ELISA-VIDITEST anti-vca EBV IgG

Transcription

1 ELISAVIDITEST antivca EBV IgG Cat. No.: ODZ265 Instruction manual PRODUCER: VIDIA spol. s r.o., Nad Safinou II/365, Vestec u Prahy, Jesenice, Czech Republic, Tel.: , Web: info@vidia.cz 1. TITLE ELISAVIDITEST antivca EBV IgG ELISA kit for the detection of IgG antibodies to viral capsid antigen of EpsteinBarr virus in human serum or plasma samples. 2. INTENDED USE ELISAVIDITEST antivca EBV is intended for in vitro diagnostic procedures in EBV induced and EBV associated diseases, such as infectious mononucleosis (IM) and the chronic active EBV infection. The test is also useful in the diagnosis of Burkitt s lymphoma, nasopharyngeal carcinoma, carcinoma of the Waldeyer s ring and in characterisation of opportunistic lymphomas (oligo and polyclonal). The other use of the test is in characterisation of chronic fatigue syndrome, in neuroinfections and immunosupression which is frequently associated with EBV reactivation. 3. TEST PRINCIPLE ELISAVIDITEST antivca EBV IgG is an enzyme linked immunosorbent assay. The wells of polystyrene strips are coated with specific antigen containing immunodominant epitopes of the VCA complex. AntiVCA antibodies if present in serum samples bind to the immobilized antigen. The antibodies that were bound to the antigen are in the next step of the assay detected with antihuman (IgG) antibodies labelled with horseradish peroxidase. The amount of the bound detection antibodies is measured by addition of a chromogenic substrate. A serum sample without antivca antibodies causes only a mild change of colour which, if occurs, may be attributed to the background of the reaction. 4. KIT COMPONENTS 12x 8 well break away strips coated with antigen, framed STRIPS Ag 1 microplate 1.3 ml Standard A=negative control serum, r.t.u. 1) ST A/CTRL 1 vial 1.3 ml Standard D=Standard 1 (calibrator), r.t.u. ST D/ST 1 1 vial 1.3 ml Standard E=Standard 2 (positive control serum), r.t.u. ST E/ST 2 1 vial 13 ml Antihuman IgG antibodiespx conjugated (Pxconjugate), r.t.u. CONJ 1 vial 55 ml Wash buffer, 10x concentrated WASH 10x 1 vial 60 ml Dilution buffer, r.t.u. DIL 1 vial 13 ml Chromogenic substrate (TMB substrate), r.t.u. TMB 1 vial 13 ml Stop solution, r.t.u. STOP 1 vial Sealable pouch for unused strips Instruction manual Certificate of quality 1

2 1) r.t.u. = ready to use Chromogenic substrate TMB is compatible and interchangeable between ELISAVIDITEST kits which contain TMB and not with other Chromogenic substrates TMBO, TMBBF. 5. MATERIALS AND EQUIPMENT REQUIRED BUT NOT PROVIDED WITH THE KIT Distilled/deionised water for diluting the concentrated Wash buffer, equipment for pipetting, dispensing and washing, test tubes for diluting samples, microplate cover or seal, microplate reader wavelenght 450 nm. 6. PREPARATION OF REAGENTS AND SAMPLES a. Allow all kit components to reach room temperature. b. Vortex samples and the Controls in order to ensure homogeneity and mix all solution well prior use. c. Dilute serum samples 101x with Dilution buffer DIL (e.g. 5 L of serum sample 500 L of Dilution buffer). Do not dilute Controls (Standards), they are ready to use. d. Prepare Wash buffer by diluting the Wash buffer concentrate 10 times with distilled/deionised water (e.g. 100 ml of the concentrated Wash buffer 900 ml of distilled water). If there are crystals of salt present in the concentrated Wash buffer, warm up the concentrate in a water bath set to 3237 C. The diluted Wash buffer is stable for one week if stored at 2 to 10 o C. e. Do not dilute Pxconjugate, TMB substrate and Stop solution, they are ready to use. Mix all solution well prior use. 7. ASSAY PROCEDURE a. Allow the vacuum sealed strips to reach room temperature before opening. It prevents moist condensation within the wells. Withdraw the needed number of strips and put the remaining strips and the desiccant device in the provided plastic bag. b. Start with filling the first well with 100 µl of Dilution buffer DIL to estimate the reaction background. Fill next two wells with 100 µl/well of Standard 1 ST D/ST 1 (it serves as calibrator) and then fill next one well with Negative control serum ST A/CTRL. Fill the remaining wells with 100 µl of diluted serum samples (S1, S2, S3,...) according to the pipetting scheme in Figure 1. It is also suitable to apply positive control serum Standard 2 ST E/ST 2 for the test control. It is sufficient to apply serum samples as singlets, however, if you wish to minimize the laboratory error apply the samples and controls as doublets, ST D/ST 1 as triplet. Incubate for 30 minutes (/ 5 min) at room temperature. c. Aspirate the contents of the wells into a collecting bottle containing appropriate disinfectant (see Safety Precautions). Wash and aspirate the wells four times with 250 µl/well of the diluted Wash buffer. Avoid well to well crosscontamination. Empty the wells by inverting the plate and tapping it against a pile of adsorbent papers. d. Mix Pxconjugate r.t.u. CONJ well and then add 100 L of the Pxconjugate into each well. Incubate for 60 minutes (/5 min) at room temperature. e. Aspirate and wash as in step d. f. Dispense 100 L of TMB substrate TMB into each well. Incubate for 10 minutes (/ 30 sec.) at room temperature. The time measurement must be started at the beginning of TMB dispensing. Pipette in a regular rhythm or use a suitable dispensing instrument. Cover the strips with an aluminium foil or keep them in the dark during the incubation. 2

