Package Insert - Sekisui Diagnostics
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1. w ashing solution in the w ells Remove residues on a cellulose pad 4 Pipette 100pl of ready to use conjugate into each w ell 5 Incubation of conjugates 30 min at 37 C w ith cover 6 Stop conjugate incubation by washing 4 times pls referto 3 above 7 Pipette 100pl of ready to use TMBinto each well 8 Incubation of substrate solution 30 min at 37 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 thor oughly 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 within 1 hour after adding the stopping solution Seite 5von 11 REV 26 Liquor Standards ELISA IgG IgM IgA GB Druckdatum 03 02 2014 7 4 Pls refer to last page for Test Procedure Scheme 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 recommende2. test run catalogue number EN108L45 7 3 Virotech ELISA Test Procedure e CSF serumpairs are principally to be analysed next to each other in the same determination row on one test plate e We recommend a double insertion for blank controls standard sera patient sera and CSF samples e To minimize matrix effects as much as possible a w orking dilution of 1 1 for CSF and 1 400 for serumis used t is recommended for IgM Diagnostics in general that a start be made with a dilution of 1 100 follow ed if necessary the 100w ME measurement point exceeded by a 1 400 dilution e Please carry outthe pre treatment w ith RF SorboTech for IgM diagnostics Please make sure to use the green dilution buffer in the case of VZV IgM 1 For eachtestrun pipette 100pl each of the dilution buffer blank the ready to use standard sera the ready to use Al controls if available or serum quality controls and the diluted CSF and serum samples Working dilutions of the serumsamples IgG 14400 e g 5ul serum 2ml dilution buffer IgM 14 100 e g 10ul serum 1ml dilution buffer IgA 14400 e g 5ul serum 2ml dilution buffer Working dilution of sera 1 400 e g 5ul serum 2mI dilutionbuffer Working dilution of CSF 1 1 e g 150pl CSF 150pl dilutionbuffer 2 After pipetting start incubation for 30 min at 37 C w ith cover 3 End incubation period by washing microtiter strips 4 times with 350 400pl washing solution per w ell Do not leave any
3. the respective Total Immunoglobuline Quotient The value is therefore independent from the condition of the individual cerebral barrier function The antibody index allow s the conclusion to the presence and dimension of a CNS ow n synthesis of pathogen specific antibodies This method is not valid in case of a poly specific intrathecal immunoglobuline synthesis as then the toal IgX quotient is no longer suitable as barrier parameter and has to be replaced by the so called Limes value refer to calculation of the Limes Quotient in point 8 3 4 B 4 Package Contents Standards for the quantification of pathogen specific antibody concentrations 4 vials 1000ul human serum w ith protein stabilizer and preservative ready to use 100w ME 25w ME 6 2w ME 1 5w ME w ME arbitrary measurement units Seite 3von 11 REV 26 Liquor Standards ELISA IgG IgM IgA GB Druckdatum 03 02 2014 5 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 col
4. CSF Standards Order No Borrelia afzelii VISEIgG IgG Borrelia afzelii IgM IgM CMV IgG EBV IgG FSME TBE IgG HSV 1 gG1 IgG HSV 2 gG2 IgG HSV Screen IgA Measles IgG Mumps IgG VZV IgG VZV IgA EC022L60 EC022L80 EC113L60 EC102L60 EC117L60 IgM EC117L80 EC130L60 EC131L60 EC108L40 IgG EC108L60 EC105L60 IgM EC105L80 EC106L60 EC110L60 IgM EC110L80 EC110L40 Please also refer to our CSF diagnostic test with separate instructions for Rubella IgG EC109L00 FOR IN VITRO DIAGNOSIS ONLY ce mdc Notified Body 0483 Druckdatum 03 02 2014 REV 26 Liquor Standards ELISA IgG IgM IgA GB Sekisui Virotech GmbH L wenplatz 5 D 65428 R sselsheim Tel 49 6142 6909 0 Fax 49 6142 966613 http www sekisuivirotech com Contents Ae intended USe cc 3 2 Diagnostic Relev nce 2zu 22u 1 sea ine casercelceivewccesstewecdsavedbeyseewccesstewecdsveeweedseteteeveerwteds 3 3 Test Principle 22222 na Bee aan A REN E EE 3 4 Package Contents eiecerunt cdots EEANN AAEE ENN EAEN ON ESKAE EENEN TN REA 3 5 Storage and Shelflife of the Testkit and the ready to use reagents 4 6 Precautions and Warnings unssnnsnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnne nn nnn nn hnius nn hnius nn nnne nn nnn 4 Tz Test Proced re iieri doctus Lice ee di aasevel gebedecdaateasdetewecdaatezabieaberest 4 TA Examination Material cioe Hrn r
