Package Insert - Sekisui Diagnostics
Contents
1. a OD values The OD value of the blank should be lt 0 15 The OD values of the low est standard 0 001 IU ml shall be gt 0 05 OD and the OD values of the highest standard 0 050 IU ml shall be lt 2 800 OD b The concentration of the low and high positive control have to be w ithin the ranges IU ml stated in the Quality Control Certificate Seite 5 von 10 REV 17 Diphtherie ELISA IgG GB Druckdatum 03 02 2014 92 9 3 Evaluation By using the standards a standard curve is plotted on the semilogarithm paper included in the kit in order to determine the Diphtheria antitoxin IgG antibody level in the serum The mean values of the extinctions are plotted on the ordinate 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 fromthe diagram must be multiplied by the factor of 100 For preparing the standard curve either a point to point procedure as w ell as a 4 paramter calculation can be used Please note Samples found to have a concentration of under 0 1lU 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 wi2. S 1090 1096 9 Naumann P Paatz R und Thomas L Diphtherie Adsorbat Impf stoff f r Erwachsene Immunologische Wirksamkeit Die gelben Hefte XXIV 1984 S 109 115 10 Pietsch M Impfserologie zur Erg nzung von Impfungen Der Allgemeinarzt 18 1993 11 Galazka A M Diphtheria The Immunological Basis for Immunization Series WHO EP GEN 93 12 Geneva pp3 12 Plikaytis et al Comparisons of Standard Curve Fitting Methods To Quantitate Neisseria meningitidis Group A Polysaccharide Antibody Levels by Enzy me Linked Immunosorbent Assay 1991 J Clin Microbiol 29 p1439 1446 Seite 9 von 10 REV 17 Diphtherie ELISA IgG 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 IgG Samples Dilution 1 101 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 Wav
3. 2 desde adec cede a aaea ma ea pa ara Aaa ee aa aa Hae adnada aana aia 10 Seite 2 von 10 REV 17 Diphtherie ELISA IgG GB Druckdatum 03 02 2014 1 Intended Use The Diphtheria ELISA is intended for the quantitative detection of IgG antibodies against the Diphtheria toxin to follow up the success of vaccinations and to determine the immunisation status Diagnostic Relevance Diphtheria is caused by Corynebacterium C diphtheriae an aerobic non sporulating rod bacterium The virulence of C diphtheriae derives from the diphtheria toxin Humans are the only reservoir for the bacterium It is transmitted by droplet infection Within a few days there is a general feeling of illness w ith fever headache abdominal pain and pains in the limbs The infectious disease occurs worldw ide but has been greatly pushed back by active immunisation There are locally limited outbreaks again and again because of gaps in immunisation and it is associated w ith high mortality In Germany only one clinical case of diphtheria w as recorded in 1998 and again in 1999 and there w as no case recorded in 2000 1 4 Active immunisation is the method of choice for the prevention of diphtheria The immunity produced by the antitoxin largely prevents the illness but not the infection so that there can also be carriers among those w ho have been vaccinated 1 Diphtheria immunisation in infants and preschoolchildren is as good as that against tetanus in Central Europe Acc
4. Diphtheria ELISA IgG Testkit Order No EC129 00 Color Coding white 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 17 Diphtherie ELISA IgG GB Contents 1 mended Use cniinne aa en ara edili rceBoilsTeraiil 3 2 Diagnostic Relev nce 2u 1i eere tuii ee ee nen er eno s Er no gear e inoh 3 3 Test Principle nune nente 3 4 Package Contents IgG Testkit unnuuunsennnnnnennnnnnennnnnnnnnnnnnnnnnnn nennen nennen anne nnne nennen 3 5 Storage and Shelflife of the Testkit and the ready to use reagents ess 3 6 Precautions and Warnings eeeeeeseeeeeesee eene enne nnne nn nnn mennun mennun nnmnnn nnmnnn 4 7 Material required but not supplied 11eeeeeeeeeeeee eene enne nennen nennen nn nnn 4 8 Test Procedure WERRRRESIEERNENEREERLNEREERRARE SE RROEAEEREEEHEEEEFEFFEERERECHEEEEFEREEEEFEFFEERERECHEEREFECEHEREEEREETERECHEFRERECEBEER 4 8 11 Examination Material nitri er p nee Shc cee ed IRR E DRE ee a eee 4 8 2 Preparation of Reagents RM MIU LUI stato A Ae DUM 5 8 3 Virotech ELISA Test Proced tre hebetes heroe teet reo eme CERES YE e Fade state tien AA 5 9 4 Usage of ELISA DIOCOSSOIS eet ere te trt Uns n a E ed ci o Dente ditta vaca oni ee er
