Package Insert - Sekisui Diagnostics
Contents
1. at 37 C with 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 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 f romall other extinctions Extinctions should be measured w ithin 1 hour after adding the stopping solution on o 20M al ODE OM 7 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 Qu2. ELISA plates Disposal box for infectious material ELISA handw asheror automated EIA plate w ashing device ELISA plate spectrophotometer w avelength 450nm reference length 620nm Reference Wavelength 620 690nm Incubator 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 2 Only fresh non inactivated sera should be used Hyperlipaemic haemolytic microbially contaminated and turbid sera should not to be used false positive 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 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 Pe eh cct Set incubator to 37 C and check proper temperature setting before start of incubation Bring all reagents to room temperature before opening
3. HIV patient sera MER 100 91 in Germany 6 59 in Germany 2 Pregnant w omen ca 90 in Spain and Italy 81 3 8 996 in Germany 6 sera n 80 6 i D Children sera n 40 Seite 8 von 10 REV 11 HSV Screen ELISA IgG IgM GB Druckdatum 03 02 2014 IgM The follow ing table gives the results found with the Virotech HSV Screen ELISA for the selected sera groups Sera group n 224 Borderline Positive in Literature Values for en the HSV Screen ELISA HSV 1 and 2 Blood donors n 80 5 1 96 Children sera n 40 7834 J SSS Prostitute sera n 40 12835 1 HiVpatentseran 24 167 10 4 Intra assay variation coefficient repeatability In one assay strips of various plates of one lot were tested with one serum This gave a variation coefficient of 9 10 5 Inter assay variation coefficient reproducibility 3 sera were tested in 10 independent tests in different laboratories and w ith different operators This gave a variation coefficient of under 15 11 Literature 1 Wutzler P et al 1999 Bundesgesundheitsbl Gesundheitsf orsch Gesundheitsschutz 10 99 766 782 2 Rabenau H F et al 2002 Seroprevalence of herpes simplex virus types 1 and type 2 in the Frankfurt am Main area Germany In Med Microbiol Immunol 190 4 153 160 3 Aurelian L Herpes Simplex Viruses 473 497 In Specter S amp G Lancz eds Clinical Virology Manual 2 Ed Elsevier New Y
4. TMB needed for the test insertion Additional conjugate or TMB taken out may not be returned but must be dismissed Material Strage Sheme Status Diluted 321048 Test Samples Undiluted 121048 20 48 g Microtitreplate After Opening 2 to 8 storage in the provided bag with desiccant bag Rheumatoid factor Absorbent After Opening After Opening After Opening COE id ene Seite 4 von 10 REV 11 HSV Screen ELISA IgG IgM GB Druckdatum 03 02 2014 6 x 8 2 09 80 OOS NO oe 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 Material required but not supplied Aqua dest demin Eight channel pipette 50ul 100 1 Micropipettes 10ul 100ul 1000uI Test tubes Paper tow els or absorbent paper Cover for
5. Wash 4times Conjugate Incubation 30 minutes at 37 C Wash 4times Substrate Incubation 30 minutes at 37 C Stopping Measure Extinctions Seite 10 von 10 HSV Screen 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 11 Druckdatum 03 02 2014
6. the CNS at the time when the CSF sample is taken may still or already be too low to increase the Al Therefore if the gG1 or gG2 test is performed alone this Seite 3von 10 REV 11 HSV Screen ELISA IgG IgM GB Druckdatum 03 02 2014 may give a false negative result in some cases or in specific cases meaning that the antibody index is neither not calculable or is normal 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 washing 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 IgG and IgM Testkit 1 Microtiter Plate consisting of 96 with antigen coated breakable single w ells lyophilised PBS Dilution Buffer blue readyto use 2x50ml pH 7 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
