Package Insert - Sekisui Diagnostics
Contents
1. 4444444HRnn nen nnnnnnnnnnnnnnn nennen nnnennn nn 10 12 Literature sale een an deceive sede cag ran eeesuveevecuavsctecsuaes 10 13 Test Procedure SCHOM iic cosd cece deceedecadeceddeececesedececceeezstecattseneccassedeagezstecedteedecceetencetetes 12 Page 2 of 12 REV 3 My coplasma pneumoniae LINE IgA IgG IgM GB Print Date 04 02 2014 1 Intended Use Line Immunoblot Testkit for the quantitative detection of specific IgG IgA and IgM antibodies in human serum Line Immunoblot is used for the serological diagnostic testing of a fresh or recent Mycoplasma pneumonia infection The kit can be used for serological diagnostic testing alone Alternatively it can be used as a confirmatory test if the result of another assay is questionable or positive The LINE has not yet been evaluated for specific questions such as the differentiated identification of pathogens in post infectious arthritis or in the Guillain Barree Syndrome 2 Diagnostic Relevance The bacteria Mycoplasma pneumoniae which 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 inthe USA 15 to 20 of all pneumonia cases are caused by Mycoplasma pneumoniae 7 The infection is endemic wit2. Procedure uunns4unsnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnsnnnnnnnanennnenn 6 9 4 Use of Immunoblot Proc eSSOrs ceceeeceeeeeee cece eee ee ee eeee cease ee ARANAS REAA EE EAAS 7 10 Interpretation of Results uennsnunssnnnnnnnnennnnnennnnnnennnnnnnennn nennen nenn anne nennen nennen nennen 7 10 1 Evaluation of the patient samples ussn4ssnnnnsnnennnnnnnnnnnnnnnennnnnnnnnnnnn nn nnnnnn nn nnnnnnn 7 10 2 Use fithe Cut Off COMPO enere E E AA AAEE AAEE RNAAR 7 10 3 Significance of the antigens 2 00 0 aiT ata ee eeaaeeeeeeaaaeeeeaaaeeeeeaaaeeeesaaeeeeeed 7 10 4 Evaluation Citerla nn ee en elek 8 10 5 Interpretation Scheme IgG IgA and IgM uss24244ssnssennnnsennnnnnnnnnnnnnnnnnnnnnnnnn nn nnnnen 8 10 6 Overall Constellations of Findings IgG IgA and IgM 44ss44444nnnn nen nnnnnn en nnnnn 9 10 7 Test Limits cece ee ann 9 T1 Performance Data BRPETSRPRREFLEERFERLTEETHEPELTEEPPEFEERFERLTEELFEFEETREFFFETEREFLTEELFEEFERERPEETLELFEFFELEREPFERRFEREETR 9 11 1 Analytical Sensitivity and Specificity 0 2 cece ene cece eee ee eres nate ee essa eeeeeaaeeeeeeaaeeeeeaaeeeeeed 9 11 2 Seroprevalence expected VAlUCS ce ceceeeneeeeeeeaeeeeeeaaeeeeeeaaeeeeeeaaeeeeeeaaeeeeeaaeeeeeeaaanees 10 11 3 Intra Assay Precision repeatability u 244444444Hnennnnnnennnnnnnennnnnnnnnnnnnnnnnnnnn nn 10 11 4 Inter Assay Precision reproducibility
3. in Medical Microbiology 2 83 90 Page 11 of 12 REV 3 My coplasma pneumoniae LINE IgA IgG IgM GB Print Date 04 02 2014 13 Test Procedure Scheme Test Procedure in short version Samples Incubation 30 minutes 15 ul patient serum plasma 100 ul control in 1 5 ml dilution washbuffer each Washing 3x5 minutes with 1 5 ml dilution washbuffer each Conjugate incubation 30 minutes with 1 5 ml working dilution 1 100 Washing 3 x 5 minutes with 1 5 ml dilution washbuffer each 1x 1 minutes with Aqua dest deionised Substrate incubation 10 3 minutes with 1 5 ml substrate solution each Stopping 3 x without incubation in between with 1 5 ml Aqua dest deionised each Conjugate Dilution table rounded Number of strips la ls Jle Jo fo Number ofstrips Im 2 pe fa ps pe r fe fo fo Number ofstrips a e fa a a se a Ja a o Number ofstrips o e2 fos foa oe fo o fe fo fo Conjugate concentrate ou asou soo siou ssou Isao seou ara soou oo Page 12 of 12 REV 3 My coplasma pneumoniae LINE IgA IgG IgM GB Print Date 04 02 2014
4. indicates earlier contact w ith Mycoplasma pneumoniae Antibodies against Mycoplasma pneumoniae are detectable Borderl Weaker reaction during convalescence with persistent antibodies or in the initial stages of an infection Orderline A follow up is recommended Positive Antibodies to Mycoplasma pneumoniae are detectable Indicates fresh or recent infection with Mycoplasma pneumoniae Negative 10 6 Overall Constellations of Findings IgG IgA and IgM Interpretation No contact with Mycoplasma pneumoniae or antibody levels have already dropped to below the cut off level ee alle ee ee Very early phase of an acute infection or re infection ZEN Very early phase of an acute infection either first infection or re infection w ithout IgM or IgM titre has not yet increased Acute infection usually first infection late phase IgG and IgM already formed IgA has not yet decreased Acute infection usually first infection late phase IgG and IgM already formed IgA has already decreased Re infection very late phase IgA still present no more IgM present or re activation or infection w ithout IgM formation Re infection very late phase IgA has already decreased or was never formed happens with some adults or re activation or infection w ithout formation of IgM or persistent IgG titre after completion of an infection KA Acute early infection IgA still missing or has already decreased IgG titre