5. ae ca rta AS e xk nba nee ADDE 5 Q gt Test Evel aa thor ec secs ss cece c ITE 5 9 1 3Test f nctior COntrol ze nv ee eave tae Mae Seat AE A ee ee 5 9 2 Evaluation ss 6 9 3 Interpretation es 6 9 4 Limits OF the Test n eon ete eere tee enfer eri rete spendere Ere vrac fevues ee Paesi vn adero de rera peces ire yia pene pep 7 10 IgG test evaluation with the 4 parameter method eese 7 10 Testf nctiorn COnIEOL s coenae ee His a Be be Fa e voz aderat AeA tie d ae i o p pa t 7 10 2 Conversion of the quantitative results to international units per milliliter IU ML eee 7 11 Performance Data ze Ice ierit ea Echte Sedeztewsuncneedelsestevsudeuedavertevttueseseeth 8 11 1 Sensitivity and Spect iCY iecore Rr ecu id Bandes dee e a ee deed re ere Deve euge ened ethane 8 INE NELICOTUEKCIO HI E en 8 11 3 Prevalence Expected Values nennen nennen nne nrnet nne trnen nein teneret restent nre 8 11 4 Intra assay Coefficient of Variation Repeatability 8 11 5 Inter assay Coefficient of Variation Reproducibility 8 11 6 PrOfICIO 1621 n t n dette av nee elves avo 1157 Detection B ss 8 12 Literature DERSRRSERERERPREERESSERERRSRFEHEFEFFEERERSTSEARFEREEAREENTEEREVETSPUEERTEEEHEEFERPPRERFEERNEERPERRERETSERTRFREPERTERESERF 8 13 Test Procedure Scheme 2 2 u
6. elength 620 690nm Seite 10 von 10 REV 17 Diphtherie ELISA IgG GB Druckdatum 03 02 2014
7. es 7T Material required but not supplied Aqua dest demin Eight channel pipette 50ul 100yl Micropipettes 10ul 100pl 1000ul 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 c XO Oc y On EDDIE _ 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 positive negative results Seite 4 von 10 REV 17 Diphtherie ELISA IgG GB Druckdatum 03 02 2014 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 standard sera high positive control and
8. g of 96 with antigen coated breakable single w ells lyophilised PBS Dilution Buffer blue readyto use 2x50ml pH 7 2 with preservative and Tw een 20 PBS Washing Solution 20x concentrated 50ml pH7 2 w ith preservative and Tw een 20 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 005IU ml 0 011U ml 0 021U ml 0 05IU ml IU international units IgG high positive Control 2 ml human serum w ith preservative ready to use 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 preservative 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 Te dq re oo 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 Seite 3 von 10 REV 17 Diphtherie ELISA IgG GB Druckdatum 03 02 2014 2 The ready to use conjugate and the TMB subs
9. 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 cor A 1 B Where A expected OD at an anti diphtheria IgG concentration of 0 B slope factor C inflection point D expected OD at an infinitely high anti diphtheria lgG concentration OD corr corrected OD of the patient serum 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 OD calibration control specified ODcorr OD patientserum OD calibration control measured See the certificate for the values of the parameters A B C and D as w ell as the specified OD of the calibration control In the case of evaluation software not compatible w ith this calculation method 6 standard value pairs that also describe the standard curve are additionally defined in the certificate Seite 7 von 10 REV 17 Diphtherie ELISA IgG GB Druckdatum 03 02 2014 The quantifiable range is betw een 0 03 IU mL and 10 IU mL Determination of the IU mL The IU mL can be determined using softw are available for purchase from Virotech Alternatively an evaluation tem
10. low positive control are only intended for this testkit Do not use in other lots dec Cor ees 8 3 on o 0 SOD ON o 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 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 Virotech ELISA Test Procedure For each test run pipette 100ul 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 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 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 kee
11. not suitable for laboratory diagnosis of an infection 3 Please refer also to vaccination certificate or information about last Diphtheria vaccination for interpretation of the antitoxin titer 4 An interpretation of antibody 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 be therefore considered in the individual case to decide if a basic immunisation or a booster vaccination should be performed 10 IgG test evaluation with the 4 parameter method By means of the Virotech Diphtheria IgG ELISA it is possible to carry outa 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 0 15 The OD value of the calibration control must lie w ithin the reference range indiciated in the quality control certificate b IU mL The anti diphtheria IgG concentrations IU mL of the weakly positive control and of the strongly positive control must lie within 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
12. ording to investigations by the RKI Robert Koch Institute approx 95 of all children are vaccinated regularly against diphtheria 4 How ever the recommended boosters are often no longer given so that deficits in vaccine immunity occur in adolescents and younger adults 1 3 4 It is estimated that about half of them are not vaccinated as recommended for their age 4 In Germany at present only about one third of adults have reliably protective antibodies 1 4 6 Similar figures are also found for other parts of Europe and the USA 5 7 The neutralisation test is the method of choice for detecting specific antibodies to the toxin How ever this is very expensive and time consuming By means of this ELISA the IgG antibody level can be measured so that conclusions can be drawn about the vaccination status Furthermore it can be used to examine the need for vaccination and to check immunity after vaccination has taken place 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 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 Testkit 1 Microtiter Plate consistin
