DN 2050-#### G - EUROIMMUN US, Inc.
Contents
1. Semin Pediatr Infect Dis 17 2006 14 19 Riffelmann M Littmann M Hellenbrand W H l e C Wirsing von K nig CH Pertussis Not Only a Disease of Childhood Dtsch Arztebl Int 105 2008 623 628 Riffelmann M Thiel K Schmetz J Wirsing von K nig CH Performance of Commercial Enzyme Linked Immunosorbent Assays for Detection of Antibodies to Bordetella pertussis J Clin Microbiol 48 2010 4459 4463 Trollfors B Taranger J Lagergard T Sundh V Bryla DA Schneerson R Robbins JB Serum IgG antibody responses to pertussis toxin and filamentous hemagglutinin in nonvaccinated and vaccinated children and adults with pertussis Clin Infect Dis 28 1999 552 559 http www cdc gov pertussis vaccines html Medizinische EUROIMMUN RES Medizinische EUROIMMUN RES Contact information for the U S EUROIMMUN US 1100 The American Road Morris Plains New Jersey 07950 973 656 1000 800 913 2022 www euroimmun us DN_2050G_A_US_CO1 doc Version 11 09 2013 09 34 002. two most important adherence factors are filament haemagglutinin FHA and pertussis toxin PT which can function as an exotoxin or as an adhesin In contrast to B pertussis B parapertussis does not produce pertussis toxin B pertussis and B parapertussis also possess on their outer membranes the membrane toxins pertactin and BrkA Bordetella resistance to killing which play a role in attachment to the host cell Further virulence factors are adenylate cyclase toxin which affects the immune response of the host and tracheal cytotoxin TCT formed from the cell wall peptidoglycan which causes stasis of cilia movement The clinical progression of whooping cough depends mainly on the formation of various virulence factors by the pathogen After an incubation time of around 7 to 14 days Bordetella pertussis infections begin with an uncharacteristic catarrhal stage which lasts for about 1 to 2 weeks Then the convulsive stage develops lasting for 2 to 3 weeks with typical paroxysmal staccato coughing attacks frequently followed by stridor with possible vomiting Nocturnal attacks are frequent During both of these stages the pathogen is coughed out Transmission via contaminated objects cannot be excluded Following this is the decrimenti stage which lasts for several weeks with continual diminishment of coughing attacks Mainly in the case of children under the age of 2 years complications such as secondary pneumonia or otitis media are
3. working strength universal buffer each 2 Incubation with conjugate Aspirate off pipette 1 5 ml of working strength enzyme conjugate 1 10 into the incubation channel Shake i Washing Aspirate off wash 3 x 5 min with 1 5 ml working strength universal buffer each 3 Incubation with substrate Aspirate off pipette 1 5 ml of substrate solution into the incubation channel Shake Gi Stopping Aspirate off rinse three times with 1 5 ml deionized water Evaluation Evaluate at same day Medizinische EUROIMMUN Labordiagnostika TUE 0 HE AG Evaluation of results After stopping the reaction using deionised or distilled water place the incubated test strips onto the corresponding field of the evaluation protocol using a pair of tweezers The position of the test strips can be corrected while they are wet As soon as all test strips have been placed onto the protocol they should be pressed hard using filter paper and left to air dry After they have dried the test strips will be stuck to the adhesive foil Visual evaluation with reaction control card Hold the reaction control card next to the incubated strip and compare the band intensity with the two color bars of the card Band intensities as weak as or weaker than the negative color bar are evaluated as negative Band intensities as strong as or stronger than the positive color bar are evaluated as positive Automated evaluation with EUROLineSc
