USER MANUAL
Contents
1. TEST PROCEDURE summary PREPARATION OF REAGENTS PREPARATION OF TESTPROTOCOL SPECIMEN DILUTION 1 21 dilution Performing multiple testing of specimens a corresponding volume is prepared in a separate tube or plate 0 100 ml OF THE SPECIMEN DILUTION ARE PIPETTED Pipetting specimens directly in the plate dispense first 0 200 ml of specimen diluent and then add 0 010 ml specimen 0 200 ml OF SPECIMEN DILUENT 0 010 ml SPECIMEN 0 100 ml OF THE CONTROLS ARE PIPETTED INCUBATION OF SPECIMENDILUTIONS AND CONTROLS 30 min at 37 C WASH 4x PIPETTING OF THE CONJUGATE 0 100 ml INCUBATION WITH CONJUGATE 30 min at 37 C WASH 4x PIPETTING OF THE TMB CHROMOGEN SUBSTRATE SOLUTION 0 100 ml INCUBATION WITH TMB CHROMOGEN SUBSTRATE SOLUTION 10 20 min at room temperature ca 20 25 C PIPETTING OF THE STOPPING SOLUTION 0 100 ml PHOTOMETRIC MEASUREMENT at 450nm Ref 630 nm TESTVALIDATION EVALUATION TESTPROCEDURE manual 1 Preparation 2 Test protocol Before starting with the test make sure that each single reagent has reached room temperature ca 20 25 C Check each single reagent to be use for its identity verify the sequence of dilution and pipetting Processing more than one strip it is recommended to identify each single strip eg 1 2 3 etc Prepare a test protocol according to the specimen identification numbers for the dilution and pipetting sequences of the specimens and t2. Specimen O D 450 nm Index Index Range Relative Units Relative Units Evaluation Diagnostic e g Index x 10 Range Ranges Significance Spec No 1 0 080 0 266 lt 0 900 2 66 lt 9 non reactive m Spec No 2 0 280 0 933 0 900 1 000 9 33 9 10 border line Spec No 3 0 350 1 167 1 000 1 500 11 67 10 15 weakly reactive Spec No 4 0 500 1 667 1 500 2 000 16 67 15 20 reactive Spec No 5 0 700 2 333 2 000 3 000 23 33 20 30 highly reactive Spec No 6 1 000 3 333 gt 3 000 33 33 gt 30 very highly reactive In general the presence of IgG IgA or IgM antibodies indicates a past infection or vaccination The detection of IgG IgA or IgM antibodies during the course of an infection may indicate a current infection if the results of a parallel determination of two specimens from the same patient taken 10 to 14 days apart indicate a seroconversion conversion from negative to positive It is to be considered that in the early stage of a seroconversion the results obtained may still fall under the values of the differential control Borderline and weakly reactive results should be retested together with an additional sample drawn 10 to 14 days apart If no differences in reactivity are detected no evidence for a current infection may be assigned if clear increments in reactivity are detected support for a current infection may be indicated Very high IgG IgA or IgM reactivities may indicate the peak o
3. After finishing pipetting shake gently the plate cover it with the adhesive foil and incubate for 30 min 2 min at 37 C 1 C Before finishing the first incubation specimen incubation period prepare the necessary volume of conjugate solution Make sure that the solution has reached room temperature 20 25 C For 8 wells 0 8 0 1 0 9 ml conjugate are needed Remove the specimen dilutions and controls form the wells add 0 300 ml washing solution to each well wait 1 min Repeat the sequence removal of the washing solution adding washing 6 Conjugate solution and waiting 1 min 3 times totally 4 washing cycles After last removal of washing solution make sure the wells have been completely voided eventually tap the plate upside down on absorbent paper 0 100 ml of the anti human IgG IgA or IgM PO conjugate solution are pipetted in all wells After finishing pipetting cover it with the adhesive foil and incubate for 30 min 2 min at 37 C 1 C 7 PREPARATION Before finishing the second incubation conjugate incubation 8 Wash step 2 prepare the corresponding volume of TMB Chromogen substrate solution and keep it in the dark until use e g for 8 wells to 1ml substrate buffer No 8 add 0 050 ml TMB Chromogen solution 21x conc No 9 Make sure the solution has reached room temperature 20 25 C Remove the conjugate solution form the wells add 0 300 ml washing solution to each w
4. 1 702 703 e Epstein M A Y M Barr and B G Achong 1965 Studies with Burkitt s Lymphoma In Wistar Inst Sympos Monogr 4 69 82 e Edwards JM Woodroof M EB virus specific IgA in serum of patients with infectious mononucleosis and of healthy people of different ages Journal of Clinical Pathology 1979 32 1036 1040 e Sumava CV Epstein Barr virus and infectious mononucleosis a review J Clin Immunoassay 1989 12 168 74 e Mazeron MC value of anti Epstein Barr antibody detection in the diagnosis and management of undifferentiated carcinoma of the nasopharynx Bull Cancer Radiother 1996 83 1 3 7 e Shimakage M Dezawa T Chatani M Proper use of serum antibody titers against Epstein Barr virus in nasopharyngeal carcinoma IgA virus capsid antigen for diagnosis and EBV related nuclear antigen 2 for follow up Acta Otolaryngol 2000 Jan 120 1 100 4 e Dardari R Khyatti M et al Antibodies to the Epstein Barr virus transactivator protein as a valuable biomarker in young patients with nasopharyngeal carcinoma Int J Cancer 2000 Apr 1 86 1 71 5 EL SA Enzyme Linked Immunosorbent Assay XQ Abiyotek ea Ankara Biyoteknoloji San ve Tic Ltd ti Ankara niversitesi Teknoloji Geli tirme B l Teknokent B evler Mah 319 sok D Blok No 7 06800 G lba Ankara Turkey Phone 90312 485 16 88 pbx Fax 90312 484 76 62 e mail info abiyotek com
