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Package Insert - Sekisui Diagnostics
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1. 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 dismissed Material Status Storage Shelflife max 6h eer T week 2 to 81 months e pee Absorbent 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 Seite 4von 9 REV 14 Helicobacter py lori IgG Helicobacter py lori IgA ELISA GB Druckdatum 03 02 2014 o og uoo Eom 8 microtiter strips should be treated as potentially infectious material Please handle products in accordance with laboratory directions Those
2. 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 Eight channel pipette 50ul 10011 Micropipettes 10ul 100ul 10004 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 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 flexi
3. results in an analytic sensitivity of 92 596 and specificity of 90 296 for IgAG 10 2 Infection expected values To obtain the expected values infection 80 sera were tested for IgG and IgA In a blood donor population an infection rate of about 30 can be expected About 90 show an IgA response Seite 7 von 9 REV 14 Helicobacter py lori IgG Helicobacter py lori IgA ELISA GB Druckdatum 03 02 2014 LJ we nso No 27 Borderime o o 2 18 225 10 3 Intra assay variation coefficient repeatability In one assay strips fromdifferent plates of one lot w ere tested with one serum The variation coefficient obtained thus is lt 9 for IgA and IgG 10 4 Inter assay variation coefficient reproducibility 3 sera were tested in 10 independent test batches in different laboratories and by different test persons The variation coefficient obtained thus is lt 15 for IgA and IgG 11 Literature 1 Helicobacter pylori Von der Grundlage zur Thearpie 1996 Herausgeber P Malfertheiner Thieme Verlag 2 Homepage Nationales Referenzzentrumf r Helicobacter pylori Institut f r Medizinische Mikrobiologie und Hygiene der Universit t Freiburg 3 Z ller et al 1993 Nachw eis der Helicobacter pylori Infektion Rolle der Immundiagnostik Klin Lab 39 45 54 4 Brandis Eggers 1994 Lehrbuch der Medizinischen Mikrobiologie 7 Auflage S 495 5 Kist M Glocker E Helicobacter pylori Infektionen Studie ResiNet
4. zur Resistenzentw icklung aktuelle Ergebnisse Epidemiologisches Bulletin 2005 Nr 24 6 KistM Glocker E SuerbaumS Pathogenese Diagnostik und Therapie der Helicobacter pylori Infektion Bundesgesundheitsblatt 2005 7 Helicobacter pyloriund gastroduodenale Ulkuskrankheit AWMF Leitlinien Register Nr 021 001 2008 Seite 8von 9 REV 14 Helicobacter py lori IgG Helicobacter py lori IgA ELISA 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 Test procedure 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 Solution Remove Residues on a Cellulose Pad Substrate Incubation 30 minutes at 37 C 100 pl Substrate Stopping 50 ul Stopping Solution shake carefully Measure Photometer at 450 620nm Extinctions Reference Wavelength 620 690nm Seite 9von 9 REV 14 Helicobacter py lori IgG Helicobacter py lori IgA ELISA GB Druckdatum 03 02 2014
5. 20 4 IgG negative Control 1300p 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 gG Conjugate anti human 11ml sheep or goat horseradish peroxidase 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 4 2 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 pH7 2 with preservative and Tw een 20 3 PBS Washing Solution 20x concentrated 50ml pH7 2 with preservative and Tw een 20 4 IgA negative Control 1300pl human serumw ith protein stabilizer and preservative ready to use 5 IgA cut off Control 1300pl human serumw ith protein stabilizer and preservative ready to use 6 IgA positive Control 1300pl human serumw ith protein stabilizer and preservative ready to use 7 IgA Conjugate 2 anti human 11ml sheep or goat horseradish peroxidase conjugate with FCS 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 5