4. Antibodies against Bordetella pertussis IgG Test instruction for the EUROLINE For In Vitro Diagnostic Use CLIA Complexity High ORDER NO ANTIBODIES AGAINST IG CLASS SUBSTRATE FORMAT DN 2050 1601 G Bordetella pertussis antigens laG Ag coated 16 x 01 16 DN 2050 24001 G FHA PT and ACT g immunoblot strips 240 x 01 240 Intended use The EUROIMMUN EUROLINE Bordetella pertussis IgG kit is an immune lineblot strip test for the qualitative detection of IgG class antibodies against Bordetella pertussis antigens FHA PT and ACT in human serum and plasma EDTA Li heparin Citrate Detection of these antibodies is used as an aid in the diagnosis of infections with Bordetella pertussis in conjunction with other laboratory and Clinical findings Summary and explanation Clinical significance The genus Bordetella B encompasses four known species B pertussis which causes whooping cough B parapertussis which causes a mild whooping cough syndrome B bronchiseptica and B avium They are distributed worldwide are highly contagious and are transmitted from person to person by droplet infection Pathogenesis The bacteria bind via various adhesins to the cells of the ciliated epithelium without reaching the epithelium or bloodstream Besides the capsule which protects the pathogen from inactivation by complement there are functionally two groups of virulence factors adhesins and toxins Adhesins and toxins The
5. De Melker HE Versteegh FGA Conyn van Spaendonck MAE Elvers LH Berbers GAM van der Zee A Schellekens JFP Specificity and Sensitivity of High Levels of Immunoglobulin G Antibodies against Pertussis Toxin in a Single Serum Sample for Diagnosis of Infection with Bordetella pertussis J Clin Microbiol 38 2000 800 806 Dragsted DM Dohn B Madsen J Jensen JS Comparison of culture and PCR for detection of Bordetella pertussis and Bordetella parapertussis under routine laboratory conditions J Med Microbiol 53 2004 749 754 Guiso N Berbers G Fry N K He Q Riffelmann M Wirsing von K nig C H What to do and what not to do in serological diagnosis of pertussis recommendations from EU reference laboratories EUR J Clin Microbiol Infect Dis 2010 Hijnen M He Q Schepp R Van Gageldonk P Mertsola J Mooi FR Berbers GA Antibody responses to defined regions of the Bordetella pertussis virulence factor pertactin Scand J Infect Dis 40 2008 94 104 Isacson J Trollfors B Hedvall G Taranger J Zackrisson G Response and decline of serum IgG antibodies to pertussis toxin filamentous hemagglutinin and pertactin in children with pertussis Scand J Infect Dis 27 1995 273 277 Khelef N Danve B Quentin Millet M J Guiso N Bordetella pertussis and Bordetella parapertussis Two Immunologically Distinct Species Inf Immun 61 1993 486 490 Munoz FM Pertussis in infants children and adolescents diagnosis treatment and prevention
6. a pertussis antigens er EOS CONG 1x0 04ml 5x0 04ml POS CONTROL 50x IgG human 50x concentrate 3 Enzyme conjugate Alkaline phosphatase labelled anti human 1x3 ml 16x 3ml CONJUGATE 10x IgG goat 10x concentrate 4 Universal buffer 1 x 50 ml 8 x 100 ml BUFFER 10x 10x concentrate gt er se 1 x 30 ml 8 x 50 ml SUBSTRATE 6 Incubation tray 2 x 8 channels TRAY 7 Test instruction 1 booklet 1 booklet LOT Lot X Storage temperature IVD In vitro determination gt Unopened usable until Additional materials and equipment not supplied Pipettes with a range of 10 ul to 1000 ul with single use tips Tweezers for handling of the strips Multistep pipette with top for dispensing up to 1 5 ml per pass Rocker Vortex mixer Distilled or de ionized water for buffer preparation stopping Glass plastic tubes for dilution of samples and reagents Lint free towelling For visual evaluation please order the required evaluation protocol and reaction control card under ZD 2050 0101 Evaluation protocol visual Anti Bordetella pertussis EUROLINE ZD 0007 0101 Reaction control card For automated evaluation with EUROLineScan only e For automation purposes it may be necessary to use incubation trays different than those included in the kit Formats with different numbers of channels are available from EUROIMMUN under the following o
7. an The evaluation protocol with the test strips is scanned using a flatbed scanner EUROIMMUN AG and evaluated with EUROLineScan For use of the EUROLineScan program please refer to the EUROLineScan user manual EUROIMMUN AG The code for entering the test into EUROLineScan is Bord pert IgG There is a control band on the strips The incubation was performed correctly if a strong color reaction is visible on this control band Additional controls may be tested in accordance with local state and or federal regulations or accreditation requirements and your laboratory s quality control procedures It is recommended that the user refers to CLSI document C24 A and 42 CFR 493 1202 c for guidance on appropriate quality control practices Interpretation of Results Antigens and their arrangement on the strips The EUROLINE test strips have been coated with the following antigens Bordetella pertussis antigens FHA FHA Filamentous hemagglutinin antigen is a cell surface protein of Bordetella pertussis which functions as an adhesin for this organism It is a component of many new acellular PT pertussis vaccines PT Pertussis toxin a virulence factor secreted by Bordetella ACT pertussis ACT Adenylate cyclase toxin of Bordetella pertussis control Medizinische EUROIMMUN Labordiagnostika TUE 0 0 AG Based on signal intensity the results can be divided into negative and positive results Visual evaluation wit
