User Manual s

ANA Detect ELISA

Enzyme Immunoassay for the qualitative screening for the presence of antinuclear antibodies (ANAs) in human serum or plasma

EIA-4117 96

Telefon: +49 (0)6421-1700 0, Fax: +49-(0)6421-1700 50 Internet: www.drg-diagnostics.de E-Mail: drg@drg-diagnostics.de

DRG Instruments GmbH, Germany Frauenbergstr. 18, D-35039 Marburg

DRG International, Inc. USA

Telephone: (908) 233-2079 Fax: (908) 233-0758 E-Mail: corp@drg-international.com

ANA Detect ELISA EIA-4117 Version 2.0 Effective, October 2010

(e-2009-08_03) (it- 0803-02)

Please use only the valid version of the package insert provided with the kit. Verwenden Sie nur die jeweils gltige, im Testkit enthaltene, Arbeitsanleitung. Si prega di usare la versione valida dell'inserto del pacco a disposizione con il kit. Por favor, se usa solo la version valida de la metodico tcnico incluido aqui en el kit.

Table of Contents / Inhaltsverzeichnis / Tabella die Contenuti / Tabla de Contenidos 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 INTENDED USE ....................................................................................................................................... 2 SUMMARY AND EXPLANATION OF THE TEST .................................................................................... 2 PRINCIPLE OF THE TEST....................................................................................................................... 2 WARNINGS AND PRECAUTIONS........................................................................................................... 3 CONTENTS OF THE KIT ......................................................................................................................... 3 STORAGE AND STABILITY..................................................................................................................... 4 MATERIALS REQUIRED.......................................................................................................................... 4 SPECIMEN COLLECTION, STORAGE AND HANDLING ....................................................................... 4 PROCEDURAL NOTES............................................................................................................................ 4 PREPARATION OF REAGENTS ............................................................................................................. 5 TEST PROCEDURE................................................................................................................................. 5 INTERPRETATION OF RESULTS ........................................................................................................... 6 PERFORMANCE CHARACTERISTICS................................................................................................... 7 LIMITATIONS OF PROCEDURE ............................................................................................................. 8 INTERFERING SUBSTANCES ................................................................................................................ 8

1 2 3 4 5 6 7 8 9 10

CONTENUTO DEL KIT............................................................................................................................. 9 AVVERTENZEE PRECAUZZIONI............................................................................................................ 9 CONSERVAZIONE E STABILIT .......................................................................................................... 10 MATERIALE NECESSARIO ................................................................................................................... 10 RACCOLTA, CONSERVAZIONE, MANIPOLAZIONE DEI CAMPIONI ................................................. 10 AVVERTENZE OPERATIVE .................................................................................................................. 11 PREPARAZIONE DEI REAGENTI ......................................................................................................... 11 ESECUZIONE DEL TEST ...................................................................................................................... 12 INTERPRETAZIONE DEI RISULTATI.................................................................................................... 12 PRESTAZIONI DEL KIT ......................................................................................................................... 12

REFERENCES / LITERATURE....................................................................................................................... 13 SYMBOLS USED WITH DRG ASSAYS.......................................................................................................... 14

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1 INTENDED USE The ANA Detect assay is a qualitative enzyme immunoassay (EIA) intended to screen for the presence of antinuclear antibodies (ANAs) in human serum or plasma as an aid in the diagnosis of certain systemic rheumatic diseases. This assay collectively detects, in one well, ANAs against double stranded DNA (dsDNA, nDNA), histones, SS-A/Ro, SS-B/La, Sm, SmRNP, Scl-70, PM-Scl-100, Jo-1, and centromeric antigens.