6. Helicobacter pylori ELISA IgG Testkit IgA Testkit Order No EC143G00 IgG Testkit EC143A00 IgA Testkit Color Coding IgG blue metallic IgA blue metallic black FOR IN VIT RO 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 C Druckdatum 03 02 2014 REV 14 Helicobacter py lori IgG Helicobacter py lori IgA ELISA GB Contents ER IM E RE 3 2 Diagnostic Relevance eege seteuccsveeweeecseetecseesweces 3 3 Testi clon 3 d Package Contents a a iaa 4 A AGO TOS MA ANA TS 4 AD LE EE tn Ge eaters 4 5 Storage and Shelflife of the Testkit and the ready to use reagents 4 6 Precautions and Warnings ccceecececeeeeeeeeeeeeeeeeeeeeeeeeee rre 4 7 Material required but not supplied uuessssnnsnnneennnnnnennnnnnnennnnnnnnnnnnnnennnnnnnennnnnnennn nennen nnn nnn 5 8 Test DE TE 5 8 11 Examination Material MERE IMEE E E 8 2 Preparation of Reagents 8 8 Virotech ELISA Test Procedure 8 4 Usage of ELISA proCess OFS vivian et 9 TestEvalutatien 0 a nn Reisen 6 9 1 Testfunctionic nti l euere A asi 9 2 Calculation of the Virotech Units VE 9 3 Interpretation Scheme IgG and IgA n 9 4 E Ee RTE 10 Performance Data geseet genge iu ed geed AER dE RER EELER AER anciano acid 7 10 1 Analytic S nsitivit
7. LISA 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 will support quality assurance in your laboratory 9 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 with this calculation method and a high reproducibility is achieved in this way 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 lt 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 ell as 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 o
8. Remove residues on a cellulose pad 4 Pipette 100ul of ready to use conjugate into each well 5 Incubation of conjugates 30 min at 37 C with cover 6 Stop conjugate incubation by w ashing 4 times pls refer to point 3 above Seite 5von 9 REV 14 Helicobacter py lori IgG Helicobacter py lori IgA ELISA GB Druckdatum 03 02 2014 7 Pipette 100ul of ready to use TMB into each w ell Incubation of substrate solution 30 min at37 C with cover keep in dark 9 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 10 Measure extinction OD at 450 620nm Reference Wavelength 620 690nm Set your photometer in such a w ay that the 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 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 E
9. all further available laboratory results 6 Both IgA and IgG Helicobacterpylori LINE results should be taken into consideration for the diagnosis of patient s sera suspected to have a Helicobacter pyloriinf ection 7 lA antibodies may persist 6 months to 3 years after successful treatment Normally IgG antibodies persist for many years 10 Performance Data 10 1 Analytic sensitivity and specificity To determine the analytic result clinically defined sera n 19 interlaboratory test sera n219 routine sera n 40 and blood donor sera n 80 were tested for IgG with a commercially available ELISA and the Virotech Helicobacter pylori LINE Samples with discrepant results w ere additionally characterised using a commercially available immunoblot Sera collective n 158 Helicobacter pylori ELISA IgG Analytic result en MN BCR positive o o 74 This results in an analytic sensitivity and specificity of gt 99 9 for IgG The borderline results w ere not included in the analysis To determine the analytic result clinically defined sera n 20 interlaboratory test sera n 19 routine sera n 32 and blood donor sera n 80 were tested for IgA with a commercially available ELISA and the Virotech Helicobacter pylori LINE Samples with discrepant results w ere additionally characterised using a commercially available immunoblot Sera collective n 151 Helicobacter pylori ELISA IgA AE CO D e borderis 5 o 7 This
10. bility regarding the possibility to use the dilution buffer washing solution TMB citrate stopping solution as w ell 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 w ith the plate lot indicated in the Quality Control Certificate 1 2 3 4 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 8 3 Virotech ELISA Test Procedure 1 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 ell as diluted patient sera We propose a double insertion blank controls and patient sera for cut off control a double insertion is absolutely necessary Working dilution of patient sera 1 100 e g 10ul serum 1ml dilution buffer 2 After pipetting start incubation for 30 min at37 C with cover 3 End incubation period by w ashing microtiter strips 4 times w ith 350 400ul w ashing solution per w ell Do not leave any w ashing solution in the w ells
