NovaLisa Zika Virus IgM µ-capture ELISA (ZVM0790) Performance Characteristics
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1 NovaLisa Zika Virus IgM µ-capture ELISA (ZVM0790) 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 ZVM0790 Performance page 2 of EH
3 1 Introduction Zika virus (ZIKV) is a single-stranded RNA virus of the Flaviviridae family (genus Flavivirus). It was first isolated in 1947 from a sentinel rhesus monkey during a yellow fever study in the Zika forest of Uganda. Since its discovery, ZIKV circulation has been detected in Africa and Asia where it has caused sporadic human infections. In 2007 its emergence on Yap Island, Micronesia was reported, marking transmission of Zika virus outside Africa and Asia. Since 2013, ZIKV has been reported from French Polynesia, New Caledonia, Cook Islands, Easter Island (Chile), Samoa and Vanuatu, and in early 2015 it spread initially to Brazil and subsequently to additional countries of the Americas. ZIKV is transmitted primarily through the bite of an infected Aedes species mosquito (A. aegypti and A. albopictus). However, there have been reports of less common transmission modes, such as blood transfusion, perinatal, and sexual contact. The incubation period of Zika virus disease is not known precisely, but is likely to be a few days. It is estimated that only one in five people infected with ZIKV develop signs or symptoms. Clinical manifestations of ZIKV infection are described as very similar to those of Dengue virus (DENV) and Chikungunya virus (CHIKV) infections, but usually milder. The most common clinical signs and symptoms are maculopapular rash, low grade fever, arthralgia, myalgia, headache, retro-orbital pain and conjunctivitis. Less frequently reported are oedema, sore throat, cough, vomiting, and haematospermia. Human infections with ZIKV are usually mild and self-limiting and the symptoms usually resolve spontaneously after 3 7 days; arthralgia may persist for up to 1 month. In rare cases, after a Zika virus infection a Guillain-Barré syndrome (GBS), a disorder of the peripheral nerves, can occur. There is now scientific consensus that Zika virus infection in pregnancy can be a cause of microcephaly. Table 1: Zika virus - Symptoms and Mechanism of Infection Species Disease Symptoms Mechanism of Infection Zika virus (ZIKV) Zika fever Fever, headache, retro-orbital pain, conjunctivitis, maculopapular rash, myalgias, arthralgias The presence of pathogen or infection may be identified by Pathogen detection: isolation in cell culture PCR Serology: Detection of antibodies by IF, ELISA Plaque Reduction Neutralization Test (PRNT) Primary mode of transmission via bite of Aedes mosquitos (less common: blood transfusion, perinatal, and sexual contact) ZVM0790 Performance page 3 of EH
4 2 Intended Use The NovaLisa Zika Virus IgM µ-capture ELISA is intended for the qualitative determination of IgM antibodies to Zika virus in human serum or plasma (citrate, heparin). 3 Principle of the Assay The qualitative immunoenzymatic determination of specific IgM-class antibodies is based on the ELISA (Enzyme-linked Immunosorbent Assay) µ-capture technique. Microplates are coated with anti-human IgM-class antibodies to bind the corresponding antibodies of the sample. After washing the wells to remove all unbound sample material, horseradish peroxidase (HRP) labelled Zika virus antigen is added. This antigen-conjugate binds to the captured specific IgM antibodies. In a second washing step unbound conjugate is removed. 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 specific IgM antibodies in the sample. Sulphuric acid is added to stop the reaction. This produces a yellow endpoint colour. Absorbance at 450/620 nm is read using an ELISA microwell plate reader. 4 Performance Characteristics 4.1 Reproducibility (Precision) Material NovaLisa Zika Virus IgM µ-capture Lot: ZVM samples Test Description The reproducibility of the NovaLisa Zika Virus IgM µ-capture ELISA kit was determined by comparing 24 replicates of 3 different samples in one assay (within-run) and by comparing 3 different samples assayed in 12 different runs (between-run). Acceptance Criterion: CV < 15 % ZVM0790 Performance page 4 of EH
5 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 (ZVM-002) Sample n Mean (E) CV [%] # # # Table 3: Between-Run Precision (ZVM-002) Sample n Mean (NTU) CV [%] # # # The acceptance criterion was met for all samples. 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 was 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. 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, ZVM0790 Performance page 5 of EH
6 4.2.2 Cross-Reactivity Material NovaLisa Zika Virus IgM µ-capture Lot: ZVM-001 NovaLisa Zika Virus IgM µ-capture Lot: ZVM potentially cross-reactive samples Test Description A panel of 84 specimens from patients with confirmed diseases other than Zika virus (ZIKV) infection was tested to establish the analytical specificity of the NovaLisa Zika Virus IgM µ-capture ELISA. The specimens were from patients infected with pathogens that may cause similar signs and symptoms to those observed for ZIKV (e.g. DENV, CHIKV) or from individuals with diseases or conditions that have the potential for cross-reactivity such as other members of the family Flaviviridae (WNV, TBEV, YFV) or pathogens involved in polyclonal B-cell activation (Epstein- Barr virus, EBV). Results Table 4: Cross-Reactivity Number of Samples Positive Result Pathogen/Disease Type n NovaLisa Dengue virus (DENV) 12 0/12 West Nile virus (WNV) 20 1/20 Tick borne encephalitis virus (TBEV) 3 0/3 Chikungunya virus (CHIKV) 5 0/5 Yellow Fever virus (YFV) 12 1/12 Epstein-Barr virus 4 0/4 Rheumatoid Factor 27 0/27 ANA 1 0/1 Total 84 2/84 Investigation of a specimen panel with antibody activities to potentially cross-reacting parameters revealed only minimal cross-reactivity to West Nile virus (WNV) and Yellow Fever virus (YFV) specimens. In endemic areas, double infection as well as past infection with other flaviviruses should be considered. ZVM0790 Performance page 6 of EH
7 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. To evaluate the diagnostic performance of the NovaLisa Zika Virus IgM µ-capture ELISA, internal studies were conducted by NovaTec with well defined samples. Samples from newborns and immunocompromised individuals were excluded from the study as in these patients serological data only have limited value. Material NovaLisa Zika Virus IgM µ-capture Lot: ZVM-001 NovaLisa Zika Virus IgM µ-capture Lot: ZVM samples positive ZIKV samples: 10 samples negative samples: 43 pregnancy samples (healthy women, Germany) 4 blood bank samples (Frankfurt, Germany) cross reactivity panel: 84 samples (incl. United States, Uganda, Guadeloupe, New Caledonia) Results Table 5: Diagnostic Sensitivity and Specificity NovaLisa Zika Virus IgM µ-capture (Equivocal results were not included in the calculations) Demand positive negative Σ positive negative Σ Diagnostic Sensitivity: 100 % (9/9) Diagnostic Specificity: 98.5 % (129/131) Agreement: 98.6 % (138/140) Dr. E. Heck ZVM0790 Performance page 7 of EH
NovaLisa (ZVM0790) Performance Characteristics
NovaLisa Zika Virus IgM µ-capture ELISA (ZVM0790) Performance Characteristics Table of Contents 1 Introduction... 3 2 Intended Use... 4 3 Principle of the Assay... 4 4 Performance Characteristics... 4
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