Treponema pallidum Screen ELISA

Transcription

1 Instructions for Use Treponema pallidum Screen ELISA Enzyme immunoassay for the qualitative detection of Treponema pallidum specific total antibody in human serum or plasma. RE58821 RE x8 10x12x8 2-8 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 Enzyme immunoassay for the qualitative detection of Treponema pallidum specific total antibody in human serum or plasma. 2. SUMMARY AND EXPLANATION Syphilis (synonym: lues), a chronic disease occurring only in humans, is caused by infection with the pathogen Treponema pallidum (T. pallidum). It is transmitted predominantly by sexual intercourse, but there is also a risk of transmission to the fetus for maternal infection during pregnancy and a risk of transmission via blood transfusion. Within a few weeks after infection Syphilis antibodies can be detected. Serological diagnosis is done with two types of antibody tests: non-specific (anti-cardiolipin, anti-reagin) and specific (anti-treponemal). One of the major reasons for the continued use of non-specific tests (RPR, VDRL, CMT, etc.) is based on the observation that in response to appropriate (antibiotic) treatment, a significant decrease in titre usually takes place. This is taken as the criterion of adequate treatment (3,4). Specific tests are based on the use of selective treponemal antigens. Prior to the HIV era, treponemal specific tests were largely used to confirm positive results obtained by non-specific screen test. Although older treponemal specific tests (such as MHA-TP and FTA-Abs are generally considered reliable for past and recurrent infection (regardless of treatment) their specificity is limited due to the absence of in vitro culture techniques for T. pallidum (2). The use of recombinant treponemal antigens in Treponema pallidum Screen ELISA results in increased diagnostic sensitivity and specificity for routine diagnostic applications. The Treponema pallidum Screen assay is used for diagnosis of T. pallidum infections in health care institutions and for screening in blood banks. 3. TEST PRINCIPLE The T. pallidum antigen is a mix of recombinant proteins from immunodominant epitopes. These antigens are considered as diagnostically conclusive in clinical routine. The antigens are immobilised on microtiterstrip wells. T. pallidum specific antibodies present in the sample and controls that are added to the wells will bind to these immobilized antigens on the plate. All non-bound proteins are removed during the washing step. Next, T. pallidum antigens labelled to peroxidase (i.e. the conjugate) will bind to free binding sites from the T. pallidum antibodies already complexed to the antigens coated on the plate. The enzyme peroxidase in the conjugate will act as the indicator for the sandwiched T. pallidum human antibodies. A second washing step removes all unbound conjugate. TMB as the chromogenic substrate induces colour relative to the amount of antibody sandwiched. There is no antibody-class dependency. Consequently, all immunoglobulin classes will be detected, making the assay a real total antibody detecting system. Due to the sensitivity of the system, the vast majority of positive samples will result in an out of range signal. 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. Avoid contact with Stop solution. It may cause skin irritations and burns. 9. 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 and therefore reagents should be treated as potential biohazards in use and for disposal. V2012_01 1 / 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 sun light. The storage and stability of specimen and prepared reagents is stated in the corresponding chapters. The microtiter strips are stable up to the expiry date of the kit in the broken, but tightly closed bag when stored at 2 8 C. 6. SPECIMEN COLLECTION AND STORAGE Serum, Plasma 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 Stability: 2 d > 2 d Keep away from heat or direct sun light. Avoid repeated freeze-thaw cycles. 7. MATERIALS SUPPLIED RE58821 Quantity RE58829 Symbol 1 x 12 x 8 10 x 12 x 8 MTP 1 x 13 ml 2 x 55 ml ENZCONJ 1 x 1.0 ml 3 x 2.0 ml CONTROL + 1 x 1.0 ml 3 x 2.0 ml CONTROL - 1 x 1.8 ml 5 x 1.8 ml CONTROL CO 1 x 100 ml 5 x 100 ml WASHBUF CONC 1 x 12 ml 2 x 55 ml TMB SUBS 1 x 12 ml 2 x 55 ml TMB STOP Component Microtiter Plate Ready to use. Break apart strips. Coated with recombinant T. pallidum antigen. Enzyme Conjugate Ready to use. Pink colored. Conjugated to HRP. Contains: recombinant T. pallidum antigen. Positive Control Ready to use. Red colored. Contains: Human serum, stabilizers, preservatives, T. pallidum specific antibodies. Negative Control Ready to use. Yellow Colored. Contains: Human serum, stabilizers, preservatives. Cut-Off Control Ready to use. Green colored. Contains: Human serum, stabilizers, preservatives, T. pallidum specific antibodies. Wash Buffer, Concentrate (10x) Contains: phosphate buffer. TMB Substrate Solution Ready to use. Contains: TMB (tetramethylbenzidine). TMB Stop Solution Ready to use. Contains: 0.5 M H 2SO MATERIALS REQUIRED BUT NOT SUPPLIED 1. Micropipettes (Multipette Eppendorf or similar devices, < 3% CV). Volume: 10; 100; 200; 1000 µl. 2. Vortex mixer 3. Incubator, 37 C 4. 8-Channel Micropipettor with reagent reservoirs 5. Wash bottle, automated or semi-automated microtiter plate washing system 6. Microtiter plate reader capable of reading absorbance at 450 nm (reference wavelength nm) 7. Bidistilled or deionised water 8. Paper towels, pipette tips and timer V2012_01 2 / 5

