Package Insert - Sekisui Diagnostics
Contents
1. 0 elele ananem anana 6 9 1 Testtunction control 11 1111011 a A GA ened ee GN ee a a A ee 9 2 Calculation of the Virotech Units VE 93 htierpretationSchemelgGandgM 9 4 Eimits OF THE TOS erer raki Ee MEKE BAREL dl e A a aaa S ka De aleme lake 10 Performance Data SERUM DIAGNOSTIC zu 2u20 u0 000000n0nununnnnunnnnunun nn nun nn nun an anna man nn sanan anana 8 10 2 Detection limits se er iy Marsa ab Rl e lada Ba l delal ni Bae Sa da a 10 3 Cross 16activity ie E awe ARMA la Sa AN MAL A AYR aaa aah 10 4 Intra assay coefficient of variation repeatability IgG and IgM 10 5 Inter assay coefficient of variation reproducibiliy eee 11 Performance Data CSF DIAGNOSTICTEST NG 11mm 12 tket cAMAalytcal S Onsitiv Ilys 1 igin adak aaa a gana ksk ez aaa ili aa l sadaka aliya a 11 2 Analytcalspefkiy A AMAMAMA A A A 11 3 Intra assay coefficient of variation repeatabiliy 11 4 Inter assay coefficient of variation reproducibility 12 LVRS MN suwadanewedtvaccen 13 13 Test Procedure Schem6E s2 u11011v1 erimi dasdmmar ia dana daa sacs daatecddeavessecavedackdwedecscdwocceeecduidediesestevedes 14 Seite 2 von 14 REV 9 CMV ELISA IgG igM GB Druckdatum 03 02 2014 1 Intended Use The CMV ELISA is used for the semiquantitative and qualitative detection of IgG and IgM antibo2. so that virus isolation must be attempted w ithin the first tw o weeks of life if there is a suspicion of infection 3 The cross reaction between CMV and other Herpes viruses can yield a false positive result This is caused by polyclonal stimulation of B lymphocytes cross reactivity between other Herpes viruses such as EBV or HHV 6 must alw ays be expected Furthermore the possibility of cross reactions between CMV and Parvovirus cannot be excluded 4 In order to reduce the risk very different types of differential diagnosis are recommended depending on the clinical situation and the symptoms presented in retinitis of the HIV infected for example toxoplasmosis in mononucleosis of immunocompetent patients for example Epstein Barr virus 10 Performance Data SERUM DIAGNOSTIC 10 1 Analytical sensitivity and specificity Analytical sensitivity and specificity were determined by testing 569 sera in CMV IgG ELISA and 670 sera in CMV IgM ELISA in comparison with a reference ELISA IgG analytical sensitivity and specificity Serum collection n 569 Ref ELISA CMV IgG ELISA zen iene negative borderline o This yields a sensitivity of 98 9 and a specificity of 99 5 for IgG Seite 8von 14 REV 9 CMV ELISA IgG igM GB Druckdatum 03 02 2014 IgM analytical sensitivity and specificity Serum collection n 672 Ref ELISA CMV IgM ELISA erence gt negative borderline negative e e borderline o e Pr en This yields a sen
3. 2 to 8C Jest Samples Undiluted 4210 48 2 2 10 8 storage in the provided bag Microtitreplate After Opening with desiccant bag 3 months Rheumatoid factor Undiluted After Opening 2 to 8 Absorbent Diluted 2 to 8 C 210 87 protect from Tight 210 BT protect from ight After Opening ne 1210 325 Seite 4von 14 REV 9 CMV ELISA IgG IgM GB Druckdatum 03 02 2014 6 Precautions and Warnings Only sera w hich have been tested and found to be negative for HIV 1 antibodies HIV 2 antibodies HCV antibodies and Hepatitis B surface antigen are used as control sera Nevertheless samples diluted samples controls conjugates and microtiter strips should be treated as potentially infectious material Please handle products in accordance with laboratory directions Those components that contain preservatives the Citrate Stopping Solution and the TMB have an irritating effect to skin eyes and mucous If body parts are contacted immediately w ash themunder flow ing water and possibly consult a doctor The disposal of the used materials has to be done according to the country specific guidelines 7 Material required but not supplied 1 Aquadest demin 2 Eight channel pipette 50ul 10011 3 Micropipettes 10ul 100pI 1000p 4 Test tubes 5 Paper tow els or absorbent paper 6 Cover for ELISA plates 7 Disposal box for infectious material 8 ELISA handw asher or automated EIA plate w ashing device 9 ELISA plate spec
