Comparable Accuracies of Nonstructural Protein 1- and Envelope Protein-Based Enzyme-Linked Immunosorbent Assays in Detecting Anti-Dengue Immunoglobulin G Antibodies
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cells and Antibodies
2.2. Plasmid Construction, Protein Expression, and Purification
2.3. Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis (SDS–PAGE) and Western Blotting
2.4. Generation of Stable Cell Lines
2.5. Antigen Titration by Ag–ELISA
2.6. NS1- and VLP-Specific MAC/GAC–ELISAs
2.7. Indirect NS1 IgM/IgG ELISA
2.8. Limits of Detection of NS1 GAC- and Indirect NS1 IgG ELISAs
2.9. Human Serum Panel
2.10. Data Processing and Statistical Analysis
3. Results
3.1. Confirmation of In-House NS1 Protein Quality
3.2. Optimal Condition for NS1 GAC–ELISA
3.3. Overall Performance of Diagnostic Assays
3.4. Limits of Anti-NS1 IgG Detection
3.5. Determination of Serostatus Using a Composite Reference Standard
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Type of Assay | Serum Panel | Classification | No. of Samples | Confirmatory Test |
---|---|---|---|---|
NS1 a and VLP b GAC c- and Indirect NS1 IgG ELISA | Community sera | |||
Primary DENV1 | Late convalescent f | 7 | FRµNT90 * | |
Primary DENV2 | Late convalescent | 10 | FRµNT90 | |
Primary DENV3 | Late convalescent | 2 | FRµNT90 | |
Primary DENV4 | Late convalescent | 1 | FRµNT90 | |
Secondary infections | Late convalescent | 41 | FRµNT90 | |
NS1 and VLP MAC d- and Indirect NS1 IgM ELISA e | Patient sera | |||
Primary DENV1 | Acute g | 5 | RT–PCR # | |
Early convalescent h | 7 | |||
Late convalescent | 5 | |||
Primary DENV2 | Early convalescent | 2 | RT–PCR | |
Primary DENV3 | Acute | 1 | RT–PCR | |
Early convalescent | 11 | |||
NS1 and VLP GAC/MAC- and Indirect NS1 IgG/IgM ELISA | Naïve sera | n/a | 51 | FRµNT90 |
FRµNT | ||||||
---|---|---|---|---|---|---|
Test | Result | Positive | Negative | AUC # (95% CI) | % Sensitivity (95% CI) | % Specificity (95% CI) |
NS1 GAC | Positive | 57 | 1 | 0.96 (0.91–1.01) a,b | 95 (86.08–98.96) | 98.04 (89.55–99.95) |
Negative | 3 | 50 | ||||
NS1 IgG | Positive | 55 | 9 | 0.93 (0.89–0.98) c | 91.67 (81.61–97.24) | 82.35 (69.13–91.60) |
Negative | 5 | 42 | ||||
VLP GAC | Positive | 54 | 7 | 0.94 (0.89–0.99) | 90 (79.49–96.24) | 86.27 (73.74–94.30) |
Negative | 6 | 44 | ||||
Composite reference standard 1 * | Positive | 57 | 2 | n/a | 95 (86.08–98.96) | 96.08 (86.54–99.52) |
Negative | 3 | 49 | ||||
Composite reference standard 2 ** | Positive | 51 | 0 | n/a | 85 (73.43–92.90) | 100 (96.38–1.00) |
Negative | 9 | 51 |
Reference | Type of Assay | Serum Dilution | Antigens Used | Gold Standard | % Sensitivity (95% CI) (or % Positive Rate) | % Specificity (95% CI) | MDC a |
---|---|---|---|---|---|---|---|
[21] | Indirect IgG ELISA | 1:50 | DENV1–4 NS1 | PRNT c | 91.89 (83.11–96.54) | 84.62 (65.85–94.47) | 2.33 EU/ml |
[24] | Indirect IgG ELISA | 1:400 | DENV1–4 NS1 | RT–PCR d | 95.6 (91.40–97.80) | 89.5 (84.10–92.30) | n/a |
[45] | Indirect IgG ELISA | 1:400 | DENV1 NS1 | RT–PCR | 81.30–95.80 positive rate | n/a e | n/a |
[46] | MIA b | 1:100 | DENV1–4 NS1 | PRNT | 89 (54.33–99.99) | 86 (46.65–99.47) | n/a |
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Galula, J.U.; Salem, G.M.; Destura, R.V.; Remenyi, R.; Chao, D.-Y. Comparable Accuracies of Nonstructural Protein 1- and Envelope Protein-Based Enzyme-Linked Immunosorbent Assays in Detecting Anti-Dengue Immunoglobulin G Antibodies. Diagnostics 2021, 11, 741. https://doi.org/10.3390/diagnostics11050741
Galula JU, Salem GM, Destura RV, Remenyi R, Chao D-Y. Comparable Accuracies of Nonstructural Protein 1- and Envelope Protein-Based Enzyme-Linked Immunosorbent Assays in Detecting Anti-Dengue Immunoglobulin G Antibodies. Diagnostics. 2021; 11(5):741. https://doi.org/10.3390/diagnostics11050741
Chicago/Turabian StyleGalula, Jedhan Ucat, Gielenny M. Salem, Raul V. Destura, Roland Remenyi, and Day-Yu Chao. 2021. "Comparable Accuracies of Nonstructural Protein 1- and Envelope Protein-Based Enzyme-Linked Immunosorbent Assays in Detecting Anti-Dengue Immunoglobulin G Antibodies" Diagnostics 11, no. 5: 741. https://doi.org/10.3390/diagnostics11050741