Package Insert - Sekisui Diagnostics
Contents
1. keep in dark 9 Stopping of substrate reaction pipette 50ul of citrate stop 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 thatthe blank value is deducted f romall other extinctions Extinctions should be measured w ithin 1 hour after adding the stopping solution Seite 5 von 10 REV 23 My coplasma pneumoniae ELISA IgG IgMWIgA GB Druckdatum 04 02 2014 8 4 9 1 9 2 9 3 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 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 will support quality assurance in yo2. 1 Intended Use The Mycoplasma pneumoniae ELISA is used for the semiquantitative and qualitative detection of IgG IgM and IgA antibodies in human serum The detection of IgG antibodies is set so that it mainly detects fresh infections 2 Diagnostic Relevance The bacteria Mycoplasma pneumoniae w hich is lacking cell wall components is the cause of atypical pneumonia and tracheobronchitis of humans and affects mostly children young adults and immunodeficient people 1 2 3 4 So called adhesins 6 enable the bacteria to attach to the epithelial cells against w hich the host develops antibodies Studies made by Foy show that in the USA 15 to 20 of all pneumonia cases are caused by Mycoplasma pneumoniae 8 The ELISA detects Mycoplasma antibodies w ith a defined antigen fraction of the strain M129 w hich is defined via monospecific sera It includes membrane proteins cytoskeleton proteins and recombinant proteins The incubation time during an infection w ith Mycoplasma pneumoniae is 10 21 days e Specific IgM antibodies occur 6 10 days after infection Basically about 80 of the patients younger than 20 years develop IgM antibodies and 40 of the patients that are older than 20 years This means a specific IgM response can be missing especially in older patients IgM antibodies may be detected referring to literature still at least one year after beginning of the symptoms e Specific lgG antibodies appear 9 14 days after infection They may
3. Staus Storage Shelflife Diluted 2 to 80 max 6h Test Samples Undiluted 2 to 8 C 1 week 3 months ng Microtitreplate After Opening fee Rn deed g bag Pen Rheumatoid factor Tweek After Opening Ritar Opening Atter Opening em After Opening 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 Nevertheless samples diluted samples controls conjugate 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 havean 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 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 100yl Micropipettes 10ul 100ul 1000pl 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 cp Ur ER 0 mw Seite 4 von 10 REV 23 My coplasma pneumoniae ELISA IgG IgM IgA GB Druckdatum 04 02 2014 9 ELISA plate spectrophotometer w avelength 450nm re
4. ater If crystals have formed in the concentrate please bring the concentrate to roomtemperature before use and shake w ell before use 5 High IgG titers or rheumatoid factors may disturb the specific detection of IgM antibodies and lead to false positive respetively false negative results For a correctlgM determination it is therefore necessary to treat the sera with RF SorboTech VIROTECH adsorption The IgM controls must not be treated w ith the pre absorption 8 3 Virotech ELISA Test Procedure 1 For eachtestrun pipette 100ul each of ready to use dilution buffer blank IgG IgM and IgA positive negative and cut off controls as wellas 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 at 37 C w ith cover 3 End incubation period by w ashing microtiter strips 4 times w ith 350 4001 w ashing solution per w ell Do not leave any washing solution in the w ells 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 7 Pipette 100ul of ready to use TMB into each well 8 Incubation of substrate solution 30 min at 37 C with cover
5. persist up to 4 years e Specific IgA antibodies appear one w eek after start of the infection and decrease about 5 weeks after start of the infection again As a rule the IgA titer exceeds the IgM titer Considering the fact that IgM antibodies persist very long in some persons and are missing in others completely it is important to detct beside the IgM also the specific IgG and IgA titer Re infections often take place w ithout any production of IgM antibodies but under significant increase of IgG and IgA antibody titers Tw o patient sera taken at an interval of 5 10 days allow a proper statement concerning the rise of the antibody titer 5 It is important to consider thata first attack of Mycoplsma pneumoniae does not leave a sufficient protection against a new colonization For diagnosis it is necessary in any case to consider the clinical picture in addition to the serological results Mycoplasma infections are generally treated successfully with antibiotics like Tetracycline and Macrolide The treatment with non suitable w g cell w all specific antibiotics penicillin leads to a serological advantage for Mycoplasma against all Penicillin sensitive microorganisms 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 immunoglobulins are again r