5. still too low 10 7 Test Limits 11 11 1 A negative Blot result does not completely exclude the possibility of infection w ith Mycoplasma pneumoniae The sample may have been taken before antibodies developed or the antibody concentration is under the limit of detection of the test 2 In rare cases patients may exhibit inverse bands dark background white bands these should not be evaluated i e the Immunoblot is not evaluable in these cases The serum should be tested w ith other serological methods Performance Data 1 Analytical Sensitivity and Specificity To determine the analytical sensitivity and specificity groups of sera were tested in the IgG IgA and IgM which had previously been determined with an ELISA and a Western Blot as reference method analytical finding The follow ing groups of sera were tested blood donors n 52 cross reactors n 69 children s sera n 27 mycoplasma sera n 52 IgG Serum Group n 200 Mycoplasma pneum oniae LINE IgG Analytical Page 9 of 12 REV 3 My coplasma pneumoniae LINE IgA IgG IgM GB Print Date 04 02 2014 Finding For the IgG this gives a sensitivity of 93 0 and a specificity of 98 4 Borderline results are excluded from the calculation IgA Mycoplasma pneumoniae LINE IgA een Analytical Finding Postwe 0 2 9 For the IgA this gives a sensitivity of gt 99 and a specificity of 92 6 Borderline results are excluded from the calculati
6. Mycoplasma pneumoniae LINE IgG IgA IgM Line Immunoblot Order No WE214A16 IgA Line Immunoblot 16 strips WE214G16 IgG Line Immunoblot 16 strips WE214M16 IgM Line Immunoblot 16 strips FOR IN VITRO DIAGNOSTIC ONLY Sekisui Virotech GmbH Lowenplatz 5 D 65428 Russelsheim Tel 49 6142 6909 0 Fax 49 6142 966613 http www sekisuivirotech com ce Print Date 04 02 2014 REV 3 Mycoplasma pneumoniae LINE IgA IgG IgM GB Contents 1 Intended Use 2 a a De a Deere 3 2 Diagnostic Relevance unsunssnnsnnnnnennnnnnnnnnnnnennnnnnnennnnnnennsnnneennnnnnennnnneennsnnnrennsnnneenn nee 3 3 Principle f Test e ene ee a essen 3 4 Package Content Srania anaa aana aaa Eee ne 4 4 1 A Kt for 16 determinations s 2 2 24422060 Hetren cs epesaddedans sedetiyss ddedoediagnbeh anne nahen onen 4 5 Storage and Stability of the Testkits and the Components 0 eeeeeeeeeeeeeeeeeeeeees 4 6 Precautions and Warnings nnsennssnnnnnnnennnnnnennnnnnnnnnnnnnnnnnnnnnnennnnnnnnnnnnnnennnnnnnennnnnrenn nn 4 7 Additional required material not supplied uursunnsnnnennnnnnnnnnnnnnnnnnnnnnnennnnnnnnnn nun 5 8 Examination M terial 2 2242202220 a aan na anna ka 5 9 Test Procedure 2 2 2 2 deed Reh 5 9 1 Preparation ofthe Samples 3 ik eetene here nenne seiko er NESA 5 9 2 Preparation of Reagents ur 2 ea 5 9 3 Immunoblot Test
7. eman 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 Page 10 of 12 REV 3 My coplasma pneumoniae LINE IgA IgG IgM GB Print Date 04 02 2014 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 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 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 8 Sasaki Y et al Detection of Mycoplasma fermentans DNA fromlymph nodes of acquired immunodeficiency syndrome patients Microb Pathog England Aug 1994 17 2 p131 5 9 DaxboeckF Krause R and WenischC Laboratory diagnosis of Mycoplasma pneumoniae infection Clin Microbiol Infect 2003 9 p263 273 10 Bebear C Biological diagnosis of Mycoplasma pneumoniae respiratory infections Diagnostic biologique des infections respiratoires a Mycoplasma pneumoniae Rev Mal Respir FRANCE 1986 3 2 p67 71 11 Jacobs E 1991 Mycoplasma pneumoniae virulence factors and immune response Reviews