3 g. Stop the reaction by adding 100 L of Stop solution STOP. Use the same regular pipetting rhythm as mentioned above to ensure the same reaction time in all wells. Check the wells for bubbles and remove them by tapping gently the microplate for a few times. h. Determine the absorbance at 450 nm in a microplate reader within 20 minutes. It is recommended to use a reference reading at nm. Figure 1: Pippetting scheme a DIL S5 b ST D/ST 1 S c ST D/ST 1 d ST A/CTRLe S1 f S2 g S3 h S4 8. PROCESSING OF RESULTS Begin the processing of results with subtraction of the background absorbance (absorbance of the DIL well) from the absorbances of all other wells Qualitative evaluation Compute the absorbance mean of Standard 1 ST D/ST 1. Compute the cutoff value by multiplying the Standard 1 mean with a Correction factor. The Correction factor value for particular Lot is written in enclosed Quality control certificate. Samples with absorbances lower than 90% of the cutoff value are considered negative and samples with absorbances higher than 110% of the cutoff value are considered positive Semiquantitative evaluation Determine the Positivity Index for each serum sample as follows: Compute the cutoff value using the Standard 1 mean and the Correction factor (see the previous paragraph). Compute the Positivity Index for each sample according to the following formula: sample absorbance sample Positivity Index = cutoff value Express serum reactivity according to Table 1 (Semiquantitative interpretation of results). Table 1: Semiquantitative interpretation of results Positivity index Interpretation < 0.90 Negative / >

4 Note! An indifferent sample reactivity, i.e. interpreted as /, requires repetition of the sample test. If the result is again indifferent then it is recommended to use an alternative testing method or to obtain another sample from the patient, usually withdrawn 12 weeks later. Example of calculation: Standard 1 absorbances = 1.119; Standard 1 mean = Correction factor = 0.16 Cutoff value = 1.132*0.16 = Sample absorbance = Sample Positivity Index = 0.800/0.181 = DIAGNOSTIC INTERPRETATION OF RESULTS VCA VCA VCA EA (D) EBNA1 EBNA1 EBV status IgG IgM IgA IgG IgG IgM EBV negative EBV primoinfection (acute phase) EBV primoinfection (postacute phase ) EBV primoinfection (convalescent phase) Seropositivity without signs of the active EBV infection EBV reactivation VIDIA offers also the other diagnostic assays to detect the antibodies against the other EBV antigens: ELISAVIDITEST antiebna1 EBV IgM and IgG ELISAVIDITEST antivca EBV IgM and IgA ELISAVIDITEST antiea (D) EBV IgM and IgG IFVIDITEST antivca EBV IgM and IgG IFVIDITEST antiea EBV (DR or D) IgG 10. TEST PERFORMANCE 10.1 Validity of the test The test is valid if: The background absorbance (the absorbance of the Dilution buffer) is less than The mean absorbance values of standards/ control sera are in the ranges stated in the Quality control certificate for this kit lot Precision of the test The intraassay variability (within the test) and the interassay variability (between tests) were determined with samples of different absorbance values. 4

