (20-26°C/64-78.8°F)
Storage: at 2-8°C/35-46°F use original vials, only
Number of determinations: 96 tests

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10. Performance Data

10.1 Specificity and sensitivity

The microplate is coated with lysed HEp2 cells. No crossreactivities to other autoantigens have been
found (tTG, PR3, TPO, TG, Gliadin). ANA are not specific for SLE but are found in a variety of rheumatic
diseases. Detection of ANA is a very sensitive marker for an active SLE and is positive in >99% of all
cases.
57 characterized sera of patients suffering from various autoimmune (AI) disases (SLE, MCTD, CREST
and Sjögrens syndrome; see table below) obtained from major hospitals which were positive on IFA
HEp-2 ANA (≥1:160) were tested on a predicate device and the AESKULISA ANA HEp-2. 2 sera which
were negative in IFA were also found to be negative in the AESKULISA ANA HEp-2. There was 100%
agreement with the predicate device.
disease # of tested sera pred. device
SLE 39 + - Total
AESKULISA + 57 0 57
MCTD 3
- 0 2 2
CREST 4
Total 57 2
Sjoegrens S. 4
various AI diseases 7
A controlgroup (n=80) were all found negative on the AESKULISA ANA-HEp-2.

10.2 Linearity
Chosen sera have been tested with this kit and found to dilute linearly. However, due to the hetero-
geneous nature of human autoantibodies there might be samples that do not follow this rule.

Sample Dilution measured expected Recovery

No. Factor concentration concentration (%)
(OD-Ratio) (OD-Ratio)

1 1 / 100 4.10 4.200 97.6

1 / 200 2.10 2.100 100.0
1 / 400 1.00 1.050 95.2
1 / 800 0.55 0.530 103.8

2 1 / 100 6.10 6.200 98.4

1 / 200 3.00 3.100 96.8
1 / 400 1.59 1.550 102.6
1 / 800 0.79 0.775 102.0

10.3 Precision
To determine the precision of the assay, the variability (intra and inter-assay) was assessed by
examining its reproducibility on three serum samples selected to represent a range over the standard
curve.
Intra-Assay Inter-Assay

Sample Mean CV Sample Mean CV

No. (OD-Ratio) (%) No. (OD-Ratio) (%)

1 4.6 1.5 1 4.7 3.1

2 2.8 2.0 2 3.0 2.5
3 1.4 1.8 3 1.2 2.4

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10.4 Calibration
The AESKULISA ANA-HEp2-S is calibrated against reference sera from the CDC (Centers for Disease
Control and Prevention) Atlanta.

11. Literature

1. Peter JB, Shoenfeld Y (1996).

Autoantibodies. Elsevier
Sciences B.V., Amsterdam.

2. Froelich CH, Wallmann H, Skosey JL and Teodorescu M (1990).

detection of 6 autoantibodies.
Clinical value of an integrated ELISA system for the detecti
The Journal of Rheumatology 17 (2): 192-200.

3. Mierau R, Genth E (1998).

Autoantikörper bei systemischem Lupus erythematodes und verwandten Erkrankungen
In: Thomas L. (Hrsg.) Labor und Diagnose
TH-Books, Frankfurt, 15. Auflage: 843-851.

4. Schmolke M, Oppermann M, Helmke K, Guder WG (2000).

Antibody determination against ENA- a challenge for the routine laboratory
Poster P59, 5 th Dresden Symposium on Autoantibodies.

5. Tan EM, (1989).

probes for cell
Antinuclear antibodies: diagnostic markers for autoimmune diseases and pr
biology.
Adv. Immunol 44: 93-151.

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 Annex A:

Pipetting scheme

We suggest pipetting calibrators, controls and samples as follows:

For qualitative interpretation use cut-off control

1 2 3 4 5 6 7 8 9 10 11 12
A NC P2
B NC P2
C CC P3
D CC P3
E PC ...
F PC ...
G P1 ...
H P1 ...

CC: Cut-off control

PC: positive control
NC: negative control
P1: patient 1
P2: patient 2
P3: patient 3

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Annex B:

Test Procedure

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Assay/Test: ____________________ Incubation / Inkub. : 1._________min Date/ Datum:________

Temperature/Temperatur:__________ °F _________ °C 2._________min

Signature/Unterschrift:.____________
Name:____________________________ 3._________min

1 2 3 4 5 6 7 8 9 10 11 12

Negative
A Control
0 U/ ml
Negative
B Control
0 U/ ml
Cut off
C Control
15 U/ ml
Cut off
D Control
15 U/ ml
Positive
E Control
100 U/ ml
Positive
F Control
100 U/ ml