8. ff band refer to the certificate supplied w ith the kit Order No IgG WE214P60 resp IgA WE214P40 or IgM WE214P80 IgG IgWIgA Negative control human serum prediluted 0 5 ml The negative control shows no bands or no bands relevant to the evaluation gt Cut off band Order No IgG Ig IgA WE214N50 1x 1x 1x 1x 1x 1x 16 strips 0 5m 50 m 0 7 ml 57 ml 1 pcs 5 Storage and Stability of the Testkits and the Components Store test kit at 2 8 C The shelf life of the single components is mentioned on the relevant label for shelf life of the Kit please refer to the Quality Control Certificate 1 Do not expose the single kit components to high temperature nor freeze them 2 Do not use the kit reagents after their expiring date 3 Do not expose reagents to strong light during storage 4 The BCIP NBT substrate solution is sensitive to light and has to be stored in dark 5 Nitrocellulose test strips Use strips immediately after taken out of the bag Close bag with the not required strips again savely and store at 2 8 C When putting the results into archives please take care that the nitrocellulose test strips are protected against direct sunlight to avoid fading of the bands Material Storage Sheiflife Test Samples Undiluted 2 to 8 C 1 week 2 to 8 C Test Strips After Opening stored in supplied bag 3 months After Opening 2 to 8 C Conjugate Diluted 2 to 8 C Substrate A
9. fter Opening 2 to 8 protect from light After Opening 2 to 8 C protect from light Washing Solution Final Dilution ready to use 2 to 8 C 6 Precautions and Warnings 1 Only sera that have been tested and found to be negative for HIV 1 ab HIV2 ab HCV ab and Hepatitis B surface antigen are used as controlsera Nevertheless samples diluted samples controls and conjugate as w ellas the antigen Page 4 of 12 REV 3 My coplasma pneumoniae LINE IgA IgG IgM GB Print Date 04 02 2014 strips should be treated as potentially infectious material Please handle products in accordance with laboratory directions 2 Use plastic foreceps and w ear protective gloves whenhandling the Immunoblot Please follow the local valid w aste disposal regulations 4 The incubation baths are designed by the manufacturer for a single use The reuse of the incubation baths is at the risk of the user If they are to be reused w e recommend that after use the incubation baths be disinfected for several hours in 1 sodium hypochlorite solution and then rinsed thoroughly with tap w ater followed by distilled or deionized w ater 7 Additional required material not supplied Incubation tray if required available w ith order no WE300 08 Rocking platform vertical not centrifugal A wash bottle for stopping Pipette or handw asher Micro pipettes 5 ul 1500 ul Pipette filler Test tubes 2 20 ml volume Plastic foreceps Aqua dest or deionised
10. h small epidemic peaks appearing all 4 5 years 7 10 Mycoplasma pneumoniae is weakly infectious and transmitted only after close contact 10 Studies have shown that Mycoplasma infections are not rare in AIDS patients 8 A past infection is no protection against a re infection 11 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 canbe 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 IgG antibodies appear 9 14 days after infection 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 as a rule 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 Two patient sera taken at an interval of 5 10 days allow a proper statement concerning the rise of the antibody titer 5 It is
11. ibodies in the Designation LINE Page 7 of 12 REV 3 My coplasma pneumoniae LINE IgA IgG IgM GB Print Date 04 02 2014 P90 is expressed on the surface and is responsible for the correct and specific integration of the P1 protein into the Highly specific bacterial membrane P400 The function of P400 is largely unknow n Low molecular w eight proteins Membrane components and Specific surface expressed proteins P RP3M On the basis of sequence differences in gene P1 isolates of amp M pneumoniae are assigned to serotype 1 M129 RP3M Highly specific RP3F or to serotype 2 FH RP3F RP14 is the rec C terminal section of the P1 adhesin Protein P1 is the main adhesin main antigen of M pneumoniae Mw 176 kDa It is expressed on the surface re 5 i Highly specific localised in the Tip region and responsible for cytoadherence Antibodies to RP14 can inhibit the cytoadherence of M Highly specific pneumoniae to HBEC human bronchial epithelial cells Pdh B is a component of pyruvate dehydrogenase Pdh B is Pdh B expressed on the surface and is one of the five most frequent proteins by w eight in M pneumoniae M pneumoniae is only coated with a double layer membrane surrounded by a lipoglycan layer In this context G it is to be expected that phospholipids and glycolipids Possible acute marker in combination essential components of membranes will be to some with highly specific M pn antigens ex
