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Instructions for useIBL International GmbHFlughafenstraße 52 aD-22335 HamburgTel. 49 (0) 40 53 28 91-0Fax 49 (0) 40 53 28 [email protected]/iblSS-A/Ro-Ab ELISAEnzyme immunoassay for the qualitative and quantitative determinationof IgG antibodies against SS-A/Roin human serum or plasma (EDTA, citrate, heparin).RE7523112x82-8 CFor research use only.Not for use in diagnostic procedures.IBL International GmbHFlughafenstrasse 52aD-22335 Hamburg, Germany

SS-A/Ro-Ab ELISA (RE75231)ENGLISH1. INTRODUCTION AND BACKGROUNDThe present ELISA is intended for the quantitative or qualitative determination of IgG antibodies directedagainst Ro60 (isolated from bovine thymus) in human serum or plasma (cf. section 7). Ro 52 is excludedfrom the immobilised antigen preparation since some Jo-1 positive sera (primarily indicative for myositis)also cause positive signals on this substrate (10, 11). The test is fast (incubation time 30 / 30 / 30 minutes)and flexible (divisible solid phase, ready-to-use reagents). Six calibrators allow quantitative measurements;a negative and a positive control check the assay performance.2. WARNINGS AND PRECAUTIONSFor research Use only. Not for use in diagnostic procedures. Not for internal or external use in humans oranimals. It must be executed by trained personnel staff.Do not use reagents beyond their expiration dates. Adherence to the protocol is strongly recommended.The Sample Diluent, calibrators and controls contain Na-azide as antimicrobial agent. The Wash Buffercontains bromonitrodioxane and the conjugate methylisothiazolone / bromonitrodioxane as preservative.The substrate contains 3, 3', 5, 5'-tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2). The stopsolution, 0,2 M sulfuric acid (H2SO4), is acidic and corrosive.The above mentioned reagents may be toxic if ingested. Follow routine precautions for handling hazardouschemicals. Avoid all body contact, wear gloves and eye protection. If one of the reagents comes intocontact with skin or mucous membrane, wash thoroughly with water. Never pipette by mouth. Dispose in amanner complying with local/national regulations.Na-Azide may react with lead and copper plumbing to form explosive metal azides. On disposal, flush with alarge amount of water to prevent azide build-up.The calibrators and controls contain components of human origin. They were tested for humanimmunodeficiency virus (HIV)-Ag, hepatitis B surface (HBs)-Ag and antibodies against HIV 1/2 and hepatitisC virus (HCV) and showed negative results; either in an FDA-approved or a CE-compliant test, according toEuropean Directive 98/79/EC.However, no test can guarantee that material of human origin is not actually infectious. The preparationsshould therefore be treated as potentially infectious and disposed of accordingly, as should the samples(and residues thereof); according to CDC (Center of Diseae Control, Atlanta, USA) or other local / nationalguidelines on laboratory safety and decontamination.3. PRINCIPLE OF THE TESTThe wells of the solid phase are coated with SS-A / Ro antigen, as described above. On this surface, thefollowing immunological reactions take place:1st reaction: SS-A / Ro-specific antibodies present in the sample bind to the immobilised antigen, formingthe antigen-antibody complex. Then, non-bound sample components are washed away from the solidphase.2nd reaction: A second antibody, directed at human IgG antibodies and conjugated with horse-radishperoxidase (HRP), is added. This conjugate binds to the complex. Then, excess conjugate is washedaway from the solid phase.3rd reaction: The enzyme-labelled complex converts a colourless substrate into a blue product. The degreeof colour development reflects the concentration of SS-A / Ro IgG in the sample.2019-08-071/8

