Package Insert - Sekisui Diagnostics
Contents
1. 1992 Use of enzyme linked immunosorbent assays with chimeric fusion proteins to titrate antibodies against Epstein Barr virus nuclear antigen 1 J Clin Micro 30 6 1442 1448 7 Lennette ET and W Henle 1987 Epstein Barr virus infections clinical and serological features Lab Manage 25 23 28 8 Bauer G 1995 Rationale und rationelle Epstein Barr V irus Diagnostik Clin Lab 41 623 634 9 Bauer G 2001 Simplicity through complexity immunoblot w ith recombinant antigens as the new gold standard in Epstein Barr virus serology Clin Lab 47 223 230 10 Modrow S und D Falke 2010 Das Epstein Barr Virus S 572 577 In Molekulare Virologie Spektrum Verlag ISBN 978 3 8274 1833 3 Seite 8von 9 REV 15 EBV ELISA IgG IgM GB Druckdatum 03 02 2014 12 Test Procedure Scheme Preparation of Patient Samples and Washing Solution V Washing Solution Fill up concentrate to 1 liter with aqua dest demin v IgG Samples Dilution 1 101 e g 10 ul serum plasma 1000 ul Dilution Buffer Serum Dilution Buffer is ready to use v 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 Ex2. blue readyto use 2x50ml pH7 2 w ith preservative and Tw een 20 PBS Washing Solution 20x concentrated 50ml pH7 2 w ith preservative and Tw een 20 IgG negative Control 1300yl human serum w ith protein stabilizer and preservative ready to use IgG cut off Control 1300pl human serum w ith protein stabilizer and preservative ready to use IgG positive Control 1300pl human serum w ith protein stabilizer and preservative ready to use IgM negative Control 1300pl human serumw ith protein stabilizer and preservative ready to use IgM cut off Control 1300pl human serumw ith protein stabilizer and preservative ready to use IgM positive Control 1300pl human serumw ith protein stabilizer and preservative ready to use IgG Conjugate anti human 11ml sheep or goat horseradish peroxidas e conjugate with protein stabilizer and preservative in Tris Buffer ready to use IgM Conjugate anti human 11ml sheepor goat horseradish peroxidase conjugate with FCS and preservative in Tris Buffer ready to use Tetramethylbenzidine s ubstrate s olution 3 3 5 5 TMB 11ml ready to use Citrate Stopping Solution 6ml contains an acid mixture CO RAN BON 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 Mirotiter strips single w ells are to be resealed i
3. 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 VE positive control x10 OD cut off control OD patient serum VE patient serum x10 OD cut off control 9 3 Interpretation Scheme IgG and IgM Resut VE Evaluation 1 1 If the measured values are above the defined borderline range they are considered to be positive 2 lf the measured VE is within the borderline range no significant high antibody concentration is present the samples are considered to be borderline 3 For the secure detection of an infection it is necessary to determine the antibody concentration of tw o serum samples 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 have 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 4 If the measured values are below the defined borderline range no measurable antigen specific IgM antibodies are present in the sample The samples are considered to be negative Notice of a primary infection 9 4 Lim
4. EBV ELISA IgG IgM Testkit Order No EC102 00 Color Coding yellow FOR IN VITRO DIAGNOSIS ONLY Sekisui Virotech GmbH L wenplatz 5 65428 R sselsheim Germany Tel 49 6142 6909 0 Fax 49 6142 966613 http www sekisuivirotech com ce Druckdatum 03 02 2014 REV 15 EBV ELISA IgG IgM GB Contents DEMIn dngr 3 2 Diagnostic Relevance 3 1i eerie tei ee ee cese reo does Took ee weno treo cc res eno e er no ga i 3 3 Test Principle enun 3 4 Package Contents IgG and IgM Testkit urnneuunennnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nenn nnn 3 5 Storage and Shelflife of the Testkit and the ready to use reagents 3 6 Precautions and Warnings eese nn nnne nn hnius nn nanus nn nnn 4 7 Material required but not supplied uuesrssnnsnnnsennnnnnennnnnnnennnnnnennnnnnnnnnn nennen nnn nn nnn 4 8 Test Procedure ciuile ERDE NEEREEEHEEEEFEFFEFEERECHESEEFEREREEEFFEERERECHERIEFECESELEEENEETERECHEREERECEEEER 4 8 1 Bami ation Mat ral se een asien TU M 4 8 2 Preparation of Reagents Bici E UNI MD toe oe A AL IAN TL 4 8 3 Virotech A SA Test Proced re roter eet reb be AR a rE E Ea ERREUR 5 8 4 Usage of ELSA prot ESSO siena toe ter ina e E PED A kcu namen rx ee dare e ua eta ten RE Ran E nga ehr 5 NI davit
5. IM 5 9 1 Test function control inet tetro Ete desde e NIH ERE EET RET Ter dena 9 2 Calculation of the Virotech Units VE 9 3 Interpretation Scheme IgG and IgM ess ux 9 4 Limits of the Test emo tee eterne Ee eer re Ela ee ree PER Te irre Ner aet manas e e TUR E Rie ee DATO NU ENe NET UR E 10 Performance Dalta 5 ae nun ose ces EN orc Reli cci eain 7 10 1 Diagnostic l SernsitiVILy onere eroe HE icr a YE ee 7 10 2 Analytical Sensitivity EE T T emcees te 7 10 3 Analytical Specficity nee BEER 7 10 4 Prevalence Expected Values ursus ts a inne DOR PR PE Dd eb ER Re I i CHR a 8 10 5 Intra assay Coefficient of Variation Repeatability 8 10 6 Inter assay Coefficient of Variation Reproducibility seen emen 8 ga NM CT CETTE DII Im 8 12 Test Procedure Scheme nn 9 Seite 2von 9 REV 15 EBV ELISA IgG IgM GB Druckdatum 03 02 2014 1 5 _ _ em em Intended Use The EBV ELISA is intended for the semiquantitative and qualitative detection of IgM and IgG antibodies against Epstein Barr Virus A diagnostic statement w ith regard to seronegativity suspicion to an EBV primary infection or past EBV infection can be made w hen combining the tw o antibody classes Diagnostic Relevance The Epstein Barr virus belongs to the family of the Herpesviridae and is transmitted mainly by saliva w hen it infects the epithelial cells of the oro
