Epstein Barr Virus VCA IgG ELISA Kit

Transcription

1 Epstein Barr Virus VCA IgG ELISA Kit Catalog Number KA assays Version: 02 Intended for research use only

2 Table of Contents Introduction... 3 Intended Use... 3 Principle of the Assay... 3 General Information... 4 Materials Supplied... 4 Storage Instruction... 4 Materials Required but Not Supplied... 4 Precautions for Use... 5 Assay Protocol... 7 Reagent Preparation... 7 Sample Preparation... 7 Assay Procedure... 7 Data Analysis... 9 Calculation of Results... 9 Performance Characteristics Resources References Plate Layout KA / 14

3 Introduction Intended Use Epstein Barr Virus VCA IgG ELISA Kit is used for the detection of IgG antibodies to viral capsid antigen to Epstein-Barr virus in serum. Epstein Barr Virus VCA IgG ELISA Kit is intended for the diagnosis of EBV-induced or -associated diseases, such as infectious mononucleosis, chronic active EBV infection, Burkitt`s lymphoma, carcinomas of Waldayer s ring, opportunistic lymphomas (oligo- or polyclonal) and nasopharyngeal carcinoma. The kit may also be used for the overall characterization of the chronic fatigue syndrome and of immunodeficiency during which EBV is often activated. Principle of the Assay Epstein Barr Virus VCA IgG ELISA Kit is a solid-phase immunoanalytical test. The polystyrene strips are coated with specific antigen that bears immunodominant epitopes of VCA. The anti-vca EBV antibodies present in serum sample bind to the immobilized antigens and the antibodies being in complexes with antigen are later on recognised by animal anti-human IgG antibodies labelled with horseradish peroxidase. The labeled antibodies are revealed by an enzymatic reaction with a chromogenic substrate. Negative sera do not react and the mild change in colour, if present, may be attributed to the reaction background. KA / 14

4 General Information Materials Supplied List of component Component Standard A, 5 AU/mL(arteficial unit), r.t.u. (ready to use) Standard B, 16 AU/mL, r.t.u.* Standard C, 45 AU/mL, r.t.u. Standard D, 95 AU/mL, r.t.u. Standard E, 150 AU/mL, r.t.u. Standard F, 360 AU/mL, r.t.u. anti-human IgG antibodies-px conjugated (Px-conjugate) 10x concentrated Washing buffer Dilution buffer, r.t.u. TMB substrate, r.t.u. Stop solution, r.t.u. * Sealable pouch * (read to use) Amount 1.3 ml, 2 vials 1.3 ml, 2 vials 1.3 ml, 2 vials 1.3 ml, 2 vials 1.3 ml, 2 vials 1.3 ml, 2 vials 15 ml 125 ml 125 ml 15 ml 15 ml 1 piece Storage Instruction Store the kit reagents at 2 to 10 C, in a dry place and protected from the light. Expiration date is indicated at the ELISA kit label and at all reagent labels. Store unused strips in the sealable pouch and keep the desiccant inside. Do not store samples and Px-conjugate in the working concentration. Always prepare fresh solutions. Store serum samples at 2 to 10 C up to one week. For longer period make aliquots of serum sample and keep them at -20 C. Avoid repeated thawing and freezing. Kits are shipped in cooling bags, the transport time up to 72 hours have no influence on expiration. If you find damage at any part of the kit, please inform the manufacturer immediately. Materials Required but Not Supplied Distilled or deionised water for dilution of the Wash buffer concentrate. Appropriate equipment for pipetting, solution dispensing and washing. Spectrophotometer/colorimeter (microplate reader wavelength 450 nm). KA / 14