8. e Deviations from the indicated volumes times etc can lead to incorrect results 4 Adaptation of this assay for use with automated sample processors and other liquid handling devices in whole or in part may yield differences in test results from those obtained using the manual procedure It is the responsibility of each laboratory to validate that their automated procedure yields test results within acceptable limits Medizinische EUROIMMUN Labordiagnostika TUE 0 0 AG Expected values Specificity and sensitivity n 29 1 reference test Anti ACT pos EUROIMMUN Ce a u E EUROLINE neg Antibodies against ACT The sensitivity and specificity determined in the investigation of 29 serologically precharacterised sera from patients with Bordetella pertussis infections amounted to 96 and 100 with respect to a reference test naa 0 CT 3 reference tests Anti PT po pos mg EUROIMMUN eee gl ee en EUROLINE Antibodies against PT The sensitivity and specificity determined in the investigation of 43 serologically precharacterised sera from patients with Bordetella pertussis infections amounted to 92 and 100 with respect to concordant results from three reference tests DEZ TEE 2 reference tests Anti FHA ps ng EUROIMMUN ps o s o E EUROLINE a EEE JE Antibodies against FHA The sensitivity and specificity determined in the investigation of 49 serologically precharacterised sera from pa
9. e diluted serum samples and incubate at room 1 step temperature 18 C to 25 C for 30 minutes on a rocking shaker Wash Aspirate off the liquid from each channel and wash 3 x 5 minutes each with 1 5 ml working strength universal buffer on a rocking shaker Conjugate incubation Pipette 1 5 ml diluted enzyme conjugate alkaline phosphatase conjugated 2 step anti human IgG into each channel and incubate for 30 minutes at room temperature 18 C to 25 C on a rocking shaker Wash Aspirate off the liquid from each channel Wash as described above Substrate incubation Pipette 1 5 ml substrate solution into the channels of the incubation tray Fa g n 3 step Incubate for 10 minutes at room temperature on a rocking shaker Stop Aspirate off the liquid from each channel and wash each strip 3 x 1 minute with deionised or distilled water Evaluate Place test strip on the evaluation protocol air dry and evaluate For automated incubation with the EUROBlotMaster select the programme Euro02 Inf WB30 Medizinische EUROIMMUN Labordiagnostika AG EUROLINE Bordetella pertussis IgG Incubation protocol Blocking Pipette 1 5 ml working strength universal buffer into the incubation channel and insert test strip Shake Gi 1 Sample incubation Aspirate off pipette 1 5 ml of diluted serum sample 1 51 into the incubation channel Shake Gi Washing Aspirate off wash 3 x 5 min with 1 5 ml
10. frequent There is no difference in morbidity between boys and girls Season and climate have no influence on the frequency of the disease An infection confers specific immunity which reduces after decades Infections in adulthood are known but are seldom diagnosed Re infections in persons older than 60 years are life threatening Prevention For the US the CDC publishes vaccination recommendations for prevention of Bordetella pertussis infections The influence of pertussis vaccination on infections with B parapertussis is not yet fully understood Neither whole cell lysates of B pertussis nor mixtures of different antigens in acellular vaccines appear to protect against infection with B parapertussis Medizinische EUROIMMUN Labordiagnostika TEU 0 6 AG Principles of the test The test kit contains test strips coated with parallel lines of highly purified antigens In the first reaction step diluted patient samples are incubated with the immunoblot strips In the case of positive samples the specific IgG antibodies also IgA and IgM will bind to the corresponding antigenic site To detect the bound antibodies a second incubation is carried out using an enzyme labelled anti human IgG enzyme conjugate catalysing a color reaction Materials supplied in the kit Contents of the test kit Component Format Format Symbol 1 Test strips coated with antigens 16 x 1 strips 15 x 16 strips STRIPS Bordetell
