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  - Đukić, Teodora
AU  - Mladenović, Maja
AU  - Stanić-Vučinić, Dragana
AU  - Radosavljević, Jelena
AU  - Smiljanić, Katarina
AU  - Sabljić, Ljiljana
AU  - Gnjatović, Marija Lj.
AU  - Cujić, Danica
AU  - Vasović, Tamara
AU  - Simović, Ana
AU  - Radomirović, Mirjana Ž.
AU  - Ćirković-Veličković, Tanja
PY  - 2021
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/5735
AB  - Serological testing is important method for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARSCoV-2) infection and for assessment of immunological response after the vaccination. Nucleocapsid (N) protein is the most abundant virus protein and strong immunogen. The aim was develop efficient, low-cost production of N protein large fragment and to characterize it with bottom-up, high-resolution tandem mass spectrometry and immunologically.
SARS-CoV-2 recombinant fragment of nucleocapsid protein (rfNP; 58–419 aa) was expressed in E. coli in soluble form and purified by several chromatographic steps and was subjected to SDS-PAGE and in-gel digested with trypsin.
rfNP was tested in immunoblot using sera of COVID-19 convalescent patients. ELISA was optimized with sera of RT-PCR confirmed positive symptomatic patients and healthy individuals. IgG detection sensitivity was 96% (47/50) and specificity 97% (67/68), while IgM detection was slightly lower. Identity of rfNP was confirmed with high scores and peptide coverage above 80%. Estimation from the value of areas under ion extracted chromatographic curves is that only up to 0,03% of the total band protein quantity belongs to host proteins, while rfNP share is well above 99,9%, resulting in highly pure nucleocapsid protein preparation.
C3  - Proteomics and Metabolomics for Personalized Medicine, XV Italian Proteomics Association Annual Meeting, Catholic University of the Sacred Heart, Roma, Italy, 8th-10th September 2021
T1  - Proteomic and immunological characterization of recombinantly expressed nucleocapsid SARS-CoV 2 protein fragment in E. coli
SP  - 50
EP  - 50
UR  - https://hdl.handle.net/21.15107/rcub_cherry_5735
ER  - 

@conference{
author = "Đukić, Teodora and Mladenović, Maja and Stanić-Vučinić, Dragana and Radosavljević, Jelena and Smiljanić, Katarina and Sabljić, Ljiljana and Gnjatović, Marija Lj. and Cujić, Danica and Vasović, Tamara and Simović, Ana and Radomirović, Mirjana Ž. and Ćirković-Veličković, Tanja",
year = "2021",
abstract = "Serological testing is important method for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARSCoV-2) infection and for assessment of immunological response after the vaccination. Nucleocapsid (N) protein is the most abundant virus protein and strong immunogen. The aim was develop efficient, low-cost production of N protein large fragment and to characterize it with bottom-up, high-resolution tandem mass spectrometry and immunologically.
SARS-CoV-2 recombinant fragment of nucleocapsid protein (rfNP; 58–419 aa) was expressed in E. coli in soluble form and purified by several chromatographic steps and was subjected to SDS-PAGE and in-gel digested with trypsin.
rfNP was tested in immunoblot using sera of COVID-19 convalescent patients. ELISA was optimized with sera of RT-PCR confirmed positive symptomatic patients and healthy individuals. IgG detection sensitivity was 96% (47/50) and specificity 97% (67/68), while IgM detection was slightly lower. Identity of rfNP was confirmed with high scores and peptide coverage above 80%. Estimation from the value of areas under ion extracted chromatographic curves is that only up to 0,03% of the total band protein quantity belongs to host proteins, while rfNP share is well above 99,9%, resulting in highly pure nucleocapsid protein preparation.",
journal = "Proteomics and Metabolomics for Personalized Medicine, XV Italian Proteomics Association Annual Meeting, Catholic University of the Sacred Heart, Roma, Italy, 8th-10th September 2021",
title = "Proteomic and immunological characterization of recombinantly expressed nucleocapsid SARS-CoV 2 protein fragment in E. coli",
pages = "50-50",
url = "https://hdl.handle.net/21.15107/rcub_cherry_5735"
}