5. 20 min the absorbance value of a particular sample will be approximately 2x the value after 10 min reaction time 3 This means practically that stopping the reaction of a given sample after 10 min giving an absorbance value of 0 8 stopping after 20 min will result in an absorbance value of approximately 1 6 4 Due to the fact that the course of the reaction is practically linear during the first 10 to 20 min for all reactivities low and high the proportions of the different reactivities to each other remain the same 5 Incubations at room temperature lead to higher absorbance values at 30 35 C than at 20 25 C during the same time period approximately 2x higher According to our test procedure the reaction time for the Chromogen Substrate incubation is set at 15 min 5 min for the manual procedure this means between 10 and 20 min reaction time 5 It is to be considered that the reactivity of the conjugate gradually decreases with time therefore reactivities are set relatively high at the beginning to assure that the validation criteria apply over the entire stability period claimed 6 Due to these facts it is possible to introduce a corrective factor in case that the absorbance values obtained for the differential control surpasses the upper limit value or remains under the lower limit value not fulfilling the validation criteria as specified under corrective measures Should this possible corrective measures not lead to
6. Although thiomerosal is also used in some vaccines as preservative in comparable concentrations in vitro reagents containing thiomerosal should be handled cautiously 9 Substrate B with substrate No 8 Substrate A concentrate No 9 and stopping solution No 11 contain irritant and corrosive substances and should be handled cautiously If contact with skin eyes or mucous tissue occurs immediately rinse with enough water and consult a physician 10 All waste solutions should be collected in adequate vessels containing disinfectants capable of inactivating human pathogenic viruses Follow the corresponding manufacturer s instructions for use 11 Disposal follow the locally ruling safety and disposal laws and regulations for disposal LIMITATIONS AND CAUSES OF ERROR It is to be considered that under certain specific laboratory working conditions adjustment of alternative incubation periods may be necessary If reagents are used too cool before reaching room temperature 20 25 C a weaker colour development will occur at the end of the test run On the other hand if room temperature is high appr 30 C or higher a stronger colour development will occur at the end of the test run Under these circumstances the validation criteria of the test run may not be achieved Periodically check functionality of pipettes and instruments used The reagents of the testkit are not to be used after its expiry date Do not use heat
7. NR Abiyotek 7 EBV Enzyme Linked Immunosorbent Assay for the cut off determination of IgA IgG or IgM Antibodies to Epstein Barr Virus EBV in human serum or plasma Microwell ELISA USER MANUAL Microwell Method 96 wells 12 x 8 well antigen coated strips individual breakaway IgA Ref 01CEBVO1 IgG Ref 01CEBV03 IgM Ref 01CEBV05 For in vitro Diagnostic Use GENERAL INFORMATION O Wavelength Measurement Filter 450 nm Optional Reference Filter 600 650 nm LI Incubation Time 70 minutes at 37 R T 30 30 15 O Enzyme Conjugate HRP Horseradish Peroxidase ready to use O Substrate Solution TMB 3 3 5 5 Tetramethyi benzidine concentrate O Sample Serum or Plasma O Stability of Samples undiluted 5 days at 2 8 C up to 6 months at 20 C LI Shelf life and Stability of Kit Components Kit 12 months from production date Kit Components see expiration date on the label KIT COMPONENTS Microwell plate 12x8 wells coated with EBV antigens in a resealable foil pouch Negative Control 1 vial 1 5 ml IgA IgG or IgM human serum ready to use Cut off Control 1 vial 3 ml IgA IgG or IgM human serum ready to use Positive Control 1 vial 1 5 ml IgA IgG or IgM human serum ready to use Enzyme Conjugate 1 vial 12 ml Anti human IgA IgG or IgM HRP Conjugate ready to use Substrate Solution A 1 vial 0 750 ml TMB Tetramethylbenzidine in DMSO Dimethylsulfoxide Subst
