Development and Evaluation of Quantitative Immunoglobulin G Enzyme-Linked Immunosorbent Assay for the Diagnosis of Coronavirus Disease 2019 Using Truncated Recombinant Nucleocapsid Protein as Assay Antigen
Abstract
:1. Introduction
2. Materials and Methods
2.1. Human Samples
2.2. Virus Inoculation and RNA Extraction
2.3. Construction of SARS-CoV-2 Truncated and Full-Length Nucleocapsid Recombinant Plasmids
2.4. Expression and Purification of Recombinant Proteins
2.5. Western Blot Analysis and Silver Staining
2.6. In-House SARS-CoV-2 N Truncated and Full-Length N Proteins-Based IgG Indirect ELISAs
2.7. Commercial SARS-CoV-2 S Protein-IgG Indirect ELISA
2.8. Plaque Reduction Neutralization Test
2.9. Statistical Analysis
3. Results
3.1. Expression and Purification of the Recombinant SARS-CoV-2 Nucleocapsid Protein
3.2. SARS-CoV-2 N Truncated Protein-Based IgG ELISA
3.3. SARS-CoV-2 SARS-CoV-2 Full-Length N Protein-Based IgG ELISA
3.4. SARS-CoV-2 S Protein-Based IgG ELISA
3.5. Plaque Reduction Neutralization Test
3.6. Comparison of SARS-CoV-2 N Truncated Protein-Based IgG ELISA with Plaque Reduction Neutralization Test
3.7. Comparison of SARS-CoV-2 N Truncated Protein-Based IgG ELISA with S IgG Protein-Based IgG ELISA
3.8. Comparison of SARS-CoV-2 S Protein-Based IgG ELISA with Plaque Reduction Neutralization Test
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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COVID-19 Suspected | PRNT50 | |||
---|---|---|---|---|
Positive | Negative | Total | ||
rNΔ121 IgG ELISA | Positive | 113 | 5 | 118 |
Negative | 11 | 48 | 59 | |
Total | 124 | 53 | 177 | |
Pre-COVID-19 | PRNT50 | |||
Positive | Negative | Total | ||
rNΔ121 IgG ELISA | Positive | 0 | 8 | 8 |
Negative | 0 | 147 | 147 | |
Total | 0 | 155 | 155 |
COVID-19 Suspected | PRNT50 | |||
---|---|---|---|---|
Positive | Negative | Total | ||
rfull-length N IgG ELISA | Positive | 120 | 4 | 124 |
Negative | 4 | 49 | 53 | |
Total | 124 | 53 | 177 | |
Pre-COVID-19 | PRNT50 | |||
Positive | Negative | Total | ||
rfull-length N IgG ELISA | Positive | 0 | 17 | 17 |
Negative | 0 | 138 | 138 | |
Total | 0 | 155 | 155 |
COVID-19 Suspected | S IgG ELISA | |||
---|---|---|---|---|
Positive | Negative | Total | ||
rfull-length N IgG ELISA | Positive | 120 | 4 | 124 |
Negative | 1 | 52 | 53 | |
Total | 121 | 56 | 177 | |
Pre-COVID-19 | S IgG ELISA | |||
Positive | Negative | Total | ||
rfull-length N IgG ELISA | Positive | 1 | 16 | 17 |
Negative | 0 | 138 | 138 | |
Total | 1 | 154 | 155 |
COVID-19 Suspected | PRNT50 | |||
---|---|---|---|---|
Positive | Negative | Total | ||
S IgG ELISA | Positive | 119 | 2 | 121 |
Negative | 5 | 51 | 56 | |
Total | 124 | 53 | 177 | |
Pre-COVID-19 | PRNT50 | |||
Positive | Negative | Total | ||
S IgG ELISA | Positive | 0 | 2 | 2 |
Negative | 0 | 153 | 153 | |
Total | 0 | 155 | 155 |
Pre-COVID-19 | COVID-19 Suspected | |
---|---|---|
PRNT50 titer | Number | Number |
1:5120 | 0 | 4 |
1:2560 | 0 | 5 |
1:1280 | 0 | 12 |
1:640 | 0 | 14 |
