Sandwich ELISA for the Quantification of Nucleocapsid Protein of SARS-CoV-2 Based on Polyclonal Antibodies from Two Different Species
Аутори
Mladenović Stokanić, MajaSimović, Ana
Jovanović, Vesna B.
Radomirović, Mirjana
Udovićki, Božidar
Krstić Ristivojević, Maja
Đukić, Teodora
Vasović, Tamara
Aćimović, Jelena
Sabljić, Ljiljana
Lukić, Ivana
Kovačević, Ana
Cujić, Danica
Gnjatović, Marija
Smiljanić, Katarina
Stojadinović, Marija
Radosavljević, Jelena
Stanić-Vučinić, Dragana
Stojanović, Marijana
Rajković, Andreja
Ćirković-Veličković, Tanja
Чланак у часопису (Објављена верзија)
Метаподаци
Приказ свих података о документуАпстракт
In this study, a cost-effective sandwich ELISA test, based on polyclonal antibodies, for routine quantification SARS-CoV-2 nucleocapsid (N) protein was developed. The recombinant N protein was produced and used for the production of mice and rabbit antisera. Polyclonal N protein-specific antibodies served as capture and detection antibodies. The prototype ELISA has LOD 0.93 ng/mL and LOQ 5.3 ng/mL, with a linear range of 1.52–48.83 ng/mL. N protein heat pretreatment (56 °C, 1 h) decreased, while pretreatment with 1% Triton X-100 increased analytical ELISA sensitivity. The diagnostic specificity of ELISA was 100% (95% CI, 91.19–100.00%) and sensitivity was 52.94% (95% CI, 35.13–70.22%) compared to rtRT-PCR (Ct < 40). Profoundly higher sensitivity was obtained using patient samples mostly containing Wuhan-similar variants (Wuhan, alpha, and delta), 62.50% (95% CI, 40.59 to 81.20%), in comparison to samples mostly containing Wuhan-distant variants (Omicron) 30.00% (6.67–65.25%). The devel...oped product has relatively high diagnostic sensitivity in relation to its analytical sensitivity due to the usage of polyclonal antibodies from two species, providing a wide repertoire of antibodies against multiple N protein epitopes. Moreover, the fast, simple, and inexpensive production of polyclonal antibodies, as the most expensive assay components, would result in affordable antigen tests.
Кључне речи:
COVID-19 diagnosis / nucleocapsid protein / antigen test / ELISA / polyclonal antibodiesИзвор:
International Journal of Molecular Sciences, 2023, 25, 1, 333-Издавач:
- MDPI
Финансирање / пројекти:
- Министарство науке, технолошког развоја и иновација Републике Србије, институционално финансирање - 200168 (Универзитет у Београду, Хемијски факултет) (RS-MESTD-inst-2020-200168)
- Министарство науке, технолошког развоја и иновација Републике Србије, институционално финансирање - 200007 (Универзитет у Београду, Институт за биолошка истраживања 'Синиша Станковић') (RS-MESTD-inst-2020-200007)
- Министарство науке, технолошког развоја и иновација Републике Србије, институционално финансирање - 200177 (Центар за имунолошка истраживања 'Бранислав Јанковић' Торлак, Београд) (RS-MESTD-inst-2020-200177)
- CAPSIDO – Developement of the assays for detection of SARS Cov-2 virus capsid proteins in biological fluids of COVID19 patients (RS-ScienceFundRS-Fond_2020_COVID19-7542203)
- Serbian Academy of Sciences and Arts GA No. F-26.
- Ghent University Global Campus and Belgian Special Research Fund BOF StG No. 01N01718.
Колекције
Институција/група
Hemijski fakultet / Faculty of ChemistryTY - JOUR AU - Mladenović Stokanić, Maja AU - Simović, Ana AU - Jovanović, Vesna B. AU - Radomirović, Mirjana AU - Udovićki, Božidar AU - Krstić Ristivojević, Maja AU - Đukić, Teodora AU - Vasović, Tamara AU - Aćimović, Jelena AU - Sabljić, Ljiljana AU - Lukić, Ivana AU - Kovačević, Ana AU - Cujić, Danica AU - Gnjatović, Marija AU - Smiljanić, Katarina AU - Stojadinović, Marija AU - Radosavljević, Jelena AU - Stanić-Vučinić, Dragana AU - Stojanović, Marijana AU - Rajković, Andreja AU - Ćirković-Veličković, Tanja PY - 2023 UR - http://cherry.chem.bg.ac.rs/handle/123456789/6436 AB - In this study, a cost-effective sandwich ELISA test, based on polyclonal antibodies, for routine quantification SARS-CoV-2 nucleocapsid (N) protein was developed. The recombinant N protein was produced and used for the production of mice and rabbit antisera. Polyclonal N protein-specific antibodies served as capture and detection antibodies. The prototype ELISA has LOD 0.93 ng/mL and LOQ 5.3 ng/mL, with a linear range of 1.52–48.83 ng/mL. N protein heat pretreatment (56 °C, 1 h) decreased, while pretreatment with 1% Triton X-100 increased analytical ELISA sensitivity. The diagnostic specificity of ELISA was 100% (95% CI, 91.19–100.00%) and sensitivity was 52.94% (95% CI, 35.13–70.22%) compared to rtRT-PCR (Ct < 40). Profoundly higher sensitivity was obtained using patient samples mostly containing Wuhan-similar variants (Wuhan, alpha, and delta), 62.50% (95% CI, 40.59 to 81.20%), in comparison to samples mostly