M h X g H. ORGENTEC Diagnostika GmbH Carl-Zeiss-Straße Mainz - Germany

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1 ORGENTEC Dagnostka GmbH Carl-Zess-Straße Manz - Germany Phone: +49 (0) / Fax: +49 (0) / Internet: Instructon For Use ORG 204G SYMBOLS USED Ant-dsDNA IgG NAME AND INTENDED USE Ant-dsDNA IgG s an ELISA-based test system for the quanttatve measurement of IgG class autoantbodes aganst double-stranded DNA n human serum or plasma samples. Ths product s ntended for professonal n vtro dagnostc use only. V M h X g H 2:r 8: w In vtro dagnostc medcal devce Manufacturer Catalogue number Suffcent for... determnatons Batch code Use by Temperature lmtaton Consult nstructons for use Keep away from sunlght Alegra Test Strps Wash Buffer System Flud Ready to use SUMMARY AND EXPLANATION OF THE TEST Connectve tssue dseases (CTD) are a group of autommune dsorders whch are characterzed by presence of antnuclear antbodes (ANA) n the blood of patents. ANA are a specfc class of autoantbodes that have the capablty of bndng and destroyng certan structures wthn the nucleus of the cells. These antbodes are nvolved n the dsease pathogeness, and they also consttute the bass for dagnoss and treatment of CTD. ANA have been categorzed nto two man groups: 1. Autoantbodes to DNA and hstones 2. Autoantbodes to extractable nuclear antgens (ENA): Sm, rbonucleoprotens (RNP), SSA/Ro, SSB/La, Scl-70, Jo-1 and PM1 Autoantbodes to DNA and hstones nclude antbodes aganst sngle and double stranded DNA (ssdna and dsdna). Sgnfcant levels of ant-dsdna antbodes are consdered to be confrmatory n the dagnoss of systemc lupus erythematosus (SLE). Ant-hstone antbodes are ndcatve of drug-nduced lupus. Besdes DNA and hstones, autoantbodes may also target other nuclear antgens. These nuclear antgens were named extractable nuclear antgens (ENA), as orgnally they were extracted from the nucle wth salne soluton. Autoantbodes to Smth antgen (Sm) whch s also consdered to be hghly specfc for SLE were the frst ant-ena detected. Thereafter, further subtypes of ENA.e. rbonucleoprotens (RNP), Sjögren antgen A or B (SSA/Ro or SSB/La), Scl-70, Jo-1 and PM1 were dentfed. Although most of these ENA are dsease specfc, a sgnfcant overlap exsts. Senstvty and specfcty may also vary dependng upon the type of underlyng CTD. Presence of autoantbodes n the sera of patents consttutes one of the crtera used for dagnoss of CTD. Together wth the clncal dagnoss ANA subtypng helps n dentfyng a specfc CTD. Indrect mmunofluorescence tests (IF) and enzyme mmunoassays (ELISA) are commonly used for ANA detecton n day to day practce. Intally, screenng s carred out by IF-ANA or a generc ELISA whch detects ANA of a broad specfcty smlar to IF- ANA. If postve, more specfc tests are performed based on clncal fndngs and the IF-ANA stanng pattern. These antgen specfc ELISA assays react wth sngle autoantgens e.g. dsdna, SS-A/Ro, SS-B/La, Scl-70, Sm, Sm/RNP etc. Autoantbodes to dsdna are specfc and dagnostc for SLE and levels are elevated durng actve dsease. Recently publshed ACR Gudelnes for Screenng, Treatment, and Management of Lupus Nephrts recommend the testng of antbodes to dsdna for montorng of lupus nephrts, rangng from monthly ntervals n pregnant patents wth actve glomerulonephrts at onset of treatment to every three months n patents wth actve nephrts at onset of treatment or pregnant patents wth prevous but not current nephrts, up to sx-monthly testng n patents wth prevous actve nephrts or no pror or current nephrts. SLE-Patents wthout antbodes aganst dsdna often produce antbodes aganst ssdna. Smlarly ant-sm s hghly specfc for SLE but s present n only 10 to 30 of SLE cases. Antbodes aganst dsdna, hstones, the 70 kd proten of the U1-snRNP complex (RNP70) and ant Sm are closely assocated