2 SUMMARY AND EXPLANATION OF THE TEST Inflammatory connective tissue diseases are characterised by idiopathic genesis along with disturbances in terms of cellular and humoral immunity, systemic organ failure and a chronic course of disease. Additionally, connective tissue diseases exhibit overlapping symptomatic features that render an accurate diagnosis difficult [1]. Considering the diversity of mixed connective tissue diseases, such disorders exhibit a common serological characteristic; the presence of antinuclear antibodies [2]. These antibodies are directed against parts of the cell nucleus and the cytoplasm, and many rheumatic diseases are characterised by the presence of one or more of these ANAs [3]. Antibodies to double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), histone, nuclear ribonucleoprotein (RNP) and Smith antigen (Sm) are associated with SLE [4], while antibodies to Sjgrens syndrome A (SSA/Ro) and Sjgrens syndrome B (SS-B/La) can occur in both SLE and Sjgrens syndrome (SS) [5, 6]. Antibodies to Jo-1 may be observed in polymyositis and dermatomyositis [6], while antibodies to sclerodermaassociated antigen (Scl-70) and centromere can occur in patients with progressive systemic sclerosis (PSS). Anti-histone antibodies are associated with SLE and drug-induced lupus [7], while anti-RNP antibodies are linked with mixed connective tissue disease (MCTD) and with SLE [2]. Antibodies directed against centromere are associated with CREST syndrome [3]. Although IFA technology was traditionally used to detect autoantibodies in conjunction with HEp2 cells, it is now widely acknowledged that ELISA technology offers an excellent alternative. IFA technology is subject to errors of interpretation and can be labour-intensive when applied to a large number of unknown samples [8]. The ANA Detect ELISA assay allows for collective and simultaneous screening for the autoantibodies of major significance in one microwell, and effectively eliminates the need for individual interpretation that is inherent in IFA technology.

3 PRINCIPLE OF THE TEST Purified antigens (SS-A 52 (Ro 52), SS-A 60 (Ro 60), SS-B (La), RNP/Sm, RNP-70, RNP-A, RNP-C, SmBB, Sm-D, Sm-E, Sm-F, Sm-G, Scl-70, Jo-1, dsDNA, ssDNA, poly-nucleosomes, mononucleosomes, histone complex, histone H1, histone H2A, histone H2B, histone H3, histone H4, PM-Scl-100, centromere B) are bound to microwells. Antibodies against these antigens, if present in diluted serum or plasma, bind to the respective antigens. Washing of the microwells removes unspecific serum and plasma components. Horseradish peroxidase (HRP) conjugated anti-human IgG immunologically detects the bound patient antibodies forming a conjugate/antibody/antigen complex. Washing of the microwells removes unbound conjugate. An enzyme substrate in the presence of bound conjugate hydrolyses to form a blue colour. The addition of an acid stops the reaction forming a yellow end-product. The intensity of this yellow colour is measured photometrically at 450nm. The assay is calibrated against the internationally recognised reference sera from CDC, Atlanta, USA and furthermore against the WHO reference preparation for human anti-dsDNA Wo/80.

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4 WARNINGS AND PRECAUTIONS 1. All reagents of this kit are strictly intended for in vitro diagnostic use only. 2. Do not interchange kit components from different lots. 3. Components containing human serum were tested and found negative for HBsAg, HCV, HIV1 and HIV2 by FDA approved methods. No test can guarantee the absence of HBsAg, HCV, HIV1 or HIV2, and so all human serum based reagents in this kit must be handled as though capable of transmitting infection. 4. Avoid contact with the TMB (3,3,5,5-Tetramethyl-benzidine). If TMB comes into contact with skin, wash thoroughly with water and soap. 5. Avoid contact with the Stop Solution which is acid. If it comes into contact with skin, wash thoroughly with water and seek medical attention. 6. Some kit components (i.e. Controls, Sample buffer and Buffered Wash Solution) contain Sodium Azide as preservative. Sodium Azide (NaN3) is highly toxic and reactive in pure form. At the product concentrations (0.09%), though not hazardous. Despite the classification as nonhazardous, we strongly recommend using prudent laboratory practices (see 8., 9., 10.). 7. Some kit components contain Proclin 300 as preservative. When disposing reagents containing Proclin 300, flush drains with copious amounts of water to dilute the components below active levels. 8. Wear disposable gloves while handling specimens or kit reagents and wash hands thoroughly afterwards. 9. Do not pipette by mouth. 10. Do not eat, drink, smoke or apply makeup in areas where specimens or kit reagents are handled. 11. Avoid contact between the buffered Peroxide Solution and easily oxidized materials; extreme temperature may initiate spontaneous combustion. Observe the guidelines for performing quality control in medical laboratories by assaying controls and/or pooled sera. During handling of all kit reagents, controls and serum samples observe the existing legal regulations.

