Package Insert - Sekisui Diagnostics
Contents
1. 9 IgA positive Control 1300pl human serumw ith protein stabilizer and preservative ready to use 10 IgG Conjugate anti human 11ml sheep or goat horseradish peroxidas e conjugate with protein stabilizer and preservative in Tris Buffer ready to use 11 IgA Conjugate anti human 11ml sheep or goat horseradish peroxidase conjugate with FCS and preservative in Tris Buffer ready to use 12 Tetramethylbenzidine substrate solution 3 3 5 5 TMB 11ml ready to use 18 Citrate Stopping Solution 6ml contains an acid mixture Seite 3von 8 REV 14 Bordetella pertussis ELISA IgG IgA GB Druckdatum 03 02 2014 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 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 dismissed2. Material Soag Shee Status Diluted 2 to 48 C max 6h Test Samples Undiluted 1210486 1 week monis Microtitreplate After Opening SET ns P i boe dd Rheumatoid factor Undiluted After Opening 2 to 8 C Absorbent Diluted 2 to 80 1 week 7210 487 protect from Tight After Opening 210 8T protect from Tight After Opening After Opening 2 to 8 months Washing Solution mal Dilution ready to use 6 Precautions and Warnings 1 Only sera which 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 2 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 3 The disposal of the used materials has to be done according to the country specific guidelines 7T Material required but not supplied Aqua dest demin Eight channel pipette 50ul 100 1 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 EI
3. A plate w ashing device ELISA plate spectrophotometer w avelength 450nm reference length 2 620nm Reference Wavelength 620 690nm Incubator DD ONE BRD 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 Seite 4 von 8 REV 14 Bordetella pertussis ELISA IgG IgA GB Druckdatum 03 02 2014 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 diro Vat 8 3 o N DOOR 10 Set incubator to 37 C and check proper temperature setting before start of incubation Br
4. Bordetella pertussis ELISA IgG IgA Testkit Order No EC115 00 Color Coding silver 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 14 Bordetella pertussis ELISA IgG IgA GB Contents We Intended cc 3 2 Diagnostic Relevance 3 1i eerie tui ee ee does Teo ark ee ee nog ere o cc res nsara 3 3 Test Principle enun Reeves Sesceec Set AREENAN E EEA 3 4 Package Contents IgG and IgA Testkit uuursnnnsnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nennen 3 5 Storage and Shelflife of the Testkit and the ready to use reagents 4 6 Precautions and Warnings urrrnnsnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnenn nnnnn nnn nn nn nn hnius nr nanus nn nnn 4 7 Material required but not supplied nuessssnnnnnnennnnnnnennnnnnnnnnnnnnennnnnnnennn anne n nnn nennen 4 82 gt Test Proced re We 4 8 1 E IIo NITE CETERORUM 4 8 2 Preparation of Reagents 5 1 Hi nina isti tede ee bete ese ie ote true edes este sete Re ena AEE AS 5 8 3 Virotech ELISA Test Proced re e etre rre t rer be Ae hou stenatt I a ERN
5. E E goo Herner 5 9 4 Usage ol ELISA DEOCOSSOIS 5e toe tern Una ta E C De een kcu covayens duc aD ran ehren tan OPER c eta Mee ADAE 5 ErIgariwtunpEmecee IER 6 9 1 Testf nction Control tee ete ae ea M erase na 9 2 Calculation of the Virotech Units VE 9 3 Interpretation Scheme IgG and IgA 9 4 Limit OF the Test seront Eee teen feeseret rte ese nde AS exer Posee S os le T E Rer PER E 10 Performance Data c ccccccc cccscccaszecessteaececssecciesetoedecetecccvscctcedccaececudetecsewcasacceeeccagsemtecetvetceqesteci 7 10 1 Analytical Sensitivity and Specfichy erret ertet trei tnn ea dea tea e e ens 10 2 Prevalence Expected Values 10 3 Intra assay Coefficient of Variation Repeatability UE OU EM E 7 12 Test Procedure Scheme 1er eene LEE cdedceneecetasebasuidcuetedsscquueveliscctetensoevecens 8 Seite 2von 8 REV 14 Bordetella pertussis ELISA IgG IgA GB Druckdatum 03 02 2014 1 Intended Use The Bordetella pertussis ELISA is a screening test It is intended for the qualitative and semiquantitative detection of IgG and IgA antibodies against PT and FHA in human serum 2 Diagnostic Relevance The main agent of the genus Bordetella B pertussis causes the clinical picture of w hooping cough The Pertussis Toxin PT is of significant importance for the pathogenesis of whooping cough It is a real exotoxin responsible for many physiological im
6. Virotech Bordetella pertussis ELISA and then compared w ith the Virotech Pertussis Toxin ELISA to determine the analytical sensitivity and specificty The sera collective comprises follow ing sera 25 sera from patients w ith suspected Bordetella pertussis infection 80 sera obtained from routine tests respiratory diseases w ithout suspicion of Bordetella pertussis 95 blood donor sera Sera collective n 200 Virotech Pertussis Virotech Bordetella pertussis ELISA IgG Virotech Bordetella pertussis ELISA IgA Toxin ELISA Negative Borderline Positive Negative Borderline Borderline o o 8 6e Positive o o ze 2 e Borderline results have not been considered for the calculation of the sensitivity and specificity Referring to the Virotech Pertussis Toxin ELISA a sensitivity of gt 99 8 resp a specificity of 78 1 for IgG and a sensitivity of 80 0 resp a specificity of 90 2 for IgA have been obtained 10 2 Prevalence Expected Values The follow ing table show s the results of the examination of 80 blood bank sera in IgG and 78 blood bank sera in IgA pr ges s Ne 10 3 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 for IgG is lt 9 and for IgA is lt 15 11 Literature Wiersbitzky S Pertussis Kosteng nstige Pravention zuw enig gen