8. V infections are transmitted via saliva occur during childhood and are subclinical Infectious agent Epstein Barr Virus EBV human herpesvirus 4 a member of the Herpesviridae family genus Lymphocryptovirus of double stranded DNA viruses TEST PRINCIPLES Human IgG IgA or IgM antibodies against EBV virus if present in the specimen bind to immobilized EBV virus antigens on the surface of the wells of the microtiterplate the human antibodies bound are then detected by specific anti human lgG IgA or IgM antibodies labelled to horse radish peroxidase and subsequently revealed by the substrate chromogen colour reaction After stopping the colour reaction the initially blue colour turns yellow and the intensity of this yellow colour is measured photometrically extinction absorbance optical density O D The intensity of the colour reaction is proportional to the corresponding antibody content CONSTITUTION OF THE REAGENTS Nr 1 Microtiterplate wells coated with EBV virus antigens Nr 2 Negative Control contains human IgG IgA und IgM antibodies in concentrations giving reactivities in the negative range of the test system Reactivity range Index range Index lt 0 60 Buffer 0 05 M PBS Tween 20 pH 7 2 Preservatives lt 0 10 sodium azide und max 0 03 Thiomerosal Colour concentrate E 104 E 132 max 0 2 v v Nr 3 Differential Control contains human IgG IgA und IgM antibodies in concentrations giving
9. acceptable results then the test run has to be repeated EVALUATION Evaluation of test results can be performed if the validation criteria apply Evaluation of the results for each specimen is done after calculating the Index value for each single specimen Calculation of the index value corresponds to a normalization of the results against the value obtained for the differential control in each single test run and may be assigned as a test reference value The Index value is obtained by dividing the absorption value extinction O D value of each single specimen by the mean value of the differential control Absorption at 450 nm of a specimen lndex meme iste ele e as eat eae ees Index of a specimen Mean Absorption at 450 nm of the differential control Index values Test reference values higher than 1 00 are scored reactive and indicate a presence of IgG IgA or IgM antibodies Index values lower than 0 90 are scored non reactive and indicate an absence of IgG IgA or IgM antibodies Index values between 0 90 and 1 00 are scored questionable For weakly reactive results it is recommended to consider a confirmatory test or to request a second specimen 10 to 14 days later to be tested in the same test run with the first specimen Example of a qualitative evaluation Qualitative evaluation is done according to the reactivity of the differential control All specimens giving Index values higher than that of the differential c
10. are transmitted via saliva occur during childhood and are subclinical In young adults EBV infection may be clinically manifested as Infectious Mononucleosis IM with typical symptoms of sore throat fever and lymphadenopathy In addition to IM EBV causes chronic active EBV infection B cell lymphoproliferative disease a complication of the immunosuppression of transplantation X linked lymphoproliferative XLP syndrome a subset of Hodgkin disease opportunistic non Hodgkin lymphoma Burkitt lymphoma unusual hepatitides nasopharyngeal and other carcinomas haemophagocytic syndromes and other diseases syndromes including a relative risk for EBV positive Hodgkin lymphoma in young adults estimated frequency 1 Hodgkin lymphoma 1000_ infectious mononucleosis infections approximately 4 years after serologically verified infectious mononucleosis Serological studies indicate that prior to the clinical onset of NPC a high antibody concentration of specific IgA to viral capsid antigens and early antigens is frequently observed The specific IgA levels increase with progression of the tumor disease and the antibody levels decline responding to therapy In patients with confirmed clinical remission elevation of specific IgA serological concentration is highly significant for prediction of relapse The diagnostic significance of IgM and IgA antibodies to EB Nuclear Antigens still has to be established Infection The majority of primary EB
11. ble corrective measures should be considered 1 Example 1 obtaining too high an absorbance value e g 1 6 for the differential control a correction factor of 0 5 can be applied to all values and the test may be revalidated This revalidation of the test run only applies if the criteria for the Index value of the positive control gt 1 4 and for the negative control lt 0 6 also apply Alternatively if some sample values are above absorbance 3 0 OVER a dilution by factor 2 dispense additionally 0 2 ml stopping solution to each of the stopped wells mix well and then withdraw 0 2 ml from each of them 1 in 2 dilution can be performed on all samples to bring OVER values in the measuring range of the photometer measuring range of the photometer should be from 0 to 3 2 Example 2 obtaining too low an absorbance value for the differential control not under 0 060 a factor of 2 may be applied and the test run revalidated This revalidation of the test run only applies if the criteria for the Index value of the positive control gt 1 4 and for the negative control lt 0 6 also apply Alternatively performing the next test run the reaction time can be extended from e g 10 min to 20 min or to 30 min General considerations on peroxidase reactivity 1 That the peroxidase reaction in our systems is initially practically linear with time and starts levelling off slowly after about 10 to 20 min 2 Therefore after a reaction time of