5. M IgA 0 77 x YQ ai 23 x107 3 1x10 8 3 4 Calculation of the Antibody Index Al A Qigx lt Qui The antibody index Al states the relation between the pathogen specific antibody quotient Qspec and the total immunoglobulin quotient Quia Thus a pathogen specific antibody synthesis can be detected and quantified In this case the total immunoglobulin quotient is used as barrier parameter IgX spec csr x dilution Al QIgX spec 9X spec Serum x dilution Q Ig X total IgX total CSF Ig X total Serum B Qigx gt Quim In case an additional poly specific intrathecal immunoglobulin synthesis is present the measured Qitota Must no longer be used for the Al calculation as an antibody synthesis searched for or eventually present at the same time may be falsified in its extent or even get totally unrecognizable In this cases the so called Limes value of the immunoglobulin quotient is calcu Seite 7 von 11 REV 26 Liquor Standards ELISA IgG IgM IgA GB Druckdatum 03 02 2014 8 4 8 5 9 lated see formula or graphically determined by using the albumin quotient w hich has to be calculated additionally This Li mes value is used instead of the immunoglobulin quotient for calculation of Al value 1 2 X spec Al QIgX spec Q Lim Interpretation Al Evaluations 4 Al lt 0 6 undetectable Should not theoretically occur Occasionally found in routine w ork No patholog ical significance Desirable to search
6. conjugate for all parameters and for all different lots The standards are parameter specific and only to use with the plate lots they are related to Please refer to Quality Control Cer tificate of the serum kit for possible combinations of plate lots and standard lots 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 with distilled or demineralised w ater If crystals have formed in the con centrate please bring the concentrate to room temperature before use and shake w ell before use 5 IgM Diagnostic Pre absorbance with RF SorboTech High IgG titer or rheumatoid factors may disturb the specific detection of IgM antibodies and may lead to false positive resp false negative results Hence for correct IgM determinationit is necessary to pre treat the sera and fluids with RF SorboTech VIROTECH adsorbent Please make sure to use the green dilution buffer in the case of VZV IgM Pre absorption is not necessary forthe IgM controls and the standards 6 HSV Screen IgA Determination Please note that the IgA conjugate catalogue number 131301 is not contained in the kit so that it must be given sepa rately w hen ordering the CSF standard We also recommend including the corresponding antibody index controls to check the
7. d 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 w ill support quality assurance in your laboratory 8 Test Evaluation 8 1 Test function control To guarantee the optimal function of the test kit the OD values of the 100 arbitrary units IgG IgM and IgA antibody standard serums must be above the minimal values given in the quality control certificates When using the Al controls keep to the ranges given in the control certificate If there are no Al controls the validity of the test run must be checked w ith the serum quality controls a OD values The OD of the blank should be 0 15 The OD values of the negative controls should be under the OD values given in the quality control certif icate The OD values of the positive controls and of the cut off controls should be above the OD values given 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 given in the quality control certificate If the requirements OD values VE are not fulfilled the test must be repeated 8 2 In
8. e Ne else 1 2 Prep ration f Re gents 2 c0 E 7 3 Virotech ELISA Test Procedure 7 4 Usage of ELISA proCess 09S oie tet eee nde ed dem rad dade nda Pu a rete e ce a are 8 Test Eva Uat OD elei RU aaae aa cae RE RN ERI aa rE MER S REC dMRA ES 6 9 4 Westf nctior controls n a N ve irren 6 PME niic E e 6 8 3 Calculation of the antibody index Al With example seseeseeseeeeeeeneneneeneneenneneen emen neret 6 8 4 Interpretation nente tene 8 5 Limits of the Test 97 UICICI I IM ETE 8 10 Test procedure Scheme Serum and CSF Diagnostic uusnnnsnnennnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nn 10 Seite 2 von 11 REV 26 Liquor Standards ELISA IgG IgM IgA GB Druckdatum 03 02 2014 1 Intended Use The CSF Standards are intended for draw ing up of a calibration curve w hichis used for detection of CNS ow n antibody syn thesis by parallel examination of Serum CSF pairs The pathogen specific quotient of CSF and serumis calculated The ratio betw een this pathogen specific antibody quotient and the total immunoglobulin quotient is called antibody index Al 2 Diagnostic Relevance During acute infections of the central nervous system CNS and also during chronical inflammatory processes e g multiple Sclerosis pathogen specific antibodies are developed in the CNS In the first case those are antibodies againstthe caus ative pathogen in the second case a poly specific i
