NovaLisa Epstein-Barr Virus (VCA) IgA ELISA (EBVA0150) Performance Characteristics

Transcription

1 NovaLisa Epstein-Barr Virus (VCA) IgA ELISA (EBVA0150) Performance Characteristics

2 Table of Contents 1 Introduction Intended Use Principle of the Assay Performance Characteristics Reproducibility (Precision) Analytical Specificity Interference from Hemoglobin, Bilirubin and Triglycerides Cross-Reactivity Diagnostic Sensitivity and Specificity... 7 EBVA0150 Performance page 2 of CF

3 1 Introduction Epstein-Barr Virus (EBV) is a member of the herpes virus family (Gamma subgroup, DNA virus of nm) and one of the most common human viruses. The virus occurs worldwide, and most people become infected with EBV sometime during their lives. Transmission of the virus is almost impossible to prevent since many healthy people can carry and spread the virus intermittently for life. Infants become susceptible to EBV as soon as maternal antibody protection disappears. Infection of children usually causes no symptoms. Infection during adolescence or young adulthood causes infectious mononucleosis 35% to 50% of the time. Infectious mononucleosis is almost never fatal. There are no known associations between active EBV infection and problems during pregnancy, such as miscarriages or birth defects. Although the symptoms of infectious mononucleosis usually resolve in 1 or 2 months, EBV remains dormant or latent in a few cells in the throat and blood for the rest of the person s life. Periodically, the virus can reactivate and is commonly found in the saliva of infected persons. This reactivation usually occurs without symptoms of illness. EBV also establishes a lifelong dormant infection in some cells of the body s immune system. A late event in a very few carriers of this virus is the emergence of Burkitt s lymphoma and nasopharyngeal carcinoma, but EBV is probably not the sole cause of these malignancies. Table 1: Epstein-Barr Virus - Symptoms and Mechanism of Infection Species Disease Symptoms Mechanism of Infection Epstein-Barr Virus (VCA) infectious mononucleosis fever, sore throat, swollen lymph glands The presence of pathogen or infection may be identified by PCR Serology: mono spot test, Detection of antibodies by ELISA 2 Intended Use Person to Person Transmission EBV requires intimate contact with the saliva of an infected person, but the virus is also found in the saliva of healthy people The Epstein-Barr Virus (VCA) IgA-ELISA is intended for the qualitative determination of IgA class antibodies against Epstein-Barr Virus viral capsid antigen (VCA) in human serum or plasma (citrate, heparin). 3 Principle of the Assay The qualitative immunoenzymatic determination of IgA-class antibodies against Epstein-Barr Virus (VCA) is based on the ELISA (Enzyme-linked Immunosorbent Assay) technique. Microtiter strip wells are coated with Epstein-Barr Virus recombinant p18 peptide to bind corresponding antibodies of the specimen. After washing the wells to remove all unbound EBVA0150 Performance page 3 of CF

4 sample material horseradish peroxidase (HRP) labelled anti-human IgA conjugate is added. This conjugate binds to the captured Epstein-Barr Virus (VCA)-specific antibodies. The immune complex formed by the bound conjugate is visualized by adding Tetramethylbenzidine (TMB) substrate which gives a blue reaction product. The intensity of this product is proportional to the amount of Epstein-Barr Virus (VCA)-specific IgA antibodies in the specimen. Sulphuric acid is added to stop the reaction. This produces a yellow endpoint colour. Absorbance at 450 nm is read using an ELISA microwell plate reader. 4 Performance Characteristics 4.1 Reproducibility (Precision) Material NovaLisa Epstein Barr Virus (VCA) IgA Lot: EBVA-102 Production date: Expiry date: Positive and negative samples Test Description The reproducibility of the NovaLisa Epstein-Barr Virus (VCA) IgA ELISA kit was determined by comparing 20 and 24 replicates of 2 different samples in one assay (within-run) and by comparing 2 different samples assayed in 12 different runs (between-run). Acceptance Criterion: CV < 15 % Results Within-run and between-run precision were estimated by analysis of variance and are presented in tables 2 and 3. Table 2: Within-Run Precision (EBVA-102) Sample n Mean (E) CV [%] # # Table 3: Between-Run Precision (EBVA-102) Sample n Mean (NTU) CV [%] # # Conclusion The acceptance criterion was met for all samples. EBVA0150 Performance page 4 of CF