5 CONTENTS OF THE KIT Package size 96 determ. Qty.1 Divisible ANA microtiter strips: 96 antigen-coated wells sealed in a foil pouch with desiccant. Ready to use. Anti-ANA controls in a serum/buffer matrix (PBS, NaN3 <0.1% (w/w)). Negative control (NC, A), cut-off control (CC, B), positive control (PC, C). Ready to use Sample buffer (Tris, NaN3 <0.1% (w/w)), yellow, concentrate (5x) Enzyme conjugate solution (PBS, Proclin 300 <0.5% (v/v)), (light red) containing polyclonal rabbit anti-human IgG; labelled with horseradish peroxidase. Ready to use TMB substrate solution. Ready to use Stop solution (contains acid). Ready to use Wash solution (PBS, NaN3 <0.1% (w/w)), concentrate (50x)

3 vials, 1.5 ml each

1 vial, 20 ml 1 vial, 15 ml

1 vial, 15 ml 1 vial, 15 ml 1 vial, 20 ml

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6 STORAGE AND STABILITY 1. Store the kit at 2-8C 2. Keep microplate wells sealed in a dry bag with desiccants 3. The reagents are stable until expiration of the kit 4. Do not expose test reagents to heat, sun or strong light during storage and usage 5. Diluted sample buffer and wash buffer are stable for at least 30 days when stored at 2-8C 7 MATERIALS REQUIRED Equipment Microplate reader capable of endpoint measurements at 450 nm Multi-Channel Dispenser or repeatable pipet for 100 l Vortex mixer Pipets for 10 l, 100 l and 1000 l Laboratory timing device Data reduction software

Preparation of reagents Distilled or deionized water Graduated cylinder for 100 and 1000 ml Plastic container for storage of the wash solution 8 SPECIMEN COLLECTION, STORAGE AND HANDLING 1. Collect whole blood specimens using acceptable medical techniques to avoid hemolysis. 2. Allow blood to clot and separate the serum by centrifugation. 3. Test serum should be clear and non-hemolysed. Contamination by hemolysis or lipemia is best avoided, but does not interfere with this assay. 4. Specimens may be refrigerated at 2-8 C for up to five days or stored at -20 C up to six months. 5. Avoid repetitive freezing and thawing of serum samples. This may result in variable loss of autoantibody activity. 6. Testing of heat-inactivated sera is not recommended. 9 PROCEDURAL NOTES 1. Do not use kit components beyond their expiration dates. 2. Do not interchange kit components from different lots. 3. All materials must be at room temperature (20-28 C). 4. Have all reagents and samples ready before start of the assay. Once started, the test must be performed without interruption to get the most reliable and consistent results. 5. Perform the assay steps only in the order indicated. 6. Always use fresh sample dilutions. 7. Pipette all reagents and samples into the bottom of the wells. 8. To avoid carryover contamination, change the tip between samples and different kit controls. 9. It is important to wash microwells thoroughly and remove the last droplets of wash buffer to achieve best results. 10. All incubation steps must be accurately timed. 11. Control sera or pools should routinely be assayed as unknowns to check performance of the reagents and the assay. 12. Do not re-use microplate wells. For all controls, the respective concentrations are provided on the labels of each vial. Using these concentrations a calibration curve may be calculated to read off the patient results semi-quantitatively.

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10 PREPARATION OF REAGENTS 10.1 Preparation of sample buffer Dilute the contents of each vial of the sample buffer concentrate (5x) with distilled or deionized water to a final volume of 100 ml prior to use. Store refrigerated: stable at 2-8C for at least 30 days after preparation or until the expiration date printed on the label. 10.2 Preparation of wash solution Dilute the contents of each vial of the buffered wash solution concentrate (50x) with distilled or deionized water to a final volume of 1000 ml prior to use. Store refrigerated: stable at 2-8C for at least 30 days after preparation or until the expiration date printed on the label. 10.3 Sample preparation Dilute all patient samples 1:100 with sample buffer before assay. Therefore combine 10 l of sample with 990 l of sample buffer in a polystyrene tube. Mix well. Controls are ready to use and need not be diluted.