9. for errors Al 0 6 1 3 normal intrathecale antibody production is improbable Al 1 4 1 5 borderline It is recommended that the sample should be retested or that a later serum CSF pair should be tested Al 21 5 pathological Indicates intrathecal antibody production Since a minimum of four different results pathogen specific CSF and serum antibody measurement units total serum and CSF IgG IgM or IgA value CSF and serum albumine in mg l are considered for the calculation of the diagnostical relevant Al value all methodic and coincidental errors add up here In the most unfavourable case a continuing mistake in the same sense is possible a double determination or better the measuring of tw o different sample dilutions are the best way to recognize this For this reason a clinical relevant limited Al value of 1 5 has established as note for a local synthesis of pathogen specific antibodies in the CSF Normally for virusspecific antibodies of the IgG IgM or IgA class there is the same ratio betw een CSF and serum as it is found for the summarised IgG IgM or IgA fraction The theoretically expected Al value is therefore 1 0 Corresponding tests have show n that for all virus specific antibodies a reference range of 0 6 1 3 is valid Al values betw een 1 4 1 5 are classified as borderline Al values above 1 5 may be considered as pathologic in case of sufficient analytical quality of all incoming single values and
10. may be characterized by a CNS ow n synthesis of the corresponding virus specific antibod ies Al values below 0 6 are theoretically impossible and do normally point out an analytical mistake Only high Al values w ithout corresponding clinical reference do not allow a definite conclusion for an acute stage of an in fectious CNS disease Long time persisting and poly specific CNS ow n antibody synthesis in particular of the IgG class but also of the IgM class are possible IgM Al increases are usually considered as prove for florid CNS infections In case of doubt the significant change of the Al value from a second determination similar to titer motion is of advantage for the judgement of an infection of the central nervous system Such a control is mandatorily bound to a further CSF w ithdrawel taken in an adequate time interval How ever for its indication only the clinical aspects are decisive as a rule Limits of the Test The interpretation of serological results shall alw ays include the clinical picture epidemiological data and all further avail able laboratory results In case of very high pathogen specific antibody concentrations in the cerebrospinal fluid or in the serum arisk that the antigen concentration in the w ells is insufficient to fulfillthe optimum conditions for a quantitative antibody detection is present If an antibody excessis suspected please consider also Heidelberg curve and total CSF result a second de termination
11. mple Abbreviations IgXtota Total lgX IgG IgM or IgA mg l lgXspec pathogen specific IgX IgG IgM or IgA Q Quotient Qib Quotient resulting from the albumin content of CSF and albumin content of the serum mg l only necessary for calc u lation of Limes value Seite 6 von 11 REV 26 Liquor Standards ELISA IgG IgM IgA GB Druckdatum 03 02 2014 8 3 1 QlgXspec pathogen specific antibody quotient Serum OD values read 0 700 thus established concentration from the reference curve 3 5 w ME dilution 1 400 CSF OD values read 0 500 thus established concentration from the reference curve 2 5 w ME dilution 1 2 QlgXspec IgXspec CSF WME x dilution _ 2 5WME x2 36 x10 3 IgXspec Serum WME x dilution 3 5wME x 400 8 3 2 Qigx Total Immunoglobulin Value Value of the clinical chemistry Xserum 10000mg l IgXtotalCSF 33mgZ 83x10 IgXtotalSerum 10 000mg l QlgXtotal 8 3 3 Calculation of the Limes Quotient Qum In case of an additional poly specific intrathecal immunoglobulin synthesis the Total IgX Quotient for the Al determination is no longer suitable The so called Qu has to be used instead of the Total IgX Quotient Therefore it is necessary to deter mine the albumin quotient additionally value of the clinical chemistry Calculation of the LIMES Value according to Reiber QLiM igG 0 93 x YQ ap 6x10 1 7x10 QLim IgM 7067 x Ja ab 120 x108 7 1x10 3 Qui