12. ed if the values of the differential control are higher than 0 080 and lower than 1 000 optimally between 0 200 and 0 600 and the deviation of the values obtained for the differential control falls within 20 of the mean value Additionally the corresponding index value of the negative control must be lt 0 6 and the corresponding index value of the positive control must be gt 1 4 These criteria apply to all our systems absorption at 450nm of the corresponding control Index value of the Controls Mean absorption at 450nm of the differential control Example of a validation mean value of the absorption of the differential control 1 value 0 280 2 value 0 320 Mean value 0 300 controls O D value 450 nm Index Absorption O D value of the negative control Nr 2 0 100 0 100 0 300 0 333 Absorption O D value of the differential control Nr 3 0 280 0 280 0 300 0 933 Absorption O D value of the differential control Nr 3 0 320 0 320 0 300 1 067 Absorption O D value of the positive control Nr 4 0 600 0 600 0 300 2 000 If the values obtained are within the range of the validation criteria then the test run is valid and evaluation can be performed If the validation criteria are not met then the test is not valid and must be repeated Corrective measures Before repeating the test the following possi
13. ell wait 1 min Repeat the sequence removal of the washing solution adding washing solution and waiting 1 min 3 times totally 4 washing cycles After last removal of washing solution make sure the wells have been completely voided eventually tap the plate upside down on absorbent paper Wipe carefully the bottom of the strips from outside with absorbent paper to remove all possible liquid residues that could interfere with photometric reading 9 TMB Chromogen Substrate Incubation 0 100 ml of the TMB Chromogen Substrate solution are pipetted in all wells After finishing pipetting incubate the plate for 15 5 min at room temperature 20 25 C avoiding exposure to light dark chamber dark box a closed drawer Reactive specimens develop a blue colour 10 Reaction stop After finishing the TMB Chromogen substrate incubation add 0 100 ml Stopping solution to all wells Reactive specimens turn from blue to yellow 11 Photometric reading Photometric reading should be done within 10 to 20 12 Validation 13 Evaluation minutes after stopping the colour reaction with a microtiterplate photometer at 450 nm if possible with the reference wavelength set at 630 nm Blanking is done against air According to the validation criteria see under Validation According to the evaluation criteria see under Evaluation TESTPROCESSING WITH AUTOMATIC DEVICES Test processing with automatic devices may be carried ou
14. eption Prolonged storage should be done at 20 C or lower Avoid repeated thawing and freezing Samples showing particles should be centrifuged prior to be processed to avoid possible erroneous results Contamination should be avoided since contaminated samples may also lead to erroneous results Handle all samples as potentially infectious PREPARATION OF REAGENTS AND TESTSAMPLES e All necessary reagents must reach room temperature 20 25 C prior to be used e f reagents are used too cool before reaching room temperature 20 25 C a weaker colour development will occur at the end of the test run On the other hand if room temperature is high appr 30 C or higher a stronger colour development will occur at the end of the test run Under these circumstances the validation criteria of the test run may not be achieved and corrective measures may be necessary prolonging or shortening the incubation period e Sample diluent No 5 No 6 2 ml additive No 6 are added to 20 ml diluent buffer No 5 Bring only the necessary volume of the ready to use specimen diluent to room temperature e Washing solution ready to use The concentrated washing solution No 10 is diluted 1 in 25 with deionised water 20 ml concentrate 480 ml deionised water use only clear solutions e Microplate strips and wells Take the necessary amount of strips or wells from the bag after they have reached room temperature Place the
15. f the acute phase of a current infection The simultaneous detection of IgM and IgA antibodies during a seroconversion very strongly support a current infection Interpretation of serological results should always only be done together with clinical data EXPECTED RESULTS REPRODUCIBILITY PERFORMANCE CHARACTERISTICS REPRODUCIBILITY Reproducibility of the results of the controls and test specimens in our test systems is calculated according to the mean of the index value MW the standard deviation SA and the variation coefficient VK Standard deviation SA Coefficient of variation VK x 100 Mean MW Repeated determinations of the same samples minimum n 4 in our test systems allow to define the following ranges for the coefficient of variation of a given index value Intraassay coefficient of variation of a given index value of a sample should be less than 10 and not greater than 20 should this occur so it is mandatory to review the test conditions and working techniques Interassay coefficient of variation of a given index value of a sample should be less than 10 and not greater than 25 should this occur so it is mandatory to review the test conditions and working techniques PERFORMANCE CHARACTERISTICS Generally the prevalence of IgG IgA or IgM antibodies in a population depends on the incidence of infection in the various subpopulations at d