1:320 | 0 | 29 |
1:160 | 0 | 33 |
1:80 | 0 | 18 |
1:40 | 0 | 4 |
1:20 | 0 | 0 |
1:10 | 0 | 5 |
<1:10 | 155 | 53 |
Total | 155 | 177 |
COVID-19 Suspected | S IgG ELISA | |||
---|---|---|---|---|
Positive | Negative | Total | ||
rNΔ121 IgG ELISA | Positive | 113 | 5 | 118 |
Negative | 8 | 51 | 59 | |
Total | 121 | 56 | 177 | |
Pre-COVID-19 | S IgG ELISA | |||
Positive | Negative | Total | ||
rNΔ121 IgG ELISA | Positive | 0 | 8 | 8 |
Negative | 2 | 145 | 147 | |
Total | 2 | 153 | 155 |
In-House rSARS-CoV-2 ELISA | Sensitivity (%) | Specificity (%) | PPV (%) | NPV (%) | Concordance (%) | Reference Test |
---|---|---|---|---|---|---|
NΔ121 | 91.1 | 93.8 | 89.7 | 94.7 | 92.8 | PRNT |
NΔ121 | 91.9 | 93.8 | 89.7 | 95.2 | 93.1 | Commercial S IgG ELISA |
Full-length N | 96.8 | 89.9 | 85.1 | 97.9 | 92.5 | PRNT |
Full-length N (Xiang et al.) | 83.3 | 95.0 | 94.8 | 83.8 | 88.9 | RT-PCR |
Full-length N (Tehrani et al.) | 89.0 | 98.0 | 96.0 | RT-PCR |
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Mutantu, P.N.; Ngwe Tun, M.M.; Nabeshima, T.; Yu, F.; Mukadi, P.K.; Tanaka, T.; Tashiro, M.; Fujita, A.; Kanie, N.; Oshiro, R.; et al. Development and Evaluation of Quantitative Immunoglobulin G Enzyme-Linked Immunosorbent Assay for the Diagnosis of Coronavirus Disease 2019 Using Truncated Recombinant Nucleocapsid Protein as Assay Antigen. Int. J. Environ. Res. Public Health 2021, 18, 9630. https://doi.org/10.3390/ijerph18189630
Mutantu PN, Ngwe Tun MM, Nabeshima T, Yu F, Mukadi PK, Tanaka T, Tashiro M, Fujita A, Kanie N, Oshiro R, et al. Development and Evaluation of Quantitative Immunoglobulin G Enzyme-Linked Immunosorbent Assay for the Diagnosis of Coronavirus Disease 2019 Using Truncated Recombinant Nucleocapsid Protein as Assay Antigen. International Journal of Environmental Research and Public Health. 2021; 18(18):9630. https://doi.org/10.3390/ijerph18189630
Chicago/Turabian StyleMutantu, Pierre Nsele, Mya Myat Ngwe Tun, Takeshi Nabeshima, Fuxun Yu, Patrick Kakoni Mukadi, Takeshi Tanaka, Masato Tashiro, Ayumi Fujita, Nobuhiro Kanie, Ryosaku Oshiro, and et al. 2021. "Development and Evaluation of Quantitative Immunoglobulin G Enzyme-Linked Immunosorbent Assay for the Diagnosis of Coronavirus Disease 2019 Using Truncated Recombinant Nucleocapsid Protein as Assay Antigen" International Journal of Environmental Research and Public Health 18, no. 18: 9630. https://doi.org/10.3390/ijerph18189630
APA StyleMutantu, P. N., Ngwe Tun, M. M., Nabeshima, T., Yu, F., Mukadi, P. K., Tanaka, T., Tashiro, M., Fujita, A., Kanie, N., Oshiro, R., Takazono, T., Imamura, Y., Hirayama, T., Moi, M. L., Inoue, S., Izumikawa, K., Yasuda, J., & Morita, K. (2021). Development and Evaluation of Quantitative Immunoglobulin G Enzyme-Linked Immunosorbent Assay for the Diagnosis of Coronavirus Disease 2019 Using Truncated Recombinant Nucleocapsid Protein as Assay Antigen. International Journal of Environmental Research and Public Health, 18(18), 9630. https://doi.org/10.3390/ijerph18189630