11. d number of bacteria eg in e marked atrophy of the gastric mucosa e gastric haemorrhage e use of proton pump inhibitors While all other methods can give false negative results in these cases detection of specific H pylori antibodies with maintained sensitivity is possible 2 3 Test Principle ELISA for detection of human serum IgG and IgA antibodies 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 washing 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 when the stopping solution is added Immunoblot procedure for the detection human serum IgG and IgA antibodies The Helicobacter pylori LINE from Virotech has the advantage that antibody production against specific virulence factors e g CagA and VacA of H pylori can be detected Seite 3von 9 REV 14 Helicobacter py lori IgG Helicobacter py lori IgA ELISA GB Druckdatum 03 02 2014 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 pH 7 2 with preservative and Tw een 20 3 PBS Washing Solution 20x concentrated 50ml pH7 2 with preservative and Tw een
12. f 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 x10 OD cut off control VE positive control VE patient serum 9 3 Interpretation Scheme IgG and IgA Result VE Evaluation 50 110 Seite 6 von 9 REV 14 Helicobacter py lori IgG Helicobacter py lori IgA ELISA GB Druckdatum 03 02 2014 1 If the measured values are above the defined borderline range they are considered to be positive 2 lf 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 3 lf 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 9 4 Limits of the Test 5 The interpretation of serological results shall always include the clinical picture epidemiological data and
13. ulcer disease and MALT lymphoma is possible with antibiotic therapy in the early stage 5 Early treatment can counteract the development of gastric carcinoma Le diagnosis of H pylori infection as soon as possible should be attempted In patients who have not had previous treatment the bacteria can be fully eliminated in 80 96 of cases 2 Recurrent infection after successful H pylori eradication is very low approx 1 per year 7 If antibiotic therapy has already been used once the success rates are low eron further treatment because of the increasing antibiotic resistance For this reason it is recommended to detect H pylorifromculture of gastric biopsies in patients w ho have previously been treated once and to carry out sensitivity testing 7 8 Invasive and non invasive methods can be used to detect H pylori infection The invasive methods include the rapid urease test histology culture and PCR In these methods the pathogen is found in biopsies The non invasive tests include the urea breath test the stool antigen test and detection of antibodies in the serum All tests have advantages and disadvantages and are not absolutely accurate on their own The method should therefore be selected according to the query 7 Serology is employed e as an initial test in patients w ho have not been previously treated 2 e in monitoring therapy long term 1 e in seroepidemiological investigations 3 Serology is indicated in cases with a reduce
14. y ANA Speck Cilicia ab 7 10 2 Infection expected values eec eere A iras 7 10 3 Intra assay variation coefficient repeatability ooonoonicnnndinnnnnnonnonnconcnnccnrcnnccnnrecnrnnn nennen nennen nenne 8 10 4 Inter assay variation coefficient reproducibility ran ennemi 8 uh De VE TEE 8 12 Test Procedure Scheme nn ia aaa 9 Seite 2von 9 REV 14 Helicobacter py lori IgG Helicobacter py lori IgA ELISA GB Druckdatum 03 02 2014 1 Intended Use The ELISA testkit is intended for the qualitative and semiquantitative detection of Helicobacter pylori specific IgG and IgA antibodies in human serum 2 Diagnostic Relevance Helicobacter H pylori is a Gram negative bacterium specialising in the gastric mucosa w hich causes about 500 000 deaths w orldw ide due to gastric carcinoma 1 6 The infection is usually acquired in childhood Transmission is fromperson to person and close contact within the family and socioeconomic status play an important part Accordingly prevalence is much higher in developing countries than in industrial nations 7 In Germany the infection rate in adults is about 30 5 H pylori infection persists throughout life and causes chronic gastritis which often remains without clinical symptoms How ever in 20 of those affected complications occur in the form of a gastric ulcer duodenal ulcer gastric carcinoma or MALT lymphoma mucosa associated lymphatic tissue 5 Healing of the
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