dsdna-ab RIA Radio immunoassay for the quantitative determination of antibodies against ds DNA in human serum.

Transcription

1 Instructions for Use dsdna-ab RIA Radio immunoassay for the quantitative determination of antibodies against ds DNA in human serum. RE C I B L I N T E R N A T I O N A L G M B H Flughafenstrasse 52a Phone: +49 (0) IBL@IBL-International.com D Hamburg, Germany Fax: +49 (0)

2 1. INTENDED USE Radio immunoassay for the quantitative determination of antibodies against double-stranded DNA (dsdna) in human serum. 2. SUMMARY AND EXPLANATION The presence of antibodies to double-stranded DNA (dsdna, native DNA) in serum is highly characteristic for patients with SLE. It is important to quantitate the amount of antibody to dsdna in order to differentiate between SLE and closely related autoimmune diseases. Antibodies to dsdna show a good correlation to the disease activity. Serial determinations of the amount of antibodies to dsdna in the serum of individual patients treated for SLE is therefore of important clinical use for the evaluation of therapy and to plan further therapeutic measurements. Antibodies to dsdna normally do not appear in patient sera with drug-induced SLE. The measurement of dsdna antibodies in serum is helpful to decide whether the patient has a lupoid-drug reaction or real SLE. 3. TEST PRINCIPLE The dsdna-ab RIA provides a simple, accurate and precise means of estimating antibodies to dsdna in serum by measuring the free dsdna binding capacity in the sample. 125 I labelled dsdna is added to the sample. After incubation, separation is achieved by the addition of ammonium sulphate solution. Tubes are centrifuged and supernatants carefully decanted. The tubes are then counted, the bound radioactivity being directly proportional to the dsdna binding capacity. 4. WARNINGS AND PRECAUTIONS 1. For in-vitro diagnostic use only. For professional use only. 2. Before starting the assay, read the instructions completely and carefully. Use the valid version of the package insert provided with the kit. Be sure that everything is understood. 3. In case of severe damage of the kit package please contact IBL or your supplier in written form, latest one week after receiving the kit. Do not use damaged components in test runs, but keep safe for complaint related issues. 4. Obey lot number and expiry date. Do not mix reagents of different lots. Do not use expired reagents. 5. Follow good laboratory practice and safety guidelines. Wear lab coats, disposable latex gloves and protective glasses where necessary. 6. Reagents of this kit containing hazardous material may cause eye and skin irritations. See MATERIALS SUPPLIED and labels for details. Material Safety Data Sheets for this product are available on the IBL- Homepage or upon request directly from IBL. 7. Chemicals and prepared or used reagents have to be treated as hazardous waste according to national biohazard and safety guidelines or regulations. 8. The cleaning staff should be guided by the professionals regarding potential hazards and handling. 9. This kit contains radioactive material, to be received, acquired, possessed and used by physicians, laboratories or hospitals only according to regulations and a specific license issued by the Nuclear Regulatory Commission or issued by a state with which the Nuclear Regulatory Commission has entered into an agreement for the exercise of regulatory authority. 10. Radioactive materials should be confined to specifically designated, regularly monitored areas in the laboratory, restricted to authorised personnel. Use disposable labware and disposable absorbent bench covers. Always wear film budges, lab coats and disposable gloves. Wipe up all spills immediately, cleaning the contaminated area with a decontaminant and dispose the contaminated materials as radioactive waste. 11. Some reagents contain thimerosal as preservative. In case of contact with eyes or skin, flush immediately with water. 12. All reagents of this kit containing human serum or plasma have been tested and were found negative for anti-hiv I/II, HBsAg and anti-hcv. However, a presence of these or other infectious agents cannot be excluded absolutely. For this reason reagents should be treated as potential biohazards in use and for disposal. Version / 5