wth SLE. Ant-SSA/Ro and ant-ssb/la antbodes are ndcatve for Sjögren's syndrome, but can also be found n up to 30 cases of SLE wth cutaneous nvolvement. Ant-SS-A/Ro antbodes pass the placenta and may cause the development of SLE n neonates. Ant-SSA/Ro antbodes are almost always present n sera of mothers wth babes wth neonatal lupus syndrome and wth complete congental heart block. Antnucleolar antbodes are a group of autoantbodes whch gve a nucleolar IF-stanng pattern. Most common of these are ant-pm-scl, ant-rna polymerase I-III and ant-u3-rnp They are found n scleroderma and polymyosts (PM). Antbodes aganst RNP and the complex RNP/Sm are lnked to mxed connectve tssue dsease (MCTD, Sharp syndrome) and to SLE. Serologcally MCTD s characterzed by the presence of autoantbodes drected aganst the 70 kd proten of the U1-snRNPcomplex. Up to 100 of MCTD patents manfest hgh tters of Ant-RNP-70 antbodes. Dseases dsdna ssdna Hstone SS-A SS-B Sm RNP/ Sm Scl-70 Jo-1 Systemc lupus erythrematosus (SLE) > 90 > Drug-nduced lupus (DIL) Sharp-syndrome / mxed connectve tssue dsease > 90 Rheumatod arthrts Sjögren s syndrome > 90 > 90 Scleroderma > 90 Photosenstve dermatts, dermatomyosts Autoantbody prevalence to (values n ) page 1 page 2

2 PRINCIPLE OF THE TEST The Alegra assay features barcoded 8-well-mcrostrps, called Alegra Test Strps. Each strp s desgned for a sngle determnaton of one patent sample. The Alegra Test Strp holds a complete set of reagents. Included are enzyme conjugate, enzyme substrate, sample buffer and a test specfc control. Furthermore each strp has two antgen-coated wells whch serve as reacton wells for one control and one patent sample. The determnaton s based on an ndrect enzyme lnked mmune reacton wth the followng steps: Antbodes present n postve samples bnd to the antgen coated on the surface of the two reacton wells formng an antbody antgen complex. After ncubaton, a frst washng step removes unbound and unspecfc bound molecules. Subsequently added enzyme conjugate bnds to the mmoblzed antbody-antgen complex. After ncubaton, a second washng step removes unbound enzyme conjugate. Addton of enzyme substrate soluton results n hydrolsaton and color development durng ncubaton. The ntensty of the blue color correlates wth the concentraton of the antbody-antgen-complex and can be measured photometrcally at 650 nm. The Alegra Test Strp s based on the propretary SMC -Technology (Sensotronc Memorzed Calbraton): nformaton about the assay, analyss and evaluaton, and the lot-specfc expry date s contaned on the barcode prnted on each Alegra Test Strp. The Alegra Test Strp can be used wth the dagnostc nstrument Alegra - a fully automated Random Access Analyser. By means of SMC -Technology data encoded on the barcode are transferred from the Alegra Test Strp to the nstrument and the assay s automatcally processed and evaluated. The nstrument reads the date of expry and rejects further processng f the Alegra Test Strp s out of date. WARNINGS AND PRECAUTIONS All reagents of ths kt are ntended for professonal n vtro dagnostc use only. Components contanng human serum were tested and found negatve 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 n ths kt must be handled as though capable of transmttng nfecton. Bovne serum albumn (BSA) used n components has been tested for BSE and found negatve. Avod contact wth the substrate TMB (3,3,5,5 -Tetramethyl-benzdne). System flud contans acd, classfacton s non-hazardous. Avod contact wth skn. Control, sample buffer and wash buffer contan sodum azde 0.09 as preservatve. Ths concentraton s classfed as non-hazardous. Enzyme conjugate contans ProCln as preservatve. Ths concentraton s classfed as non-hazardous. Durng handlng of all reagents, controls and serum samples observe the exstng regulatons