16. h single reagent ensures their correct identification and an accurate automatic processing of the test DIAGNOSTIC RELEVANCE DISEASE AND RECOMMENDED LITERATURE Besides detection of the infectious agent the detection of antibodies to EBV significantly contributes to the serological diagnosis of these infections The detection of EBV specific IgG IgM and IgA antibodies to its major immunodominant antigens has become an important and useful tool for the monitoring and follow up of patients showing EBV associated diseases several years after primary infection In the acute phase of infectious mononucleosis IgM and also IgA and then IgG IgA or IgM antibodies to EBV early antigens EA to viral capsid antigen VCA and IgG IgA or IgM to nuclear antigens EBNA appear in sequence Therefore the presence of IgM IgA_ antibodies to VCA or IgM IgA IgG antibodies to EA with low or absent EBNA antibodies is indicative of current or recent infection IgM VCA antibodies are almost always detectable at the early stage of infection and lasts 4 to 8 weeks The presence of EBNA antibodies are indicative for past infection and appear after 2 to 3 months after infection Epstein Barr Virus EBV infects the majority of people worldwide gt 90 and persists as a latent lifelong infection of long lived memory B lymphocytes with virus production EBV infection may demonstrate a wide spectrum of clinical symptoms The majority of primary EBV infections
17. hat besides the wells needed to be processed in their corresponding positions empty positions are filled with empty wells to prevent overflow of washing solution in the washing chamber After inserting the plate it is brought to the pipetting area and the assay assays job jobs is are started The job is processed in the following way 1 Dispensing specimen diluent 0 200 ml in the wells assigned for the specimens 2 Dispensing specimens in the wells assigned for specimens 0 010 ml in 0 200 ml 1 21 dilution 3 Dispensing the controls in the wells assigned for the controls 0 100 ml 4 Incubation of the specimens and controls 30 min 1 min bei 37 1 C 5 Washing 6 Dispensing the conjugate 0 100 ml 7 Incubation with conjugate 30 min 1 min bei 37 1 C 8 Washing 9 Dispensing the TMB Chromogen Substrate solution 1 cycle 0 025 2 cycle immediately there after 0 050 ml 10 Incubation with TMB Chromogen Substrate solution 15 min 1 min at room temperature 20 25 C 11 Stop of the reaction by adding stopping solution 0 100 ml 12 Photometric reading in integrated photometer 13 Results may be printed out or further transferred online The corresponding protocols include validated and evaluated results VALIDATION OF THE TEST CORRECTIVE MEASURES GENERAL CONSIDERATIONS Validation Results obtained in absorbance units extinction units O D units for the controls are us
18. he controls to be tested One well is assigned to the negative control two wells to the differential control and one well to the positive control If necessary more controls may be scheduled 3 SPECIMEN PREPARATION DILUTION PIPETTING OF SPECIMENS AND 4 Preparation 5 Wash step 1 CONTROLS INCUBATION Specimens liquid serum or plasma are prepared to be tested at a 1 21 dilution Performing multiple testing of specimens a corresponding dilution volume is prepared in a separate tube or plate then 0 100 ml of the diluted specimen is pipetted into the corresponding well according to the pipetting protocol it is recommended to pipette in replicates at the beginning to establish the own pipetting accuracy and reproducibility Are the specimens directly diluted into the wells then 0 200 ml specimen diluent are dispensed first into each well and then 0 010 ml of each specimen are added into the corresponding wells mix well it is recommended to pipette in replicates at the beginning to establish the own pipetting accuracy and reproducibility In addition 0 100 ml of each control are pipetted into their corresponding wells Pipetting longer series of specimens it is recommended to pipette the controls after reaching half of the series to compensate for pipetting delays The corresponding controls may also be pipetted at the beginning and at the end of a longer series for evaluation the mean of the corresponding results is used