12. important to consider that a 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 w ith non suitable w g cell wall specific antibiotics penicillin leads to a serological advantage for Mycoplasma against all Penicillin sensitive microorganisms Thus a fast and specific laboratory diagnosis of this infection is very important for the beginning of a suitable therapy In a comparative overview of mycoplasma diagnostic testing in 2003 the then current Virotech Mycoplasma pneumoniae Western Blot was described as possessing the highest specificity of any commercially available method 9 The Mycoplasma pneumoniae LINE is an improved follow up version of this Western Blot product 3 Principle of Test Pathogen antigen proteins are transferred onto a nitrocellulose membrane by a special spraying process The nitrocellulose membrane is then cut up into individual strips Incubation of the antigen coated nitrocellulose strips with samples of human serum or plasma permits the detection of specific antibodies These antibodies develop immuncomplexes w ith the antigen fixed on the test strip After removing the unbound antibodies by washing steps the single nitrocellulose stri
13. irate w ashing buffer alw ays completely Before ending of the last w ashing step prepare the needed amount of fresh conjugate dilution refer to table 8 Aspirate or pour aw ay the liquid completely out of the channels please refer to point 6 9 Pipette 1 5 ml of the prepared conjugate dilution each into the corresponding incubation channel and incubate for 30 minutes on the rocking platform 10 Pour away or aspirate liquid completely out of the channels 11 Washing of the strips Incubate with 1 5 ml ready to use dilution w ashbuffer each for 3 x 5 minutes on the rocking platform Pour away or aspirate the washbuffer always completely Afterwards rinse 1 x 1 minute with Aqua dest deionised 12 Pour away or aspirate the liquid completely out of the channels refer to point 6 13 Pipette 1 5 ml portions of ready to use substrate solution into the channels and develop for 10 3 minutes on the rocking platform 14 Stop the color reaction by pouring aw ay the substrate solution Afterwards washthe strips w ithout incubation in betw een for 3 x with 1 5 ml Aqua dest deionised each 15 Pour awaythe aqua dest deionised and let the strip dry on a clean cellulosis paper The background coloring that may be observed on the moistured antigen strips disappears completely when the strips are completely dry Solid antigen strips need a little longer than the conventional antigen strips until they are completely dry 16 Use the enclosed evalua
14. lues 2 Bring the corresponding concentrate to roomtemperature 20 25 C before preparing the dilution Use only high quality Aqua dest deionised and bring up to room temperature 20 25 C before usage 3 Mix dilutions w ell before starting the test 4 Dilution Washbuffer The dilution w ashbuffer is provided as a 10 fold concentrate Dilute the dilution w ashbuffer concentrate 1 10 with distilled or deionised w ater 10ml 50ml 100ml concentrate 90m 450m 900ml A distilled or deionised w ater mix well The dilution w ash buffer concentrated or already diluted may eventually show a yellow dye This yellow dye has no influence to the shelf life of the dilution w ash buffer nor does it influence the functionality or diagnostic meaning of the test run Page 5 of 12 REV 3 My coplasma pneumoniae LINE IgA IgG IgM GB Print Date 04 02 2014 5 IgG IgA resp IgM Conjugate Dilute the conjugate 1 100 with finally diluted dilution w ashing buffer and mix thoroughly 1 5 ml conjugate w orking solution is required for each serumsample See conjugated dilution table item Test Procedure 6 Substrate Solution The substrate solution is delivered ready to use 9 3 Immunoblot Test Procedure Attention The nitrocellulose teststrips may only be tested in the approved Ig class see label on blot bookletand indication on each individual test strip For the correct performance and evaluation of the Mycoplasma pneumoniae LINEs each test
15. of the cut off control are not included in the evaluation The P1 band must be of low intensity Evaluation of the band intensities consider exceptions Pdh B GL I Protein RP3M RP3F and P1 EPI P1 band The evaluation of all protein bands in the IgG IgA and IgMis related to the intensity of the P1 band of the cut off control as follow s Lower intensity than the P1 band of the cut off control 0 Same intensity as the P1 band of the cut off control 1 Greater intensity than the P1 band of the cut off control 2 The sum of the band intensities gives the overall evaluation Important exceptions In the IgM the bands Pdh B GL and l Protein are only evaluated if at least one of the bands P1 P90 or P400 is gt the cut off band i e it is evaluated with 1 or 2 In the IgG only one of the bands RP3M and RP3F is evaluated The more strongly reactive band is used for the evaluation In the IgG the seroprevalence band P1 EPI is not included in the sum This is evaluated as positive w hen its intensity is gt P1 band of the cut off control If the overall evaluation in all IgG classes is also negative this indicates that the patient had contact w ith Mycoplasma pneumoniae in the distant past 10 3 Significance of the antigens List of the recombinants used P1 P90 P400 RP3M RPSF RP14 P200 and purified native antigens NMP Pdh B GL Protein Antigen Significance of the Antigens Specificity of the Ant