7. 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 substrate solution 3 3 5 5 TMB 11ml ready to use Citrate Stopping Solution 6ml contains an acid mixture O O 00 NOY Ove coco _ TE D N 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 Mirotiter 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 dark Should there be a color reaction of the substrate dilution due to incidence of light it is not useable anymore 3 Take out only the amount of ready to use conjugate or
8. E A OPER xL eta M EDAD IE 6 cr iqariwtunpEmece Ims 6 9 1 Testf nction Control cte haa Aas etae ede en ERU SERRE ERREUR 9 2 Calculation of the Virotech Units VE 9 3 Interpretation Scheme IgG and IgM ess us 9 4 Limits of the Testi emot ATEN Ee enfer re Eia se nde Pe e arre Pope aetas e TUR Rie e DATE P Een feed eod 10 Performance Data ici lt icccic ccccccatzecesstecececeseccneseceedecencetevscctoedcasacecuseiedceccasaccereccaasentececvetcenestecse 7 10 1 Analytical Sensitivity and specif City erect tete ecrire eret ttt tait eec dann pa ca 7 10 2 CrOsssresctvily 22 en tia ee rode tede mee teen aee 8 10 3 Prevalence expected Values 9 ectetuer t dete IH 8 10 4 Intra assay variation coefficient repeatability sseesesseeeeeeeneeeennennnen nennen nennen 9 10 5 Inter assay variation coefficient reproducibility nennen nennen nen 9 JE NICHIL MAP secon sedans oe ccassdetoscweceseteetonccavecszocsecss EEE aeea raaa eE ee aT 9 12 Test Procedure Scheme itii 22 Lee a ec trend cheese eee aes 10 Seite 2von 10 REV 11 HSV Screen ELISA IgG IgM GB Druckdatum 03 02 2014 1 Intended Use The HSV Screen ELISA is used for the semi quantitative and qualitative detection of specific IgG and IgM antibodies to Herpes simplex Virus HSV 1 2 in human serum The HSV Screen ELISA uses a combination of purified HSV 1 and HSV 2 antigens Differentiation betw een HSV 1 and HSV 2 is only p
9. HSV Screen ELISA IgG IgM Testkit Order No EC108 00 Color Coding red metallic 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 ce Druckdatum 03 02 2014 REV 11 HSV Screen ELISA IgG IgM GB Contents DEMIn dOngr M n 3 2 Diagnostic Relevance 3 reiecit te ee t eec cese reo does Teo ao ree weno trao cc ree weno e er novae rei co 3 3 Test Principle ennuinrinID enel 4 4 Package Contents IgG and IgM Testkit urnnuuunennnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nennen 4 5 Storage and Shelflife of the Testkit and the ready to use reagents 4 6 Precautions and Warnings eese esee nnne nennen nennen nenn 5 7 Material required but not supplied unssssnnsnnnennnnnnnennnnnnnnnnnnnnennnnnnnennn anne nn nnn nn nnn 5 8 Test Procedure WERRRRESHEREUENERNERARREEFRARE ERROR REEREEEHEEEEEFFEFRERECHESEEEEFEEEEEFFEERERECHEEIEFECESELEEENEENERECHEREERECEREER 5 8 1 Examination Material icri per e Eee ORE een 5 8 2 Preparation of Reagents J saetveceesdveds 5 8 3 Virotech ELISA Test Proced re roter eet reb Ae shou stenatt E SEE ERIT SEO OET 5 9 4 Usage ol ELISA DIOCOSSOIS 5e toe ter Eas e E PE aaae eA EA EEE NE
10. M cannot be given 10 2 Cross reactivity IgG 40 potentially cross reactive sera with positive serological findings for at least one of the following pathogens EBV CMV VZV parvovirus and measles were tested with the Virotech HSV 1 gG1 HSV 2 gG2 ELISA and the Virotech HSV LINE All seraw hich were negative in both HSV reference systems were also negative in the HSV Screen ELISA This gives the specificity of 100 for the above panel IgM 40 potentially cross reactive sera with positive serological findings for at least one of the following pathogens EBV CMV VZV parvovirus and measles were also tested for IgM with the Virotech HSV 1 gG1 and HSV 2 gG2 ELISA 2 of 40 sera with negative findings in the HSV reference system gave positive findings in the HSV Screen This gives a specificity of 95 for the above panel 10 3 Prevalence expected values IgG The follow ing table gives the results found with the Virotech HSV Screen ELISA for the selected sera groups They are compared w ith epidemiological data taken from the literature Borderline Sera group Positive in the Literature Values for Literature Values for n 256 HSV Screen HSV 1 HSV 2 ELISA 79 in Germany 6 15 in Germany 6 30 1 5 years lt 2 1 5 years 45 6 11 years lt 3 6 11 years 50 12 16 years ca 8 12 16 years in Germany 6 in Germany 6 64 for STD patients in the Prostitute sera n 40 91 95 in Germany 7 USA 5 78 in Germany 2