Đukić, T., Mladenović, M., Stanić-Vučinić, D., Radosavljević, J., Smiljanić, K., Sabljić, L., Gnjatović, M. Lj., Cujić, D., Vasović, T., Simović, A., Radomirović, M. Ž.,& Ćirković-Veličković, T.. (2021). Proteomic and immunological characterization of recombinantly expressed nucleocapsid SARS-CoV 2 protein fragment in E. coli. in Proteomics and Metabolomics for Personalized Medicine, XV Italian Proteomics Association Annual Meeting, Catholic University of the Sacred Heart, Roma, Italy, 8th-10th September 2021, 50-50.
https://hdl.handle.net/21.15107/rcub_cherry_5735

Đukić T, Mladenović M, Stanić-Vučinić D, Radosavljević J, Smiljanić K, Sabljić L, Gnjatović ML, Cujić D, Vasović T, Simović A, Radomirović MŽ, Ćirković-Veličković T. Proteomic and immunological characterization of recombinantly expressed nucleocapsid SARS-CoV 2 protein fragment in E. coli. in Proteomics and Metabolomics for Personalized Medicine, XV Italian Proteomics Association Annual Meeting, Catholic University of the Sacred Heart, Roma, Italy, 8th-10th September 2021. 2021;:50-50.
https://hdl.handle.net/21.15107/rcub_cherry_5735 .

Đukić, Teodora, Mladenović, Maja, Stanić-Vučinić, Dragana, Radosavljević, Jelena, Smiljanić, Katarina, Sabljić, Ljiljana, Gnjatović, Marija Lj., Cujić, Danica, Vasović, Tamara, Simović, Ana, Radomirović, Mirjana Ž., Ćirković-Veličković, Tanja, "Proteomic and immunological characterization of recombinantly expressed nucleocapsid SARS-CoV 2 protein fragment in E. coli" in Proteomics and Metabolomics for Personalized Medicine, XV Italian Proteomics Association Annual Meeting, Catholic University of the Sacred Heart, Roma, Italy, 8th-10th September 2021 (2021):50-50,
https://hdl.handle.net/21.15107/rcub_cherry_5735 .

TY  - CONF
AU  - Maja, Mladenović
AU  - Đukić, Teodora
AU  - Vasović, Tamara
AU  - Stanić-Vučinić, Dragana
AU  - Smiljanić, Katarina
AU  - Radosavljević, Jelena
AU  - Sabljić, Ljiljana
AU  - Dević, Marija
AU  - Cujić, Danica
AU  - Ćirković-Veličković, Tanja
PY  - 2021
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/5737
AB  - Serological testing is important method for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARSCoV-2) infection. Nucleocapsid (N) protein is the most abundant virus derived protein and strong immunogen. We aimed to find its efficient, low-cost production, suitable for serological diagnosis. SARS-CoV-2 recombinant fragment of nucleocapsid protein (rfNP; 58–419 aa) was expressed in E. coli in soluble form, purified by immobilized metal ion affinity chromatography and strong cation exchange chromatography after which it was analyzed by Mass and CD spectrometry and characterized biochemically and immunologically. Purified rfNP has secondary structure of full-length recombinant N protein, with high percentage of disordered structure (34.2%) and of β-sheet (40.7%). rfNP was tested in immunoblot using sera of COVID-19 convalescent patients. ELISA was optimized with sera of RT-PCR confirmed positive symptomatic patients and healthy individuals. IgG detection sensitivity was 96% (47/50) and specificity 97% (67/68), while IgM detection was
slightly lower (94% and 96.5%, respectively). Cost-effective approach for soluble recombinant N protein fragment production was developed, with reliable IgG and IgM antibodies detection of SARS-CoV-2 infection.
C3  - FoodEnTwin Symposium: Novel analytical approaches in food and environmental sciences, June 16-18, 2021 Belgrade, Serbia
T1  - Expression, purification and immunological characterization of recombinant protein fragment from SARS-CoV-2
SP  - 33
EP  - 33
UR  - https://hdl.handle.net/21.15107/rcub_cherry_5737
ER  - 