16. on IgM Serum Group n 200 Mycoplasma pneumoniae LINE IgM Analytical Negative 126 7 8 Finding Borderline 4 1 8 For the IgM this gives a sensitivity of 97 7 and a specificity of 94 0 Borderline results are excluded from the calculation 11 2 Seroprevalence expected values The cut off setting was performed in such a w ay that fresh or recent Mycoplasma pneumoniae infections were detected The follow ing table show s the results from 52 blood donor sera C oe oa Ta Positive 0 4 2 Seroprevalence band P1 EPI in the IgG Of 148 sera blood donor sera n 52 cross reactive sera n 69 and children s sera n 27 89 exhibited a P1 EPI band gt cut off 60 1 11 3 Intra Assay Precision repeatability At each batch release a strip with a specific human serum w as tested in the IgG IgA and IgM in the quality control Thus 100 of all Immunoblots w ere controlled The intensities of the bands may deviate fromthe mean by maximally one step on a 1 5 point scale 11 4 Inter Assay Precision reproducibility To determine the reproducibility 4 sera each were tested in the IgG IgA and IgM The determination w as performed in 10 test batches on 6 independent test days The serological requirements were fulfilled in all tests 12 Literature 1 Clyde WAJ 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 Bas
17. ps are incubated w ith alcalic phosphatasis conjugated anti human IgG IgA respectively IgM antibodies After unbound conjugated antibodies have been removed by a further washing step a visualisation of the antigen antibody complex of the bound antibodies is accomplished by the addition of a non coloured substrate which forms blue violette precipitates at each site antigen bands where the conjugated anti human antibodies have bound The enzyme substrate reaction is stopped through washing the nitrocellulose strips with aqua Page 3 of 12 REV 3 My coplasma pneumoniae LINE IgA IgG IgM GB Print Date 04 02 2014 dest deionised Depending on the observed band pattern one can interpret the presence of specific IgG IgA respectively IgM antibodies 4 Package Contents 4 1 Kit for 16 determinations 1 w IgG resp IgM or IgA Nitrocellulose test strips with sprayed antigen solid strips stabilised on a plastic foil sorted in a booklet ready to use IgG resp IgM or IgA Cut off Control human serum prediluted Dilution washbuffer pH 7 3 10x conc with Tris and preservative IgG resp IgM or IgA Conjugate 100x conc Anti human goat Alcalic Phosphatasis with preservative Substrate BCIP NBT ready to use Evaluation Record sheet for the notation and deposit of the results Also available on request IgG resp IgM or IgA Positive control human serum prediluted 0 5 ml The positive bands gt For the cut o
18. run should include the appropriate parameter and batch specific cut off controls For reliable diagnostic testing for Mycoplasma pneumoniae the LINE should be performed in IgG IgA and IgM 1 Test has to be proceeded at room temperature 2 For each sample put 1 strip into the channel of a clean incubation tray Hold strip only at the marked upper end 3 Pipette 1 5ml ready to use dilution washbuffer each and put onto the rocking platform Take care that the antigen strips are consistently covered with liquid the strips must not dry out during the w hole test procedure 4 The solid antigen strips are being moistured completely w ithin one minute and can be incubated in supine lateral position or face dow n position 5 15 ul patient serum or plasma or 100 ul of the cut off or positive negative control added by pipetting if at all possible at the upper marked end of the strip Incubate the patient serumand control for 30 minutes on the rocking platform Ensure that no cross contamination occurs between individual patient samples during pipetting and subsequent pouring aw ay 6 Aspirate or carefully pour away the liquid out of the channels completely During the pour aw ay of the liquid the antigen strips remain at the bottom of the channel Drain the remaining liquid onto a cellulosis paper 7 Washing of strips Incubate with 1 5 ml ready to use dilution w ashbuffer each for 3 x 5 minutes on the rocking platform Pour aw ay or asp