4 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. Use a pipetting scheme to verify an appropriate plate layout. 6. Incubation time affects results. All wells should be handled in the same order and time sequences. It is recommended to use an 8-channel Micropipettor for pipetting of solutions in all wells. 7. Microplate washing is important. Improperly washed wells will give erroneous results. It is recommended to use a multichannel pipette or an automatic microplate washing system. Do not allow the wells to dry between incubations. Do not scratch coated wells during rinsing and aspiration. Rinse and fill all reagents with care. While rinsing, check that all wells are filled precisely with Wash Buffer, and that there are no residues in the wells. 8. Humidity affects the coated wells/tubes. Do not open the pouch until it reaches room temperature. Unused wells/tubes should be returned immediately to the resealed pouch including the desiccant. 10. PRE-TEST SETUP INSTRUCTIONS Preparation of Components The contents of the kit for 96 determinations can be divided into 3 separate runs. The volumes stated below are for one run with 4 strips (32 determinations). Dilute / dissolve 30 ml Component Diluent Relation Remarks Storage Stability WASHBUF CONC 270 ml bidist. water 1:10 Warm up at 37 C to dissolve crystals, if necessary. 2-8 C 4 w V2012_01 3 / 5

5 11. TEST PROCEDURE 1. Dispense 100 µl of the controls and samples into the respective wells of the Microtiter Plate. We recommend to pipette the positive (RED) and negative (YELLOW) in duplicates, the cut-off control (GREEN) in quadruplicates. 2. Cover plate with adhesive foil. Incubate at 37 C for 60 min. 3. Remove adhesive foil. Discard incubation solution. Wash plate 5 x with 300 µl of diluted Wash Buffer. Remove excess solution by tapping the inverted plate on a paper towel. 4. Pipette 100 µl of Enzyme Conjugate into each well. 5. Cover plate with adhesive foil. Incubate at 37 C for 30 min. 6. Remove adhesive foil. Discard incubation solution. Wash plate 5 x with 300 µl of diluted Wash Buffer. Remove excess solution by tapping the inverted plate on a paper towel.. 7. For adding of Substrate and Stop Solution use, if available, an 8-channel Micropipettor. Pipetting should be carried out in the same time intervals for Substrate and Stop Solution. Use positive displacement and avoid formation of air bubbles. 8. Pipette 100 µl of TMB Substrate Solution into each well. 9. Incubate 15 min at 37 C Avoid direct sun light. 10. Stop the substrate reaction by adding 100 µl of TMB Stop Solution into each well. Color changes from blue to yellow. 11. Measure optical density with a photometer at 450 nm (Reference-wavelength: nm) within 10 min after pipetting of the Stop Solution. 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 other applicable standards/laws. 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 The Cut-off value is given by the optical density (OD) of the Cut-off control. The Cut-off index (COI) is calculated from the mean optical densities of the sample and Cut-off value. If the optical density of the sample is within a range of 20% around the Cut-off value (grey zone), the sample has to be considered as borderline. Samples with higher ODs are positive, samples with lower ODs are negative. For a quantification, the Cut-off index (COI) of the samples can be formed as follows: COI = OD Sample OD Cut-off control 14. INTERPRETATION OF RESULTS Method Range Interpretation < 0.8 negative Qualitative equivocal (Cut-off Index, COI.) > 1.2 positive 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. Negative: No antibodies against T. pallidum detected. Equivocal: No clear interpretation possible. In such case it is advised to confirm the results by testing that sample again in duplicate. In the case the repeated result is again equivocal, a second sample should be tested and judged for a change in result. Positive: Specific antibodies against T.pallidum detected. V2012_01 4 / 5

