Package Insert - Sekisui Diagnostics
Contents
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 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 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 EZ Clogs 1 melle Test Samples EE 2 to 8C Undiluted 210 8 After Opening 210 48 2 to 8 storage in the provided bag with desiccant bag 3 months Microtitreplate After Opening Rheumatoid factor Undiluted After Opening 2 to 8 C Absorbent Diluted 2 to 8C Conjugate After Opening 2 to 8 C protect from light Tetramethylbenzidine After Opening 2 to 8 C protect from light Stop Solution After Opening 2 to 8 After Opening 2 to 8C Washing OU Final Dilution ready to use 3210 3257 6 T d 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 tre2. 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 and this w ill support quality assurance in your laboratory 9 Qualitative and semiquantitative test evaluation The ready to use controls serve for a semiquantitative determination of specific IgG and IgA antibodies Their concentration can be expressed in Virotech units VE Fluctuations resulting from the test procedure can be balanced w ith 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 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
3. 9 5 Limits of the Test 1 The interpretation of serological results shall always include the clinical picture epidemiologic al data and all further available laboratory results Seite 7 von 12 REV 13 Pertussis Toxin IgG Pertussis Toxin IgA ELISA GB Druckdatum 04 02 2014 10 Quantitative IgG test evaluation in IU ml The ready to use calibration control is separately available w ith the IgG Quantification Set EN215Q60 and is used for the quantitative determination in IU ml of anti PT IgG antibodies in patient serum The calibration control corrects for fluctuations fromthe test performance The mean of the OD values is used for the calculation 10 1 Control of test function 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 range given in the corresponding certificate b Ui The anti PT IgG concentrations IU ml of the w eakly reactive control and of the strongly reactive control must lie w ithin the ranges given in the quality control certificate If the requirements are no fulfilled OD values IU ml the test must be repeated 10 2 Calculation of the quantitative results in International Units per ml IU ml The extinction of the blank 450 620nm must be subtracted from all extinctions Using the Virotech IgG Quantification Set the patient sera are quantified by correlation w ith the WHO International Standard Extensive tests have been performed with each pla
4. 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 4 1 IgG Testkit 1 1 Microtiter Plate consisting of 96 with antigen coated breakable single w ells lyophilised 2 PBS Dilution Buffer blue readyto use 2x50ml pH7 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 negative Control 2000ul human serum w ith protein stabilizer and preservative ready to use 5 IgG cut off Control 20001 human serum w ith protein stabilizer and preservative ready to use 6 IgG positive Control 2000HI human serum w ith protein stabilizer and preservative ready to use 7 lgG 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 Seite 3von 12 REV 13 Pertussis Toxin IgG Pertussis Toxin IgA ELISA GB Druckdatum 04 02 20
5. s Borderline results have not been considered for the calculation of the sensitivity and specificity Relative to Bordetella pertussis LINE the sensitivity for IgG w as 96 6 and the specificity 98 7 96 11 2 Cross Reactivity In order to testfor any cross reaction between Virotech Pertussis Toxin ELISA and antibodies from respiratory diseases 37 sera was tested for IgG and 33 sera was tested for IgA The results are shown in the follow ing table borderline 4 0o positive 4 o The result show s that the Virotech Pertussis Toxin ELISA is a very good tool for the differential diagnostic usage Seite 9von 12 REV 13 Pertussis Toxin IgG Pertussis Toxin IgA ELISA GB Druckdatum 04 02 2014 11 3 Prevalence Expectet Values The follow ing table show s the test results of blood donators for 80 IgG samples and 78 IgA samples borderine 3 0 poste 4 o 11 4 Intra assay Coefficient of Variation Repeatability In this assay strips from various plates of a batch w ere tested in a chessboard pattern w ith tw o sera The coefficients of variation are as follow ed lt 9 for IgG and 15 for IgA 11 5 Inter assay Coefficient of Variation Reproducibility A minimum of 3 seraw eretested in minimum 10 independent test batches on 3 differenttest days This gave a coefficient of variation of lt 15 11 6 Distribution of the antibody concentrations in VE of sera with w