4. 7 von 14 REV 9 CMV ELISA IgG IgM GB Druckdatum 03 02 2014 9 3 Interpretation Scheme IgG and IgM Result VE Evaluation 30 110 1 IF the measured values are above the defined borderline range they are considered to be positive 2 o the measured VE is within the borderline range no significant high antibody concentration is present the samples are considered to be borderline For the secure detection of an infection it is necessary to determine the antibody concentration of tw o serumsamples One sample shall be taken directly at the beginning of the infection and a second sample 5 10 days later convalescent serum The antibody concentration of both samples has to be tested in parallel that means in one test run A correct diagnosis based on the evaluation of a single serum sample is not possible 3 f the measured values are below the defined borderline range no measurable antigen specific antibodies are present in the samples The samples are considered to be negative 9 4 Limits of the Test 1 The interpretation of serological results should alw ays include the clinical picture epidemiological data and any other laboratory findings that are available 2 The ELISA is not designed to diagnose a CMV infection with risk patients suspected of acute infection A direct detection procedure is to be preferred forimmune compromised patients and pregnant w omen Neonates w ith congenital CMV infections can be serologically normal
5. CMV ELISA IgG IgM Testkit Including performance data for CSF diagnosis Order No EC113 00 IgG IgM Testkit Order No EC113L60 IgG CSF standards Colour code yellow transparent FOR IN VITRO DIAGNOSIS ONLY Sekisui Virotech GmbH Lowenplatz 5 65428 Russelsheim Germany Tel 49 6142 6909 0 Fax 49 6142 966613 http www sekisuivirotech com mdc Notified Body 0483 Druckdatum 03 02 2014 REV 9 CMV ELISA IgG IgM GB Contents 1 Intended EE EE EE a Ri 3 2 Diagnostic Rele Vane cece sc 1 1 1110011 avsar ee yenilen cd savedbayseewccasstewecdsveeweedseteteeveerwtens 3 3 TesiPrinciplez er ak nn Ben aaa la a l sayti yalli adada Alli 4 4 Package Contents IgG IQM Testkit 10 eee aaa 4 5 Storage and Shelflife of the Testkit and the ready to usereagenis 0000 0 2200 4 6 Precautions ANd WarningS ceececeeeeeeeee ence ee eeeee ee eeeeee ee eeeeee aaa anana mennun nennen nenn 5 7 MaterialreguireJdbutnotsupplied 0 eee aaa manen nennen nennen 5 8 Test Procedure SERUM DIAGNOSTIC eee aaa anaa aa ananananan 5 8 1 Examination Material agrenme eyi a an Da AE E A E AN een eher 82 PreparatiolnofReagents 8 3 Virotech ELISA Test Procedure 84 Usage of ELISA process ONS ga a Ra Ra ga vala cud laaan ma il 9 Test Evaluation SERUMDIAGNOSTIC
6. d this will support quality assurance in your laboratory 9 Test Evaluation SERUM DIAGNOSTIC The ready to use controls serve for a semiquantitative determination of specific IgG and IgM antibodies Their concentration canbe expressed in Virotech units VE Fluctuations resulting from the test procedure can be balanced with this calculation method and a high reproducibility is achieved in this w ay Use the means of the OD values for calculation of the VE 9 1 Test function control a OD values The OD of the blank should be lt 0 15 The OD values of the negative controls should be low er than the OD values mentioned in the Quality Control Certificate The OD values of the positive controls as w ellas of the cut off controls should be above the OD values mentioned in the Quality Control Certificate b Virotech Units VE The Virotech Units VE of the cut off controls are defined as 10 VE The calculated VE of the positive controls should be within the ranges mentioned in the Quality Control Certificate If those requirements OD values VE are not fulfilled the test has to be repeated 9 2 Calculation of the Virotech Units VE The extinction of the blank value 450 620nm has to be subtracted from all other extinctions OD positive control x VE positive control OD cut off control OD patient serum VE patient serum OD cut off control Seite 6 von 14 REV 9 CMV ELISA IgG IgM GB Druckdatum 03 02 2014 Seite