5 4.2 Analytical Specificity Interference from Hemoglobin, Bilirubin and Triglycerides Introduction Increased concentrations of possible interference materials such as bilirubin, triglycerides and hemoglobin may interfere with immunoassays creating false-negative or false-positive results. In order to investigate this topic a literature research in combination with investigation of nearly 100 parameters were performed. Material and Test Condition Different members of the NovaLisa ELISA line were used including assays for the detection of different antibody isotypes (IgA, IgG, IgM, IgG + IgM) to bacteria, viruses, fungi, parasites and worms as well as an antigen ELISA for the detection of TSH. Defined positive resp. negative or equivocal samples were used. A certain amount of the potentially interfering substance was added to each sample. The final concentration of each substance was in a pathological range as also described by competitors (10 mg/ml hemoglobin, 0.5 mg/ml bilirubin and 5 mg/ml triglycerides). The results obtained with the sample with added interfering substance should be % of the result of the untreated sample in order to fulfil the specifications. Conclusion The internal specifications of % were always fulfilled. Interferences with hemolytic, lipemic or icteric samples were not observed up to a concentration of 10 mg/ml hemoglobin, 0.5 mg/ml bilirubin and 5 mg/ml triglycerides. These results are also in agreement with literature data. Dimeski, G. (2008) Interference Testing, Clin Biochem Rev 29, S43 S48 Tate, J. and Ward, G. (2004) Interferences in Immunoassay, Clin Biochem Rev 25, EBVA0150 Performance page 5 of CF

6 4.2.2 Cross-Reactivity Material NovaLisa Epstein Barr Virus (VCA) IgA Lot: EBVA-102 Production date: Expiry date: potentially cross-reactive samples Test Description A panel of 19 specimens from patients with confirmed diseases other than Epstein Barr Virus (VCA) was tested to establish the analytical specificity of the NovaLisa Epstein Barr Virus (VCA) IgA ELISA. The specimens were from patients infected with pathogens that may cause similar signs and symptoms to those observed for Epstein Barr Virus (VCA) or from individuals with diseases or conditions that have the potential for cross-reactivity. Results Table 4: Cross-Reactivity Disease Type Sample NTU Evaluation Adenovirus neg CMV neg neg neg neg neg EBV neg Echinococcus neg HBV neg Influenza Virus A neg Influenza Virus B neg Leptospira neg M. pneumoniae neg Picorna neg RSV neg Rubella neg Syphilis neg Toxoplasma neg VZV neg EBVA0150 Performance page 6 of CF

7 Conclusion Investigation of a specimen panel with antibody activities to potentially cross-reacting parameters (antibodies to several infectious agents) did not reveal significant evidence of falsepositive results due to cross-reactions. 4.3 Diagnostic Sensitivity and Specificity Introduction The purpose of this study was to determine the efficiency of the assay to discriminate between positive and negative clinical samples. Sera from newborns and immunocompromised individuals were excluded from the study as in these patients serological data only have limited value. To evaluate the diagnostic performance of the NovaLisa Epstein Barr Virus (VCA) IgA ELISA, internal studies were conducted by NovaTec with well defined samples from External Quality Control Schemes (INSTAND e.v.) and in comparison to an immunoassay already established on the market (IBL Epstein Barr Virus (VCA) IgA). Samples from newborns and immunocompromised individuals were excluded from the study as in these patients serological data only have limited value. Material NovaLisa Epstein Barr Virus (VCA) IgA Lot: EBVA-102 Production date: Expiry date: IBL Epstein Barr Virus (VCA) IgA Lot: EA samples (incl. 6 EQAS samples) Results Table 5: Diagnostic Sensitivity and Specificity NovaLisa Epstein Barr Virus (VCA) IgA (Equivocal results were not included in the calculations) Conclusion IBL/Instand positive negative Σ positive negative Σ Diagnostic Sensitivity: % (11/11) Diagnostic Specificity: % (40/40) Agreement: % (93/93) Dr. C. Flechsig EBVA0150 Performance page 7 of CF