11 TEST PROCEDURE 1. Prepare a sufficient number of microplate modules to accommodate controls and prediluted patient samples. 2. Pipet 100 l of controls and prediluted patient samples in duplicate into the wells. 1 A B C D E F G H CC CC NC NC PC PC P1 P1 2 P2 P2 P.. P.. P1, P2... patient samples 1, 2 ... CC: NC: PC calibrator control negative control positive control 3 4 5 6

3. Incubate for 30 minutes at room temperature (20-28C) 4. Discard the contents of the microwells and wash 3 times with 300 l of wash solution. 5. Dispense 100 l of enzyme conjugate into each well 6. Incubate for 15 minutes at room temperature 7. Discard the contents of the microwells and wash 3 times with 300 l of wash solution 8. Dispense 100 l of TMB substrate solution into each well 9. Incubate for 15 minutes at room temperature 10. Add 100 l of stop solution to each well of the modules and incubate for 5 minutes at room temperature 11. Read the optical density at 450 nm and calculate the results. Bi-chromatic measurement with a reference at 600-690 nm is recommended. The developed colour is stable for at least 30 minutes. Read optical densities during this time.

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12 INTERPRETATION OF RESULTS 12.1 Quality Control This test is only valid if the optical density at 450 nm for negative control (NC, A), cut-off control (CC, B) and positive control (PC, C) complies with the respective range indicated on the Quality Control Certificate enclosed to each test kit! If any of these criteria is not fulfilled, the results are invalid and the test should be repeated. The assays is calibrated against the internationally recognised reference sera from CDC, Atlanta, USA and furthermore against the WHO reference preparation for human anti-dsDNA Wo/80. 12.2 Calculation of results For detailed semi-quantitative results, each patient-OD value can be expressed as an "Index Value". The Index Value is calculated by dividing the sample-OD by the cut-off-OD: Index Value = ODSample / ODCut-Off The calculation of Index Values is not influenced by variations of the sample-OD and/or cut-off-OD. Index Values are recommended for long term validations (i.e. internal quality control samples). 12.3 Interpretation of results 1. Evaluation of the ANA Detect ELISA test is easily carried out by direct comparison of the optical density of each patient sample with the optical density of the cut-off control (B). Patient samples exhibiting optical densities higher than the optical density of the cut-off control are considered to be positive. Negative: OD patient < OD Cut-off Elevated: OD patient > OD Cut-off 2. Index Values are interpreted as follows: ANA Detect ELISA: (Index-value) Negative: < 1.0 Borderline: 1.0 -1.2 Positive: 1.2 Example: The table shows typical results for an ANA Detect ELISA assay. These data are intended for illustration only and should not be used to calculate results from a laboratory assay. Sample OD 1 2 3 4 0.107 0.435 1.294 2.496 OD Cut-Off 0.435 0.435 0.435 0.435 Index Value 0.25 1.00 2.97 5.74 Interpretation negative borderline positive positive

12.4 Expected Values The approximate incidence of positive ANA is 5% in the general normal population, 40% in normal old age and 25% in healthy relatives of SLE patients. ANA positivity has been reported in: SLE (systemic Lupus erythematosus) > 95% SS (Sjgrens syndrome) 50-65% PSS (progressive systemic sclerosis) 40-60% RA (rheumatoid arthritis) 12-24% Juvenile RA (juvenile rheumatoid arthritis) 20%

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13 PERFORMANCE CHARACTERISTICS 13.1 Parallelism Three dilutions of three patient samples were assayed using two kit batches. The following table shows the mean values and the dilution-corrected recovery Sample No. 1 Dilution 1:100 1:200 1:400 2 1:100 1:200 1:400 3 1:100 1:200 1:400 Index Value 4.8 2.2 1.3 2.8 1.5 0.8 3.5 1.7 0.8 Dilution corrected recovery [%] 100 92 108 100 107 114 100 97 91

13.2 Precision (Reproducibility) Statistics for coefficients of variation (CV) were calculated for each of four samples from the results of 32 determinations in a single run for Intra-Assay precision. Run-to-run precision was calculated from the results of 3 different runs with 24 determinations of each sample: Intra-Assay Sample No 1 2 3 4 Mean (Index Value) 1.8 2.4 2.8 3.1 CV [%] 6.9 9.1 10.4 7.4 Sample No 1 2 3 Inter-Assay Mean (Index Value) 1.6 3.7 4.1 CV [%] 13.7 10.4 11.2