AESKU.Diagnostics GmbH 55234 Wendelsheim - Mikroforum Ring 2, Germany Phone: + 49-6734-96270, Fax: + 49-6734-962727
ANA-Hep 2
♦ Diagnosi in vitro ♦ For in vitro diagnostic use
♦ Pour diagnostic in vitro ♦ Para uso diagnóstico in vitro
♦ Ιn Vitro Diagnostikum ♦ In Vitro Διαγνωστικό μέσο
♦ Para uso Diagnóstico in vitro
♦ Numero d’ordine ♦ Cataloge number
♦ Référence Catalogue ♦ Numéro de catálogo
♦ Bestellnummer ♦ Αριθμός παραγγελίας
♦ Número de catálogo
♦ Descrizione lotto ♦ Lot
♦ Lot ♦ Lote
♦ Chargen Bezeichnung ♦ Χαρακτηρισμός παρτίδας
♦ Lote
♦ Conformità europea ♦ EC Declaration of Conformity
♦ Déclaration CE de Conformité ♦ Declaración CE de Conformidad
♦ Europäische Konformität ♦ Ευρωπαϊκή συμφωνία
♦ Déclaracão CE de Conformidade
♦ 96 determinazioni ♦ 96 tests
♦ 96 tests ♦ 96 pruebas
♦ 96 Bestimmungen ♦ 96 προσδιορισμοί
♦ 96 Testes
♦ Rispettare le istruzioni per l’uso ♦ See instructions for use
♦ Voir les instructions d‘utilisation ♦ Ver las instrucciones de uso
♦ Gebrauchsanweisung beachten ♦ Λάβετε υπόψη τις οδηγίες χρήσης
♦ Ver as instrucões de uso
♦ Da utilizzarsi entro ♦ Use by
♦ Utilise avant le ♦ Utilizar antes de
♦ Verwendbar bis ♦ Χρήση μέχρι
♦ Utilizar antes de
♦ Conservare a 2-8°C ♦ Store at 2-8°C (35-46°F)
♦ Conserver à 2-8°C ♦ Conservar a 2-8°C
♦ Lagerung bei 2-8°C ♦ Φυλάσσεται στους 2-8°C
♦ Conservar entre 2-8°C
♦ Prodotto da ♦ Manufactured by
♦ Fabriqué par ♦ Fabricado por
♦ Hergestellt von ♦ Κατασκευάζεται από
♦ Fabricado por
♦ Calibratore cut-off ♦ Cut off Control
♦ Etalon Seuil ♦ Calibrador de cut-off
♦ Grenzwert Kalibrator ♦ Οριακός ορός Αντιδραστήριο βαθμονόμησης
♦ Calibrador de cut-off
♦ Controllo positivo ♦ Positive Control
♦ Contrôle Positif ♦ Control Positivo
♦ Positiv Kontrolle ♦ Θετικός ορός ελέγχου
♦ Controlo positivo
♦ Controllo negativo ♦ Negative Control
♦ Contrôle Négatif ♦ Control Negativo
♦ Negativ Kontrolle ♦ Αρνητικός ορός ελέγχου
♦ Controlo negativo
♦ Calibratore ♦ Calibrator
♦ Etalon ♦ Calibrador
♦ Kalibrator ♦ Αντιδραστήριο βαθμονόμησης
♦ Calibrador
♦ Recupero ♦ Recovery
♦ Corrélation ♦ Recuperado
♦ Wiederfindung ♦ Ανάκτηση
♦ Recuperacão
♦ Coniugato ♦ Conjugate
♦ Conjugé ♦ Conjugado
♦ Konjugat ♦ Σύζευγμα
♦ Conjugado
♦ Micropiastra rivestita ♦ Coated microtiter plate
♦ Microplaque sensibilisée ♦ Microplaca sensibilizada
♦ Beschichtete Mikrotiterplatte ♦ Επικαλυμμένη μικροπλάκα
♦ Microplaca revestida
♦ Piastra ad aghi rivestita ♦ Coated pinplate
♦ Pinplate sensibilisée ♦ Pinplate sensibilizada
♦ Beschichtete Pinplatte ♦ Επικαλυμμένη πλάκα Pin
♦ Pinplate revestida
♦ Tampone di lavaggio ♦ Wash buffer
♦ Tampon de Lavage ♦ Solución de lavado
♦ Waschpuffer ♦ Ρυθμιστικό διάλυμα πλύσης
♦ Solucão de lavagem
♦ Tampone substrato ♦ Substrate buffer
♦ Substrat ♦ Tampón sustrato
♦ Substratpuffer ♦ Ρυθμιστικό διάλυμα υποστρώματος
♦ Substrato
♦ Reagente bloccante ♦ Stop solution
♦ Solution d‘Arrêt ♦ Solución de parada
♦ Stopreagenz ♦ Αντιδραστήριο διακοπής αντίδρασης
♦ Solucão de paragem
♦ Tampone campione ♦ Sample buffer
♦ Tampon Echantillons ♦ Tampón Muestras
♦ Probenpuffer ♦ Ρυθμιστικό διάλυμα δειγμάτων
♦ Diluente de amostra
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