19. ifferent age groups incidence is also dependent on the mobility of these subpopulations on the availability of adequate vaccines on the geographical location and last but not least on the locally given socio economic conditions Reference population The expected values for IgG IgA und IgM antibody reactivity as adjusted in our systems correspond to the expected distribution of negative and positive samples in a Swiss blood donor population Depending on the infectious agent the proportion of positive samples for IgG IgA und IgM may vary between 0 10 10 30 30 50 50 70 and 70 90 or more The proportion of IgG IgA und IgM positive samples for EBV virus is adjusted between 70 and 80 in our blood donor population Fig 1 shows the distribution of ELISA results in a blood donor population Reactivity IgG IgM IgA IgMA n N n N n N n N EBV VCA 79 17 57 72 12 5 9 72 0 00 0 72 n d EBV EBNA 1 70 88 51 72 12 5 9 72 0 00 0 72 n d EBV EAd 75 00 54 72 12 5 9 72 0 00 0 72 n d EBV EAr 75 00 54 72 12 5 9 72 0 00 0 72 n d Legend n N number of positive samples total number of tested samples n d not done Specificity To determine specificity reactivity inhibition tests with inactivated homologous infected cell suspensions and also with non infected cell suspensions of positive reacting samples are performed The reactivity of specific positive samples in
20. inactivated specimens Avoid testing contaminated samples strong hemolytic icteric or lipemic samples since erroneous results may be obtained To ensure the performance of the testkit storage conditions and stability of the opened and diluted reagents must be strictly respected as depicted under storage and stability Reagent No 1 antigen coated wells No 2 negative control No 3 differential control No 4 positive control and No 7 conjugate solution are lot specific and are not allowed to be used together with corresponding reagents from another lot Reagent No 5 Sample Diluent No 6 additive for Sample Diluent No 8 Substrate B with substrate No 9 chromogen concentrate No 10 washing solution and No 11 stopping solution are not lot specific and may be used if necessary and respecting the corresponding expiry date in a test run together with corresponding lot specific reagents Nr 1 Nr 2 Nr 3 Nr 4 from another lot Avoid cross contaminations during manipulations Never use the same vessel for the ready to use conjugate dilution and the ready to use substrate chromogen solution Since TMB turns blue coloured upon oxidation any contact of the reagents No 8 No 9 and No 11 with heavy metals should be avoided Also protect TMB solutions from direct light exposure SAMPLE COLLECTION AND HANDLING Plasma or serum collected by venipuncture should be tested within 2 days if stored at 8 C after rec
21. logarithmic scale Relatonship between O D values Index values and unit values for the above mentioned results Spec No 1 Spec No 2 Spec No 3 Spec No 4 Spec No 5 Spec No 6 O D values 0 080 0 280 0 350 0 500 0 700 1 000 Index values 0 266 0 933 1 167 1 667 2 33 3 333 Units values 2 66 9 33 11 67 16 67 23 33 33 33 Further mathematical evaluation methods of the results like using a standard curve with serum dilutions as a reference or with the help of the lt one point quantification gt are also possible However it has to be kept in mind that all these additional evaluation methods use one common basic operation calculating a reference value of the basic reactivity with at least one standard before further mathematical transformation logarithmic exponential polynomial 4 PL Model etc is done to obtain the corresponding relative units The scales of the relative units found are also divided in reactivity ranges with increasing reactivity that can be related to an increasing probability of a diagnostic indication In principle however all these evaluation methods operate with the same originally measured values absorption extinction O D value and corresponding differentiating reactivity ranges INTERPRETATION OF RESULTS The probability to assign a diagnostic significance to a given reactivity increases with increasing absorption value or increasing Index value or increasing value of relative units EXAMPLE
22. ly minor poisonous and must be handled cautiously Nr 10 Washing solution concentrate 25x conc contains 200gr Liter sodium chloride 1 25 Tween 20 max 0 01 thiomerosal Nr 11 Stopping solution sulfuric acid max 1 v v lt 0 2 M Sulfuric acid is corrosive and must be handled cautiously REAGENTS DESCRIPTION STORAGE STABILITY COMMENTS Closed components of the test kit 2 8 C until expiry date Opened Microplate strips 2 8 C 6 weeks keep storage bag tightly closed avoiding high humidity Opened components No 2 3 4 5 6 7 2 8 C 12 weeks avoid Temperature stress and contamination Opened substrate buffer No 8 2 8 C 12 weeks avoid direct exposure to light Opened TMB Chromogen solution 21x conc No 9 2 8 C 12 weeks avoid direct exposure to light Specimen diluent No 5 No 6 ready to use 2 8 C 12 weeks prepare only the necessary volume and avoid contamination Substrate A B solution No 8 No 9 2 8 C max 24h prepare only the necessary volume ready to use and avoid direct exposure to light Washing solution ready to use 2 8 C 12 weeks use only a clear solution 20 25 C max 2 weeks use only a clear solution Stop solution 2 8 C until expiry date SAFETY MEASURES Warnings Precautions Disposal 1 GLP RULES should always be followed GLP Good Laboratory Practice 2 The testkit is only to be used for in vitro diagnostic purposes and by professi
