Package Insert - Sekisui Diagnostics
Contents
1. 8 months 7210 425 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 and standards Nevertheless samples diluted samples standards controls conjugates and microtiter strips should be treated as potentially infectious material Please handle products in accordance with laboratory directions eyes and mucous If body parts are contacted immediat Material required but not supplied Aqua dest demin Eight channel pipette 50ul 100yl Micropipettes 10ul 100pl 1000uI Test tubes Paper tow els or absorbent paper Cover for ELISA plates Disposal box for infectious material c ou gr gu ED ING es _ Incubator 8 Test Procedure Those components that contain preservatives the Citrate Stopping Solution and the TMB have an irritating effect to skin ely wash 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 ELISA handw asheror automated EIA plate w ashing device ELISA plate spectrophotometer w avelength 450nm reference length 620nm Reference Wavelength 620 690nm 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 th2. antibody concentration of 20 1 IU ml is indicated as immune protection 2 11 or safe immune protection 9 10 Booster vaccinations are not indicated w here antibody concentrations exceed0 5 IU ml 10 In the information below we w ould like to point out the follow ing vaccination recommendations These have been drafted based on the recommendations of the w orking group for immunoprophylaxis 13 lU mI Interpretation and next steps 0 01 No vaccine protection Depending on medical history initial vaccination or booster vaccination required Serological test after 4 to 8 w eeks 0 01 0 1 Vaccine protection uncertain Booster vaccination required Serologicaltest after 4 to 8 w eeks 0 11 0 5 Vaccine protection still ensured for a short time Booster vaccination recommended Booster vaccination will provide long termvaccine protection 0 51 1 0 Vaccine protection provided Booster vaccination or serological test recommended after 3 years Note Vaccination in the case of antibody concentrations gt 0 5 IU ml could lead to Seite 6 von 10 REV 18 Tetanus ELISA IgG GB Druckdatum 04 02 2014 fF undesired reactions to the vaccine gt 1 0 5 0 Long termvaccine protection provided Booster vaccination or serologicaltest recommended at the earliest after 5 years 5 0 10 0 Long termvaccine protection provided Booster vaccination or serological test recommended at the earliest after 8 years gt 10 Long
3. 10 For each test run pipette 100yl each of ready to use dilution buffer blank standard and control sera as w ellas diluted patient sera We propose a double insertion blank standards controls and patient sera Working dilution of patient sera 1 100 e g 10ul serum 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 400pl 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 conjugate 30 min at 37 C with cover Stop conjugate incubation by w ashing 4 times pls refer to point 3 above Pipette 100yl 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 501 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 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 Pls refer to last page for Test Procedure Scheme 8 4 Usage of ELISA processors All Sekisui Virotech ELISAs can be used on ELISA process
4. RES e EPA ER Ke REA 5 9 4 Usage of HSA process OFS ced eee Ce s e DE Ee Te E cients cunt peer acct etree E ne ER ea nde ennt 5 9 Test Evaluation a 2 2 eS SE A Ae 5 951 3Test f riction Control VA ee ER ere ER teen Were est Se ea Porter eS eee 5 9 2 Evaluation n5 9 3 Interpretation ss 6 9 4 Limits OF the Test an eon eret eere eene cose rea ER Een tei Y PECES Reeve PRESE FREE YE Eque a H Ve ea arce A perle pep 7 10 IgG test evaluation with the 4 parameter Method mnsenneennnnnnnennnnnnennnnnnnennnnnnnennnnnnnenn nn 7 10 1 Test f hction COMO Poner ee Yr ne ern eine reinen nenne avr Rede EN Teen 7 10 2 Conversion of the quantitative results to international units per milliliter IU ML een 7 11 Performance aic 8 11 1 Sensitivity and SpeckieiyY an see Sp rete ence ede eee en ge e neige rh ege nein einher 8 11 2 Detection Limit 8 11 8 Proficiency nh Ln 11 4 Recovery rate ee sn 11 5 Prevalence Expected Values 8 11 6 Intra assay Coefficient of Variation Repeatability 9 11 7 Inter assay Coefficient of Variation Reproducibility sees rne nnns 9 12 Literature NR ii 9 13 Test Procedure Scheme 2 2 u 2 2222 22 ea a aea ma aea pa ara Aaea ree aa aa pae adari aandaa ia 10 Seite 2 von 10 REV 18 Tetanus ELISA IgG GB Druckdatum 04 02 2014 1 Intended Use Th