11. h reaction control card Reaction control card compared to color bars Test result As weak as or weaker than negative color bar Negative As strong as or stronger than positive color bar Positive Automated evaluation with EUROLineScan EUROLineScan grey scale units Test result 0 10 Negative gt 10 Positive Antigen o O Significance and characteristics Specific for Bordetella pertussis Exclusion of B parapertussis infections Anti PT antibodies are detectable both after infection and vaccination PT is contained in many acellular vaccines Pertussis toxin PT Bordetella specific B pertussis and B parapertussis Anti FHA antibodies are detectable both after infection and vaccination FHA is contained in many acellular vaccines Filamentous haemagglutinin FHA Bordetella specific B pertussis and B parapertussis Adenylate cyclase toxin Antigen is currently not used in acellular vaccines ACT Anti ACT antibodies can be an indication of a naturally acquired infection Limitations of the procedure 1 This kit is used as an aid in diagnosis only A positive result should be interpreted with clinical findings and other diagnostic tests 2 The results obtained from this assay are not diagnostic proof of the presence or absence of a disease 3 The test is only to be performed by laboratory professionals in a clinical laboratory setting Strictly adhere to the test procedur
12. ncture following CLSI formerly NCCLS document H03 A6 Use of hemolyzed hyperlipemic hemolytic heat treated or contaminated samples should be avoided Stability CLSI document H18 A3 recommends the following storage conditions for samples Samples should be stored at room temperature no longer than 8 hours If the assay will not be completed within 8 hours the samples should be refrigerated at 2 8 C If the assay will not be completed within 48 hours or for shipment of the sample samples should be frozen at 20 C or lower Frozen samples must be mixed well after thawing and prior to testing Diluted samples should be incubated within 8 hours Do not use bacterially contaminated samples Sample dilution The patient samples for analysis are diluted 1 51 in diluted universal buffer For example add 30 ul of sample to 1 5 mi diluted universal buffer and mix well by vortexing Sample pipettes are not suitable for mixing Procedure Blocking Fill the channels of the incubation tray according to the number of serum samples to be tested with 1 5 ml ready for use diluted universal buffer each Remove the required amount of test strips from the packaging using a pair of tweezers and place them one by one in the channels containing the buffer The number on the test strip should be visible Incubate for 15 minutes at room temperature on a rocking shaker Afterwards aspirate off all the liquid Sample incubation Fill each channel with 1 5 ml of th
13. ration of lt 0 09 Sodium azide has been reported to form lead or copper azides in laboratory plumbing which may cause explosions Rinse sink thoroughly with water after disposing of solutions containing azide Avoid skin contact 6 Some of the reagents are poisonous buffer substrate solution Avoid skin contact 7 Storage and stability The test kit has to be stored at a temperature between 2 C to 8 C Do not freeze Unopened all test kit components are stable until the indicated expiry date 8 Waste disposal Patient samples control and incubated test strips should be handled as infectious waste All reagents must be disposed of in accordance with local disposal regulations Preparation and stability of the reagents Note All reagents must be brought to room temperature 18 C to 25 C approx 30 minutes before use After first use the reagents are stable until the indicated expiry date if stored at 2 C to 8 C and protected from contamination unless stated otherwise below Coated test strips Ready for use Open the packing with the test strips only when the strips have reached room temperature to prevent condensation on the strips After removal of the strips the packing should be sealed tightly and stored at 2 C to 8 C Positive control The control is a 50x concentrate For the preparation of the ready for use control the amount required should be removed from the bottle using a clean pipette and diluted 1 51