7. dies against cytomegalovirus CMV in human serum It can also be used to perform parallel tests of serum CSF pairs for the quantitative detection of endogenous CNS synthesis of IgG antibodies Diagnostic Relevance Epidemiology and incidence CMV is an ubiquitously distributed virus The rate of infection is dependant on the socio economic status of the patient examined It is about 50 in industrial countries w hile in developing countries it is almost 100 The routes of transmission are droplet and contact intimate intercourse transfusions transplantations and prenatal infections An infection w ith CMV can occur in three different stages as primary infection as latency or as reactivation 1 2 3 As with other herpes viruses the primary infection is follow ed by life long latency when no symptoms occur Latency is defined as a form of reversible nonproductive infection of the host by virus capable of reproduction Reactivation can be brought about by a pow erful renewed multiplication of the virus or by renew ed infection A CMV infection usually progresses subclinically in healthy persons The symptoms of a CMV infection are those of a viral syndrome The symptoms are fever fatigue sore throat heterophilic lymphocytosis and liver malfunction In addition there can also be direct organ damage such as pneumonia retinitis colitis oesophagitis hepatitis mononukleosis and meningoencephalitis 2 3 5 Pregnant women The most severe ef
8. fects are those on neonates who have been infected in utero This occurs mainly as a result of primary infection of the woman during pregnancy Such a congenital infection can lead to severe consequences for the neonate such as severe mental damage deafness or death CMV is the most frequent infection of the new born The disease itself breaks out in appreciably less than 10 of infected neonates The outbreak of the disease in the foetus or neonate is dependent on many variables that have not yet been studied 3 4 6 1 4 primary infections 40 transmission to foetus 10 15 CMV disease with 85 80 symptoms asymptomatic 10 90 5 15 85 95 normal development consequential damage consequential damage normal development Figure 1 Proportion of primary reactions in all pregnancy and proportion of subsequent damage Transplant recipients and immunosuppressed patients The immune system of transplant recipients is suppressed in order to avoid rejection reactions against the transplanted organ This becomes critical if the donor is CMV positive and the recipient CMV negative CMV infections precipitate rejection reactions in organ tranplantation The second group is made up of patients with insufficiency of the immune system such as occurs for example after HIV infection Here too the immune system is too weak to prevent the outbreak of a disease caused by CMV Typical damage such as retinitis occurs in th
9. ibited values gt 9 0 for signal cut off in the IgG values for the last three samples taken 10 3 Cross reactivity The follow ing sera were tested in IgG to determine cross reactivity Pathogen Number tested neg bor pos Pov 5 4 2 1 KE m en Ea BE EE All positive results w ere confirmed in the reference test Only one serum exhibited a false positive result This was an EBV positive serum Seite 10 von 14 CMV ELISA IgG igM GB REV 9 Druckdatum 03 02 2014 The follow ing sera w ere tested in IgM to determine the cross reactivity Pathogen Number tested neg bi pos e 3 vx 1 5 yy gt xa e Panowie ae aa The positive results also reacted as positive in a reference test 10 4 Intra assay coefficient of variation repeatability IgG and IgM The intra assay coefficient of variation was determined by using 12 strips from various plates of a batch in a test series All 96 wells were tested with one serum PT wccv mov borderline 80 57 10 2 10 5 Inter assay coefficient of variation reproducibility In the case of IgG 12 different test series w ere carried out in different laboratories and by different testers with 7 positive sera 1 borderline and one negative borderline serum IgG inter assay coefficient of variation MENE VE value negative borderline 90 91 positive 268 100 positive 384 88 positie 250 85