3 5. STORAGE AND STABILITY The kit is shipped at ambient temperature and should be stored at 2-8 C. Keep away from heat or direct sunlight. The storage and stability of specimens and prepared reagents is stated in the corresponding chapters. 6. SPECIMEN COLLECTION AND STORAGE Serum The usual precautions for venipuncture should be observed. It is important to preserve the chemical integrity of a blood specimen from the moment it is collected until it is assayed. Do not use grossly hemolytic, icteric or grossly lipemic specimens. Samples appearing turbid should be centrifuged before testing to remove any particulate material. Storage: 2-8 C -20 C (Aliquots) Keep away from heat or direct sunlight. Stability: 48 h 6 months Avoid repeated freeze-thaw cycles. 7. MATERIALS SUPPLIED Quantity Symbol Component 2 x 7.5 ml TRACER 1 x 6 x 0.25 ml CAL A-F LYO 1 x 2 x 0.25 ml CONTROL 1+2 LYO 1 x 2.5 ml SAMPLEDIL LYO 2 x 50 ml AMMSULF dsdna 125 I-Tracer Ready to use. Activity: < 37 kbq (1 µci). Contains: 125 I-dsDNA, 0.01% Thimerosal. Standard A-F, lyophilized Contains: dsdna antibodies, Human serum, 0.01 % Thimerosal. Exact concentrations see vial labels or QC certificate. Control 1+2, lyophilized Contains: dsdna antibodies, Human serum. Concentrations / acceptable ranges see QC certificate. Sample Diluent, lyophilized Contains: Human serum, 0.01% Thimerosal. Ammonium Sulfate Ready to use. Contains: Ammonium Sulfate, 0.01% Thimerosal. 8. MATERIALS REQUIRED BUT NOT SUPPLIED 1. Micropipettes (Multipette Eppendorf or similar devices, < 3 % CV). Volume: 25; 150; 250; 1000 µl. 2. Round-bottom polystyrene test tubes (12 x 75 mm) 3. Rack to decant supernatants 4. Vortex mixer 5. Water bath, 37 C 6. Centrifuge (preferably refrigerated); 1500 x g 7. Gamma Counter 8. Bidistilled or deionised water 9. Paper towels, pipette tips and timer 9. PROCEDURE NOTES 1. Any improper handling of samples or modification of the test procedure may influence the results. The indicated pipetting volumes, incubation times, temperatures and pretreatment steps have to be performed strictly according to the instructions. Use calibrated pipettes and devices only. 2. Once the test has been started, all steps should be completed without interruption. Make sure that required reagents, materials and devices are prepared ready at the appropriate time. Allow all reagents and specimens to reach room temperature (18-25 C) and gently swirl each vial of liquid reagent and sample before use. Mix reagents without foaming. 3. Avoid contamination of reagents, pipettes and wells/tubes. Use new disposable plastic pipette tips for each component and specimen. Do not interchange caps. Always cap not used vials. Do not reuse wells/tubes or reagents. 4. It is advised to determine samples in duplicate to be able to identify potential pipetting errors. 5. Labelling of all tubes is recommended. 6. The relative centrifugal force (g) is not equivalent to rounds per minute (rpm) but it has to be calculated depending on the radius of the centrifuge. Version / 5

4 10. PRE-TEST SETUP INSTRUCTIONS Preparation of lyophilized or concentrated components Dilute / dissolve Component CAL A-F LYO 250 µl bidist. water Cap and mix gently to dissolve. CONTROL 1+2 LYO 250 µl bidist. water Let stand for 10 min. SAMPLEDIL LYO 2.5 ml bidist. water Mix without foaming. with Diluent Remarks Storage Stability -20 C (Aliquots) 9 weeks Dilution of Samples Samples suspected to contain concentrations higher than the highest standard have to be diluted with Sample Diluent. 11. TEST PROCEDURE The Ammonium Sulfate solution must be kept at 2-8 C until use. 1. Pipette 25 µl of each Standard, Control, sample and/or diluted sample into the respective tubes. Leave two tubes empty for Total Activity (T). 2. Pipette 150 µl of dsdna 125 I-Tracer into each tube. Vortex. 3. Incubate 60 min at 37 C in a waterbath. 4. Pipette 1000 µl of cold Ammonium Sulfate into each tube. (Except T.) Vortex. 5. Centrifuge all tubes for 15 min at 1500 x g. (Except T.) 6. Decant the tubes carefully. Drain 10 min with blotting paper. (Except T.) 7. Count the tubes in a Gamma counter for 1 min. (Including T.) 12. QUALITY CONTROL The test results are only valid if the test has been performed following the instructions. Moreover the user must strictly adhere to the rules of GLP (Good Laboratory Practice) or comparable standards/laws. User and/or laboratory must have a validated system to get diagnosis according to GLP. All kit controls must be found within the acceptable ranges as stated on the labels and the QC certificate. If the criteria are not met, the run is not valid and should be repeated. Each laboratory should use known samples as further controls. It is recommended to participate at appropriate quality assessment trials. In case of any deviation the following technical issues should be proven: Expiration dates of (prepared) reagents, storage conditions, pipettes, devices, incubation conditions and washing methods. 13. CALCULATION OF RESULTS Plot a curve on semi-log graph with the corrected counts of the standards on the y-axis (linear) against the corresponding concentration on the x-axis (logarithmic). The concentrations of the samples can be read directly from this standard curve. Alternatively the percentage value of the counts of the standards and samples may be compared to the counts of the highest standard. % B/B max = cpm(sample or standard)- cpm(zero cpm(highest standard) - cpm(zero standard) x 100 standard) Plot a curve of % B/Bmax of the standards for the standards (y-axis, linear) against the standardconcentrations (x-axis, logarithmic) on semi-log graph paper. A good fit is provided by automated methods like cubic spline, 4 Parameter Logisitcs or Logit-Log. For the calculation of the standard curve, apply each signal of the standards. In case of diluted samples the values have to be multiplied with the corresponding dilution factor. Samples showing concentrations above the highest standard have to be diluted as described in PRE-TEST SETUP INSTRUCTIONS and reassayed. Version / 5