6 15. LIMITATIONS OF THE PROCEDURE Specimen collection has a significant effect on the test results. See SPECIMEN COLLECTION AND STORAGE for details. The following substances do not have a significant effect on the test results up to the below stated concentrations. 16. PERFORMANCE Analytical Specificity (Cross Reactivity) No cross-reactivities were found to: Hemoglobin Bilirubin Triglyceride Precision COI CV (%) Sample Intra-Assay Inter-Assay Inter-Lot Clinical specificity 100% (n = 728) Clinical sensitivity 99% (n = 160) 0.07 mg/ml 1.25 mg/ml 5.70 mg/ml H.pylori, TORCH, EBV, Measles, parvovirus, borrelliosis, ds-dna antibodies, ANA-ANCA, autoimmune antibodies and Serum from Pregnant women. 17. PRODUCT LITERATURE REFERENCES 1. Report of a WHO Scientific group (1982). Treponemal infections. Technical Report Series Fieldsteel A.H., Cox D.L., Moeckli R.A. (1981). Cultivation of virulent Treponema pallidum in tissue culture. Infect. Immun. 32: Baker - Zander S.A., Roddy R.E. (1986). IgG and IgM antibody reactivity to antigens of Treponema pallidum after treatment of syphilis. Sex.Trans.Dis. 13 (4): Schroeter A. L., Lucas J.B. (1972). Treatment for early syphilis and reactivity of serologic tests. JAMA 221 (5): Farshy C., Hunter E. (1984). Double-Conjugate Enzyme-Linked Immunosorbent Assay for Immunoglobulins G and M against Treponema pallidum, J. Clin, Microbiol. 20(6): Lee. J., Larsen S. (1986). Detection of Immunoglobulin M in Cerebrospinal Fluid from Syphilis Patients by Enzyme-Linked Immunosorbent Assay, J. Clin, Microbiol. 24(5): Norris S. J. (1993). Polypeptides of Treponema pallidum: Progress toward understanding their Structural, Functional, and Immunologic Roles, Microbiological Reviews 57(3): Ito F., Larsen S. (1992). Specific Immonofluorescent Staining of Pathogenic Treponemes with a Monoclonal Antibody. J. Clin, Microbiol. 30(2): Larsen S, Steiner B. (1995). Laboratory Diagnosis and Interpretation of Tests for Syphilis, Clinical Microbiology Reviews. 8(1): Zrein M., Maure I. (1995). Recombinant Antigen-Based Enzyme Immunoassay for the Screening of Treponema pallidum Antibodies in Blood Bank Routine. J.Clin.Microbiol. 33(2): Young H., Moyes A. (1998). Novel Recombinant-Antigen Enzyme Immunoassay for Serological Diagnosis of Syphilis. J.Clin.Microbiol. 36 (4): Ijsselmuiden O., Schouls L. (1998). Sensitivity and Specificity of an Enzyme-Linked Immunosorbent Assay Using the Recombinant DNA-Derived Treponema pallidum Protein TmpA for Serodiagnosis of Syphilis and the Potential Use of TmpA for Assessing the Effect of Antibiotic Therapy, J.Clin.Micobiol. 27(1): Notenboom R., Meddens M. (2006). Evaluation of Trep-Sure; a new first-line enzyme immunoassay for the detection of total anti-treponemal antibodies. Int. J. STD&AIDS. 17(S1): 15 V2012_01 5 / 5

7 Symbols / Symbole / Symbôles / Símbolos / Símbolos / Σύµβολα REF LOT 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: CONC Concentrate / Konzentrat / Concentré / Concentrar / Concentrado / Concentrato / Συµπύκνωµα LYO IVD 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. IBL AFFILIATES WORLDWIDE IBL International GmbH Flughafenstr. 52A, Hamburg, Germany IBL International Corp. 194 Wildcat Road, Toronto, Ontario M3J 2N5, Canada Tel.: + 49 (0) Fax: IBL@IBL-International.com WEB: Tel.: +1 (416) Fax: Sales@IBL-International.com WEB: LIABILITY: Complaints will be accepted in each mode written or vocal. Preferred is that the complaint is accompanied with the test performance and results. 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. Regardless, in the event of any claim, the manufacturer s liability is not to exceed the value of the test kit. Any damage caused to the kit during transportation is not subject to the liability of the manufacturer Symbols Version 3.5 /