6. 00 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 IgM 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 pl Stop Solution shake carefully Measure Photometer at 450 620nm Extinctions Reference Wavelength 620 690nm Seite 10 von 10 REV 23 My coplasma pneumoniae ELISA IgG IgW IgA GB Druckdatum 04 02 2014
7. ET 5 94 cExamination Materlal 15 Artnr Rei MIRI edi EE DURUM 8 2 Preparation of Reagents 8 8 Virotech ELISA Test Procedure 8 4 Usage of ELISA processor P M 9 TestsEval Wath On m 6 9 11 Test Punction COMMON PEE 6 9 2 Calculation of the Virotech Units VE sesseessssesseeeeeee nennen nennen enne nnn nnne nnne enne enne 6 9 3 Interpretation of Results 6 9 4 Interpretation Scheme Luxe sce iive sete reti enc TTE 7 PESE COERISIIPEREMED EHE 7 10 Performance Data siisii eaea setseewoatsecevaccuseeccsecccvseiedseccasaccdeetcaneceteccevetecacsteche 7 10 1 Analytical Sensitivity and Specficily eene te eren tt eee dna tt t rt n 7 10 2 Diagnostic Sensitivity m 10 3 Diagnostic Specificity 10 4 Prevalence Expected Values coiere Baer En e ed te Pet pte era pd ee idee 8 10 5 Intra Assay Coefficient of Variation Repeatability sessesesseeeeeeeeeenennneneenne nennen nnne 9 10 6 Inter Assay Coefficient of Variation Reproducibility eeceeceesceeseeneeeeeeeeeesneeeeesaeecaeesaeeeaeeeaeesaeeeaeeeaeeeaeseaeeeaeenaeeeaees 9 MEE CUIU IM 9 12 Test Procedure Schemata 2c c sccceeeceeeeeeee ee ee ee ence ee eeee ee eeeaeeeaeseeeseeeaseaeeeseeeeeeeneseeeeeeeeneoes 10 Seite 2von 10 REV 23 My coplasma pneumoniae ELISA IgG IgW IgA GB Druckdatum 04 02 2014
8. IgA GB Druckdatum 04 02 2014 12 Tetramethylbenzidine substrate solution 3 3 5 5 TMB 11ml ready to use 13 Citrate Stopping Solution 6ml contains an acid mixture 4 2 IgA Set IgA negative Control 1300ul human serumw ith protein stabilizer and preservative ready to use IgA cut off Control 1300pl human serumw ith protein stabilizer and preservative ready to use IgA positive Control 1300pl human serumw ith protein stabilizer and preservative ready to use IgA Conjugate 2 anti human 11ml sheep or goat horseradish peroxidase with FCS and preservative in Tris Buffer ready to use PONS 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 shown 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
9. Mycoplasma pneumoniae ELISA recombinant IgG IgM Testkit IgA Set Order No EC114 00 IgG IgM Testkit Order No EC114 08 IgA Set Color Coding dark blue FOR IN VITRO DIAGNOSIS ONLY Sekisui Virotech GmbH Lowenplatz 5 65428 Russelsheim Germany Tel 49 6142 6909 0 Fax 49 6142 966613 http www sekisuivirotech com CE Druckdatum 04 02 2014 REV 23 Mycoplasma pneumoniae ELISA IgG IgM IgA GB Contents He Intended US 3 2 Diagnostic Relev nce 2u 1i eret tei laserceleeitewcnevedewecesseecbdestewece seteuccesseeweuecseetecseesweeds 3 3 Test Principle ERE san ae AANEEN EE 3 4 Package Contents ia ccs ci c2esi cccchecbecceh cea ceactennetsahenducecdecdeteauedetetendute sveudubevvetuuaesiveseevesevaesvenstnes 3 A IgorlgM Testki o ERO ee a ee ey 3 42 IgA SGL s cta iecee te ern t cix mr iere fried rase ratam fob trece ae Db t ta e Da re e P LO 4 5 Storage and Shelflife of the testkit and the ready to use reagents esee 4 6 Precautions and Warnings eeeeeeseeeeeesee eene eene nnn nn nnn nn hnius nr nnne nnn nnns nr nnn 4 7 Material required but not supplied 1 eeeeeeeeeesee eene nennen nennen nennen nnn nnn nnn 4 8 Test Procedute gt IEPEBREFISRTSRFPRREEHRFESFETPERFEETERLETEFLLEFFTFEEBELEFEELFEFETFFEEEFFETBEFFPEFERREFFFEOPFEFEFFPECFEERFPREFTTETLFEF
10. and IgA 9 0 11 0 2110 b In IgG for children 0 14 years if IgM and or IgA positive For children betw een 0 and 14 years the threshold cut off in the IgG can be reduced as the Virotech ELISA for IgG is adjusted so that it mainly detects acute infections How ever the condition for using this scheme is that the serum gives a positive IgM and or IgA result Result VE Evaluation IgG 0 14 years Seite 6 von 10 REV 23 My coplasma pneumoniae ELISA IgG IgWIgA GB Druckdatum 04 02 2014 7 0 8 0 threshold 1 If the measured values are above the defined borderline range they are considered to be positive 2 For the secure detection of an infection it is necessary to determine the antibody concentration of two serum samples 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 have 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 The highest sensitivity is reached if all 3 antibody classes IgG IgM and IgA are tested as it has to be considered that some persons do not develop IgM 3 _ If the measured values are below the defined borderline range no measurable antigen specific antibodies are present in the sample The samples are considered to be negative 9 4 Interpretation Scheme No contact with Mycoplasma pneu