5. Tetanus ELISA IgG Testkit Order No EC 124 00 Color Coding white 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 ce Druckdatum 04 02 2014 REV 18 Tetanus ELISA IgG GB Contents DEMIn necngr Mm 3 2 Diagnostic Relev nce 2zu 1i eerie tei ee t eec cese Onn NEAMEN KARANA NNA ninnaa naana 3 3 Test Principle niue neben AANE EA 3 4 Package Contents IgG Testkit ccceeeeceeeee ence cece eee eeeeeeeeeeee eene nennen nennen nennen enhn nnn nhan 3 5 Storage and Shelflife of the Testkit and the ready to use reagents 3 6 Precautions and Warnings eeeeseeeeeeeesee eene enne nennen nn nnn nennu nnn nnns nnmnnn sn nnn nnn 4 7 Material required but not supplied uesrssnnsnnnsennnnnnennnnnnnnnnnnnnennnnnnnnnnn nennen nnne nnn nnn nnn nnn 4 8 Test Procedure WERRRRESIRRENEENEE LARREEF REES ERDE NEEREEEUEEEEFEFFEERERECBEREEFEFEFEETELFEERERECBEEREFECESELEEENEETERECHERFERECRERER 4 8 1 Examination Material oret nn ara EROR E eua rar een sie ete ds HERR E c a aee edd ded 4 8 2 Preparation of Reagents Rino MI RN Mee M Me DIM MEINE 4 8 3 Virotech ELISA TesEProcedure eo eher heri i renean ene ue nOD R e E EFE E
6. any Vaccine 17 7 8 844 50 1999 Pietsch M et al Influence of information campaigns on the vaccination immunity among the population of a small town area seroepidemiological results of the Wittlich V accination Study Gesundheitswesen 64 1 60 4 2002 Epidemiologisches Bulletin 7 2002 Epidemiologisches Bulletin 19 1999 Epidemiologisches Bulletin 40 1998 Epidemiologisches Bulletin 28 2001 Epidemiologisches Bulletin 23 1999 Werner G T et al Tetanusimmunitat im Alter Zeitschrift f r Gerontologie 16 130 133 1983 M ller H E et al Tetanus Schutzimpf ung Indikation und Kontraindikation Dtsch med Wsch 113 1988 1326 1328 Schr der J P etal Vermeidung hyperergischer Reaktionen bei Tetanus Impfungen durch Einsatz eines wissensbasierten Systems bei Fragen der Impfnotw endigkeit Klin Lab 1992 38 229 233 Plikaytis et al Comparisons of Standard Curve Fitting Methods To Quantitate Neisseria meningitidis Group A Polysaccharide Antibody Levels by Enzyme Linked Immunosorbent Assay 1991 J Clin Microbiol 29 p1439 1446 Arbeitskreis Immunprophylaxe Koordinator M Pietsch Infektionsschutz durch Impfprophylaxe Storck Medien amp Verlag KG Bruchsal 1999 Seite 9 von 10 REV 18 Tetanus ELISA IgG GB Druckdatum 04 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 IgG Samples Dilution 1 101
7. e Tetanus ELISA is intended for the quantitative detection of IgG antibodies against the Tetanus toxoid to follow up the success of vaccinations and to determine the immunisation status 2 Diagnostic Relevance Tetanus is caused by Clostridium C tetani an obligate anaerobic spore forming bacterium Its spores are ubiquitous in soil and are extremely resistant to heat and disinfectants The vegetative form of C tetani produces two exotoxins tetanolysin and tetanospasmin of which the tetanospasmin causes the typical clinical symptoms such as increased muscle tone and spasms The clinical forms of tetanus that can be distinguished are generalised local and neonatal disease 1 The bacteria can get into the human body through contaminated wounds which thus represent an infection risk The detection of specific antibodies is unimportant in diagnosing the infection The serological measurement of the IgG antibodies is more important and therefore more suitable for obtaining evidence of tetanus immunity 1 Up to date vaccination is an absolute necessity for everyone especially for elderly people as these are often inadequately immunised 4 5 Overall tetanus vaccination rates are higher than those of other vaccinations such as diphtheria 8 After basic immunisation periodic boosters should be given at intervals of 10 years How ever the booster is often neglected so that the immunisation is often inadequate 1 The IgG antibody level can be meas
8. e g 10 ul serum plasma 1000 yl Dilution Buffer Serum Dilution Buffer is ready to use Testprocedure Samples Incubation 30 minutes at 37 C 100 pl Patient Samples blank value Dilution Buffer standards and controls Wash 4times 400 ul Washing Solution Remove Residues on a Cellulose Pad Conjugate Incubation 30 minutes at 37 C 100 ul Conjugate IgG Wash 4times 400 ul Washing Solution Remove Residues on a Cellulose Pad Substrate Incubation 30 minutes at 37 C 100 pl Substrate Stopping 50 ul Stopping Solution shake carefully Measure Photometer at 450 620nm Extinctions Reference Wavelength 620 690nm Seite 10 von 10 REV 18 Tetanus ELISA IgG GB Druckdatum 04 02 2014
9. e instructions Any type of anticoagulant can be used for plasma The patient samples can be stored for 1 week at 2 8 C Alw ays prepare patient dilution freshly Maximum shelf life 6h at 2 8 C For a longer storage the sera must be frozen Repeated def 1 _ Only fresh non inactivated sera should be used 2 results 8 2 Preparation of Reagents The Sekisui Virotech System Diagnostica offers a high degre rosting should be avoided Hyperlipaemic haemolytic microbially contaminated and turbid sera should not to be used false positive negativ e e 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 standard sera high positive control and low positive control 1 Seite 4 von 10 Tetanus ELISA IgG GB are only intended for this testkit Do not use in other lots Set incubator to 37 C and check proper temperature setting before start of incubation REV 18 Druckdatum 04 02 2014 e Bring all reagents to room temperature before opening package of microtiter strips Shake all liquid components w ell before use Make up the washing 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 8 3 Virotech ELISA Test Procedure 1 om oo nn ar