23. onal staff only 3 The use of protective laboratory clothes protective hand gloves and also protective glasses during the actual manual procedure is recommended Do not pipet by mouth 4 All tested samples should be regarded as potentially infectious and should be handled accordingly The controls have been derived from donations which have been tested for anti HIV 1 2 anti HCV and HBsAg on a single donor basis and have been found non reactive Nevertheless they should also be handled as potentially infectious Do not use heat inactivated test specimens 5 Material of bovine origin used as ingredients in reagents originate from countries known to be BSE free at the time of purchase 6 The controls and the additive for the diluent buffer contain lt 0 1 sodium azide and 0 05 thiomerosal as preservatives The dilution buffer contains 0 05 thiomerosal as preservative 7 Precautions to be considered using on vitro diagnostic devices containing sodium azide as preservative Sodium azide is poisonous swallowing and contact with skin eyes and mucous tissue is to be avoided Sodium azide generates explosive azides with heavy metals like copper or lead Disposing sodium azide containing waste solutions always rinse with enough water 8 Precautions to be considered using in vitro diagnostic devices containing thiomerosal as preservative Thiomerosal is poisonous swallowing and contact with skin eyes and mucous tissue should be avoided
24. ontrol are considered as reactive and all giving lower Index values are considered as non reactive The entire Index range may be divided in ranges with increasing reactivity and to these ranges a diagnostic meaning may be assigned The higher the reactivity the higher the diagnostic meaning Mean value of the differential control 1 value O D 0 280 2 value O D 0 320 Mean value O D 0 300 Specimen O D 450 nm Index Test reference value Index range Evaluation Ranges Spec No 1 0 080 0 080 0 300 0 266 lt 0 900 non reactive Spec No 2 0 280 0 280 0 300 0 933 0 900 1 000 border line Spec No 3 0 350 0 350 0 300 1 167 1 000 1 500 weakly reactive Spec No 4 0 500 0 500 0 300 1 667 1 500 2 000 reactive Spec No 5 0 700 0 700 0 300 2 333 2 000 3 000 highly reactive Spec No 6 1 000 1 000 0 300 3 333 3 000 5 000 very highly reactive Example of a quantitative evaluation after introduction of relative units For clinical reports quantitative results in relative units are usually requested to better assess and assign the results obtained For this purpose the simplest way is to multiply the Index value with a simple factor and assign the new range of values a new range of units It is to be considered that these relative units are also based on a logarithmic scale Example multiplying the Index values of the specimens in above table by 10 gives the new unit values
25. rate Solution B 1 bottle 15 ml contains urea peroxide corrosive Sample Diluent 2 bottles 20 ml each Additive for Sample Diluent 2 vials 2ml each Wash Buffer 1 bottle 20 ml Contains NaCl and Tween 20 25x Concentrate Stop Solution 1 bottle 15 ml 1 v v sulphuric acid corrosive Ready to use MATERIALS REQUIRED BUT NOT PROVIDED LI Deionized or distilled water O Graduated cylinders and beakers O Wash trays O Macropipettes capable of delivering 5 ul to 1000 ul O Multichannel Micropipette O Stepper O Incubator 37 C 2 C dry incubator make sure to correctly seal the wells with the adhesive foil to prevent evaporation which may lead to erroneous results LI Microplate reader capable of reading absorbance values at 450 nm If dual wavelength microplate reader is available the reference filter should be set at 600 690 nm O Automatic microplate washer capable of dispensing 300 ul Microplate reader and microplate washers are available from Abiyotek Company SUMMARY AND EXPLANATION Indirect enzyme immunoassay for the detection of antibodies to Epstein Barr virus EBV in human serum and plasma The enzyme immunoassay is intended for testing individual specimens not pooled specimens In vitro diagnosticum only to be used for in vitro diagnostic purposes by correspondingly educated laboratory personnel The test can be processed manually or automatically The barcode identification of eac