5 Precautions for Use Safety Precautions All ingredients of the kit are intended for laboratory use only. Standard contain human sera that has been tested negative for HBsAg, anti-hiv-1,2 and anti-hcv. However they should be regarded as contagious and handled and disposed of according to the appropriate regulations. Autoclave all reusable materials that were in contact with human samples for 1 hour at 121 C, burn disposable ignitable materials, decontaminate liquid wastes and nonignitable materials with 3% chloramine. Liquid wastes containing acid (Stop solution) should be neutralized in 4% sodium bicarbonate solution. Handle Stop solution with care. Avoid contact with skin or mucous membranes. In case of contact with skin, rinse immediately with plenty of water and seek medical advice. 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 or specimens and wash your hands thoroughly afterwards. Avoid spilling or producing aerosol.. Handling Precautions Avoid contamination of samples and kit reagents. Avoid cross-contamination of reagents. Substrate, TMB solution, Dilution buffer and Px-conjugate contain preservative ProClin 300. Avoid contact of the TMB substrate with oxidizing agents or metal surfaces. Variations in test results are usually due to: * Insufficient mixing of reagents and samples * Inaccurate pipetting and inadequate incubation times * Poor washing technique or spilling of sample or Px-conjugate at the rim of well * Use of identical pipette tip for different solutions KA / 14

6 Flow Chart Prepare reagents and samples Dispense 100 µl/ well of Standard and samples Incubate 30 min at room temperature Wash 4 times (250µL/ well), aspirate Dispense100 µl/ well of Px conjugate Incubate 60 min at room temperature Wash 4 times (250 µl/well), aspirate Dispense 100 µl/well TMB substrate Incubate 10 minutes at room temperature Dispense 100 µl/well of Stop solution Read optical density at 450/630 nm till 20 minutes KA / 14

7 Assay Protocol Reagent Preparation Prepare Wash buffer by diluting the concentrate 10 times with an appropriate volume of distilled or deionized water (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 vial to 32 to 37 C in a water bath. Diluted Wash buffer is stable for one week if stored at 2 to 10 C. Do not dilute Px-conjugate, TMB substrate and Stop solutions, they are ready to use. Sample Preparation Allow samples and all kit components to reach room temperature prior to the assay. Vortex samples and Standards in order to ensure homogeneity and mix the solutions well prior use. Dilute serum samples 101x with Dilution buffer (e.g. 5 µl of serum sample 500 µl of Dilution buffer). Do not dilute Standards, they are ready to use. Assay Procedure 1. Allow the microwell strips sealed inside the aluminium bag to reach room temperature. Withdraw an adequate number of strips and put the unused strips into the provided pouch and seal it carefully with the desiccant kept inside. 2. Fill the first well only with 100 µl of Dilution buffer (DB) to determine the reaction background. For qualitative and semiquantitative assay fill another two wells with 100 µl of Standard D (ST D), next one well with 100 µl of Standard A (ST A), Pipette 100 µl of diluted serum samples to the remaining wells (S1, S2, S3 and so one, see Fig. 1). For quantitative assay fill always 100 µl each Standards per two wells. Remaining wells fill with 100 µl diluted patients sera (S1, S2, S3 and so one, see Fig. 2). If you want to minimize the pipetting error, use duplicates for serum samples. 3. Incubate for 30 (+/- 2) minutes at room temperature. 4. Aspirate the liquid 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. If some liquid remains in the wells, invert the plate and tap it on an adsorbent paper to remove the last remaining drops. 5. Shake the bottle with Anti-human IgG Px-conjugate and Add 100 µl of the Anti-human IgG Px-conjugate into each well. 6. Incubate for 60 (+/-5) minutes at room temperature. 7. Aspirate and wash as in the step d). 8. Dispense 100 µl of the TMB substrate into each well; pipette in a regular rhythm or use an appropriate dispensing instrument. Incubate for 10 (+/-5 sec.) minutes at room temperature. The time measurement KA / 14

8 must be started right at the beginning of TMB dispensing. Cover the strips with an aluminium foil or keep them in the dark during the enzymatic reaction. 9. Stop the reaction by adding 100 µl of Stop solution. Use the same pipetting rhythm as with the TMB substrate to ensure the same reaction time in all wells. Tap gently the microplate for a few times to ensure complete mixing of the reagents. 10. Determine the absorbance at 450 nm in a microplate reader within 20 minutes. It is recommended to use reference reading at 630 nm. KA / 14