@conference{
author = "Maja, Mladenović and Đukić, Teodora and Vasović, Tamara and Stanić-Vučinić, Dragana and Smiljanić, Katarina and Radosavljević, Jelena and Sabljić, Ljiljana and Dević, Marija and Cujić, Danica and Ćirković-Veličković, Tanja",
year = "2021",
abstract = "Serological testing is important method for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARSCoV-2) infection. Nucleocapsid (N) protein is the most abundant virus derived protein and strong immunogen. We aimed to find its efficient, low-cost production, suitable for serological diagnosis. SARS-CoV-2 recombinant fragment of nucleocapsid protein (rfNP; 58–419 aa) was expressed in E. coli in soluble form, purified by immobilized metal ion affinity chromatography and strong cation exchange chromatography after which it was analyzed by Mass and CD spectrometry and characterized biochemically and immunologically. Purified rfNP has secondary structure of full-length recombinant N protein, with high percentage of disordered structure (34.2%) and of β-sheet (40.7%). rfNP was tested in immunoblot using sera of COVID-19 convalescent patients. ELISA was optimized with sera of RT-PCR confirmed positive symptomatic patients and healthy individuals. IgG detection sensitivity was 96% (47/50) and specificity 97% (67/68), while IgM detection was
slightly lower (94% and 96.5%, respectively). Cost-effective approach for soluble recombinant N protein fragment production was developed, with reliable IgG and IgM antibodies detection of SARS-CoV-2 infection.",
journal = "FoodEnTwin Symposium: Novel analytical approaches in food and environmental sciences, June 16-18, 2021 Belgrade, Serbia",
title = "Expression, purification and immunological characterization of recombinant protein fragment from SARS-CoV-2",
pages = "33-33",
url = "https://hdl.handle.net/21.15107/rcub_cherry_5737"
}

Maja, M., Đukić, T., Vasović, T., Stanić-Vučinić, D., Smiljanić, K., Radosavljević, J., Sabljić, L., Dević, M., Cujić, D.,& Ćirković-Veličković, T.. (2021). Expression, purification and immunological characterization of recombinant protein fragment from SARS-CoV-2. in FoodEnTwin Symposium: Novel analytical approaches in food and environmental sciences, June 16-18, 2021 Belgrade, Serbia, 33-33.
https://hdl.handle.net/21.15107/rcub_cherry_5737

Maja M, Đukić T, Vasović T, Stanić-Vučinić D, Smiljanić K, Radosavljević J, Sabljić L, Dević M, Cujić D, Ćirković-Veličković T. Expression, purification and immunological characterization of recombinant protein fragment from SARS-CoV-2. in FoodEnTwin Symposium: Novel analytical approaches in food and environmental sciences, June 16-18, 2021 Belgrade, Serbia. 2021;:33-33.
https://hdl.handle.net/21.15107/rcub_cherry_5737 .

Maja, Mladenović, Đukić, Teodora, Vasović, Tamara, Stanić-Vučinić, Dragana, Smiljanić, Katarina, Radosavljević, Jelena, Sabljić, Ljiljana, Dević, Marija, Cujić, Danica, Ćirković-Veličković, Tanja, "Expression, purification and immunological characterization of recombinant protein fragment from SARS-CoV-2" in FoodEnTwin Symposium: Novel analytical approaches in food and environmental sciences, June 16-18, 2021 Belgrade, Serbia (2021):33-33,
https://hdl.handle.net/21.15107/rcub_cherry_5737 .