5 Intraassay variability The coefficient of intraassay variability is max. 5%. It is measured for each particular Lot at least on 12 parallels of the same microtitration plate. Example: (N = number of parallels of the same microtitration plate, SD = standard deviation) N Mean absorbance SD CV% % % Interassay variability The coefficient of interassay variability is max. 15%. It is measured for each particular Lot as comparison of the OD values of the same serum sample in several consecutive tests. Example: (N = number of an independent examination of the same serum sample, SD = standard deviation): N Mean Absorbance SD Range (minmax) CV% % % Recovery test Measured values of recovery test for every Lot are between 80120% of expected values Diagnostic specificity and sensitivity The diagnostic sensitivity was measured by testing the population sample that is expected to have antivca IgG antibodies (blood donors, patients with active EBV infection). The results were confirmed by other commercial test. The diagnostic sensitivity was 98.1 %. The specificity of the test was measured by testing EBV negative healthy blood donors. The specificity of the test was 97.1% Interference Haemolytic, icteric and lipaemic samples showed no influence on results up to the concentration of 50 mg/ml of hemoglobin, 5 mg/ml of bilirubin and 50 mg/ml of triglycerides. 11. SAFETY PRECAUTIONS All components of the kit are intended for laboratory use only. Standards contain human sera that has been tested negative for HBsAg, antihiv1,2 and anti HCV. However they should be regarded as contagious and handled and disposed of according to the appropriate regulations, i.e. autoclave for 1 hour at 121 C all reusable materials that were in contact with Standards or samples, burn the disposable ignitable materials, decontaminate liquid wastes and nonignitable materials with 3% chloramine. Liquid wastes containing Stop solution (0.4M sulphuric acid) should be neutralized with 4% sodium bicarbonate. Handle Stop solution with care. Avoid contact with skin or mucous membranes. In case of contact with skin or mucous membranes, rinse immediately with plenty of water. The waste from strip washing disinfect in waste container using appropriate disinfection solution (e.g. Incidur, Incidin, Chloramin,...) in concentrations recommended by the producer. Do not pipette by mouth. Do not smoke, eat or drink where specimens or kit reagents are handled. Wear disposable gloves while handling kit reagents and specimens and wash your hands thoroughly afterwards. 5

6 Avoid spilling or producing aerosol. 12. HANDLING PRECAUTIONS Manufacturer guarantees the performance of the ELISA kit. Wash solution, TMB substrate, Stop solution and Dilution buffer are compatible and interchangeable between ELISAVIDITEST kits unless otherwise stated in the instruction manual. Avoid microbial contamination of serum samples and kit reagents. Avoid crosscontamination of reagents. Controls (Standards), TMB solution, Dilution buffer and Pxconjugate contain preservative ProClin 300. Avoid contact of the TMB substrate with oxidizing agents or metal surfaces. Follow the assay procedure indicated in the Instruction manual. Unreliable results of the test are usually due to: * Insufficient mixing of reagents and samples * Inaccurate pipetting and inadequate incubation times * Poor washing technique, spilling drops of the samples or Pxconjugate to the rim of wells * Use of the same pipette tip for different solutions 13. STORAGE AND EXPIRATION The kits are shipped cooled in cool bags. The transport time up to 72 hours has no influence on the expiration date. If you find damage to any kit component, please inform the manufacturer. Store the kit and the reagents at 2 to 10 C, in a dry place, protected from the light. The date of expiration is indicated on the ELISA kit label and on labels of the components. Store the unused strips in the provided plastic bag with the desiccant kept inside. Store serum samples at temperature 18 to 28 C, undiluted in small aliquots. Avoid repeated freezing and thawing of samples. Short term storage (up to one week) of thawed/fresh serum samples is possible at 2 to 10 C. Do not store the diluted samples, always prepare fresh. References: 1. GorgievskiHrisoho M.,Hinderer W.,NebelSchickel H.,Horn J.,Vornhagen R., Sonneborn H., Wolf H., Siegl G.: Serodiagnosis of Infectious Mononucleosis by Using Recombinant EpsteinBarr Antigens and EnzymeLinked Immunosorbent Assay Technology. J.of Clinical Microbiology 28: , Bowdre J.H.: EpsteinBarr virus serology, Clin.Imunol Newsletter 1991;11, Roubalová K., Roubal J., Staňková M., Vrbová K., Jandová M. & Kouba K.: Protilátková odpověď proti EB viru u pacientů s infekční mononukleosou, Čas. lék. čes. 125: ,

7 14. FLOW CHART: Step 1 Prepare/dilute reagents and samples Step 2 Pipette 100 L/well of Standards and samples Incubate for 30 minutes at laboratory temperature Wash 4 times (250 L/ well) Step 3 Dispense 100 L/ well of Px conjugate r.t.u. Incubate for 60 minutes at laboratory temperature Wash 4 times (250 L/well) Step 4 Dispense 100 L/well TMB substrate Incubate for 10 minutes laboratory temperature in the dark Step 5 Step 6 Dispense 100 L/well of Stop solution Read optical density at 450/ nm within 20 minutes References: 1. GorgievskiHrisoho M.,Hinderer W.,NebelSchickel H.,Horn J.,Vornhagen R., Sonneborn H., Wolf H., Siegl G.: Serodiagnosis of Infectious Mononucleosis by Using Recombinant EpsteinBarr Antigens and EnzymeLinked Immunosorbent Assay Technology. J.of Clinical Microbiology 28: , Bowdre J.H.: EpsteinBarr virus serology, Clin.Imunol Newsletter 1991;11, Roubalová K., Roubal J., Staňková M., Vrbová K., Jandová M. & Kouba K.: Protilátková odpověď proti EB viru u pacientů s infekční mononukleosou, Čas. lék. čes. 125: , Last revision: 06/2014 7

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