6. et al What to do and what notto do in serological diagnosis of pertussis recommendations from EU reference laboratories 2010 Eur J Clin Microbiol Infect Dis DOI 10 1007 s10096 010 1104 y 13 RKI Ratgeber Infektionskrankheiten Merkbl tter f r rzte Pertussis Keuchhusten 03 09 2010 14 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 15 Podbielski et al MO 13 2010 Mikrobiologisch infektiologische Qualitatsstandards MiQ Teil II Heft 13b Bakterielle Erreger Bordetella pertussis 2 Auflage p98 106 Seite 11 von 12 REV 13 Pertussis Toxin IgG Pertussis Toxin IgA ELISA 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 IgA Samples Dilution 1 101 1 100 e g eg 10 ul serum plasma 1000 ul Dilution Buffer 5 ul serum plasma 450 ul Dilution Buffer Serum Dilution Buffer is ready to use 1 drop RF SorboTech incubate for 15 min at room temperature Testprocedure Samples Incubation 30 minutes at 37 C 100 pl Patient Samples blank value Dilution Buffer and controls Wash 4times 400 ul Washing Solution Remove Residues on a Cellulose Pad Conjugate Incubation 30 minutes at 37 C 100 ul Conjugate I
7. sera should be used Hyperlipaemic haemolytic microbially contaminated and turbid sera should not to be used false positive negative results 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 are parameter specific and only to use with the plate lot indicated in the Quality Control Certificate T 2 3 4 8 3 wp OO OY UY dx 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 In order to determine the pertussis toxin IgA correctly it is necessary to pre treat the serausing RF SorboTech VIROTECH adsorption medium Pre adsorption is omitted in IgA controls Virotech ELISA Test Procedure For each test batch pipette 100ul each of the ready to use dilution buffer blank the controls and the diluted patient sera We recommend the use of duplicates blank controls and patient sera It is absolutel
8. uuusunsnsnaannnnannnnannnnnnnnnannnnnnnnnnnnnnn nn 6 9l clestfunction cont ol emere ester ide ere APER tirer ertt i A Re esee SEEN 6 9 2 Calculation of the Virotech Units WD 6 9 97 interpretation Scheme GG EE 6 9 4 Interpretation Scheme A 7 9 5 Limits of the Test rue cen ee di e Rinne ra C ee oh deed ein 7 10 Quantitative IgG test evaluation in IU ml eeeeeeee eren 8 10 1 Control of test UNCHONI s idt t time ee e da 10 2 Calculation of the quantitative results in International Units per ml IU ml 10 3 Interpretation schieme for NG centre tente tede ree certare ded de de d de Fa eaa i eae Ea re deet 11 Performance D ta ue LER A 9 11 1 Sensitivity and Speckicily nece a TER ERR ANE 9 A122 CrOSS RAGIN EE 9 11 3 Prevalence Expectet Valles eerte e ree a eek 10 11 4 Intra assay Coefficient of Variation Repeatability essent nter nnne 10 11 5 Inter assay Coefficient of Variation Reproducibility eese eene nnn 10 11 6 Distribution of the antibody concentrations in VE of sera withAvithout suspected PertusSisS 111111111111 10 11 7 Correlation betw een declared and measured Um 10 12 Literature ERE c RE 11 13 Test Procedure Scheme NEESS an nnd 12 Seite 2von 12 REV 13 Pertussis Toxin IgG Pertussis Toxin IgA ELISA GB Druckdatum 04 02 201
9. 04 02 2014 significant high antibody titer against Pertussis Toxin e Note tow ards an acute or recent infection gt 11 positive e Detection of vaccine antibodies Absolutely consider vaccine management as the testis not able to differentiate between vaccine antibodies and antibodies of an infection Significant high antibody titer against Pertussis Toxin an argument for an gt 100 gt 17 infection BE DEP acute infection if the last vaccination is longerthan 12 months ago 9 4 Interpretation Scheme IgA The Pertussis Toxin IgA ELISA has been adjusted to the WHO International Standard This gives the correlation in the evaluation betw een Virotech Units VE and International Units per Milliliter IU ml for IgA 11 12 Wim VE IgA antibodies Interpretation WHO gt no increased Ab titre to pertussis toxin 1 e No suspicion of B pertussis infection 9 negative JM it If there are clinical symptoms repeat measurements later or clarify via 12 differential diagnosis IU ml increased Ab titre to pertussis toxin e Persisting Ab froma previous infection 9 11 borderline a e Abfromthe initial stages of an immune response e Vaccine antibodies significantly increased Ab titre to pertussis toxin Accompanied by positive IgG Ab titre gt 11 VE e Evidence for new or recent infection gt 12 sii positive e Detection of vaccine antibodies Essential to consider vaccine management IU ml as the test