9 Data Analysis Calculation of Results Processing of Results At first subtract the optical absorbance of the background of the reaction (DB) from the absorbancies of all other wells. Qualitative evaluation 1. Compute the mean of the three parallels with Standard D. 2. Compute the cut-off value by multiplying the Standard D mean with a Correction factor. The Correction factor value for particular Lot is written in Quality control certificate. 3. Samples with absorbances lower than 100% of the cut-off value are considered negative and samples with absorbances higher than 130% the cut-off value are considered positive. Semiquantitative evaluation Determination of sample Positivity Index: 1. Compute the cut-off value 2. Compute the Positivity Index for each sample according to the following formula: sample Positivity Index = sample absorbance cut - off value 3. Express serum reactivity according to Table 1 (Interpretation of results) Table 1: Interpretation of results Positivity Index Interpretation < 1.00 Negative / > Note! Indifferent sample reactivity, i.e. interpreted as +/-, requires repetition of the sample testing. If the result is repetitively indifferent then it is recommended to use an alternative testing method or to obtain another sample from the patient withdrawn 1-2 weeks later. Example of calculation: Standard D absorbances = 0.996; 0.951; Standard D mean = Correction factor = 0.22 KA / 14

10 Cut-off value = 0.970*0.22 = Sample absorbance = Sample Positivity Index = 0.800/0.213 = 3.76 Quantitative evaluation 1. Compute the sample antibody titre in artificial units (AU/mL) as follows: 2. Construct the calibration curve by plotting the units of Standards (x-axis) to absorbance of Standards (y-axis). We recommended to use logarithmic axis.the concentration of each Standard (A-F) is mentioned in paragraph 4 Kit components. 3. Find the place where the absorbance of tested samples intersect calibration curve and find the corresponding values (AU/mL) on the axis x. It is possible to use various softwares for the standard curve fitting and for the calculation of the unknowns, e.g. Winliana, KimQ. For better fitting, the polynomic (four-parameter) function is the most convenient 4. Resul interpretation: Concentration (AU/mL) Evaluation < 15 negative 15,0 20,0 +/- 20,1 45,0 + 45,1 110, ,1 240,0 +++ > Note: Range between AU/mL is a grey zone. In such case it is recommended to repeat the assay. If the result of sample is in the grey zone, again use an alternative diagnostic method or initiate taking another blood sample 1-2 weeks later. Diagnostic Interpretation of Results VCA VCA VCA EA (D) EBNA- EBNA- Stages of EBV infection IgM IgG IgA IgG 1 IgG 1 IgM EBV negative Primoinfection EBV (acute phase) /- + Primoinfection EBV (post-acute phase ) Primoinfection EBV (convalescent phase) Eliminated infection EBV Reactivation of EBV KA / 14

11 Performance Characteristics Validity of the test The test is valid when: - The mean absorbance of the Standard D > 0, The background of the reaction (absorbance of the Dilution buffer well) is less than The absorbance of Standards follows this order: ST A < ST B < ST C < ST D < ST E < ST F Precision of the test The intraassay variability (within the test) and the interassay variability (between tests) were determined with samples of different absorbance values. 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 variation The coefficient of intraassay 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 (min max) CV(%) Recovery test Measured values of recovery test for every Lot are between % of expected values. Diagnostic specificity and sensitivity Diagnostic sensitivity of the test is 98.1%. Evaluation was performed on blood samples that were expected to be positive for anti-vca EBV IgG antibodies (blood donors, patients with history of infectious mononucleosis). Results were confirmed with another commercially available diagnostic test. Diagnostic specificity of the test is 97.1%. Specificity was determined on blood samples from healthy EBV negative blood donors. KA / 14

12 Limit of quantification The limit of quantification is 1.6 AU/mL The limit of quantification was defined as the lowest measurable concentration, which can be distinguished with 95% confidence from zero. 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. Resources References 1. Gorgievski-Hrisoho M.,Hinderer W.,Nebel-Schickel H.,Horn J.,Vornhagen R., Sonneborn H., Wolf H., Siegl G.: Serodiagnosis of Infectious Mononucleosis by Using Recombinant Epstein-Barr Antigens and Enzyme- Linked Immunosorbent Assay Technology. J.of Clinical Microbiology 28: , Bowdre J.H.: Epstein-Barr 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: , KA / 14

13 Plate Layout Qualitative and semiquantitative antibody determination A B C D E F G H 1 DB ST D ST D ST A S1 S2 S3 S KA / 14

14 Qualitative antibody determination A B C D E F G H DB ST A ST B ST C ST D ST E ST F S1 1 S2 S3 S KA / 14