for laboratory safety regulatons and good laboratory practce: Frst ad measures: In case of skn contact, mmedately wash thoroughly wth water and soap. Remove contamnated clothng and shoes and wash before reuse. If system flud comes nto contact wth skn, wash thoroughly wth water. After contact wth the eyes carefully rnse the opened eye wth runnng water for at least 10 mnutes. Get medcal attenton f necessary. Personal precautons, protectve equpment and emergency procedures: Observe laboratory safety regulatons. Avod contact wth skn and eyes. Do not swallow. Do not ppette by mouth. Do not eat, drnk, smoke or apply makeup n areas where specmens or kt reagents are handled. When splled, absorb wth an nert materal and put the splled materal n an approprate waste dsposal. Exposure controls / personal protecton: Wear protectve gloves of ntrl rubber or natural latex. Wear protectve glasses. Used accordng to ntended use no dangerous reactons known. Condtons to avod: Snce substrate soluton s lght-senstve. Store Alegra strps n the dark. For dsposal of laboratory waste the natonal or regonal legslaton has to be observed. Observe the gudelnes for performng qualty control n medcal laboratores by assayng controls and/or pooled sera. CONTENTS OF THE KIT X24 X12 ORG 204G-24 ORG 204G-12 Suffcent for 24 determnatons Suffcent for 12 determnatons Alegra Test Strps are modules of 8 wells each composed of: Wells 1 + 2: empty and not coated (wells for the sample dluton) Wells 3 + 4: coated wth antgen (reacton wells) Well 5: Control; yellow; contanng test specfc antbodes, PBS, BSA, detergent, preservatve sodum azde Well 6: Enzyme Conjugate; lght red; contanng ant-human IgG antbodes, HRP labelled; PBS, BSA, detergent, preservatve ProCln Well 7: Sample Buffer: yellow; contanng PBS, BSA, detergent, preservatve sodum azde Well 8: TMB Substrate: clear; contanng 3,3, 5,5 - Tetramethylbenzdn. Human recombnant double-stranded DNA (dsdna) s bound to mcrowells. Product code on barcode: dsdna IgG 1x 20 ml Wash Buffer, contanng Trs, detergent, preservatve sodum azde 0.09; 50 x conc. 1x 2.5 ml System Flud, contans acd; 1000 x concentrate STORAGE AND STABILITY Store test kt at 2-8 C n the dark. Do not expose reagents to heat, sun, or strong lght durng storage and usage. Store Alegra Test Strps sealed and desscated n the clp bag provded. Shelf lfe of the unopended test kt s 15 months from day of producton. Unopened reagents are stable untl expraton of the kt. See labels for ndvdual batch. Dluted Wash Buffer and System Flud are stable for at least 30 days when stored at 2-8 C. Once transferred to the reagent contaner we recommend consumpton on the same day. MATERIALS REQUIRED Vortex mxer Ppettes for 10 µl Measurng cylnder for 1000 ml and 2500 ml Dstlled or deonzed water 1 Alegra Instructon for Use: Alegra Mn-CD 1 Certfcate of Analyss SPECIMEN COLLECTION, STORAGE AND HANDLING Collect whole blood specmens usng acceptable medcal technques to avod hemolyss. Allow blood to clot and separate the serum or plasma by centrfugaton. Test serum should be clear and non-hemolyzed. Contamnaton by hemolyss or lpema should be avoded, but does not nterfere wth ths assay. Specmens may be refrgerated at 2-8 C for up to fve days or stored at -20 C up to sx months. Avod repettve freezng and thawng of serum or plasma samples. Ths may result n varable loss of antbody actvty. Testng of heat-nactvated sera s not recommended. PROCEDURAL NOTES Do not use kt components beyond ther expraton dates. All materals must be at room temperature (20-28 C) pror to use. To avod carryover or contamnaton, change the ppette tp between samples. page 3 page 4