10. 14 4 2 10 11 12 4 3 NEO BRD ars IgG Quantification Set IgG calibration control 2000 ul human serum w ith protein stabilisers and preservative ready to use IgG weakly reactive control 2000 ul human serum w ith protein stabilisers and preservative ready to use IgG strongly reactive control 2000 ul human serumw ith protein stabilisers and preservative ready to use IgA 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 50m 1 pH 7 2 w ith preservative and Tw een 20 IgA negative Control 2000ul human serumw ith protein stabilizer and preservative ready to use IgA cut off Control 2000ul human serumw ith protein stabilizer and preservative ready to use IgA positive Control 2000pl human serumw ith protein stabilizer and preservative ready to use IgA Conjugate 2 anti human 11ml sheep or 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 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
11. 2 Literature 1 Medizinische Mikrobiologie Hahn Falke Klein Springerverlag 1991 p361 363 2 Wiersbitzky Pertussis Kosteng nstige Pr vention zuw enig genutzt 1995 Therapiewoche 25 p1485 1486 3 Lehrbuch der Medizinischen Mikrobiologie Brandis K hler Eggers Pulverer 7 Auflage p483 4 Mastrantonio et al Bordetella parapertussis infections 1997 Dev Biol Stand 89 p255 259 5 Mastrantonio et al Antibody kinetics and long term sero prevalence in the Italian clinical trial of acellular pertussis vaccines 1997 Dev Biol Stand 89 p275 278 6 Wirsing von K nig et al Evaluation of a single Sample Serological Technique for Diagnosing Pertussis in Unvaccinated Children 1999 Eur J Clin Microbiol Infect Dis 18 p341 345 7 De Melker etal Specificity and sensitivity of high levels of immunoglobulin G against pertussis toxin in a single serum sample for diagnosis of infection w ith Bordetella pertussis 2000 J Clin Microbiol 38 p800 806 8 Swidsinski Diagnostische Bibliothek Nr 47 April 1997 9 Meade etal Serodiagnosis of Pertussis 1994 Center for Biologics and Research Food and Drug Administration Bethesda Maryland 20892 10 Meijer Numerical Comparison of 4 Pertussis Toxin IgG ELISAs 2002 nicht publiziert Krankenhaus Groningen NL 11 Riffelmann et al Performance of Commercial Enzyme Linked Immunosorbent Assays for Detection of Antibodies to Bordetella pertussis 2010 J Clin Microbiol 48 p4459 4463 12 Guiso
12. 4 1 Intended Use The Pertussis Toxin ELISA is intended for the semiquantitative and qualitative detection of IgG or IgA antibodies in human serum It is intended for the detection of an acute or recent infection respectively forthe detection of vaccination antibodies check of vaccination success It is also possible to quantify the IgG in international units per ml IU ml by using the IgG Quantification Set EN215Q60 w hich is supplied separately 2 Diagnostic Relevance The main agent of the genus Bordetella B pertussis causesthe clinical picture of w hooping cough Milder forms are caused by B parapertussis those are not detected w ith the Pertussis Toxin ELISA The Pertussis Toxin is of significant importance for the pathogenesis of w hooping cough It is a real exotoxin responsible for many physiological immunological and pharmacological effects In contrastto other exotoxins of the species Bordetella that show high cross reactivities in serum diagnostics the Pertussis Toxin is high specific 4 During primary infection the IgM antibodies can be detected at the earliest 5 10 days after the beginning of the convulsive stage and persist for 6 12 w eeks they are the expression of an acute disease IgA antibodies can be detected 11 days after disease started at the earliest IgA antibodies can persist 6 24 months They are also developed in vaccinated adults during a natural re infection w ithout clinical disease and are therefo
13. 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 x10 OD cut off control OD patient serum OD cut off control VE positive control VE patient serum 9 3 Interpretation Scheme IgG The Virotech units VE of the Pertussis Toxin IgG ELISA enzyme linked immunosorbent assay have been calibrated using the WHO International Standard This leads to the follow ing calibration betw een Virotech Units VE and International Units per ml IU ml for IgG 7 IU ml VE IgG antibodies Interpretation WHO gt no increased antibody titer against Pertussis Toxin i e No suspicion of a B pertussis infection 9 negative n 3 In case of clinical symptoms a follow up control or a differential diagnosis is recommended increased antibody titer against Pertussis Toxin 36 44 9 11 borderline e Persistent antibodies of a recent infection e antibodies of a starting immune response Seite 6 von 12 REV 13 Pertussis Toxin IgG Pertussis Toxin IgA ELISA GB Druckdatum