6. d for all different lots The Seite 4 von 9 REV 15 EBV ELISA IgG IgM GB Druckdatum 03 02 2014 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 Pe ER vos 8 3 o N OP CO N OLOA 10 Set incubator to 37 C and check proper temperature setting before start of incubation Bring all reagents to room temperature before opening package of microtiter strips Shake all liquid components w ell before use Make up the w ashing solution concentrate to 1 L w ith 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 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 Virotech ELISA Test Procedure For each test run pipette 100ul each of ready to use dilution buffer blank IgG and IgM positive negative and cut off controls as w ellas diluted patient sera We propose a double insertion blank controls and patient sera for cut off controla double insertion is absolutely necessary Working dilution of patient sera 1 100 e g 10ul s
7. erum 1ml dilution buffer After pipetting start incubation for 30 min at 37 C w ith 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 washing solution in the w ells Remove residues on a cellulose pad Pipette 100ul of ready to use conjugate into each well Incubation of conjugates 30 min at 37 C w ith cover Stop conjugate incubation by w ashing 4 times pls refer to point 3 above Pipette 100ul of ready to use 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 Measure extinction OD at 450 620nm Reference Wavelength 620 690nm Set your photometer in sucha w ay that the blank value is deducted fromall other extinctions Extinctions should be measured w ithin 1 hour after adding the stopping solution 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 3 Sekisui Virotech recommends to proceed the validation of device referring to the instructions of the device
8. its of the Test 1 The interpretation of serological results shall always include the clinical picture epidemiological data and all further available laboratory results 2 A negative ELISA result does not completely exclude an EBV infection 3 Infective agents with similar clinical picture should be considered in differential diagnosis Seite 6 von 9 REV 15 EBV ELISA IgG IgM GB Druckdatum 03 02 2014 4 Crossreactivities of Epstein Barr Virus against the Herpes Virus family are know n With a positive IgM result specially crossreactivities against CMV should be excluded 5 A negative IgM result does not exclude the possibility of a primary infection as in some cases no IgM is built during an acute infection IgM nonresponder 8 9 6 With clinical suspicion of an EBV infection and a negative serology a second blood sample should be taken 7 A positive EBV ELISA IgM result should be checked by specific EBV confirmation tests Virotech ELISA versus VCA IgM VCA IgG EBNA 1 IgG EA D IgG or through Virotech EBV LINE 8 Antibodies which w ere passively transmitted shortly before examination may influence the EBV serological result This might be e g by blood transfusion or maternally transmitted antibodies to the infant 10 Performance Data 10 1 Diagnostical Sensitivity 19 clinically characterized sera frompatients w ith primary infection were tested to determine the diagnostical sensitivity sera from Dr G rtner Homburg Saar I
9. manufac turer during the implementation of the ELISA processor respectively after bigger reparations 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 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 and this w ill support quality as surance in your laboratory Test Evaluation The ready to use controls serve for a semiquantitative determination of specific IgG and IgM antibodies Their concentration can be 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 Seite 5von 9 REV 15 EBV ELISA IgG IgM GB Druckdatum 03 02 2014 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
10. mmunoblots and or IFA were used as reference tests VT EBV ELISA Sera Collective n 19 IgG IgM overall result Primary Infection Past Infection Analytical Finding Past Infection The results of the Virotech EBV ELISA correspond with the clinical and analytical findings in all cases 10 2 Analytical Sensitivity Follow ing sera collectives w ere tested to determine the analytical sensitivity 1 Sserafrompatients w ith EBV primary infection n 20 serafromDr Gartner Homburg Saar and proficiency testings 2 Sserafromdonors w ith past EBV infection n 41 serafromDr G rtner Homburg Saar Immunoblots and or IFA w ere used as reference tests VT EBV ELISA Sera collective n 61 IgG IgM overall result Primary Infection Past Infection Primary Infection Analytical y Infection The results of the Virotech EBV ELISA correspond with the reference findings in all cases 10 3 Analytical Specificity Follow ing sera collective w as tested to determine the analytical specificity 20 seronegative sera sera from Dr G rtner Homburg Saar Immunoblots and or IFA w ere used as reference tests Sera collective n 20 TE IgG IgM overall result Seite 7 von 9 REV 15 EBV ELISA IgG IgM GB Druckdatum 03 02 2014 Primary Infection Seronegative Analytical seronegative 2 Finding Past Infection The results of the Virotech EBV ELISA correspond with the reference findings in all cases 10 4 Prevalence E