11. ality 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 assurance in your laboratory 9 Test Evaluation The ready to use controls serve for asemiquantitative 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 10 VE positive control OD cut off control OD patient s
12. e genital area the infections are mostly caused by HSV 2 Only a small part 5 30 is generated by HSV 1 1 The genital HSV 1 infections recurrent considerably more rarely than HSV 2 infections A previous infection w ith genital HSV1 seems to give a certain protection of infections with HSV 2 respectively allays the symptoms or entirely prevent them 1 A previous oral HSV 1 infection does not protect against a genital HSV 2 infection 2 The clinical picture of genital herpes corresponds those of other ulceration of the sexual organs and has therefore to be differentiated against Haemophilus durcreyi Treponema pallidum and Chlamydia trachomatis Viral isolation direct fluorescent antibody DFA testing and serology can be used to diagnose HSV infections Disadvantages of the first tw o methods are how ever length of culture time specimen collection and transport difficulties procedural complexity and other variables that are associated w ith DFA and culture 3 4 How ever due to the significant cross reactivity betw een HSV 1 and HSV 2 the serological assays that use virus lysates as antigens are not sufficiently Suited to differentiate HSV 1 infections from HSV 2 infections Due to the high contamination w ith HSV 1 the serological status for HSV 2 can be detected hardly reliable w ith such methods 1 Intrathecal IgG antibodies occur only 8 10 days after the clinical symptoms in a present Herpes encephalitis IgM antibodies are not
13. e herpes group cannot be totally excluded 10 Performance Data 10 1 Analytical sensitivity and specificity IgG The detection of IgG in 336 sera was compared with the Virotech HSV 1 gG1 and HSV 2 gG2 ELISA ea Veen SV 1001 and Bande 1 Io S HSV 2 g62 ELISA enge ee 10 sera w ith borderline findings w ere excluded fromthe analysis On the basis of these results the analytical sensitivity for IgG was calculated as 97 9 and the analytical specificity as 89 2 In addition the detection of IgG in 336 sera w as compared with the Virotech HSV LINE HSV Screen ELISA ene mete 88 2 2 nein Boi e 86 7 eo Virotech HSV LINE borderline 18 3 11 posive 3 0o 29 34 sera with borderline findings w ere excluded from the analysis On the basis of these results the analytical sensitivity for IgG was calculated as 98 7 and the analytical specificity as 97 1 IgM Seite 7 von 10 REV 11 HSV Screen ELISA IgG IgM GB Druckdatum 03 02 2014 The detection of IgM in 344 sera was compared with the Virotech HSV 1 gG1 and HSV 2 gG2 ELISA Sera group n 344 HSV Screen ELISA gar ee AE Eee PERS TEC soorseeine a ae rev ZIERT none 9 0 17 sera with borderline findings w ere excluded from the analysis On the basis of these results the analytical specificity for IgM w as calculated as 91 3 Because of the low number of positive sera the analytical sensitivity for Ig
14. erum OD cut off control VE patient serum Seite 6 von 10 REV 11 HSV Screen ELISA IgG IgM GB Druckdatum 03 02 2014 9 3 Interpretation Scheme IgG and IgM Result VD Evaluation 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 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 If 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 4 In case of a positive IgM result a review of the result by observing the IgG titercourse is recommended 9 4 Limits of the Test 1 The interpretation of serological results shall always include the clinical picture epidemiological data and all further available laboratory results 1 Therapy with acyclovir can influence antibody formation 5 2 Cross reactions w ith other members of th