14. rder numbers These trays are for single use only ZD 9899 0130 white tray for 30 strips ZD 9898 0130 black tray for 30 strips ZD 9898 0144 black tray for 44 srips ZD 9898 0148 black tray for 48 strips e For the creation of work protocols and the evaluation of incubated test strips using EUROLineScan green paper and adhesive plastic foil are required ZD 9880 0101 Green paper 1 sheet ZD 9885 0116 Adhesive foil for approx 16 test strips ZD 9885 0130 Adhesive foil for approx 30 test strips Medizinische EUROIMMUN Labordiagnostika TEU 0 0 AG Warnings and precautions 1 For in vitro diagnostic use For use by laboratory professionals in a clinical or research laboratory setting 2 Before starting the assay carefully read the instructions Use only the valid version provided with the kit 3 Observe prudent laboratory practice and safety guidelines Avoid eye and skin contact with specimens and reagents In case of eye or skin contact wash off thoroughly with plenty of clean running water Remove and wash contaminated clothing In case of ingestion obtain medical attention 4 The serum contained in the control has been tested negative for HBsAg anti HCV anti HIV 1 and anti HIV 2 using FDA cleared or European CE approved test systems Nonetheless all materials should be treated as being a potential infection hazard and should be handled with care 5 Some of the reagents contain sodium azide at a concent
15. tients with Bordetella pertussis infections amounted to 100 with respect to two concordant results from reference tests Note It is recommended that each laboratory determine its own normal range based on the population and equipment used Performance characteristics Measurement range The EUROLINE is a qualitative method No measurement range is provided The titre limit is given at a dilution of 1 51 Inter and intra assay variation The inter assay variation was determined by multiple analyses of characterised samples over several days The intra assay variation was determined by multiple analyses of characterised samples on one day In every case the intensity of the bands was within the specified range This EUROLINE displays excellent inter and intra assay reproducibility Cross reactions The high analytical specificity of the test system is guaranteed by the quality of the antigen substrates used antigens and antigen sources This EUROLINE specifically detects IgG class antibodies against Bordetella pertussis antigens FHA PT and ACT Interference Haemolytic lipaemic and icteric sera up to a concentration of 5 mg ml for haemoglobin of 20 mg ml for triglycerides and of 0 4 mg ml bilirubin showed no effect on the analytical results of the present EUROLINE Medizinische EUROIMMUN Labordiagnostika TEU 0 0 AG 10 11 12 Literature references Cherry J D Immunity to Pertussis CID 44 2007 1278 1279
16. with ready for use universal buffer Example add 30 ul of control to 1 5 ml of ready for use diluted universal buffer and mix thoroughly The ready for use diluted control should be used at the same working day Enzyme conjugate The enzyme conjugate is supplied as a 10x concentrate For the preparation of the ready for use enzyme conjugate the amount required should be removed from the bottle using a clean pipette and diluted 1 10 with ready for use diluted universal buffer For 1 test strip dilute 0 15 ml anti human IgG concentrate with 1 35 ml ready for use diluted universal buffer The diluted enzyme conjugate should be used at the same working day Universal buffer The universal buffer is supplied as a 10x concentrate For the preparation of the ready for use universal buffer the amount required should be removed from the bottle using a clean pipette and diluted 1 10 with deionised or distilled water For the incubation of 1 test strip 1 5 ml buffer concentrate should be diluted with 13 5 ml deionised or distilled water The ready for use diluted universal buffer should be used at the same working day Substrate solution Ready for use Close bottle immediately after use as the contents are sensitive to light Medizinische EUROIMMUN Labordiagnostika TUU 0 HE AG Preparation and stability of the serum or plasma samples Sample material Human serum or EDTA Li heparin or citrate plasma Samples are to be obtained by venipu
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