10. ors 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 manufacturer 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 will support quality assurance in your laboratory 9 Test Evaluation 9 1 Testfunction control 8 b OD values The OD value of the blank should be 0 15 The OD values of the low est standard 0 001 IU ml shall be 20 05 OD and the OD values of the highest standard 0 050 IU ml shall be lt 2 800 OD The concentration of the low and high positive control have to be within the ranges IU ml stated in the Quality Control Certificate 9 2 Evaluation By using the standards a standard curve is plotted on the semilogarithm paper included in the kit in order to determine the Tetanus antitoxoid IgG antibody level in the serum The mean values of the extinctions are plotted on the o
11. rdinate and the concentrations IU ml of the ready to use standards of the standard sera on the abscissa You have to be aware that the patient sera intended for the test procedure have been diluted 1 100 This is w hy the result read from the diagram must be Seite 5 von 10 REV 18 Tetanus ELISA IgG GB Druckdatum 04 02 2014 multiplied by the factor of 100 For preparing the standard curve either a point to point procedure as well as a 4 paramter calculation can be used Please note Samples found to have a concentration of under 0 11U ml can be retested with a 1 10 dilution The difference in the dilution must be considered in the evaluation Samples with an extinction above the value of the 0 05lU ml standard have to be used in a higher dilution in the test e g 1 200 1 400 etc At OD values above 2 00 the measuring precision decreases with increasing optical density It is therefore recommended that sera w hich attain OD values above 2 00 at a dilution of 1 100 be used in higher dilution e g 1 200 1 400 etc in the test These dilutions have to be considered during the test evaluation Example of Tetanus standard curve Tt Col 0 001 0 002 0 005 0 010 0 020 0 050 concentration lo g lU ml Lg in al tn S N e e ui a o E ul 9 3 Interpretation The Tetanus antitoxoid concentrations are expressed in International Units IU ml follow ing the WHO Standards A Tetanus antitoxoid IgG
12. termvaccine protection provided Booster vaccination or serological test recommended at the earliest after 10 years 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 2 The Virotech Tetanus ELISA is not suitable for laboratory diagnosis of an infection 3 Please refer also to vaccination certificate or information about last Tetanus vaccination for interpretation of the antitoxo id titer 7 4 Aninterpretation of antitoxoid titers below 0 1 IU ml is not recommendable as they are below the technically reproducible sensitivity limit when using ELISA test systems The vaccination anamnesis should therefore be considered in the individual case to decide if a basic immunisation or a booster vaccination shall be performed gt 10 2 10 IgG test evaluation with the 4 parameter method By means of the Virotech Tetanus IgG ELISA it is possible to carry out a quantitative assay with the 4 parameter method For this purpose the 0 01 IU mL standard is used for calibration control The calibration control compensates for the fluctuations caused by performance of the test Mean values of the OD readings are used for the calculation 10 1 Test function control a OD values The OD value of the blank should be lt 0 15 The OD value of the calibration control must lie within the reference range indiciated in the quality con
13. tion method 6 standard value pairs that also describe the standard curve are additionally defined in the certificate The quantifiable range is betw een 0 01 IU mL and 15 IU mL Determination of the IU mL The IU mL can be determined using software available for purchase from Virotech Alternatively an evaluation template for common tabular estimates can be provided The calculated concentrations alw ays indicate the actual concentrations of the undiluted serum w here this has been diluted for test purposes using a 1 100 dilution If a different dilution w as used during testing the concentration values must be adjusted accordingly Performance Data 11 1 Sensitivity and Specificity It is not possible to determine diagnostical sensitivity and specificity because this ELISA is a quantitave test which is not intended to differentiate betw een positve and negative results 11 2 Detection Limit In internal tests a lower reproduceable determination limit of 0 06 IU ml at a coefficient of variation of 3 9 could be determined 11 3 Proficiency Within the period from March 2002 to November 2009 32 sera from interlaboratory comparisons and of known concentrations w ere measured in the Virotech ELISA 30 sera corresponded to the declared value Tw o sera did not meet the specifications 11 4 Recovery rate The international standard of the WHO for Tetanus Immunglobuline human TE 3 was tested on the Virotech ELISA to determine the recover