14. Pertussis Toxin ELISA IgG Testkit IgA Testkit Order No EC215G00 IgG Testkit EN215Q60 IgG Quantification Set EC215A00 IgA Testkit Color Coding IgG silver dark blue IgA silver black 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 04 02 2014 REV 13 Pertussis Toxin IgG Pertussis Toxin IgA ELISA GB Contents He lnte ded US 3 2 Diagnostic Relevangpe eege Eege eege ENEE eege EE 3 3 Test Principle nune CE AO OPO O OTO OTO 3 d Package Contents ieiunii eege Dese eege 3 dl VERTU TEE DINEM 3 AD IgG Quantification ET 4 43 NICO EE 4 5 Storage and Shelflife of the Testkit and the ready to use reagents EREE EEN 4 6 Precautions and Warnings ursrnnsnnnennnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnenn nenn nn hnius nn hnius nr nnne nn nnn 4 7 Material required but not supplied uueusssnnsnnnennnnnnnennnnnnennnnnnnnenn eene nennen nnne nnn nnne nnn nnn 4 Bb Test Procedure me DE 5 9 Examination Material eta E e ed Ri 8 2 Preparation of Reagents we 8 3 Virotech ELISA Test Procedure eterne erede Ath ol Te ee Nae at vere pL dd A 5 8 4 Usage of ELIGA proCessSOIS cuire anne ep a hp Ina nn 6 9 Qualitative and semiquantitative test evaluation
15. Toxin IgA ELISA GB Druckdatum 04 02 2014 10 3 Interpretation scheme for IgG The international units IU ml of the Pertussis Toxin IgG ELISAs were calibrated w ith WHO International Standard 335 The interpretation is in accordance w ith the recommendations of European Reference Centres 7 11 12 13 15 IU mI Interpretation WHO No evidence for recent contact with the pathogen Doubtful result Repeat measurements later or determine IgA anti PT IgA anti PT lt 11 VE corresponding to 12 IU ml No evidence for recent pathogen contact IgA anti PT gt 11 VE corresponding tox 12 IU ml Evidence for recent pathogen contact assuming that the last vaccination w as more than 12 months previously Pay attention to vaccine management Evidence for recent pathogen contact on the assumption that the last vaccination was more than 12 months previously Check vaccination management 11 Performance Data 11 1 Sensitivity and Specificity In an internal study 151 sera with suspected Bordetella pertussis infection w ere tested for anti PT IgG These sera have been predefined in Neutralisationtest NT by a a former reference centre The Virotech Bordetella pertussis LINE Line Immuno Assay has been used as comparative test The results are summarised in the follow ing table Sera Collective nz 151 negative _ borderline positive negative 76 1 1 borderline 6 1 7 positive 2 13
16. ated 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 Seite 4 von 12 REV 13 Pertussis Toxin IgG Pertussis Toxin IgA ELISA GB Druckdatum 04 02 2014 SOMNDAAR WY 8 Eight channel pipette 50ul 10011 Micropipettes 10ul 100ul 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 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 82 Only fresh non inactivated
17. cannot distinguish betw een vaccine antibodies and infection antibodies Accompanied by negative or threshold IgG Ab titres lt 11 VE e Repeat measurements later Notice IgA antibodies are not alw ays developed and are therefore a less reliable marker for a Bordetella pertussis infection than IgG antibodies 1 Absolutely consider vaccine management as the test is not able to differentiate betw een vaccine antibodies and antibodies of an infection 2 _ If the measured values are above the defined borderline range they are considered to be positive 3 If 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 4 ffthe 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 5 Ata borderline IgA result and the presence of an IgG result 17 VE a second serumsample is necessary to check for an acute infection
18. gG IgA Wash 4times 400 ul Washing Solution Remove Residues on aCellulose 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 12 von 12 REV 13 Pertussis Toxin IgG Pertussis Toxin IgA ELISA GB Druckdatum 04 02 2014