11. ate Clin Infect Dis 23 671 684 5 Jacobs E Mycoplasmen Infektionen mta 1997 12 236 239 6 Jacobs E Das Adh sin von Mycoplasma pneumoniae Seine Bedeutung als Virulenzfaktor in der Pathogenese und in der Diagnostik Klin Lab 1994 40 228 229 7 Dumke R A Strubel C Cyncynatus H Nuyttens R Herrmann C L ck and E Jacobs 2012 Optimized serodiagnosis of Mycoplasma pneumoniae infections Diagnostic microbiology and infectious disease Elsevier Inc 73 200 203 8 Foy HM Infections caused by Mycoplasma pneumoniae and possible carrier state in different populations of patients J Clin Infect Dis 1993 17 suppl 1 37 47 9 Baum H v et al Mycoplasma pneumoniae pneumonia revisited w ithin the German Competence Netw orkfor Community acquired pneumonia CAPNETZ BMC Infectious Diseases 2009 9 62 Seite 9 von 10 REV 23 My coplasma pneumoniae ELISA IgG IgWIgA GB Druckdatum 04 02 2014 12 Test Procedure Schemata Preparation of Patient Samples and Washing Solution V Washing Solution Fill up concentrate to 1 liter with aqua dest demin v IgG IgA Samples Dilution v IgM Samples Dilution 1 101 1 101 Rheumafactor absorption with RF SorboTech e g e g 10 ul serum plasma 1000 ul Dilution Buffer 5 pl 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 1
12. atory diseases 21 B pertussis positive sera and 17 Legionella pneumophila positive sera Analytical Sensitivity Analytical Specificity Reference Mycoplasma pneumoniae LINE Reference Mycoplasma pneumoniae LINE 10 2 Diagnostic Sensitivity To determine the diagnostic sensitivity 49 clinically characterized sera w ere tested from patients w ith established atypical pneumonia These sera came from the stocks of the CAPNETZ Foundation All patient samples had previously given a positive PCR result for Mycoplasma pneumoniae The previous serological findings w ith ELISA gave a positive finding for IgM in 34 sera and a negative finding in 15 sera 7 9 Although Mycoplasma pneumoniae DNA w as detectable in these patient samples antibodies may not be detectable if the immune response is delayed This explains the low sensitivity of the previous findings Diagnostic Sensitivity Previous findings w ith the CAPNETZ sera PCR positive and 69 serologically positive Total gG IgA and IgM If an overall evaluation is performed with IgG IgA and IgM for this critical serum panel the sensitivity is clearly raised to a value of 96 10 3 Diagnostic Specificity To determine the diagnostic specificity the follow ing sera were tested from the stocks of the CAPNETZ Foundation 26 sera from patients with clinically confirmed community acquired pneumonia not caused by Mycoplasma pneumoniae CAP Community acquired Pneumonia with a positiv
13. e PCR result for the corresponding pathogen and a negative PCR result for Mycoplasma pneumoniae Diagnostic Specificity Previous findings w ith the CAPNETZ sera M pneumoniae PCR negative 10 4 Prevalence Expected Values 71 blood donor sera w ere tested for IgG IgA and IgM oc LP OM WEN oa negative 65 92 68 6 70 9 Seite 8 von 10 REV 23 My coplasma pneumoniae ELISA IgG IgWIgA GB Druckdatum 04 02 2014 threshold positive 1 gt 3 o 0 10 5 Intra Assay Coefficient of Variation Repeatability In a single assay strips fromdifferent plates froma batch w ere tested in a chessboard pattern The resulting coefficients of variation w ere under 9 n 2x48 10 6 Inter Assay Coefficient of Variation Reproducibility 3 sera were tested in 10 independent test batches on 3 different test days This gave a coefficient of variation of lt 15 11 Literature 1 Clyde WA J Clinical overview of typical Mycoplasma pneumoniae infections J Clin Infect Dis 1993 17 suppl 1 32 37 2 Hu P C Collier A M and Baseman J B 1977 Surface parasitismby Mycoplasma pneumoniae of respiratory epithelium J of Experimental med 145 1328 13343 3 Razin S 1992 Peculiar properties of mycoplasmas the smallest self replicating prokaryotes FEMS Microbiol Lett 100 423 432 4 Taylor Robinson D 1996 Infections due to species of Mycoplasma and Ureaplasma an upd