5 Typical Calibration Curve (Example. Do not use for calculation!) dsdna (IU/mL) Mean cpm Corrected cpm T A B C D E F Standard B/Bmax (%) CPM IU / ml 14. EXPECTED VALUES The results themselves should not be the only reason for any therapeutical consequences. They have to be correlated to other clinical observations and diagnostic tests. Apparently healthy subjects show the following values: < 7 IU/mL (n = 590) It is recommended that each laboratory establishes its own range of normal values. n dsdna-ab RIA Antibody [IU/mL] 15. LIMITATIONS OF THE PROCEDURE Specimen collection and storage have a significant effect on the test results. See SPECIMEN COLLECTION AND STORAGE for details. For cross-reactivities, see PERFORMANCE. The following blood components do not have a significant effect (+/-20% of expected) on the test results up to the below stated concentrations: Hemoglobin Bilirubin Triglyceride 16. PERFORMANCE Analytical Specificity (Cross Reactivity) Analytical Sensitivity (Limit of Detection) 4.0 mg/ml 0.5 mg/ml 30 mg/ml No cross-reactivities were found with the following substances tested. Substance Source Conc. Rheumatoid arthritis serum WHO 100 IU/mL Anti-nuclear ribonucleoprotein (nrno) autoantibody WHO 1:2 Dilution IgM class anti-nuclear antibody WHO 10 mg/ml Glycoprotein C1q Calbiochem 500 µg/ml 2.5 IU/mL Mean signal (Zero-Standard) - 2SD Precision Sample No. Mean ± SD (IU/mL) CV (%) Intra-Assay (n = 20) Inter-Assay (n = 20) ± ± ± ± ± ± ± ± ± Version / 5

6 Linearity Recovery Method Comparison versus RIA Sample No Sample No Dilution Meas. Conc. (IU/mL) Expected Conc. (IU/mL) Recovery (%) 1: : : : : : : : : : : : : : Endogenous Added Rec. Expected Rec. dsdna (IU/mL) dsdna (IU/mL) dsdna (IU/mL) dsdna (IU/mL) (%) IBL-Assay = 1.14 x AMERLEX RIA (Ortho Clinical Diagnostics) r = 0.951, n = PRODUCT LITERATURE REFERENCES 1. Hamann D., Smeenk R.J.T. dsdna Autoantibodies. In Autoantibodies 2nd Edition Linnik M.D., Hu J.Z., Heilbrunn K. R., Strand V., Hurley F. L., Joh T., LJP 394 Investigator Consortium. Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus. Arthritis and rheumatism Apr, 52(4): Riboldi P., Gerosa M., Moroni G., Radice A., Allegri F., Sinico A., Tincani A., Meroni PL. Anti-DNA antibodies: a diagnostic and prognostic tool for systemic lupus erythematosus? Autoimmunity Feb, 38(1): Isenberg D. Anti-dsDNA antibodies: still a useful criterion for patients with systemic lupus erythematosus? Lupus 2004, 13: Wasmuth J.-C., Grün B., Terjung B., Homrighausen A., Spengler U. ROC Analysis Comparison of Three Assays for the Detection of Antibodies against Double-Stranded DNA in Serum for the Diagnosis of Systemic Lupus Erythematosus. Clinical Chemistry 2004, 50: Smeenk R.J.T. Detection of antibodies to dsdna: Current insights into its relevance. Clin Exp Rheumatol 2002; 20: Egner W. The use of laboratory tests in the diagnosis of SLE. J. Clin. Pathol. 2000; 53; Takeuchi Y., Ishikawa O., Miyachi Y. The comparative Study of Anti-Double Stranded DNA Antibody Levels Measured by Radioimmunoassay and Enzyme-Linked Immunosorbent Assay in Systemic Lupus Erythematosus. The Journal of Dermatology Vol. 24: Ter Borg E.J., Horst G., Hummel E.J., Limburg P.C., Kallenberg C.G.M. Measurement of increases in anti-double-stranded dna antibody levels as a predictor of disease exacerbation in systemic lupus erythematosus. Arthritis & Rheumatism May, 33 (5): Sutjita M., Hohmann A., Boey M.L., Bradley J. Microplate ELISA for Detction of Antibodies to DNA in Patients with Systemic Lupus Erythematosus: Specificity and Correlation with Farr Radioimmunoassay. Journal of Clinical Laboratory Analysis. 1989, 3: Version / 5