SS-A/Ro-Ab ELISA (RE75231)ENGLISH4. CONTENTS OF THE KITa. MTP 1 Microtiter Plate, coated with the SS-A / Ro antigen described above and hermetically packed ina foil laminate pouch together with a desiccant bag. The plate consists of 12 strips, each of which can bebroken into 8 individual wells.b. ENZCONJ IgG Enzyme Conjugate IgG, 14 mL, ready-to-use, red coloured. Buffered solutioncontaining stabilising protein, methylisothiazolone and bromonitrodioxane.c. CAL A–F Calibrator A-F, 2,0 mL each, 0 - 0,70 – 2,0 - 7,0 - 20 and 70 U SS-A / Ro IgG / mL, ready-touse, gradually blue coloured. Contain TBS, BSA, Tween and Na-azide.d. CONTROL - & CONTROL Negative and positive control, 2,0 mL each, ready-to-use, green andred coloured, respectively. Contain TBS, BSA, Tween and Na-azide.e. SAMPLEDIL Sample Diluent, 100 mL, ready-to-use, orange coloured. Contains Tris-buffered saline(TBS), bovine serum albumin (BSA), Tween and Na-azide.f. WASHBUF CONC Wash Buffer, 100 mL, 10x-concentrate, blue coloured. Contains TBS, Tween andbromonitrodioxane.g. TMB SUBS TMB Substrate Solution, 14 mL, ready-to-use, colourless. Contains a buffered solution ofTMB and H2O2. Contained in a vial impermeable to light.h. STOP TMB Stop Solution (0,2 M H2SO4), 14 mL, colourless, ready-to-use.Caution: sulfuric acid is corrosive.i. Directions for usej. Lot-specific certificate of analysis5. MATERIALS REQUIRED BUT NOT SUPPLIEDa. Deionised or distilled waterb. Graduated cylinder, 1000 mLc. Tubes for sample dilution (transfer tubes in the microwell plate format recommended)d. Pipettes for 10, 100 and 1000 µL (1- and 8-channel pipettes recommended)e. Microwell plate washer (optional)f. Microwell plate photometer fitted with a 450 nm filterg. ELISA evaluation program (recommended)6. STORAGE OF THE KITStore kit at 2 - 8 C. It is stable up to the expiry date stated on the label of the box. Do not use kit beyond itsexpiry date.2019-08-072/8

SS-A/Ro-Ab ELISA (RE75231)ENGLISH7. REAGENT AND SAMPLE PREPARATION / SPECIMEN REQUIREMENTSDo not exchange or pool corresponding components from different kits, due to possibly different shipping orstorage conditions. If the kit is to be used for several tests, only the currently needed amount of reagentsshould be withdrawn. It is crucially important that no cross-contamination between the reagents occurs.Use only clean pipettes and do not pour back residues into the original flasks.a. The solid phase must reach room temperature before opening the pouch. Remove the supernumerarymicrowells from the frame and immediately put them back into the pouch, together with the desiccantbag. Reseal the pouch hermetically and keep it refrigerated for future use.b. Dilute the Wash Buffer 10x-concentrate (100 mL, blue) with 900 mL deionised water. Mix thoroughly.The diluted buffer is stable for several weeks if stored refrigerated (2 - 8 C).c. Preparation of the samples: handle specimens as potentially infectious agents. Besides serum, EDTA-,citrate- or heparin-treated plasma is suitable sample material as well.Specimen requirements: highly lipemic, haemolysed or microbially contaminated samples may causeerroneous results and should be avoided.Prepare samples using normal laboratory techniques. Turbid samples must first be clarified(centrifuged). The clarified or clear samples are mixed and then diluted 1/100, e.g. 10 µL serum orplasma 990 µL sample buffer. Also mix the dilution.For rapid dispensing during the assay procedure, preparation of the calibrators, controls and samples inmicrowell transfer tubes is recommended. This allows the operation of an 8-channel pipette during theassay procedure.If samples are not assayed immediately, they should be stored at 2 - 8 C and assayed within 3 days. Forlonger storage, -20 C or lower temperatures are recommended. Repeated freezing and thawing ofsamples should be avoided. Thawed samples must be mixed prior to diluting.8. ASSAY PROCEDUREBefore starting the assay, all components of the kit must have reached room temperature (23 3 C).To achieve best results, i.e. the maximum ratio between specific and background signal, careful washingis essential (steps a, c and e). It is crucially important to remove the wash solution completely. For thatpurpose, tap the plate firmly on several layers of absorbent tissue. Automated washers must be verifiedaccording to results obtained by manual washing.a. Immediately prior to use, wash the solid phase once: fill wells with 350 µL Wash Buffer each, let soak forabout 10 seconds in the wells and remove.b. Dispense the calibrators (2,0 mL each, ready-to-use, gradually blue), controls (2,0 mL each, ready-touse, green and red) and the diluted samples rapidly into the microwells; 100 µL per well. Duplicatemeasurements are recommended.Incubate the plate for 30 minutes at room temperature (23 3 C).c. Wash the wells 4 times as in step a.d. Rapidly (preferably using an 8-channel pipette) dispense the conjugate (14 mL, ready-to-use, red);100 µL per well. Incubate the plate as in step b.e. Repeat wash step c.f. Rapidly (preferably using an 8-channel pipette) dispense the substrate solution (14 mL, ready-to-use,colourless, black vial); 100 µL per well. Incubate the plate as in step b. As the substrate isphotosensitive, avoid intense light exposure (e.g. direct sunlight) during incubation.g. Rapidly (preferably using an 8-channel pipette) dispense the stop solution (14 mL, ready-to-use,colourless. Caution: corrosive!); 100 µL per well. Use the same sequence as for the substrate. Thecolour changes from blue to yellow. Agitate the plate, preferably on an orbital shaker, for about10 seconds.h. Immediately read the absorbance in the microwell plate photometer at 450 nm.Refrigerate the remainder of the reagents (2 - 8 C) if they are to be used again.2019-08-073/8