26. reactivities in the borderline range of the test system Reactivity range Absorbance range OD range 0 080 1 000 Index 1 Buffer 0 05 M PBS Tween 20 pH 7 2 Preservatives lt 0 10 sodium azide und max 0 03 thiomerosal Colour concentrate E 110 max 0 1 v v Nr 4 Positive Control contains human IgG IgA und IgM antibodies in concentrations giving reactivities in the positive range of the test system Reactivity range Index range Index gt 1 40 Puffer 0 05 M PBS Tween 20 pH 7 2 Preservatives lt 0 10 sodium azide and max 0 03 thiomerosal Colour concentrate E 123 max 0 1 v v Nr 5 Diluent buffer 0 05 M PBS Tween 20 pH 7 2 Preservatives max 0 05 thiomerosal Colour Bromphenolblue max 20 mg Liter Nr 6 Additive for diluent buffer contains newborn calf serum NCS Preservatives lt 0 10 sodium azide and max 0 05 thiomerosal Nr 7 Anti human lgG IgA or IgM Peroxidase conjugate Sheep antibodies against human IgG IgA or IgM antibodies conjugated with horse radish peroxidase Contains bovine serum ingredients max 1 5 Preservatives thymol max 0 1 g Liter Bronidox L max 0 4 v v Colour Bromphenolblue Nr 8 Substrate buffer with substrate contains urea peroxide in succinate borate buffer Urea peroxide is irritant and must be handled cautiously Nr 9 o Chromogen concentrate 21x conc TMB tetra methyl benzidine in Dimethylsulfoxide DMSO Dimethylsulfoxide and TMB are corrosive respective
27. required strips or wells firmly in the frame make sure they are evenly arrayed in the frame If required fill the empty plate positions with empty wells or strips not antigen coated according to the pipetting or washing device used to avoid overflow of fluid during pipetting or washing steps of the test run e Not required strips or wells antigen coated must be transferred into the storage bag well sealed avoiding humidity and reset for storage at 2 8 C e Specimens Specimens are tested at a 1 21 dilution Although testing of other body fluids than serum or plasma is possible specific adjustment of the conditions is needed e Conjugate always prepare only the amount of conjugate needed plus lt 0 1 ml e Chromogen Substrate solution always prepare only the necessary amount of chromogen substrate solution plus lt 0 1 ml e As an example to 1 ml substrate buffer 0 050 ml TMB Chromogen concentrate is added 1 21 dilution TEST PROCEDURE The test should only be performed by properly trained professional laboratory staff All reagents must have reached room temperature 22 25 C prior to start performing the test run Incubation periods Specimens and controls 30 min at 37 C Conjugate 30 min at 37 C and TMB Chromogen Substrate solution 10 to 20 min at room temperature 22 25 C Adaptation of incubation periods to specific internal laboratory needs is possible according to GLP they should be validated
28. t according to the assay definition programs of the automatic device in use e g BEP 2000 EtiMax3000 Evolis Quickstep among others The assay definition program allows the bar code identification of the reagents and of the specimens and their sequential process assignment for the entire process of the test After defining the jobs to be done a list of the corresponding reagents needed is generated including the necessary reagent volumes and their corresponding containers Specimen diluent controls conjugate TMB Chromogen Substrate solution stop solution and wash solution For each single reagent needed the minimal calculated quantities have to be present in the corresponding amounts and in the corresponding bar coded vials to be processed The barcoded reagent vials prepared are placed in the corresponding reagents rack for processing The racks containing the barcoded specimens and the racks containing the barcoded reagents can then be introduced in the processing area During introduction of the racks barcode reading is affected and the position of each barcoded reagent and specimen registered After verifying the necessary amounts of reagents the plate to be processed is requested it is possible to align a variety of different tests the only requirement is that the different assays to be processed must have all the same single incubation periods for each incubation Before inserting the plate in the plate holder make sure t
29. the ELISA test is blocked after pre incubation with homologous infected cell suspensions but not after pre incubation with non infected cell suspensions If the reactivity is also blocked after pre incubation with the non infected cell suspension a non specific reactivity is indicated Sensitivity To determine sensitivity selected samples when ever possible from patient populations seroconversion paired samples vaccination studies epidemic studies different endemic regions etc are tested to review and optimize the test settings Relative specificity and sensitivity When comparing different ELISA test systems one should always bear in mind that obtained results very much depend on the composition of the tested sample population and also on the characteristics of the antigen preparation used therefore these results are only indicative for the population of samples selected for this comparative testing REFERENCES e Mackie PL The Classification of viruses infecting the respiratory tract Paediatr Respir Rev 2003 4 457 61 e Babcock GJ Decker LL Volk M Thorley Lawson DA EBV persistence in memory B cells in vivo Immunity 1998 9 395 404 e Yao QY Rickinson AB Epstein MA A reexamination of the Epstein Barr virus carrier state in healthy seropositive individuals Int J Cancer 1985 35 35 42 e Epstein M A B B Achong and Y M Barr 1964 Virus particles in Cultured Lymphoblasts from Burkitt s Lymphoma In Lancet
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