7 Symbols / Symbole / Symbôles / Símbolos / Símbolos / Σύμβολα REF LOT CONC LYO IVD Cat.-No.: / Kat.-Nr.: / No.- Cat.: / Cat.-No.: / N.º Cat.: / N. Cat.: / Αριθμός-Κατ.: Lot-No.: / Chargen-Bez.: / No. Lot: / Lot-No.: / Lote N.º: / Lotto n.: / Αριθμός -Παραγωγή: Use by: / Verwendbar bis: / Utiliser à: / Usado por: / Usar até: / Da utilizzare entro: / Χρησιμοποιείται από: No. of Tests: / Kitgröße: / Nb. de Tests: / No. de Determ.: / N.º de Testes: / Quantità dei tests: / Αριθμός εξετάσεων: Concentrate / Konzentrat / Concentré / Concentrar / Concentrado / Concentrato / Συμπύκνωμα Lyophilized / Lyophilisat / Lyophilisé / Liofilizado / Liofilizado / Liofilizzato / Λυοφιλιασμένο In Vitro Diagnostic Medical Device. / In-vitro-Diagnostikum. / Appareil Médical pour Diagnostics In Vitro. / Dispositivo Médico para Diagnóstico In Vitro. / Equipamento Médico de Diagnóstico In Vitro. / Dispositivo Medico Diagnostico In vitro. / Ιατρική συσκευή για In-Vitro ιάγνωση. Evaluation kit. / Nur für Leistungsbewertungszwecke. / Kit pour évaluation. / Juego de Reactivos para Evaluació. / Kit de avaliação. / Kit di evaluazione. / Κιτ Αξιολόγησης. Read instructions before use. / Arbeitsanleitung lesen. / Lire la fiche technique avant emploi. / Lea las instrucciones antes de usar. / Ler as instruções antes de usar. / Leggere le istruzioni prima dell uso. / ιαβάστε τις οδηγίες πριν την χρήση. Keep away from heat or direct sun light. / Vor Hitze und direkter Sonneneinstrahlung schützen. / Garder à l abri de la chaleur et de toute exposition lumineuse. / Manténgase alejado del calor o la luz solar directa. / Manter longe do calor ou luz solar directa. / Non esporre ai raggi solari. / Να φυλάσσεται μακριά από θερμότητα και άμεση επαφή με το φως του ηλίου. Store at: / Lagern bei: / Stocker à: / Almacene a: / Armazenar a: / Conservare a: / Αποθήκευση στους: Manufacturer: / Hersteller: / Fabricant: / Productor: / Fabricante: / Fabbricante: / Παραγωγός: Caution! / Vorsicht! / Attention! / Precaución! / Cuidado! / Attenzione! / Προσοχή! Symbols of the kit components see MATERIALS SUPPLIED. Die Symbole der Komponenten sind im Kapitel KOMPONENTEN DES KITS beschrieben. Voir MATERIEL FOURNI pour les symbôles des composants du kit. Símbolos de los componentes del juego de reactivos, vea MATERIALES SUMINISTRADOS. Para símbolos dos componentes do kit ver MATERIAIS FORNECIDOS. Per i simboli dei componenti del kit si veda COMPONENTI DEL KIT. Για τα σύμβολα των συστατικών του κιτ συμβουλευτείτε το ΠΑΡΕΧΟΜΕΝΑ ΥΛΙΚΑ. COMPLAINTS: Complaints may be submitted initially written or vocal. Subsequently they need to be filed including the test performance and results in writing in case of analytical reasons. WARRANTY: The product is warranted to be free from material defects within the specific shelf life and to comply with product specifications delivered with the product. The product must be used according to the Intended use, all instructions given in the instructions for use and within the product specific shelf life. Any modification of the test procedure or exchange or mixing of components of different lots could negatively affect the results. These cases invalidate any claim for replacement. LIMITATION OF LIABILITY: IN ALL CIRCUMSTANCES THE EXTENT OF MANUFACTURER S LIABILITY IS LIMITED TO THE PURCHASE PRICE OF THE KIT(S) IN QUESTION. IN NO EVENT SHALL MANUFACTURER BE LIABLE FOR ANY INCIDENTAL OR CONSEQUENTIAL DAMAGES, INCLUDING DAMAGES FOR LOST PROFITS, LOST SALES, INJURY TO PERSON OR PROPERTY OR ANY OTHER INCIDENTAL OR CONSEQUENTIAL LOSS. IBL International GmbH Flughafenstr. 52A, Hamburg, Germany Tel.: + 49 (0) Fax: IBL@IBL-International.com WEB: Symbols Version 4.5 /