12. nd CSF forms an immune complex w ith the antigen coated on the micr o titer plate Unbound immunoglobulins are removed by w ashing 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 pro duced by the bound enzyme peroxidase The color changes to yellow w hen the stopping solution is added The extinction OD of the color solution is directly proportional to the concentration of the analysed pathogen specific IgG IgM respectively IgA antibody in Serum and CSF For the detection of CNS ow n antibody synthesis it is necessary to pro ceeda quantification of the antibody concentrations that are available in extinctions initially For this the arrays of standard seraw ith graded pathogen specific antibody concentration are provided Those standard sera serve for the creation of a ref erence curve that can be made manually or by using a suitable programme and allow s the conversion of the detected OD values into arbitrary defined non dimensional measurement units w ME By allocating of the obtained measurement units w ME w ith the nephelometrical measured Sera and CSF Total IgG IgM respectively IgA concentrations the so called an tibody index Al is detected refer to the calculation of the Al in point 8 3 This antibody index mentions the searched patho gen specific antibody quotient as a multiple respectively as a fraction of
13. ntrathecal immune response against possibly several pathogens without actual pathogen presence is possible 1 2 Bacterial infections of the CNS are mainly distinguished by highly pathologic and quite characteristic CSF results The diag nostic detection of viral CNS infections in cerebral spinal fluid CSF is depending on stage of infection and individual status of immunity possible in tw o ways by direct antigen detection or by detection of pathogen specific antibodies Itis w ellknown that culture of viral pathogens unlike bacterial pathogens is complicated alternative pathogen specific testings are bound to high methodical efforts The pathogen specific antibody detection usually takes effect earliest 6 days after onset of disease but is routinely used in CSF diagnosis by now 3 The antibodies detected in the CSF may either diffuse out of the plasma into the CSF area or be result of a local synthesis intrathecal antibody production The specific antibody index Al describing the relation betw een the specific antibody quo tient and the total immunoglobulin quotient serves for the clarification of a CNS infection A local antibody synthesis is pre sent if the pathogen specific antibody quotient of a certain antibody class is bigger than the corresponding total immunglobulin quotient Otota Please refer to our CSF diagnostic booklet for further information 3 Test Principle The antibody searched for in the human serum a
14. or 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 TVB needed for the test insertion Additional conjugate or TVB taken out may not be returned but must be dismissed Material Soag Shee Status Diluted 2 to 48 C max 6h Test Samples Undiluted 1210486 1 week monis Microtitreplate After Opening SET ns P i nE BA dd Rheumatoid factor Undiluted After Opening 2 to 8 C Absorbent Diuted Tweek Kier Opening After Opening ter Opening 2 to 48 months Washing Solution imal Dilution ready to use 6 Precautions and Warnings 1 Only serawhich 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 standards Nevertheless samples diluted samples standards 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 effect to 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 Test Procedure Working exactly referring to the Sekisui Virotech user manual is
15. te 5 ul serum plasma 450 ul dilution buffer 1 drop RF absorbent RT for 15 min 1 401 100 ul serum V P RF SorboTech mixture 300 ul di lution buffer 45 ul RF SorboTech 180 ul Dilution Buffer 225 ul CSF sample incubate for 15 min at room tempera ture Test procedure Samples Incubation 30 Minutes at 37 C Wash 4 times Conjugate Incubation 30 Minutes at 37 C Wash 4 times Substrate Incubation 30 Minutes at 37 C Stopping Measure Extinctions Seite 10 von 11 Liquor Standards ELISA IgG IgM IgA GB 100 pl Patient Samples blank Dilution Buffer and Standards 400 ul Washing Solution Remove Residues on a Cellulose Pad 100 pl Conjugate IgG IgM IgA 400 ul Washing Solution Remove Residues on a Cellulose Pad 100 pl Substrate 50 ul Stopping Solution shake carefully Photometer at 450 620nm Reference Wavelength 620 690nm REV 26 Druckdatum 03 02 2014 Seite 11 von 11 REV 26 Liquor Standards ELISA IgG IgWIgA GB Druckdatum 03 02 2014