13.3 Performance Comparison to Predicate Assay Performance of the ANA Detect assay was compared to a commercially available ELISA screen assay utilizing 94 previously characterized autoimmune positive samples and 148 "presumed normals" from a blood bank facility. Two of the presumed normals screened as borderline and were subsequently deleted from the data analysis. Results of the comparison study are summarized as follows: Predicate ANA Screen Pos ANA Detect EIA-4117 Pos Neg 88 7 95 Neg 6 139 145 94 146 240 Relative Sensitivity: 92.6 % Relative Specificity: 95.8 % Relative Agreement: 94.5 %

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13.4 Specificity Specificity can be defined as the ability of a test to give a negative result for "normal" sera. The specificity performance of the ANA Detect screen assay was established using 148 "presumed normal" sera obtained from a blood donor centre. One hundred forty five of the sera were normal in the ANA Detect assay; one screened as positive and two screened as borderline (the two borderline results were not included in data analysis) thus yielding 99.3% specificity. This data is not in conflict with published data suggesting that 1-4% of the apparently healthy, asymptomatic population may contain ANA in their serum.

14 LIMITATIONS OF PROCEDURE 1. The ANA Detect ELISA is a diagnostic aid. A definite clinical diagnosis should not be based on the results of a single test, but should be made by the physician after all clinical and laboratory findings have been evaluated. 2. Due to the potential for a cumulative effect of each of the coated antigens, sera with positive results in the ANA Detect ELISA test may be determined to be negative when confirmatory testing is performed. 3. Positive ANA may be found in apparently healthy people. 4. SLE patients undergoing steroid therapy may have negative test results. 5. Commonly prescribed drugs may induce ANA.

15 INTERFERING SUBSTANCES No interference has been observed with haemolytic (up to 1000 mg/dL), lipemic (up to 3 g/dL triglycerides) or bilirubin (up to 40 mg/dL) containing sera. Nor have any interfering effects been observed with the use of anticoagulants. However for practical reasons it is recommended that grossly hemolysed or lipemic samples should be avoided.

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Test immunometrico per la determinazione qualitativa degli autoanticorpi anti-nucleo (ANA) della classe IgG Per uso diagnostico in vitro esclusivo.

1 1

CONTENUTO DEL KIT Micropiastra a pozzetti separabili costituita da 12 strip da 8 pozzetti ciascuno,sensibilizzata con dsDNA,Istoni,SS-A/Ro,SS-B/La,RNP 70,Sm,RNP/Sm,Scl-70,PM-Scl-100,Jo-1 e centromero B. Pronta all'uso. ANA Controllo negativo (NC,A,controllo di Cut-Off (CC,B)) e controllo positivo (PC,C) in matrice serica tamponata (PBS,BSA,Sodio Azide<0,1% (p/p);le rispettive concentrazioni sono riportate nel foglietto illu-strativo. Pronti all'uso. Diluente campioni (TRIS, Sodio Azide <0,1% p/p), giallo, concentrato 5X Coniugato (PBS, Proclin 300 <0.5 % v/v), rosa, contenente anticorpi policlonali di coniglio anti-IgG umane coniugati con perossidasi di rafano. Reagente pronto all'uso TMB Substrato. Reagente pronto all'uso. Soluzione Stoppante (contiene acido). Reagente pronto all'uso. Tampone di lavaggio (PBS, Sodio Azide <0,1% p/p), concentrato 50X

3 flaconi da 1,5 mL cad.

