Package Insert - Sekisui Diagnostics
Contents
1. protein stabilizer and preservative in Tris Buffer ready to use OTO OU m Qv m t5 N Seite 3von 10 REV2 My coplasma pn polyvalent ELISA IgG IgA M GB Druckdatum 04 02 2014 11 IgA M_ conjugate anti human IgA IgM mixture 11ml sheep or goat horseradish peroxidase conjugate with protein stabilizers and preservatives in Tris buffer ready to use 12 Tetramethylbenzidine substrate solution 3 3 5 5 TMB 11ml ready to use 13 Citrate Stopping Solution 6ml contains an acid mixture 5 Storage and Shelf Life of the test kit 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 Diluted 2 to 48 C max 6h Test Samples Undiluted 32. 10 Seite 2von 10 REV2 My coplasma pn polyvalent ELISA IgG IgA M GB Druckdatum 04 02 2014 1 4 4 1 Intended Use Mycoplasma pn polyvalent ELISA is used for the semiquantitative and qualitative detection of IgG and the combined detection of IgA and IgM antibodies in human serum The detection of IgG antibodies is adjusted so that fresh infections are mainly detected To enhance the efficiency of the diagnostic testing IgA and IgM antibodies are determined together w ith polyvalent IgA lgM mixed conjugate 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 p
3. 21048 C 1 week Smonihs ng Microtitreplate After Opening tae E deeem h dd 96299 Rheumatoid factor 1 week ea Nee eae 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 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 have an 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 7 Material required but not supplied Aqua dist demin Eight channel pipette 50ul 100yl Micropipettes 10ul 100pl 1000uI Test tubes Paper tow els or absorbent paper Cover for ELISA plates Disposal box for infectious material ELISA handw asheror automated EIA plate w ashing device ELISA plate spectrophotometer w avelength 450nm reference length 620nm Reference Wavelength 620 690nm Incubator RF Sorbotech for Virotech IgA M ELISA with anti human IgA IgM mixed conjugate see too Preparation of Reagents DOO OO 9 Or BONN ab a Seite 4von 10 REV 2 My co
4. Mycoplasma pn polyvalent ELISA recombinant with polyvalent IgA M conjugate IgG IgA M Test Kit Order No EC214 00 IgG IgA M Test Kit Color Coding dark blue 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 2 Mycoplasma pn poly valent ELISA IgG IgA M GB Contents 1 Intended Use cniinn aE c lcu ceo Loci cr na sierra lle cce pepliTeculiire edili iceBoilsTeraiii 3 2 Diagnostic Relevance 1i ecce teil inet eic cete EANN innn NNN NURANA i e rad ne s er eno der ia 3 3 Test Principle nune EE A tee venice decctesdecn cub EENE E A 3 4 Package Corients ieiunii nAra Aan AEAN EENE AARE REREN UA EA RAAN EEANN KEA 3 42 BE BANTS ai oet cte rece teet tub e ene U E tete nnn 3 5 Storage and Shelf Life of the test kit and the ready to use reagents 4 6 Precautions and Warnings eeeeeeseeeeeeeee eene enne nnn nnn nn hnius nn nnne nr nnne nr nnn 4 7 Material required but not supplied eeeeeeeeeesee eene enne nennen nennen enhn nn nnn 4 8 Test Procedure eiie ecu eii turco eret cetiee cotsstaduee KESEN EAA AONAN RT 5 98 31 Examination Materi l a rae enn attese na eile SE aidai 8 2 Preparation of Reagents 8 3 Virotech ELISA Test Proce
5. all patients in the IgG for patients gt 14 years Result IgG gt 14 years IgA M lt 9 0 negative 9 0 11 0 b In the IgG for children 0 14 years w hen IgA Mis positive For children betw een 0 and 14 years the borderline range cut off in the IgG can be displaced dow nwards as the Virotech ELISA in the IgG is adjusted so that it predominantly detects acute infections How ever the condition for using this procedure is that the serumgives a positive IgA M result Seite 6 von 10 REV2 My coplasma pn polyvalent ELISA IgG IgA M GB Druckdatum 04 02 2014 Result IgG 0 14 years 70 80 1 If the measured values are above the defined borderline range they are considered to be positive 2 lf an infection is to be reliably detected the antibody content of tw o serum samples must be determined One serum sample must be tested directly after the start of the infection The second sample must be tested 5 10 days later convalescence sample The antibody concentrations of the tw o samples must be tested in parallel in the same test batch A correct diagnosis cannot be made on the basis of a single serum sample As some individuals do not form IgM and in general all 3 antibody classes IgG IgM and IgA are not tested a high measure of diagnostic sensitivity can be achieved with the IgG and the IgA M determinations together 3 lf the measured values are below the defined borderline range no measurable antigen specif ic a
6. atients that are older than 20 years This means a specific IgM response can be missing especially in older patients lgM 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 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 My coplasma 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 sensiti