SS-A/Ro-Ab ELISA (RE75231)ENGLISH9. EVALUATION AND QUALITY CONTROLQuantitative evaluation: the data obtained are quantitatively evaluated with the standard curve, as shownbelow. However, the depicted curve can only serve as a model. It can not substitute the measurement ofthe calibrators, together with the controls and actual samples. The curve has been constructed with aconventional ELISA evaluation program, using a 4-parameter function. The Spline approximation is alsoappropriate.If no computer-supported evaluation is possible, the standard curve may be drawn by hand. It allowstransformation of the absorbance value of a sample into its concentration, i.e. into U SS-A / Ro IgG per mLsample.Qualitative evaluation: the test may also be evaluated in a qualitative manner. This requires measurementof the positive control only. Nevertheless, measurement and examination of the negative control isrecommended (see below: quality control).In qualitative test evaluation, the absorbance of the samples is compared with the borderline absorbance ( cut-off). It is determined according to the following formula:absorbanceborderline absorbancepositive control x factorThe factor depends on the kit lot and is quoted in the lot-specific certificate of analysis which is includedwith each test kit. Example:absorbancepositive control 1250 mODfactor 0,35absorbanceborderline 1250 mOD x 0,35 438 mODIn order to gain an impression of how positive a particular sample is for SS-A / Ro-Ab IgG, one maycalculate the ratio, according to the formula:ratio absorbancesample / absorbanceborderlineExample:absorbanceborderline 438 mODabsorbancesample 1480 mODratio 1480 mOD / 438 mOD 3,4Quality control: the positive and negative control check the assay performance. Their authorised values andacceptable ranges, respectively, are quoted in the lot-specific certificate of analysis. Values of the controlsmust within the indicated ranges; otherwise, the results of the assay are invalidated.2019-08-074/8