10. is group of patients Seite 3von 14 REV 9 CMV ELISA IgG IgM GB Druckdatum 03 02 2014 When there is reactivation an IgM respose is not to be expected fromimmunocompetent patients but rather in the immunosuppressed 2 3 3 Test Principle The antibody searched for in the human serum forms an immune complex with the antigen coated on the microtiter plate Unbound immunoglobulins are removed by washing processes The enzyme conjugate attaches to this complex Unbound conjugate is again removed by w ashing processes After adding the substrate solution TMB a blue dye is produced by the bound enzyme peroxidase The color changes to yellow when the stopping solution is added 4 Package Contents IgGWigM Testkit 1 1 Microtiter Plate consisting of 96 with antigen coated breakable single w ells lyophilised 2 PBS Dilution Buffer blue readyto use 2x50m1 pH7 2 with preservative and Tw een 20 3 PBS Washing Solution 20x concentrated 50m1 pH 7 2 w ith preservative and Tw een 20 4 IgG negative Control 130041 human serum w ith protein stabilizer and preservative ready to use 5 IgG cut off Control 1300p1 human serum w ith protein stabilizer and preservative ready to use 6 IgG positive Control 130041 human serum w ith protein stabilizer and preservative ready to use 7 IgM negative Control 130041 human serumw ith protein stabilizer and preservative ready to use 8 IgM cut off Control 1300p human serumw ith protein stabilize
11. positie 280 108 positive 274 121 positive 273 141 positive 429 ne In the case of IgM 10 different test series w ere carried out in different laboratories and by different testers with 5 positive sera 1 borderline negative and 3 negative sera Seite 11 von 14 REV 9 CMV ELISA IgG IgM GB Druckdatum 03 02 2014 IgM inter assay coefficient of variation coefficient Mean of arom vee O negative borderline 85 69 positive positive 143 41 positive 443 42 positive 810 43 positie 196 28 11 Performance Data CSF DIAGNOSTIC TESTING 11 1 Analytical sensitivity To determine the analytical sensitivity of the CMV CSF IgG ELISA 25 CSF serum pairs were compared with a reference ELISA CSF sera samples n 25 CMV CSF IgG ELISA Reference ELISA pathological ae EEE nome fi pathological 24 This gives sensitivity of gt 99 9 The false positive is a pathological serum CSF pair w hich was not recognized in the reference test 11 2 Analytical specificity To determine the analytical specificity of the CMV CSF IgG ELISA 26 CSF serum pairs w ere compared with a reference ELISA CSF sera group n 26 CMV CSF IgG ELISA Reference ELISA pathological EEE eee aa pathologic oO J To This gives specificity of gt 99 9 11 3 Intra assay coefficient of variation repeatability To determine the intra assay coefficient of
12. r and preservative ready to use 9 IgM positive Control 1300p human serumw ith protein stabilizer and preservative ready to use 10 IgG Conjugate anti human 11ml sheep or goat horseradish peroxidase conjugate with protein stabilizer and preservative in Tris Buffer ready to use 11 IgM Conjugate anti human 11ml sheepor goat horseradish peroxidase conjugate with FCS and preservative in Tris Buffer ready to use 12 Tetramethylbenzidine substrate solution 3 3 5 5 TMB 11m1 ready to use 13 Citrate Stopping Solution 6mI contains an acid mixture 5 Storage and Shelflife of the Testkit and the ready to use reagents Store the testkit at 2 8 C The shelf life of all components is show n on each respective label for the kit shelf life please see Quality Control Certificate 1 oMicrotiter strips single w ells are to be resealed in package after taking out single w ells and stored w ith desiccant at 2 8 C Reagents should immediately be returned to storage at 2 8 C after usage 2 The ready to use conjugate and the TMB substrate solution are sensitive to light and have to be stored in the dark Should there be a color reaction of the substrate dilution due to incidence of light it is not useable anymore 3 o Take outonly the amount of ready to use conjugate or TMB needed for the test insertion Additional conjugate or TMB taken out may not be returned but must be dismissed oo Material Status Storage Shelflife Diluted