7. dure 8 4 Usage of PESA Process OSs irse rtc td hne ni t erra ertet NTE o ere nete erue ree vede ITEAN ERT 9 Test Eval ation neneda siei aasar a Ena ANAE AEEA E ENESA EA AR EES ENEA 6 931 Test function Control siin a da o ce pe aa a 6 9 2 Calculation of the Virotech Units VE ssessssssssssseeseeeeeee nnne nnnm nnn nnne nnne nnne 6 9 3 Interpretation of Results H tat 6 9 4 Interpretation Scheme EET 7 9 5 E LITE BU SRESARRRRDERESSEITIOIROUDT UTI LOU TRE 7 10 Performance Data 2 a 2 ii a a es saxesisdessees cedecesscetceezedsecessegdeceasdesccaeaceezs ieectceaceszsd 8 10 1 Analytical Sensitivity and Specificity oy D 8 10 2 Diagnostic Sensitivity Sts 8 10 3 Prevalence expected values ss Mie nete ree hin T e IE ERE 8 10 4 Intra Assay Coefficient of Variation Repeatability 2 ee eeeeeeeeseeeseeeeeeeeseeeeeeeeeeeeeaeeeaeeeaeeeaeeeaeesaeeeaeseaeseaeseeeeeeeseaeeeates 8 10 5 Inter Assay Coefficient of Variation Reproducibility eccececeecsesseeseceeeeeeeeeseeeeseeceecseseseaeeeesaeseesaeseseeseeeeaesaesaesaeeeatees 9 11 Literature 2 o p eco Seed cc adacel cee tei aec eel anec casero i etes 9 12 Test Procedure Schemata cssccssccssccseeeeeeeneeseeeeeeeeeseneceneceeeeuseeesseseeeeeeaseseesseesenesenssensaee
8. into each w ell 8 Incubation of substrate solution 30 min at 37 C with cover 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 REV2 My coplasma pn polyvalent ELISA IgG IgA M 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 tis recommended to check the ELISA processor with the Validationkit EC250 00 afterw ards A regular check using the Validation kit 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 test run With this procedure you
9. is by molecular biological procedures to support the serology 3 Cross reactions with M genitalium or M hominis can not be excluded Also EBV positive sera can cross react Seite 7 von 10 REV2 My coplasma pn polyvalent ELISA IgG IgA M GB Druckdatum 04 02 2014 10 Performance Data 10 1 Analytical Sensitivity and Specificity To determine the analytical sensitivity 120 sera were tested in the IgG and IgA M in comparison to Virotech Mycoplasma pneumoniae LINE The serumpanel w as made up as follow s 70 clinically characterized sera from patients w ith established Mycoplasma pneumoniae induced atypical pneumonia provided by the CAPNETZ Foundation 34 sera from children aged 1 to 14 years suspected of having a Mycoplasma pneumoniae infection and 16 sera from adult patients w ith suspected Mycoplasma pneumoniae infection To determine the analytical specificity 131 sera were tested in the IgG and IgA M in comparison to the Virotech Mycoplasma pneumoniae LINE The serumpanel w as made up as follow s 67 blood donor sera 26 neonatal sera patients aged 0 to 3 months and 38 sera from patients with other respiratory 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 sensitivty 70 clinically characterized sera w ere
10. neralised w 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 can interfere with the specific detection of IgM antibodies and lead to false positive or false negative results For the correct detection of combined IgA and IgM with IgA IgM mixed conjugate it is therefore necessaryto pretreatthe sera with RF SorboTech VIROTECH Adsorption Material Pre adsorption is not used for the IgA M controls 8 3 Virotech ELISA Test Procedure 1 For each test mixture pipette 100pl of the ready to use dilution buffer blank together with the negative cut off and positive IgG IgA M controls as w ellas the 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 with cover 3 End incubation period by w ashing microtiter strips 4 times w ith 350 400pl 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
11. ntibodies are present in the sample The samples are considered to be negative 9 4 Interpretation Scheme IgG IgA M Interpretation No increase in antibody titer to M pneumoniae No suspicion of M pneumonia infection If the clinical symptoms persist repeat test later or consider differential diagnosis Increased antibody titer to M pneumoniae in the IgA and or IgM Suspicion of early phase of acute M pneumonia infection lsolated false positive IgA or IgM results are alw ays possible in principle and may both play a role here As confirmation the IgG titer must be checked in 5 10 days A check by Immunoblot LINE may also be recommended Increased antibody titer to M pneumoniae Suspicion of an acute infection w ith M pneumoniae Suspicion of an infection w ith M pneumonia in the recent past IgA M have already dropped Raised levels from a long past infection are also in principle possible 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 Even after taking the medical history a clinical examination standard laboratory tests and an X ray a Mycoplasma infection is difficult to distinguish from other infections of the upper and lower respiratory tract or other atypical pneumonia If the case is uncertain or if the symptoms persistin spite of negative findings w e recommend diagnos