1 flacone da 20 mL 1 flacone da 15 mL

1 flacone da 15 mL 1 flacone da 15 mL 1 flacone da 20 mL

2 AVVERTENZEE PRECAUZZIONI 1. Tutti i reagenti del kit si intendono per esclusivo uso diagnostico in vitro 2. Non scambiare reagenti del kit con altri aventi lotto diverso da quelli presenti nel kit stesso 3. Reagenti contenenti siero umano sono stati testati con esito negativo per la presenza di HBsAg e HIV, con kit approvati dalla FDA. 4. Evitare il contatto con TMB (3,3', 5-5'-Tetrametilbenzidina); in caso di contatto di TMB con la pelle, lavare accuratamente con acqua e sapone 5. Evitare il contatto con la Stop Solution, che contiene acido; in caso di contatto con la pelle, lavare accuratamente con acqua e richiedere l'intervento medico. 6. Determinati reagenti (controlli, tampone del campione, soluzione di lavaggio tamponata) contengono sodio azide come conservante. Sodio azide un composto altamente tossico e reattivo allo stato puro, tuttavia alle concentrazioni di utilizzo non pericoloso. Nonostante la classificazione di materiale non pericoloso, vengono raccomandate procedure di laboratorio prudenti (vedi punti 8., 9., 10.) 7. L'eliminazione dei reagenti contenenti Proclin 300 come conservante, deve avvenire con una abbondante lavaggio delle tubature idrauliche al fine di diluire il componente sotto il livello di attivit. 8. Indossare guanti monouso nella manipolazione di campioni umani e dei reagenti contenuti nel kit, e lavare quindi le mani abbondantemente con acqua. 9. Non pipettare con la bocca 10. Non mangiare, bere, fumare, usare cosmetici nelle aree dove i reagenti vengono usati 11. Evitare il contatto tra la soluzione tamponata di Acqua Ossigenata e materiali facilmente ossidabili; temperature elevate possono provocare combustione spontanea Osservare le line guida per l'esecuzione delle procedure di controllo qualit nei laboratori medici, utilizzando materiali di controllo e pool di sieri di controllo. Osservare tutte le disposizioni di legge vigenti nella manipolazione dei reagenti contenuti nel kit, controlli, e campioni da analizzare.

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3 CONSERVAZIONE E STABILIT 1. Conservare il kit a 4-8C 2. Mantenere la micropiastra sigillata in una busta, con essicante, a tenuta di umidit 3. I reagenti sono stabili fino alla scadenza riportata sul kit 4. Mantenere i reagenti al riparo da calore, sole, luce solare diretta durante la conservazione e l'uso 5. Il tampone diluente il campione e il tampone di lavaggio sono stabili almeno 30 gg, se conservati a 2-8C, dopo la loro preparazione.

4 MATERIALE NECESSARIO Strumentazione Lettore di micropiastre con possibilit di misurazione end point, a 450 nm Dispensatore multicanale o pipetta sequenziale da 100 l Pipette da 10, 100, 1000 l Agitatore di tipo vortex Orologio da laboratorio Software per l'elaborazione dei dati

Preparazione dei reagenti acqua distillata o deionizzata cilindri graduati da 100 ml e 1000 mL bottiglie di plastica per la conservazione della soluzione di lavaggio

5 RACCOLTA, CONSERVAZIONE, MANIPOLAZIONE DEI CAMPIONI 1. Il prelievo di sangue deve essere eseguito con le modalit necessarie per evitare l'emolisi del campione 2. Attendere che il campione sia coagulato e separare il siero per centrifugazione 3. Verificare che il siero sia non emolizzato. Sebbene emolisi e lipidi non interferiscono nella determinazione, opportuno l'uso di campioni non lipemici e non emolizzati. 4. I campioni possono essere conservati a 4-8C fino a 5 giorni, oppure a -20C fino a 6 mesi 5. Evitare di congelare e scongelare ripetutamente i campioni di siero. Ci pu causare una perdita di attivit auto anticorpale. 6. Non raccomandato testare sieri inattivati col calore.