19. tent presented on the cell surface of the bacterium w here they are recognised by the human immune system Proteins are erythrocyte antigens which are recognised by cold agglutinins CA The CAs are induced by M Possible acute marker in combination pneumoniae and are of IgMtype and are directed against with highly specific M pn antigens protein in more than 90 of cases A mixture of the P1 antigens of strains FH and M129 and Highly specific show s the seroprevalence in the IgG 10 4 Evaluation Criteria The interpretation of serological results should always incorporate the clinical picture epidemiological data and other available laboratory findings IgG or IgA Evaluation Sum of the Band Intensities Possible acute marker in combination with highly specific M pn antigens P200 is involved as a structural protein in the formation of the cytoskeleton of M pneumoniae It permits the bacterium A ae eee Highly specific to slide on surfaces so that successful host colonisation is then possible IgM Evaluation Sum of the Band Intensities lt Negative 10 5 Interpretation Scheme IgG IgA and IgM Page 8 of 12 REV 3 My coplasma pneumoniae LINE IgA IgG IgM GB Print Date 04 02 2014 Evaluation Interpretation No serological evidence for Mycoplasma pneumoniae infection A positive seroprevalence band P1 or status after an infection in the distant past EPI in the IgG gt cut off band
20. tion record sheet for evaluation The high specificity bands annotated on the rec ord sheet facilitate evaluation of the patient samples For test procedure scheme pls refer to last page Page 6 of 12 REV 3 My coplasma pneumoniae LINE IgA IgG IgM GB Print Date 04 02 2014 9 4 Use of Immunoblot Processors The follow ing instruments have been validated for the automatic processing of the Blots and LINEs Apollo and Profiblot All commercially available Blot machines are suitable in principle 10 Interpretation of Results For a secure interpretation each LINE strip is fitted out with two controls 1 Serum control Only after the incubation w ith patient serum the serum incubation band appears below the markline 2 Conjugate control The LINE strip is fitted out w ith aconjugate control band which appears after incubation w ith the respective conjugate The test procedure is valid if the serum control as well as the internal conjugate control appears clearly visible on the developed nitrocellulose test strip The position of the serum band and conjugate control band may be found on the record sheet 10 1 Evaluation of the patient samples Please refer to the protocol sheet for the position and designation of the reactive bands IgG bands P1 P90 P400 NMP RP3M RP3F and P1 EPI IgA bands P1 P90 P400 RP14 P200 IgM bands P1 P90 P400 Pdh B GL Prot 10 2 Use of the cut off control Bands which are weaker than the cut off band P1
21. w ater Filter paper DON BIRD 8 Examination Material Either serum and plasma may be used as test materials even when the package leaflet only mentions serum Plasma samples may contain any anticoagulant For CSF samples please refer to the separate instructions for the CSF LINE 9 Test Procedure Working exactly according to the Sekisui Virotech user manual is the prerequisite for obtaining correct results 9 1 Preparation of the Samples 1 15 ul serum or plasma are needed for each patient sample For CSF serum processing use only the separate individually calculated CSF serum dilution for each IgG class see instructions for the CSF LINE 2 Blood samples should be taken separately by venous puncture Serumis separated after complete coagulation not applicable to plasma 3 Repeated freezing and thaw ing should be avoided 4 Sera that are heat inactivated lipaemic haemolytic or microbiologically contaminated may lead to faulty results and shall therefore not be used 5 Do notuse turbid samples especially after thawing centrifuge if necessary 5 minutes at 1000 x g pipette clear supernatant and use for testing 9 2 Preparation of Reagents 1 To facilitate routine laboratory w ork all LINEs and EcoBlots canbe processed ina single test run w ith the same incubation times and the same component when these are independent of the parameters and batches The cut off controls now have parameter and batch specific va
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