15. ork 1992 4 CDC 1998 Guidelines for Treatment of Sexually Transmitted Diseases MMWR 47 1998 5 LiZetal 1999 Acyclovir treatment of skin lesions results in immune deviation in mice infected cutaneously with herpes simplex virus Dep of Virology Toyama Medical and Pharmaceutical University Japan 6 Wutzler P et al 2000 Seroprevalence of Herpes Simplex Virus Type 1 and type 2 in Selected German Populations Relevance for the Incidence of Genital Herpes In Journal of Medical and Virology 61 201 207 7 Smith J amp Robinson N 2002 The Journal of Infectious Diseases 186 3 28 8 Sauerbrei A Wutzler P 2006 Herpes simplex and varicella zoster virus infections during pregnancy current concepts of prevention diagnosis and therapy Part 1 Herpes simplex virus infections In Med Microbiol Immunol Springer Verlag Seite 9 von 10 REV 11 HSV Screen 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
16. ossible if glycoproteins G are used If there is a positive finding in HSV screening w e therefore recommend subsequent differentiation w ith a type specific ELISA or HSV LINE gG1 gG2 The serology is suitable for the detection of the immune status and as Herpes exclusion The IgM result must not be observed isolated from the IgG result The diagnosis of the genital herpes must be confirmed w ith the pathogen detection The serology is not suitable for the detection of new born Herpes as the immune system of a baby is not completely developed at the date of its birth How ever itcan be usedin retrospect to measure the transplacentally transferred anti HSV IgG antibodies 2 Diagnostic Relevance Herpes simplex viruses are widely spread throughout the population The transmitted results fromdirect contact w ith infected secretions fromeither a symptomatic or an asymptomatic host Therefore the contamination starts already in the early child age How ever these primary infections remain asymptomatical in over 9096 of the cases a latent infection is established in the regional ganglia as a rule For the understanding of the pathogenesis of HSV infection the fact that latent persistent viruses in the ganglia cells may be reactivated is of important meaning The further spreading of the virus is favourabled by the asymptomatical virus expression throughout saliva and genital secretion In the orafacial area the HSV1 infections prevail whereas in th
17. package of microtiter strips Shake all liquid components w ell before use Make up the w ashing solution concentrate 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 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 each test run pipette 100ul each of ready to use dilution buffer blank lgG 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 Seite 5 von 10 REV 11 HSV Screen ELISA IgG IgM GB Druckdatum 03 02 2014 control a double insertion is absolutely necessary Working dilution of patient sera 1 100 e g 10ul serum 1ml dilution buffer 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
18. regularly developed but if so it is in short term appearance and in very low concentration This means the serology can be used as a confirmatory tool of the clinical diagnosis retroactively Herpes simplex CSF diagnosis In contrastto the serological diagnosis of HSV infections w hatis most important in CSF diagnosis is the reliable detection of endogenous synthesis of pathogen specific antibodies in the CNS rather than any differentiation betw een the pathogen species HSV 1 and HSV 2 As aresult of the combination of highly purified HSV 1 and HSV 2 lysate antigens in the VT HSV screening test this test systemprovides a very suitable screening test for the CSF diagnosis of HSV infections of the CNS as it is highly sensitive A broad spectrumof highly purified HSV antigens is used in the VT HSV screen This not only leads to the desired high sensitivity but also to the equally desirable specificity w ith respect to differentiation from CNS infections with other neutrotropic pathogens of the herpes virus group We therefore recommend that the antibody index Al should initially be determined in Herpes simplex diagnostic testing w ith the HSV Screen ELISA If there is the additional aim of achieving differentiation betw een HSV 1 and HSV2 after detection of an HSV CNS infection this can be achieved with the help of the tw o species specific gG1 and gG2 ELISA tests Limits The level of pathogen specific antibodies against the gG1 or gG2 epitopes in
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