12. pical 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 s elf replicating prokaryotes FEMS Microbiol Lett 100 423 432 4 Taylor Robinson D 1996 Infections due to species of Mycoplasma and Ureaplasma an update Clin Infect Dis 23 671 684 5 Jacobs E Mycoplasmen Inf ektionen mta 1997 12 236 239 6 Jacobs E Das Adhasin von Mycoplasma pneumoniae Seine Bedeutung als Virulenzfaktor in der Pathogenese und in der Diagnostik Klin Lab 1994 40 228 229 7 Baum H V Strubel A Nollert J Layh Schmitt G Tw o Cases of Fulminant Mycoplasma Pneumoniae Pneumonia within 4 Month Infection 28 2000 No 3 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 REV2 My coplasma pn polyvalent ELISA IgG IgA M GB Druckdatum 04 02 2014 12 Test Procedure Schemata Preparation of Patient Samples and Washing Solution V Washing Solution Fill up concentrate to 1 lite
13. plasma pn polyvalent ELISA IgG IgA M GB Druckdatum 04 02 2014 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 2 8 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 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 demi
14. r ELISA processor will function properly and this w ill support quality assurance in your 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 n OD cut off control Interpretation of Results a I the IgA M for
15. r with aqua dest demin v IgG Samples Dilution v IgA M Samples Dilution 1 101 1 100 Rheumafactor absorption with RF SorboTech e g e g 10 ul serum plasma 1000 ul Dilution Buffer 5 ul serumplasma 450 ul Dilution Buffer Serum Dilution Buffer is ready to use 1 drop RF SorboTech incubate for 15 min at room temperature Test Procedure Samples Incubation 30 minutes at 37 C 100 pl Patient Samples blank value Dilution Buffer and controls Wash 4times 400 pl Washing Solution Remove Residues on a Cellulose Pad Conjugate Incubation 30 minutes at 37 C 100 pl Conjugate IgG IgA M Wash 4times 400 pl 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 REV2 My coplasma pn polyvalent ELISA IgG IgA M GB Druckdatum 04 02 2014
16. tested from patients w ith established atypical pneumonia These sera w ere taken fromthe stocks of the CAPNETZ Foundation All patient samples had previously given a positive PCR result for Mycoplasma pneumoniae in the prior finding The prior ELISA tests had been IgM positive for 35 sera and negative for 35 sera 9 Although Mycoplasma pneumoniae DNA can be detected in these patient samples antibodies may not yet be detectable if the immune response is delayed This explains the low sensitivity of the prior findings Diagnostic Sensitivity Prior finding of the CAPNETZ sera PCR positive and 5096 serologically positive total E As a result of the combined evaluation of IgG and IgA M a markedly increased sensitivity of 80 was attained for this critical serum panel 10 3 Prevalence expected values 67 blood donor sera were tested in the IgG and IgA M PG i positive 1 1 1 1 10 4 Intra Assay Coefficient of Variation Repeatability Strips of different plates of a batch w ere tested with two serain a chessboard pattern This gave a coefficient of variation of lt 9 n 2x48 Seite 8von 10 REV 2 My coplasma pn polyvalent ELISA IgG IgA M GB Druckdatum 04 02 2014 10 5 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 1596 11 Literature 1 Clyde WAJ Clinical overview of ty
17. ve microorganisms 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 removed 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 Package Contents IgG IgA M Testkit 1 Microtiter Plate consisting of 96 with antigen coated breakable single w ells lyophilised PBS Dilution Buffer blue ready to use 2x50ml pH7 2 w ith preservative and Tw een 20 PBS Washing Solution 20x concentrated 50ml pH7 2 w ith preservative and Tw een 20 IgG negative Control 1300pl human serum w ith protein stabilizer and preservative ready to use IgG cut off Control 1300pl human serum w ith protein stabilizer and preservative ready to use IgG positive Control 1300pl human serum w ith protein stabilizer and preservative ready to use IgA M negative Control 1300pl human serumw ith protein stabilizer and preservative ready to use IgA M cut off Control 1300pl human serumw ith protein stabilizer and preservative ready to use IgA M positive Control 1300pl human serumw ith protein stabilizer and preservative ready to use IgG Conjugate anti human 11ml sheep or goat horseradish peroxidas e conjugate with
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