7. he 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 kis recommended to use the Virotech Bordetella pertussis LINE Immunoblot to confirm a positive IgG or positive IgA result 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 Filamentous haemagglutinin is a group antigen w hich is also found in other agents of the species Bordetella e g Bordetella parapertussis Bordetella bronchiseptica 5 6 A cross reactivity is therefore to be expected Seite 6von 8 REV 14 Bordetella pertussis ELISA IgG IgA GB Druckdatum 03 02 2014 10 Performance Data 10 1 Analytical Sensitivity and Specificity 200 sera were tested in the
8. ibodies Their concentration can be expressed in Virotech units VE Fluctuations resulting from the test procedure can be balanced with this calculation method and a high reproducibility is achieved in this w ay Use the means of the OD values for calculation of the VE 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 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 VE positive control OD patient serum OD cut off control VE patient serum 9 3 Interpretation Scheme IgG and IgA A Result VE Evaluation 1 9 0 negative 30 110 1 If the measured values are above the defined borderline range they are considered to be positive please take notice of vaccination management 2 lf t
9. id 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 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 Seite 5von 8 REV 14 Bordetella pertussis ELISA IgG IgA GB Druckdatum 03 02 2014 9 Test Evaluation The ready to use controls serve for a semiquantitative determination of specific IgG and IgA ant
10. ing 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 IgG and IgA positive negative and cut off controls as w ellas diluted patient sera We propose a double insertion blank controls and patient sera for cut off controla double insertion is absolutely necessary Working dilution of patient sera 1 100 e g 10ul 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 w ith 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 501 of citrate stopping solution into each w ell Shake plate carefully and thoroughly until liqu
11. munological 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 3 An important adherence protein for attaching B pertussisto the mucosal cells of the respiratory tract is filamentous haemagglutinin FHA 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 atthe earliest and can persist 6 24 months They are also developed in vaccinated adults during a natural re infection without clinical disease and are therefore found in healthy adults as well Infected infants up to an age of 12 months do usually not develop IgA antibodies against Pertussis Toxin Infants between 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 4 In this case the detection of specific IgM can be a notice for a recent infection 2 IgG antibodies occur 2 3 weeks after onset of the 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 immunit
12. n 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 8von 8 REV 14 Bordetella pertussis ELISA IgG IgA GB Druckdatum 03 02 2014
13. utzt Therapiew oche 25 1995 p 1485 1486 Lehrbuch der Medizinischen Mikrobiologie H Brandis W K hler H J Eggers G Pulverer 7 Auflage p 483 Mastrantonio et al 1997 Bordetella parapertussis inf ections Dev Biol Stand 89 255 259 Wirsing von K nig et al 1999 Evaluation of a single Sample Serological Technique for Diagnosing Pertussis in Unvaccinated Children Eur J Clin Microbiol Infect Dis 18 341 345 5 Elisabeth Bergfors MD et al Parapertussis and Pertussis Differences and Similarities in Incidence Clinical Course and Antibody Responses Intern J Infet Dis 3 3 1999 6 Jacob Dubuisson F et al Molecular characterization of Bordetella bronchiseptica filamentous haemagglutinin and its secretory machinery Microbiology 2000 146 1211 1221 PrO gem Seite 7 von 8 REV 14 Bordetella pertussis ELISA IgG IgA 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 IgG IgA Samples Dilution 1 101 e g 10 ul serum plasma 1000 ul 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 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 IgA Wash 4times 400 ul Washing Solutio
14. y 1 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 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 and IgA Testkit 1 1 Microtiter Plate consisting of 96 with antigen coated breakable single w ells lyophilised 2 PBS Dilution Buffer blue readyto use 2x50ml 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 negative Control 1300pl human serum w ith protein stabilizer and preservative ready to use 5 IgG cut off Control 1300pl human serum w ith protein stabilizer and preservative ready to use 6 IgG positive Control 1300pl human serum w ith protein stabilizer and preservative ready to use 7 IgA negative Control 1300pl human serumw ith protein stabilizer and preservative ready to use 8 IgA cut off Control 1300pl human serumw ith protein stabilizer and preservative ready to use
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