11. n 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 Theready to use conjugate and the TMB substrate solution are sensitive to light and have to be stored in dark Should there be a color reaction of the substrate dilution due to incidence of light it is not useable anymore Seite 3von 9 REV 15 EBV ELISA IgG IgM GB Druckdatum 03 02 2014 3 Take out only 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 Material Storage Sheme Status Diluted 7210 8T Lesh Samples Undiluted 1210 48 n Microtitreplate After Opening IT um Psal bag 1900 2a9 Rheumatoid Tactor i 3 After Opening 2 to 8 M 3210 325 6 Precautions and Warnings 1 Only seraw hich have been tested and found to be negative for HIV 1 antibodies HIV 2 antibodies HCV antibodies and Hepatitis B surf ace 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 2 Those components that contain preservatives the Citrate Stopping Solution and the TMB have an irritating effectto skin eyes and mucous If body parts are contacted immediately w ash themunder flo
12. pharynx initially and then the B lymphocytes The virus is the cause of infectious mononucleosis IM and chronic active EBV infection There is also an association betw een EBV infections and Burkitt s lymphoma and also nasopharyngeal carcinomas in Africa and Asia According to serological investigations approx 95 of adults are seropositive for EBV Primary EBV infections are normally asymptomatic but can be the cause of infectious mononucleosis in adolescents and young adults IM is a self limiting illness and is characterised by lymphadenopathy fever hepatosplenomegaly and leukocytosis w ith atypical lymphocytes 1 7 The role of EBV serology consists of differentiating betw een or confirming seronegativity primary infection and previous infection and in making a differential diagnosis from clinically similar symptomatic diseases 8 Test Principle The antibody searched for in the human serum forms an immune complex w ith the antigen coated on the microtiter plate Unbound immunoglobulins are removed by w ashing 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 w hen the stopping solution is added Package Contents IgG and IgM Testkit 1 Microtiter Plate consisting of 96 with antigen coated breakable single w ells lyophilised PBS Dilution Buffer
13. tinctions Seite 9 von 9 EBV 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 400 ul Washing Solution Remove Residues on a Cellulose Pad 100 pl Substrate 50 ul Stopping Solution shake carefully Photometer at 450 620nm Reference Wavelength 620 690nm REV 15 Druckdatum 03 02 2014
14. w ing water and possibly consult a doctor 3 The disposal of the used materials has to be done according to the country specific guidelines 7T Material required but not supplied Aqua dest demin Eight channel pipette 50ul 100yl Micropipettes 10ul 100ul 1000uI Test tubes Paper tow els or absorbent paper Cover for ELISA plates Disposal box for infectious material ELISA handw asher or automated EIA plate w ashing device ELISA plate spectrophotometer w avelength 450nm reference length 2 620nm Reference Wavelength 620 690nm Incubator OAO Q0 c Lg Or dx QUIS _ 8 Test Procedure 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 Hyperlipaemic haemolytic microbially contaminated and turbid sera should not to be used false positiv e negative 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 an
15. xpected Values 77 sera were tested to determine the prevelance rate of 95 in adults past EBV infection as described in literature 10 Den Da Seracollektive n 77 10 5 Intra assay Coefficient of Variation Repeatability In one assay strips of different plates of one batch have been tested w ith the same serum sample The obtained coefficient of variation is 996 10 6 Inter assay Coefficient of Variation Reproducibility Three sera were tested in 10 independent test runs by different persons in different laboratories EBV ELISA IgG Serum Average Value VE ___CoefficientofVarition Positive 2 11 7 11 Literature 1 Evans A S J C Niedermann L C Cenabre B West and V A Richards 1975 A prospective evaluation of heterophile and EBV specific IgM antibody tests in clinical and subclinical infectious mononucleosis J Infect Dis 132 546 554 2 Nikoskelainen J J Lelkola and E Lelkola 1974 IgM antibodies specific for EBV in IM without heterophile agglutination test Br Med J 4 72 75 3 Klemola E R von Essen G Henle and W Henle 1970 IV like disease w ith negative heterophile agglutination test J Infect Dis 121 608 614 4 Sumaya C V and Y Ench 1985 EBV mononucleosis in children Il paediatrics 75 1011 1019 5 Schmitz H D Volz C H Krainlek Richert and M Schere 1972 Acute EBV infections in Children Med Microbiol Immunol 158 58 63 6 Inoue N et al
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