SS-A/Ro-Ab ELISA (RE75231)ENGLISH10. PERFORMANCE CHARACTERISTICS10.1. StandardisationThe test is standardised with a purified serum preparation containing IgG antibodies specifically directed atthe SS-A / Ro 60 kDa antigen. This preparation is calibrated against a set of gradually positive sera, solelyreserved for this purpose. The degree of sample reactivity is measured in arbitrary units (U/mL) since nointernational standard is available.10.2. Analytical specificityThe test allows the specific determination of human IgG antibodies directed against SS-A / Ro. It has beenvalidated (among other parameters) by means of the commercially available human reference sera of the"Center of Disease Control" (CDC, Atlanta, USA). The following results are typical:serum12345678910CDCresultdsDNASS-B mo- speck- speck- --nuc-centro- --gen / ledledleolarmererimELISA (U/mL)1,119371,15,00,6 700,52,60,510.3. Detection limit (analytical sensitivity)The detection limit is defined as that concentration of analyte that corresponds to the mean absorbance ofSample Diluent plus 3-fold standard deviation (s). It was determined as 0,2 U SS-A / Ro IgG per mLsample (n 24).Recommended measuring range: 0,5 - 50 U / mL10.4. Homogeneity of the solid phaseMeasurement of the solid phase homogeneity is a regular QC part of each production lot. This isdetermined by 288-fold measurement of a positive but non-saturating sample on 3 selected plates.Acceptance criterion: mOD-coefficient of variation (cv) over the plates 8%. The figure below shows arepresentative excerpt (solid phase lot no. 3004S) of such an eancv%line a 1382 1301 1345 1347 1288 1297 1326 1302 1291 1282 1297 1254 13092,7line b 1389 1309 1331 1350 1313 1336 1380 1325 1323 1347 1322 1395 13432,2line c 1362 1307 1314 1359 1306 1312 1342 1305 1302 1319 1313 1322 13221,6line d 1358 1309 1328 1371 1317 1317 1363 1333 1280 1322 1305 1308 13262,0line e 1399 1353 1335 1357 1336 1332 1355 1336 1316 1342 1338 1337 13451,5line f1391 1314 1353 1365 1322 1338 1365 1351 1340 1357 1352 1336 13491,5line g 1403 1342 1344 1378 1362 1344 1371 1347 1345 1368 1374 1340 13601,4line h 1331 1293 1338 1329 1292 1284 1332 1265 1244 1253 1259 1257 12902,7mean 1377 1316 1336 1357 1317 1320 1354 1321 1305 1324 1320 1319 1330cv 5/8

SS-A/Ro-Ab ELISA (RE75231)ENGLISHline aline bline cline dline eline fline gline h0200400600800100012001400mOD 450nm14001200mOD 24n4plate #row #10.5. LinearityIn order to assess the dose-response relationship of the test, positive sera were measured in serial 2-folddilution. Acceptance criterion: linear regression of 4 successive dilutions must yield a correlation factor 0,98.A typical result is depicted below.U SS-A / Ro IgG / mL .20serum 1serum 2serum 3lin.regr.1lin.regr.2lin.regr.315105001020304050nL serum / well2019-08-076/8

SS-A/Ro-Ab ELISA (RE75231)ENGLISH10.6. PrecisionFor the assessment of the test precision, the variability of results under the following conditions wasdetermined: a. within 1 assay and between 3 assays, b. between 3 operators and c. between 2 kit lots.a. Intra- and inter-assay variability (n 24 and 72, respectively)samplemeanvariability (cv, 1,72,4b. Operator to operator variability (n 12)samplemeanvariabilityU/mL(cv, %)15,62,42111,33221,8c. Variability between 2 kit lots (n 6)samplemeanvariabilityU/mL(cv, %)15,62,22113,83232,611. WARRANTYIBL International GmbH guarantees that the product delivered has been thoroughly tested to ensure that itsproperties specified herein are fulfilled. No further warranties are given.The performance data presented here were obtained using the procedure indicated. Any modification in theprocedure may affect the results in which case IBL disclaims all warranties whether expressed, implied orstatutory. Moreover, IBL accepts no liability for any damage, whether direct, indirect or consequential, whichresults from inappropriate use or storage of the product.2019-08-077/8