14. tive in Tris Buffer ready to use 8 Tetramethylbenzidine substrate solution 3 3 5 5 TMB 11ml ready to use 9 Citrate Stopping Solution 6ml 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 Microtiter 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 TVB 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 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 Seite 3von 10 REV 18 Tetanus ELISA IgG GB Druckdatum 04 02 2014 Materii Status Diluted Undiluted ng Microtitreplate After Opening ing Test Samples After Open Diluted After Opening After Opening After Opening After Opening inal Dilution ready to use Rheumatoid factor Washing Solution Precautions and Warnings Storage Shoe 210 48 2 to 48 storage in the provided bag with desiccant bag Expiry date 4210
15. trol certificate b IU mL The anti tetanus IgG concentrations IU mL of the w eakly positive control and of the strongly positive control must lie w ithin the ranges indicated in the quality control certificate If the requirements OD readings IU mL are not satisfied the testis to be repeated 10 2 Conversion of the quantitative results to international units per milliliter IU mL The extinction of the blank value 450 620 nm must be subtracted from all extinctions The patient sera are quantified by expressing them in international units The standard curve is determined by non linear regression on the basis of extensive tests and is described mathematically by the follow ing formula 12 IU mL e In 0 A OD corr A 1 B Where A expected OD at an anti tetanus IgG concentration of 0 B slope factor C inflection point D expected OD at an infinitely high anti tetanus IgG concentration OD corr corrected ODof the patient serum Seite 7 von 10 REV 18 Tetanus ELISA IgG GB Druckdatum 04 02 2014 11 To allow for fluctuations within the course of the tests the measured OD of the patient serum is corrected on the basis of a calibration control Obr Odin OD calibration control specified OD calibration control measured See the certificate for the values of the parameters A B C and D as w ellas the specified OD of the calibration control In the case of evaluation software not compatible w ith this calcula
16. ured by means of this ELISA so that conclusions can be drawn about immunisation status Furthermore it can be used to determine the need for vaccination and to check immunity after vaccination has taken place 3 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 4 Package Contents IgG Testkit 1 1 Microtiter Plate consisting of 96 with antigen coated breakable single w ells lyophilised 2 PBS Dilution Buffer blue readyto use 2x50m pH 7 2 with preservative and Tw een 20 3 PBS Washing Solution 20x concentrated 50ml pH 7 2 w ith preservative and Tw een 20 4 IgG Ab standard sera for drawing a standard curve 6 vials 2ml ready to use human serumw ith preservative 0 0011U ml 0 0021U ml 0 005lU ml 0 011U ml 0 021U ml 0 05IU ml IU international units 5 IgG high positive Control 2 ml human serum w ith preservative ready to use 6 IgG low positive Control 2 ml human serumw ith preservative ready to use 7 gG Conjugate anti human 11ml sheep or goat horseradish peroxidas e conjugate with protein stabilizer and preserva
17. y rate of the Tetanus ELISA By using the standard curve the concentration in IU ml w as calculated considering the respective dilution and then compared with the expected initial value The results show that the starting concentration of 120 IU ml could exactly be recovered in the diagnostic relevant low er area of the standard curve 0 1 to 0 5IU ml 11 5 Prevalence Expected Values 117 blood bank sera were tested and the IU ml concentrations w ere calculated Seite 8von 10 REV 18 Tetanus ELISA IgG GB Druckdatum 04 02 2014 The sera distribution corresponds approximately to the prevelance rate described in some literature 3 6 The data can only partly be compared as no information about the age of the blood donors is available 11 6 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 low er then 9 11 7 Inter assay Coefficient of Variation Reproducibility Three sera were tested in 10 independent test runs by different persons in diff erent laboratories The obtained variation coefficient values are low er than 15 12 Literature i 2 E al D gi e rto 0 00 c x 0T Epidemiologisches Bulletin 27 2002 Stark K Schonfeld C Barg J Molz B Vornw ald A Bienzle U Seroprevalence and determinants of diphtheria tetanus and poliomyelitis antibodies among adults in Berlin Germ
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