3 PREPARATION OF REAGENTS Dlute the content of the Wash Buffer concentrate (50x) wth dstlled or deonzed water to a fnal volume of 1000 ml pror to use. Transfer the dluted Wash Buffer nto the nstrument reagent contaner. If only one Alegra run s to be performed on one day we recommend transferrng only 500 ml dluted Wash Buffer. Dlute the content of the System Flud concentrate (1000x) wth dstlled or deonzed water to a fnal volume of 2500 ml pror to use. Transfer the dluted System Flud nto the nstrument reagent contaner. Take the requred number of Alegra Test Strps out of the clp bag and let them reach room temperature (20-28 C). Do not remove fol coverng the empty wells untl you are ready to start the assay. TEST PROCEDURE Alegra Test Strps wth SMC technology are used wth the dagnostc nstrument Alegra. Detaled nformaton about operatng the nstrument can be taken from the Instrument User Manual. (1) Remove the fol from the empty wells 1 to 4 of the Alegra Test Strp. Do not remove fol wth prnted barcode, coverng wells 5 to 8. (2) Ppette 10 µl undluted patent sample (serum or plasma) at the bottom of well 1. (3) Insert the strp nto the SysTray. Place loaded SysTrays nto the correct poston n the Alegra nstrument and start run. All further steps wll be done automatcally. The test run s completed when the nstrument starts prntng the results. CALIBRATION The assay system s calbrated aganst the nternatonal reference preparaton WHO Wo/80 for human ant-dsdna IgG antbodes as 200 IU/ml. CALCULATION OF RESULTS By means of SMC Technology (Sensotronc Memorzed Calbraton), all test data are transferred to the system through ndvdual barcodes on the Alegra Test Strp. Calculaton and nterpretaton of results wll be performed automatcally. PERFORMANCE CHARACTERISTICS Measurng range The calculaton range of ths Alegra assay s U/ml Expected values In a normal range study wth samples from healthy blood donors the followng ranges have been establshed wth ths Alegra assay: Cut-off 20 IU/ml Interpretaton of results Normal: Elevated: < 20 IU/ml 20 IU/ml Lnearty Three patent samples contanng hgh levels of specfc antbody were serally dluted n sample buffer to demonstrate the dynamc range of the assay. Actvty for each dluton was calculated by means of SMC Tecnology. Sample Dluton.. 1 1: : :100 Senstvty Functonal senstvty was determned to be: 1 IU/ml Reproducblty Intra-assay precson: Coeffcent of varaton (CV) was calculated for each of three samples from the results of 24 determnatons n a sngle run. Results for precson-wthn-assay are shown n the table below. Inter-assay precson: Coeffcent of varaton (CV) was calculated for each of three samples from the results of 6 determnatons n 5 dfferent runs. Results for run-to-run precson are shown n the table below. Interferng substances No nterference has been observed wth haemolytc (up to 1000 mg/dl) or lpemc (up to 3 g/dl trglycerdes) sera or plasma, or blrubn (up to 40 mg/dl) contanng sera or plasma. Nor have any nterferng effects been observed wth the use of antcoagulants (Ctrate, EDTA, Heparne). However for practcal reasons t s recommended that grossly hemolyzed or lpemc samples should be avoded. Study results Intra-Assay Sample Mean. [IU/ml] CV [] Observed Expected O/E [IU/ml] [IU/ml] [] Inter-Assay Sample Mean. [IU/ml] CV [] Study populaton n n pos SLE Other autommune dseases Normal human sera page 5 page 6