SS-A/Ro-Ab ELISA (RE75231)ENGLISH12. SUMMARY FLOW CHARTa. Dilute the samples 1/100 in Sample Diluent (100 mL, ready-to-use, orange) and mix.b. Dilute the Wash Buffer 10x-concentrate (100 mL, blue) with water and mix.c. Wash the wells once with 350 µL Wash Buffer each. Dispense 100 µL of the calibrators (2,0 mL each,ready-to-use, gradually blue) and controls (2,0 mL each, ready-to-use, green and red) and of the dilutedsamples into the wells of the solid phase. Duplicate measurements are recommended. Incubate for 30minutes at room temperature (23 3 C).d. Wash the wells 4 times with 350 µL Wash Buffer each.e. Dispense 100 µL of the conjugate (14 mL, ready-to-use, red) into the wells. Incubate as in step c.f. Repeat washing step d.g. Dispense 100 µL of the substrate solution (14 mL, ready-to-use, black vial) per well. Incubate as in stepc. Then, add 100 µL stop solution (14 mL, ready-to-use, colourless) per well and agitate the plate briefly.h. Immediately measure the absorbance at 450 nm.i. Quantitative evaluation: determine the standard curve and, using this curve, transform the absorbance ofthe samples into their respective antibody concentration (U/mL).j. Qualitative evaluation: determine the borderline absorbance by multiplying the absorbance of the positivecontrol with the factor shown in the certificate of analysis. Then, calculate the ratio of the samples bydividing their absorbance by the borderline absorbance.2019-08-078/8

Symbols / Symbole / Symbôles / Símbolos / Símbolos / ΣύμβολαREFCat.-No.: / Kat.-Nr.: / No.- Cat.: / Cat.-No.: / N.º Cat.: / N.–Cat.: / Αριθμός-Κατ.:LOTLot-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:CONCLYOIVDConcentrate / Konzentrat / Concentré / Concentrar / Concentrado / Concentrato / ΣυμπύκνωμαLyophilized / Lyophilisat / Lyophilisé / Liofilizado / Liofilizado / Liofilizzato / ΛυοφιλιασμένοIn Vitro Diagnostic Medical Device. / In-vitro-Diagnostikum. / Appareil Médical pour Diagnostics InVitro. / Dispositivo Médico para Diagnóstico In Vitro. / Equipamento Médico de Diagnóstico InVitro. / Dispositivo Medico Diagnostico In vitro. / Ιατρική συσκευή για In-Vitro Διάγνωση.Evaluation kit. / Nur für Leistungsbewertungszwecke. / Kit pour évaluation. / Juego de Reactivospara 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 istruzioniprima dell’uso. / Διαβάστε 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 laluz solar directa. / Manter longe do calor ou luz solar directa. / Non esporre ai raggi solari. / Ναφυλάσσεται μακριά από θερμότητα και άμεση επαφή με το φως του ηλίου.Store at: / Lagern bei: / Stocker à: / Almacene a: / Armazenar a: / Conservare a: / Αποθήκευσηστους: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 thetest 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 withproduct specifications delivered with the product. The product must be used according to the Intended use, allinstructions given in the instructions for use and within the product specific shelf life. Any modification of the testprocedure or exchange or mixing of components of different lots could negatively affect the results. These casesinvalidate any claim for replacement.LIMITATION OF LIABILITY: IN ALL CIRCUMSTANCES THE EXTENT OF MANUFACTURER’S LIABILITY IS LIMITEDTO THE PURCHASE PRICE OF THE KIT(S) IN QUESTION. IN NO EVENT SHALL MANUFACTURER BE LIABLEFOR ANY INCIDENTAL OR CONSEQUENTIAL DAMAGES, INCLUDING DAMAGES FOR LOST PROFITS, LOSTSALES, INJURY TO PERSON OR PROPERTY OR ANY OTHER INCIDENTAL OR CONSEQUENTIAL LOSS.IBL International GmbHFlughafenstrasse 52aD-22335 Hamburg, GermanySymbols Version 5.6 / 2019-12-30Phone: 49 (0)40-53 28 91-0Fax: 49 (0)40-53 28 [email protected]/ibl

IBL International GmbH Flughafenstraße 52 a D-22335 Hamburg Tel. 49 (0) 40 53 28 91-0 Fax 49 (0) 40 53 28 91-11 [email protected] . according to CDC (Center of Diseae Control, Atlanta, USA) or other local / national guidelines on laboratory safety and decontamination. 3. PRINCIPLE OF THE T