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6 AVVERTENZE OPERATIVE 1. Non usare kit scaduti. 2. Non intercambiare componenti di kit con diversi numeri di lotto. 3. Tutti i materiali devono essere conservati a temperatura ambiente. 4. Preparare tutte le soluzioni di lavoro prima di iniziare il ciclo analitico; questo deve essere completato senza interruzioni al fine di ottenere risultati affidabili e coerenti. 5. Utilizzare la procedura analitica indicata. 6. Usare sempre campioni freschi. 7. Pipettare reagenti e campioni sul fondo del pozzetto. 8. Lavare accuratamente i pozzetti e rimuovere tutte le goccioline di tampone di lavaggio al fine di ottenere i risultati pi corretti. 9. Rispettare accuratamente i tempi di incubazione. 10. Cambiare i puntali dopo la dispensazione di ciascun campione e dei controlli, al fine di evitare fenomeni di trascinamento. 11. I sieri e i pool serici di controllo vanno trattati come campioni anonimi al fine di valutare le prestazioni dei reagenti. 12. Non riutilizzare piastre gi usate. Le concentrazioni dei controlli sono riportate sulle rispettive etichette; utilizzando queste concentrazioni possibile costruire una curva di taratura ed ottenere dei risultati semiquantitativi.

PREPARAZIONE DEI REAGENTI

7.1 Preparazione del Diluente campioni Prima dell'uso, diluire il contenuto di ciascun flacone di Diluente campioni concentrato 5X con acqua distillata o deionizzata fino a un volume finale di 100 ml. Conservare in frigorifero: la stabilit di almeno 30 gg a 2-8C dalla data di preparazione, o fino alla data di scadenza stampata in etichetta. 7.2 Preparazione del Tampone di Lavaggio Prima dell'uso, diluire il contenuto di ciascun flacone di tampone di lavaggio concentrato 50X con acqua distillata o demonizzata fino a un volume finale di 1000 ml. Conservare in frigorifero: la stabilit di almeno 30 gg a 2-8C dalla data di preparazione, o fino alla data di scadenza stampata in etichetta. 7.3 Preparazione dei campioni Diluire tutti i campioni 1:100 con il Diluente campioni. Dispensare 10 l di campione in 990 l Diluente campioni in una provetta di plastica. Miscelare bene. I controlli sono pronti per l'uso e non necessitano di diluizioni.

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8 1. 2. 3. 4. 5. 6. 7. 8. 9.

ESECUZIONE DEL TEST Prelevare il numero di strip necessario per l'analisi in funzione del numero di campioni, controlli e calibratori Dispensare nei rispettivi pozzetti 100 l dei calibratori, controlli e campioni prediluiti. Incubare per 30' a temperatura ambiente (20-28C). Svuotare i pozzetti e lavare 3 volte con 300 l di soluzione di lavaggio. Dispensare 100 l di coniugato in ciascun pozzetto. Incubare per 15' a T.A. Svuotare i pozzetti e lavare 3 volte con 300 l di soluzione di lavaggio. Dispensare 100 l di TMB in ciascun pozzetto. Incubare per 15' a T.A.

10. Dispensare 100 l di Soluzione Stoppante a ciascun pozzetto. 11. Leggere la Densit Ottica a 450 nm e calcolare il risultato. E' raccomandata una lettura in bicromatismo con una lunghezza d'onda di riferimento a 600-690 nm. Il colore stabile per almeno 30'. Leggere la Densit Ottica in questo periodo di tempo.

INTERPRETAZIONE DEI RISULTATI

9.1 Controllo di Qualit Il test valido solo se la D.O.a 450 nm per il controllo positivo (PC,C) e controllo negativo(NC,A),e il controllo di Cut-off ( CC,B)cadono nei limiti indicati nel Certificato di Controllo di Qualit allegato a ciascun kit. Se tutti questi criteri non sono riscontrati,i risultati devono esse-re considerati non validi e il test deve essere ripetuto. 9.2 Calcolo dei risultati Per ottenere dettagliate risposte semiquantitative,il valore di D.O. di ciascun paziente puessere espresso come "Index Value"; l'Index Value calcolato dividendo la D.O. di ciascun cam-pione per la D.O. del controllo Cut-Off. Il calcolo di Index Value non influenzato da variazioni della D.O. del campione e del control-lo di Cut-Off. Index Value particolarmente utile per validazioni del reagente a lungo termine (p.e. per i controlli di qualit interni). 9.3 Interpretazione dei risultati 1. La interpretazione del test pu essere eseguita per diretta correlazione tra la D.O. di cias-cun paziente e la D.O. del controllo di cut-off. Campioni di pazienti con D.O maggiore della D.O del controllo di Cut-off sono considerati positivi. Negativo: campione con D.O. < della D.O. del controllo di cut-off Positivo: campione con D.O. > della D.O. del controllo di cut-off. 2. Index Values sono interpretati come segue: Index Value di ANA ELISA: Negativo: < 1.0 Borderline: 1.0-1.2 Positivo: > 1.2