19. ithout suspected Pertussis The main part of the negative Pertussis Sera in IgG is found below the 10VE limit the main part of the positive ones above 40 0 40 0 the 17VE limit S In the area between 10 and 17 u VE positive sera are found E whose antibodies may result from post or just starting cr 300 4 infection LM Significant high antibody titer z responsible for an acute A a infection if the vaccination is g a longer than 12 months ago are E 2 po found above 17 VE 100 Um amp OL 10 A The main part of the negative 8 E A sera of blooddonors is found x A below the 10 VE limit 40 Um i 40 IU ml 10 0 4 d r 5 Po a m uis ok pi T as 2 R a hut E A WA Ee 0 0 00 A A in LINE predefined positive sera A blooddonors E in LINE predefined negative sera 1 Line Immuno Assay ot Virotech 11 7 Correlation between declared and measured IU ml The follow ing diagramshow s the correlation of the declared IU ml of several dilutions of the WHO International Standard w ith the IU ml determined using the Virotech Pertussis Toxin IgG ELISA The calculated Pearson correlation coefficient demonstrates the very good agreement betw een the calculated and declared values Seite 10 von 12 REV 13 Pertussis Toxin IgG Pertussis Toxin IgA ELISA GB Druckdatum 04 02 2014 Ki o E S o E Pearson correlation coefficient 0 998 100 150 200 250 300 IU ml declared 1
20. re found in healthy adults as w ell Infected infants up to an age of 12 months do usually not develop IgA antibodies against Pertussis Toxin Infants betw een 1 4 years rarely develop IgA antibodies against Pertussis Toxin at an age between 5 10 years they develop only very small concentrations of IgA antibodies against Pertussis Toxin 6 In this case the detection of specific IgM can be a notice for a recent infection 3 IgG antibodies occur 2 3 w eeks after beginning disease in the serum at the earliest Re infections are marked by increased antitoxin IgG and IgA antibodies as a rule IgG and secrete IgA antibodies are beside the specific sensibilised T lymphocytes the carrier of the long term immunity 2 The pertussis serology cannot replace antigen detection but should ber performed in addition The anti pertussis antibodies are produced later in comparison to other infectious diseases With the WHO World Health Organization International Standard Pertussis Antiserum NIBSC National Institute for Biological Standards and Control code 06 140 a reference serum enabling the standardised and precise quantification of the anti PT IgG concentration of a patient serum in International Units ml has been in existence since 2009 The Bordetella reference centres in several countries have suggested that this standard serumshould be used for a tw o cut off system w ith a low er limit of 40 50 IU ml and an upper limit of 100 120 IU ml 11 12 13
21. te batch This has led to a standard non linear regression curve expressed mathematically by the follow ing formula 14 IU ml Cln D A OD corr A 1 B Where A expected OD w ith an anti PT IgG concentration of 0 B gradient C point of inflexion D expected OD at an infinitely high anti PT IgG concentration OD corr corrected OD of the patient serum To correctfor fluctuations within test processing the measured OD of the patient serum is corrected w ith a calibration control OD Calibration control given OD Calibration control measured ODcorr OD Patient serum The values of parameters A B C and D as w ellas the given value for the OD of the calibration control are to be found in the certificate 6 additional standard value pairs are defined in the certificate These also describe the standard curve and can be used if the evaluation software is not compatible w ith this calculation method Determination of the IU mI value The IU ml can be determined with a program that can be ordered from Virotech Alternatively an evaluation template for common table calculations can be supplied The borderline range for the quantification w ith the Virotech Pertussis Toxin IgG Quantification Set is defined as being from gt 40 lU ml to lt 100 IU ml corresponding to the VE range from gt 10 VEto lt 17 VE The quantifiable range lies from 5 IU ml to 500 IU ml Seite 8von 12 REV 13 Pertussis Toxin IgG Pertussis
22. y essentialto use duplicates for the cut off and calibration control Working dilution of the patient sera 1 100 e g 10l 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 conjugates 30 min at 37 C with cover Stop conjugate incubation by w ashing 4 times pls refer to point 3 above Pipette 100pl 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 such a w ay thatthe blank value is deducted f romall other extinctions Extinctions should be measured within 1 hour after adding the stopping solution Seite 5 von 12 REV 13 Pertussis Toxin IgG Pertussis Toxin IgA ELISA GB Druckdatum 04 02 2014 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
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