14. emoved by washing 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 IgM Testkit 1 1 Microtiter Plate consisting of 96 with antigen coated breakable single w ells lyophilised 2 PBS Dilution Buffer blue ready to use 2x50ml pH 7 2 w ith 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 IgM negative Control 1300pl human serumw ith protein stabilizer and preservative ready to use 8 IgM cut off Control 1300pl human serumw ith protein stabilizer and preservative ready to use 9 IgM 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 IgM Conjugate anti human 11ml sheepor goat horseradish peroxidase conjugate with FCS and preservative in Tris Buffer ready to use Seite 3von 10 REV 23 My coplasma pneumoniae ELISA IgG IgM
15. ference length 2 620nm Reference Wavelength 620 690nm 10 Incubator 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 shall 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 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 1 Set incubator to 37 C and check proper temperature setting before start of incubation 2 Bring all reagents to room temperature before opening package of microtiter strips 3 Shake all liquid components w ell before use 4 Make up the washing solution concentrate to 1 L w ith distilled or demineralised w
16. he symptoms persist in spite of a negative finding we recommend that the serological diagnosis should be supported by a molecular biological test 3 Cross reactions with M genitalium or M hominis can not be excluded Also EBV positive sera can cross react 10 Performance Data 10 1 Analytical Sensitivity and Specificity To determine the analytical sensitivity 99 sera were tested for IgG IgA and IgM in comparison to the Virotech Mycoplasma pneumoniae LINE The serum panel was made up as follows 49 clinically characterized sera from patients w ith confirmed Mycoplasma pneumoniae induced atypical pneumonia M pneumoniae PCR positive provided by the CAPNETZ Foundation 34 sera from children aged 1 to 14 years suspected of having a Mycoplasma pneumoniae infection 16 sera from adults suspected of having a Mycoplasma pneumoniae infection Seite 7 von 10 REV 23 My coplasma pneumoniae ELISA IgG IgWIgA GB Druckdatum 04 02 2014 To determine the analytical specificity 161 sera were tested for IgG IgA and IgM in comparison to the Virotech Mycoplasma pneumoniae LINE The serum panel was made up as follows 71 blood donor sera 26 sera from clinically confirmed community acquired pneumonia not caused by Mycoplasma pneumoniae CAP community acquired Pneumonia with a positive PCR result for the corresponding pathogen and a negative PCR result for Mycoplasma pneumoniae 26 sera from neonates aged 0 3 months and 38 sera from patients with other respir
17. moniae or antibody level has already decreased below the c o level Very early stage of an acute infection or re infection Very early stage of an acute infecction either primary infection or reinfection w ithout IgM or IgM titer is still to come already developed IgA not yet decreased already developed IgA already decreased Re infection very late stage IgA still present IgM no longer present or reactivation or infection w ithout development of IgM Re infection very late stage IgA already decreased or not developed at al happens in some adults or reactivation or infection without development of IgM or persistent IgG titer after past infection Acute early infection IgA still missing or already decreased IgG titer still too low Im portantnote Isolated false positive IgA or IgM results can never be totally excluded As confirmation it is recommended that the IgG titer should be checked in 5 10 days or that a control should be performed using Immunoblot LINE 9 5 Limits of the Test 1 The interpretation of serological results shall alw ays include the clinical picture epidemiological data and all further available laboratory results 2 Evenif a medical history is taken and a clinical examination is performed w ith standard clinical chemistry and X rays it is difficult to distinguish a Mycoplasma infection fromother infections of the upper and low er respiratory tracts or from atypical pneumonias In unclear cases or if t
18. ur laboratory Test Evaluation The ready to use controls serve for a semiquantitative determination of specific IgG IgM 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 w ay Use the means of the OD values for calculation of the VE 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 The calculated VE of the positive controls should be w ithin the ranges mentioned in the Quality Control Certificate If those requirements OD values VE are not fulfilled the test has to be repeated 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 OD cut off control VE positive control OD patient serum VE patient serum OD cut off control Interpretation of Results a In IgM and IgA for all patients in IgG for patients gt 14 years Result VE IgG gt 14 y ears IgM
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