TY  - 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 . .

TY  - CONF
AU  - Sibinčić, Nikolina
AU  - Krstić-Ristivojević, Maja
AU  - Stojanović, Marijana
AU  - Mladenović Stokanić, Maja
AU  - Vasović, Tamara
AU  - Ćirković-Veličković, Tanja
AU  - Stojadinović, Marija
PY  - 2023
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/6051
AB  - The SARS-CoV-2 nucleocapsid (N) protein plays a significant role in the coronavirus life cycle and participates in a variety of critical events following viral invasion1. In infected patients, high titers of immunoglobulin G (IgG) targeting N protein were detected and correlated with the clinical course of the disease2. Therefore, N protein and anti-N protein IgGs were recognized as important diagnostic indicators of COVID-19 infection in serological and quick antigen tests3. In this study, we optimized the expression of the recombinant form of SARS-CoV-2 N protein in a mammalian cell line HEK293T by comparing the transfection efficiency between Polyethylenimine (PEI) and Calcium Phosphate (CaP) DNA-complexing agents. Transfection potency was tested at different cell confluency and passage number, in several cell culture media, pre-transfection and post-transfection media change and in conditions of reduced serum. Chloroquine and glycerol treatments were included to enhance transfection efficiency as they might inhibit DNA degradation in lysosomes or increase membrane permeability. Protein expression was monitored in cell supernatants up to 7 days post-transfection in dot-bot and Western blot using anti-N protein antibodies. Both transfection methods have shown moderate to relatively high transfection efficiency dependent on the applied conditions, making them affordable and easy to use techniques for recombinant N protein production on a small-scale in adherent mammalian systems. PEI acts as a good delivery system regardless of the presence of the fetal bovine serum (FBS), while CaP transfection is more dependent on the presence of FBS which in turn favors N protein degradation. However, we have optimized both methods to achieve optimal expression of unfragmented N-protein in serum-free conditions. Apart from setting up a cost-effective platform for expression of N protein in mammalian cells, we plan on investigating the mechanisms behind the PEI and CaP non-viral gene delivery systems as there are still some uncertainties in the scientific community.
PB  - Beograde : Serbian Biochemical Society
C3  - "Biochemistry in Biotechnology", Twelfth Conference, International scientific meeting, September 21-23, 2023, Belgrade, Serbia
T1  - Expression of recombinant SARS-CoV-2 nucleocapsid protein in mammalian cells
SP  - 91
EP  - 91
UR  - https://hdl.handle.net/21.15107/rcub_cherry_6051
ER  - 

@conference{
author = "Sibinčić, Nikolina and Krstić-Ristivojević, Maja and Stojanović, Marijana and Mladenović Stokanić, Maja and Vasović, Tamara and Ćirković-Veličković, Tanja and Stojadinović, Marija",
year = "2023",
abstract = "The SARS-CoV-2 nucleocapsid (N) protein plays a significant role in the coronavirus life cycle and participates in a variety of critical events following viral invasion1. In infected patients, high titers of immunoglobulin G (IgG) targeting N protein were detected and correlated with the clinical course of the disease2. Therefore, N protein and anti-N protein IgGs were recognized as important diagnostic indicators of COVID-19 infection in serological and quick antigen tests3. In this study, we optimized the expression of the recombinant form of SARS-CoV-2 N protein in a mammalian cell line HEK293T by comparing the transfection efficiency between Polyethylenimine (PEI) and Calcium Phosphate (CaP) DNA-complexing agents. Transfection potency was tested at different cell confluency and passage number, in several cell culture media, pre-transfection and post-transfection media change and in conditions of reduced serum. Chloroquine and glycerol treatments were included to enhance transfection efficiency as they might inhibit DNA degradation in lysosomes or increase membrane permeability. Protein expression was monitored in cell supernatants up to 7 days post-transfection in dot-bot and Western blot using anti-N protein antibodies. Both transfection methods have shown moderate to relatively high transfection efficiency dependent on the applied conditions, making them affordable and easy to use techniques for recombinant N protein production on a small-scale in adherent mammalian systems. PEI acts as a good delivery system regardless of the presence of the fetal bovine serum (FBS), while CaP transfection is more dependent on the presence of FBS which in turn favors N protein degradation. However, we have optimized both methods to achieve optimal expression of unfragmented N-protein in serum-free conditions. Apart from setting up a cost-effective platform for expression of N protein in mammalian cells, we plan on investigating the mechanisms behind the PEI and CaP non-viral gene delivery systems as there are still some uncertainties in the scientific community.",
publisher = "Beograde : Serbian Biochemical Society",
journal = ""Biochemistry in Biotechnology", Twelfth Conference, International scientific meeting, September 21-23, 2023, Belgrade, Serbia",
title = "Expression of recombinant SARS-CoV-2 nucleocapsid protein in mammalian cells",
pages = "91-91",
url = "https://hdl.handle.net/21.15107/rcub_cherry_6051"
}

Sibinčić, N., Krstić-Ristivojević, M., Stojanović, M., Mladenović Stokanić, M., Vasović, T., Ćirković-Veličković, T.,& Stojadinović, M.. (2023). Expression of recombinant SARS-CoV-2 nucleocapsid protein in mammalian cells. in "Biochemistry in Biotechnology", Twelfth Conference, International scientific meeting, September 21-23, 2023, Belgrade, Serbia
Beograde : Serbian Biochemical Society., 91-91.
https://hdl.handle.net/21.15107/rcub_cherry_6051

Sibinčić N, Krstić-Ristivojević M, Stojanović M, Mladenović Stokanić M, Vasović T, Ćirković-Veličković T, Stojadinović M. Expression of recombinant SARS-CoV-2 nucleocapsid protein in mammalian cells. in "Biochemistry in Biotechnology", Twelfth Conference, International scientific meeting, September 21-23, 2023, Belgrade, Serbia. 2023;:91-91.
https://hdl.handle.net/21.15107/rcub_cherry_6051 .

Sibinčić, Nikolina, Krstić-Ristivojević, Maja, Stojanović, Marijana, Mladenović Stokanić, Maja, Vasović, Tamara, Ćirković-Veličković, Tanja, Stojadinović, Marija, "Expression of recombinant SARS-CoV-2 nucleocapsid protein in mammalian cells" in "Biochemistry in Biotechnology", Twelfth Conference, International scientific meeting, September 21-23, 2023, Belgrade, Serbia (2023):91-91,
https://hdl.handle.net/21.15107/rcub_cherry_6051 .