4 ORG 204G Ant-dsDNA IgG. Senstvty: Specfcty: Overall agreement: Pos Neg Clncal Dagnoss Pos Neg LIMITATIONS OF THE PROCEDURE Ths assay s a dagnostc ad. A defnte clncal dagnoss should not be based on the results of a sngle test, but should be made by the physcan after all clncal and laboratory fndngs have been evaluated concernng the entre clncal pcture of the patent. Also every decson for therapy should be taken ndvdually. The above pathologcal and normal reference ranges for antbodes n patent samples should be regarded as recommendatons only. Each laboratory should establsh ts own ranges accordng to ISO or other applcable laboratory gudelnes. 16. Revelle JD. Predctve value of autoantbodes for actvty of systemc lupus erythematosus. Lupus JID ; 13(5): Smon JA, Cabedes J, Ortz E, Alcocer-Varela J, Sanchez-Guerrero J. Ant-nucleosome antbodes n patents wth systemc lupus erythematosus of recent onset. Potental utlty as a dagnostc tool and dsease actvty marker. Rheumatology (Oxford ) 2004; 43(2): Snclar D, Saas M, Wllams D, Hart M, Goswam R. Can an ELISA replace mmunofluorescence for the detecton of ant-nuclear antbodes?--the routne use of ant-nuclear antbody screenng ELISAs. Cln Lab 2007; 53(3-4): Tozzol R, Bzzaro N, Tonutt E, Vllalta D, Bassett D, Manon F et al. Gudelnes for the laboratory use of autoantbody tests n the dagnoss and montorng of autommune rheumatc dseases. Am J Cln Pathol 2002; 117(2): Madhof W., Hlas O. Lupus: an pvervew of the dsease and management optons. P T 2012; 37(4): Hahn BH, McMahon MA, Wlknson A, Wallace WD, Dakh DI, Ftzgerald JD et al. Amercan College of Rheumatology gudelnes for screenng, treatment, and management of lupus nephrts. Arthrts Care Res (Hoboken ) 2012; 64(6): REFERENCES 1. Alba P, Bento L, Cuadrado MJ, Karm Y, Tungekar MF, Abbs I et al. Ant-dsDNA, ant-sm antbodes, and the lupus antcoagulant: sgnfcant factors assocated wth lupus nephrts. Ann Rheum Ds 2003; 62(6): Antco A, Platzgummer S, Bassett D, Bzzaro N, Tozzol R, Vllalta D. Dagnosng systemc lupus erythematosus: new-generaton mmunoassays for measurement of ant-dsdna antbodes are an effectve alternatve to the Farr technque and the Crthda luclae mmunofluorescence test. Lupus 2010; 19(8): Brouwer R, Hengstman GJ, Vree EW, Ehrfeld H, Bozc B, Ghrardello A et al. Autoantbody profles n the sera of European patents wth myosts. Ann Rheum Ds 2001; 60(2): Castro C, Gourley M. Dagnostc testng and nterpretaton of tests for autommunty. J Allergy Cln Immunol 2010; 125(2 Suppl 2):S238-S Defendent C, Atzen F, Spna MF, Grosso S, Cereda A, Guerclena G et al. Clncal and laboratory aspects of Ro/SSA-52 autoantbodes. Autommun Rev 2011; 10(3): Erksson C, Kokkonen H, Johansson M, Hallmans G, Wadell G, Rantapaa-Dahlqvst S. Autoantbodes predate the onset of Systemc Lupus Erythematosus n northern Sweden. Arthrts Research & Therapy 2011; 13(1):R Haugbro K, Nossent JC, Wnkler T, Fgenschau Y, Rekvg OP. Ant-dsDNA antbodes and dsease classfcaton n antnuclear antbody postve patents: the role of analytcal dversty. Ann Rheum Ds JID ; 63 (4): Ippolto A, Wallace DJ, Gladman D, Fortn PR, Urowtz M, Werth V et al. Autoantbodes n systemc lupus erythematosus: comparson of hstorcal and current assessment of seropostvty. Lupus 2011; 20(3): Isenberg DA, Manson JJ, Ehrensten MR, Rahman A. Ffty years of ant-ds DNA antbodes: are we approachng journey's end? Rheumatology (Oxford) 2007; 46(7): Kattah NH, Kattah MG, Utz PJ. The U1-snRNP complex: structural propertes relatng to autommune pathogeness n rheumatc dseases. Immunol Rev 2010; 233(1): Kumar Y, Bhata A, Mnz RW. Antnuclear antbodes and ther detecton methods n dagnoss of connectve tssue dseases: a journey revsted. Dagn Pathol 2009; 4: Meron PL, Schur PH. ANA screenng: an old test wth new recommendatons. Ann Rheum Ds 2010; 69: Petr M, Magder L. Classfcaton crtera for systemc lupus erythematosus: a revew. Lupus 2004; 13(11): Poole BD, Schneder RI, Guthrdge JM, Velte CA, Rechln M, Harley JB et al. Early targets of nuclear RNP humoral autommunty n human systemc lupus erythematosus. Arthrts Rheum 2009; 60(3): Putova I, Dostal C, Becvar R. Prevalence of antnucleosome antbodes by enzyme-lnked mmunosorbent assays n patents wth systemc lupus erythematosus and other autommune systemc dseases. Ann N Y Acad Sc 2007; 1109: page 7 page 8

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