13. se TMB into each well Incubation of substrate solution 30 min at 37 C with cover keep in dark Stopping of substrate reaction pipette 50ul of citrate stopping solution into each w ell Shake plate carefully and thoroughly until liquid is completely mixed and a homogeneous yellow color is visible 10 Measure extinction OD at 450 620nm Reference Wavelength 620 690nm Set your photometer in such a w ay that the blank value is deducted fromall other extinctions Extinctions should be measured within 1 hour after adding the stopping solution om OAL OLA Pls refer to last page for Test Procedure Scheme 8 4 Usage of ELISA processors All Sekisui Virotech ELISAs can be used on ELISA processors The user is bound to proceed a validation of the devices processors on a regular basis Sekisui Virotech recommends the follow ing procedure 1 Sekisui Virotech recommends to proceed the validation of device referring to the instructions of the device manufacturer during the implementation of the ELISA processor respectively after bigger reparations 2 It is recommended to check the ELISA processor with the Validationkit EC250 00 afterw ards A regular check using the Validationkit shall be proceeded minimum once a quarter to test the accuracy of the processor 3 The release criteria of the Quality Control Certificate of the product must be fulfilled for each testrun With this procedure your ELISA processor will function properly an
14. sitivity of 96 5 and a specificity of 98 5 for IgM Borderline sera w ere not included in the calculation Diagnostic sensitivity The diagnostic sensitivity was determined by testing 81 clinically characterized sera in CMV IgM ELISA and 83 in CMV IgM ELISA IgG diagnostic sensitivity Serum collection reactivations n 81 as ee ef This yields a sensitivity of 97 5 IgM diagnostic sensitivity Serum collective reactivations n 80 and primary infections n 3 res 5 ef This yields a sensitivity of 93 9 Of the 5 negative sera one serum also tested negative and one serum as borderline in the reference test Seite 9 von 14 REV 9 CMV ELISA IgG IgM GB Druckdatum 03 02 2014 10 2 Detection limits The seroconversion panel PTC 901 was tested The VT CMV ELISA exhibited the expected sensitivity in IgG and in IgM BBI seroconversion IgG BBI seroconversion IgM 9 0 14 0 8 0 12 0 7 0 10 0 5 6 0 5 3 5 0 3 8 0 5 40 60 2 a 3 0 4 0 2 0 2 0 1 0 0 08 in 0 20 40 60 80 0 20 40 60 80 Days after first sampling Days after first sampling VT ELISA VE 10 a Abbott AxSYM VT ELISA VE 10 a Abbott Imx a Abbott Imx bioMerieux Vidas a bioMerieux Vidas Centocor EIA Gull EIA Zeus EIA Gull EIA Zeus EIA Original data from BBI The bioMerieux Vidas exh
15. trate to 1 L with distilled or demineralised w ater If crystals have formed in the concentrate please bring the concentrate to roomtemperature before use and shake w ell before use 5 High IgG titer or rheumatoid factors may disturb the specific detection of IgM antibodies and may lead to false positive resp false negative results For acorrectigM determination it is therefore necessary to pre treat the sera with RF SorboTech VIROTECH adsorbent For IgM controls a pre absorbent treatment is not necessary 8 3 Virotech ELISA Test Procedure 1 For eachtestrun pipette 100ul each of ready to use dilution buffer blank IgG and IgM positive negative and cut off controls as w ell as diluted patient sera We propose a double insertion blank controls and patient sera for cut off control a double insertion is absolutely necessary Working dilution of patient sera 14100 e g 10ul serum 1ml dilution buffer Seite 5von 14 REV 9 CMV ELISA IgG IgM GB Druckdatum 03 02 2014 After pipetting start incubation for 30 min at 37 C with cover End incubation period by w ashing microtiter strips 4 times w ith 350 400ul w ashing solution per w ell Do not leave any w ashing solution in the w ells Remove residues ona cellulose pad Pipette 100ul of ready to use conjugate into each well Incubation of conjugates 30 min at37 C with cover Stop conjugate incubation by w ashing 4 times pls refer to point 3 above Pipette 100u of ready to u