containing Wuhan-distant variants (Omicron) 30.00% (6.67–65.25%). The developed product has relatively high diagnostic sensitivity in relation to its analytical sensitivity due to the usage of polyclonal antibodies from two species, providing a wide repertoire of antibodies against multiple N protein epitopes. Moreover, the fast, simple, and inexpensive production of polyclonal antibodies, as the most expensive assay components, would result in affordable antigen tests. PB - MDPI T2 - International Journal of Molecular Sciences T1 - Sandwich ELISA for the Quantification of Nucleocapsid Protein of SARS-CoV-2 Based on Polyclonal Antibodies from Two Different Species VL - 25 IS - 1 SP - 333 DO - 10.3390/ijms25010333 ER -
@article{ author = "Mladenović Stokanić, Maja and Simović, Ana and Jovanović, Vesna B. and Radomirović, Mirjana and Udovićki, Božidar and Krstić Ristivojević, Maja and Đukić, Teodora and Vasović, Tamara and Aćimović, Jelena and Sabljić, Ljiljana and Lukić, Ivana and Kovačević, Ana and Cujić, Danica and Gnjatović, Marija and Smiljanić, Katarina and Stojadinović, Marija and Radosavljević, Jelena and Stanić-Vučinić, Dragana and Stojanović, Marijana and Rajković, Andreja and Ćirković-Veličković, Tanja", year = "2023", abstract = "In this study, a cost-effective sandwich ELISA test, based on polyclonal antibodies, for routine quantification SARS-CoV-2 nucleocapsid (N) protein was developed. The recombinant N protein was produced and used for the production of mice and rabbit antisera. Polyclonal N protein-specific antibodies served as capture and detection antibodies. The prototype ELISA has LOD 0.93 ng/mL and LOQ 5.3 ng/mL, with a linear range of 1.52–48.83 ng/mL. N protein heat pretreatment (56 °C, 1 h) decreased, while pretreatment with 1% Triton X-100 increased analytical ELISA sensitivity. The diagnostic specificity of ELISA was 100% (95% CI, 91.19–100.00%) and sensitivity was 52.94% (95% CI, 35.13–70.22%) compared to rtRT-PCR (Ct < 40). Profoundly higher sensitivity was obtained using patient samples mostly containing Wuhan-similar variants (Wuhan, alpha, and delta), 62.50% (95% CI, 40.59 to 81.20%), in comparison to samples mostly containing Wuhan-distant variants (Omicron) 30.00% (6.67–65.25%). The developed product has relatively high diagnostic sensitivity in relation to its analytical sensitivity due to the usage of polyclonal antibodies from two species, providing a wide repertoire of antibodies against multiple N protein epitopes. Moreover, the fast, simple, and inexpensive production of polyclonal antibodies, as the most expensive assay components, would result in affordable antigen tests.", publisher = "MDPI", journal = "International Journal of Molecular Sciences", title = "Sandwich ELISA for the Quantification of Nucleocapsid Protein of SARS-CoV-2 Based on Polyclonal Antibodies from Two Different Species", volume = "25", number = "1", pages = "333", doi = "10.3390/ijms25010333" }
Mladenović Stokanić, M., Simović, A., Jovanović, V. B., Radomirović, M., Udovićki, B., Krstić Ristivojević, M., Đukić, T., Vasović, T., Aćimović, J., Sabljić, L., Lukić, I., Kovačević, A., Cujić, D., Gnjatović, M., Smiljanić, K., Stojadinović, M., Radosavljević, J., Stanić-Vučinić, D., Stojanović, M., Rajković, A.,& Ćirković-Veličković, T.. (2023). Sandwich ELISA for the Quantification of Nucleocapsid Protein of SARS-CoV-2 Based on Polyclonal Antibodies from Two Different Species. in International Journal of Molecular Sciences MDPI., 25(1), 333. https://doi.org/10.3390/ijms25010333
Mladenović Stokanić M, Simović A, Jovanović VB, Radomirović M, Udovićki B, Krstić Ristivojević M, Đukić T, Vasović T, Aćimović J, Sabljić L, Lukić I, Kovačević A, Cujić D, Gnjatović M, Smiljanić K, Stojadinović M, Radosavljević J, Stanić-Vučinić D, Stojanović M, Rajković A, Ćirković-Veličković T. Sandwich ELISA for the Quantification of Nucleocapsid Protein of SARS-CoV-2 Based on Polyclonal Antibodies from Two Different Species. in International Journal of Molecular Sciences. 2023;25(1):333. doi:10.3390/ijms25010333 .
Mladenović Stokanić, Maja, Simović, Ana, Jovanović, Vesna B., Radomirović, Mirjana, Udovićki, Božidar, Krstić Ristivojević, Maja, Đukić, Teodora, Vasović, Tamara, Aćimović, Jelena, Sabljić, Ljiljana, Lukić, Ivana, Kovačević, Ana, Cujić, Danica, Gnjatović, Marija, Smiljanić, Katarina, Stojadinović, Marija, Radosavljević, Jelena, Stanić-Vučinić, Dragana, Stojanović, Marijana, Rajković, Andreja, Ćirković-Veličković, Tanja, "Sandwich ELISA for the Quantification of Nucleocapsid Protein of SARS-CoV-2 Based on Polyclonal Antibodies from Two Different Species" in International Journal of Molecular Sciences, 25, no. 1 (2023):333, https://doi.org/10.3390/ijms25010333 . .