13. ovember 2005 to April 2010 20 sera frominterlaboratory comparisons and of know n concentrations were measured in the Virotech ELISA All 20 declared sera were correctly identified 11 7 Detection Limit Tests within the company have found a low er reproducible limit of detection of 0 04 IU ml w ith a coefficient of variation of 2 6 12 Literature 1 Epidemiologisches Bulletin 6 2001 2 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 Germany Vaccine 17 7 8 844 50 1999 3 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 Vaccination Study Gesundheitswesen 64 1 60 4 2002 Seite 8 von 10 REV 17 Diphtherie ELISA IgG GB Druckdatum 03 02 2014 4 Epidemiologisches Bulletin 19 1999 5 Pachon Amela C De Ory F Age specific seroprevalence of poliomyelitis diphtheria and tetanus antibodies in Spain Epidemiol Infect 2002 129 3 535 41 6 rztezeitung 23 04 2003 Norddeutsche sind Impfmuffelin Deutschland 7 Gupta RK Griffin P Jr Xu J Rivera R Thompson C Siber GR Diphtheria antitoxin levels in US blood and plasma donors J Infect Dis 1996 173 6 1493 7 8 Naumann P Hagedorn H J Paatz R 1983 Diphtherie Immunit t und ihre epidemiologische Bedeutung Dtsch med Wschr 108
14. p 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 such a 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 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 w ill support quality assurance in your laboratory 9 Test Evaluation 9 1 Test function control
15. plate for common tabular estimates can be provided 11 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 Recovery rate The standard curve of the VIROTECH Diphtheria ELISA has been checked with the Diphtheria Antitoxin Human Serum 00 496 of the Institute for Biological Standards and Control WHO International Laboratory for Biological Standards in Great Britain 11 3 Prevalence Expected Values 117 blood bank sera were tested and the IU ml concentrations w ere calculated Number of sera found lt 0 1 Ui 0 1 1 0 Umi 1 0 1 4 Um 1 4 2 Um gt 2 Um The sera distribution corresponds approximately to the prevelance rate described in some literature 1 2 3 4 6 The data can only partly be compared as no information about the age of the blood donors is available 11 4 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 5 Inter assay Coefficient of Variation Reproducibility Three sera w ere tested in 10 independent test runs by different persons in different laboratories The obtained variation coefficient values are low er than 15 11 6 Proficiency Within the period from N
16. th 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 Diphtheria standard curve Extinction OD 450 620nm 2 5 0 001 0 002 0 005 0 010 0 020 0 050 concentration log IU ml Interpretation The Diphtheria antitoxin concentrations are expressed in International Units IU ml follow ing the WHO Standard According to references in literature a sufficient immune protection can be expected above 0 11U ml 2 8 9 11 But this figure has to be considered as recommendation only We w ould like to draw your attention to vaccination recommendation mentioned below that w as taken from the publication Pietsch M 10 recommended vaccination 14 20 booster vaccination after 10 years The detection of the Diphtheria antitoxin IgG antibody level can be a decisive help for determining the necessity of vaccination and for checking the success of vaccination In addition information on the different reactions of the individual immune system can be obtained Seite 6 von 10 REV 17 Diphtherie ELISA IgG GB Druckdatum 03 02 2014 9 4 Limits ofthe Test 1 The interpretation of serological results shall always include the clinical picture epidemiological data and all further available laboratory results 2 The Virotech Diphtheria ELISA is
17. trate 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 TVB needed for the test insertion Additional conjugate or TMB taken out may not be returned but must be dismissed Matera Saus O Sorge Sheme Diluted 4210480 Test Samples Undiluted 1210 18C n Microtitreplate After Opening Fe log Ks Sa Doo 109099 Rheumatoid factor Undiluted After Opening 2 to 8 C Absorbent Diluted 210 48C 7210 48C protect from ight 210 8 C protect fromight After Opening ea Pan 32 10 3253 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 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 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 flow ing water and possibly consult a doctor 3 The disposalof the used materials has to be done according to the country specific guidelin
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