16. trophotometer w avelength 450nm reference length 620nm Reference Wavelength 620 690nm 10 Incubator 8 o Test Procedure SERUM DIAGNOSTIC Working exactly referring to the Sekisui Virotech user manual is the prerequisite for obtaining correct results 8 1 Examination Material Either serum or plasma can be used as test material even if only serum is mentioned in the instructions Any type of anticoagulant can be used for plasma Alw ays prepare patient dilution freshly For a longer storage the sera must be frozen Repeated defrosting should be avoided 1 Only fresh non inactivated sera should be used 2 o Hyperlipaemic haemolytic microbially contaminated and turbid sera should not to be used false positive results 8 2 Preparation of Reagents The Sekisui Virotech System Diagnostica offers a high degree of flexibility regarding the possibility to use the dilution buffer washing solution TMB citrate stopping solution as well as the conjugate for all parameters and for all different lots The ready to use controls positive control negative control cut off control are parameter specific and only to use with the plate lot indicated in the Quality Control Certificate 1 Set incubator to 37 C and check proper temperature setting before start of incubation 2 Bring all reagents to room temperature bef ore opening package of microtiter strips 3 Shake all liquid components w ell before use 4 Make up the washing solution concen
17. variation a CSF serum pair with normal Al value and a CSF serum pair with pathological Al w ere tested 10 times in a run ev SEE Seite 12 von 14 REV 9 CMV ELISA IgG IgM GB Druckdatum 03 02 2014 11 4 Inter assay coefficient of variation reproducibility To determine the inter assay coefficient of variation a CSF serum pair w ith normal Al value w as tested 10 times in different laboratories by different w orkers A CSF serum pair w ith normal Al value w ith pathological Al value w as tested 11 times in different laboratories by different workers a re Normal Al Pathological Al 12 Literature 1 Darai G M Handermann and E Hinz 2003 Lexikon der Infektionskrankheiten des Menschen 2 ed Springer Berlin 2 Gold E Nankervis G 1989 Cytomegalovirus p 169189 InA Evans ed Viral Infections of Humans 3 ed Plenum Medical Book Company New York London 3 Mocarski E 1999 Cytomegaloviruses p 344357 In A W Granoff R ed Encyclopedia of Virology 2 ed vol 1 Academic Press San Diego San Francisco New York Boston London Sydney Tokio 4 Revello M G and G Gerna 2002 Diagnosis and management of human cytomegalovirus infection in the mother fetus and new born infant Clin Microbiol Rev 15 680715 5 Froberg M K 2004 Review CMV escapes Ann Clin Lab Sci 34 12330 6 Lazzarotto T L Gabrielli M Lanari B Guerra T Bellucci M Sassi and M P Landini 2004 Congenital cytomegalo
18. virus infection recent advances in the diagnosis of maternal infection Hum Immunol 65 4105 Seite 13 von 14 REV 9 CMV ELISA IgG IgM GB Druckdatum 03 02 2014 13 Test Procedure Scheme Preparation of Patient Samples and Washing Solution V Washing Solution Fill up concentrate to 1 liter with aqua dest demin y IgG Samples Dilution 1 101 e g 10 ul serum plasma 1000 ul Dilution Buffer Serum Dilution Buffer is ready to use y IgM Samples Dilution 1 101 Rheumafactor absorption with RF SorboTech e g 5 ul serum plasma 450 ul Dilution Buffer 1 drop RF SorboTech incubate for 15 min at room temperature Testprocedure Samples Incubation 30 minutes at 37 C Wash 4times Conjugate Incubation 30 minutes at 37 C Wash 4times Substrate Incubation 30 minutes at 37 C Stopping Measure Extinctions Seite 14 von 14 CMV ELISA IgG IgM GB 100 pl Patient Samples blank value Dilution Buffer and controls 400 ul Washing Solution Remove Residues on a Cellulose Pad 100 ul Conjugate IgG IgM IgA 400 ul Washing Solution Remove Residues on a Cellulose Pad 100 ul Substrate 50 ul Stopping Solution shake carefully Photometer at 450 620nm Reference Wavelength 620 690nm REV 9 Druckdatum 03 02 2014
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