16. terpretation For CSF diagnostic testing calculation through the cut off control as in serology is not possible To quantify the pathogen specific antibody content of serum CSF pairs a reference curve is prepared either manually or in strumentally with the help of the IgG IgM or IgA antibody standard sera The OD values of the standard sera are plotted on the y axis and the antibody concentrations in w ME on the x axis The reference curve created by hand or by instrument 100w ME 25w ME 6 2w ME 1 5w ME shall have a sufficiently steep curve a curve origin near the zero coordination point and acceptable deviation of all curve points from the extrapolished curve course The OD values of the serum CSF pairs can now be expressed in w ME by reading off fromthe curve and after multiplication by the appropriate dilution factor 100 400 for serum 2 for CSF they correspond to the concentrations of the pathogen specific IgG IgM or IgA antibody in serum and CSF To obtain plausible antibody indices OD values below 0 05 and w ME values below 1 5 or above 100 shall not be considered for the calculation When the OD values measured lead to values greater than 100w ME then greater serum dilutions than 1 100 1 400 or a greater CSF dilution than 1 1 can be used while taking the changed dilution ratios into account Virotech provides user friendly CSF softw are solutions to simplify the Al calculation 8 3 Calculation of the antibody index Al with exa
17. the prerequisite for obtaining correct results 7 1 Examination Material Either serumor plasma can be usedas test material even if only serum is mentioned in the instructions Any type of antic o agulant can be used for plasma Consider the following for the serum samples Alw ays prepare patient dilution freshly For a longer storage the sera must be frozen Repeated defrosting should 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 r e sults Consider the following for the CSF samples Alw ays prepare patient dilution freshly If the CSF samples are to be stored for an extended period it is best to aliquot them and then to freeze them at 80 C This avoids repeated thaw ing 1 Vein and lumbar puncture should alw ays be performed at approx the same time 2 Only optically clear and uncelled and not inactivated CSF may be used 3 Do notuse haemolytic or microbiologically contaminated or turbid CSF Seite 4 von 11 REV 26 Liquor Standards ELISA IgG IgM IgA GB Druckdatum 03 02 2014 4 7 2 The use of deepfrozen CSF is possible if after thaw ing the conditions of items 2 and 3 are fulfilled 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
18. w ith higher dilution of serum respectively CSF has to follow Notice the detailed performance data sensitivity and specificity for Borrelia and CMV CSF diagnostic in the regular instruction sheetin addition to the serology Literature Zimmermann K Liquordiagnostik MTA 11 1996 4 258 260 Reiber H Lange P Virus spezifische Antik rper in Liquor und Serum ELISA Analytik und Auswertung mittels Antik r per Index und Quotientendiagramm Lab med 15 204 1991 204 207 Seite 8 von 11 REV 26 Liquor Standards ELISA IgG IgM IgA GB Druckdatum 03 02 2014 3 Linke E Zimmermann K Liquordiagnostik hauseigene Liquorbrosch re 2003 4 Petereit Sindern Wick 2007 Leitlinien der Liquordiagnostik und Methodenkatalog der Deutschen Gesellschaft f r Li quordiagnostik und Klinische Neurochemie Springer Verlag ISBN 978 3 540 39017 6 Seite 9 von 11 REV 26 Liquor Standards ELISA IgG IgM IgA GB Druckdatum 03 02 2014 10 Test procedure Scheme Serum and CSF Diagnostic Preparation of the Patient Samples and Washing Solution V Washing Solution Fill up concentrate to 1 liter with aqua dest demin v IgG IgA Samples Dilution 1 401 W csr ilution 1 2 e g 5 ulserum plasma 2000 ul Dilution Buffer Serum Dilution Buffer is ready to use 150 ul CSF sample 150 ul Dilution Buffer W igM Samples Dilution 1 101 1 401 W csr Dilution 1 2 Rheumatoid factor absorption with RF SorboTech e g 1 101 Incuba
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