10 PRESTAZIONI DEL KIT Parallelismo: 86-100% Precisione: Intra-assay: 6.9 - 10.4 %; Inter-assay: 10.4 - 13.7 %

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REFERENCES / LITERATURE 1. Hiepe F., Burmester G. R. Klinik und Diagnostik des systematischen Lupus erythematodes. Dtsch. med. Wschr.;Vol. 121; 1129-1133; 1996. 2. Nakamura R. M., Tan E. M. Update on autoantibodies to intracellular antigens in systemic rheumatic diseases. Clin. Lab. Med.; Vol. 12; 1-23; 1992. 3. Barland P., Lipstein E.. Selection and use of laboratory test in the rheumatic diseases. Am. J. Med.; Vol.100 (suppl 2A); 16s-23s. 4. Isenberg D. A., Ravirajan C. T., Rahman A., Kalsi J. The role of antibodies to DNA in systemic lupus erythematosus A review and introduction to an international workshop on DNA antibodies held in London, May 1996. Lupus; 6; 290-304; 1997. 5. Alexander E., Buyon J. P., Provost T. T., Guarnieri T. Anti-Ro/SS-A antibodies in the pathophysiology of congenital heart block in neonatal lupus syndrome, an experimental model. Arthritis and Rheumatism; Vol. 35 No.2; 176-189; 1992. 6. Hietarinta M., Lassila O. Clinical significance of antinuclear antibodies in systemic rheumatic disease. Ann. Med.; Vol. 28; 283-291; 1996. 7. Fritzler M. J. Clinical relevance of autoantibodies in systemic rheumatic diseases. Mol. Biol. Rep.; Vol.23; 133-145; 1996. 8. Feltkamp T. E. W. Antinuclear antibody determination in a routine laboratory. Ann. Rheum. Dis.; Vol. 55; 723-727; 1996. 9. Amoura Z., Koutouzov S., Chabre H., Cacoub P., Amoura I., Musset L., Bach J.-F., Piette J.-C., Presence of anti-nucleosome autoantibodies in a restricted set of connective tissue diseases. Arthritis and Rheumatism; Vol. 43 (1); 76-84; 2000. 10. Lundberg U. N., Hedfors E., Pettersson I. Recombinant 70-kD protein for determination of autoantigenic epitopes by anti-RNP sera. Clin. Exp. Immunol.; 81; 52-58; 1990.

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SYMBOLS USED WITH DRG ASSAYS Symbol

English Consult instructions for use European Conformity In vitro diagnostic device Deutsch Gebrauchsanweisung beachten CE-Konfirmittskennzeichnung In-vitro-Diagnostikum Nur fr Forschungszwecke Katalog-Nr. Chargen-Nr. Ausreichend fr n Anstze Lagerungstemperatur Mindesthaltbarkeitsdatum Hersteller Vertreiber Inhalt Volumen/Anzahl Franais Consulter les instructions dutilisation Conformit aux normes europennes Usage Diagnostic in vitro Seulement dans le cadre de recherches Numro de catalogue Numro de lot Contenu suffisant pour n tests Temprature de conservation Date limite dutilisation Fabricant Distributeur Conditionnement Volume/Quantit Espaol Consulte las instrucciones de uso Conformidad europea Para uso Diagnstico in vitro Slo para uso en investigacin Nmero de catlogo Nmero de lote Contenido suficiente para <n> ensayos Temperatura de conservacin Fecha de caducidad Fabricante Distribuidor Contenido Volumen/Nmero Italiano Consultare le istruzioni per luso Conformit europea Per uso Diagnostica in vitro Solo a scopo di ricerca Numero di Catalogo Numero di lotto Contenuto sufficiente per n saggi Temperatura di conservazione Data di scadenza Fabbricante Distributore Contenuto Volume/Quantit

RUO

For research use only Catalogue number Lot. No. / Batch code Contains sufficient for <n> tests/ Storage Temperature Expiration Date Legal Manufacturer

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