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 .

TY  - GEN
AU  - Ćirković-Veličković, Tanja
AU  - Gnjatović, Marija
AU  - Ćujić, Danica
AU  - Todorović, Aleksandra
AU  - Stanić-Vučinić, Dragana
AU  - Đukić, Teodora
AU  - Mladenović, Maja
AU  - Vasović, Tamara
AU  - Stojadinović, Marija
AU  - Krstić-Ristivojević, Maja
AU  - Jovanović, Vesna
AU  - Simović, Ana
AU  - Radosavljević, Jelena
AU  - Aćimović, Jelena M.
AU  - Radomirović, Mirjana Ž.
AU  - Stojanović, Marijana
PY  - 2023
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/6014
AB  - Нови корона вирус (SARS CoV-2) који се појавио у Вухану 2019. године припада групи једноланчаних РНК вируса [1]. Представља нови инфективни агенс за хуману популацију и веома је брзо детектован у великом броју земаља. Узрочник је респираторних инфекција које могу да буду праћене и веома тешком клиничком сликом. Брзо ширење, одсуство имунитета на овај вирус и одсуство поузданих тестова за детекцију вируса у тренутку избијања пандемије су болест изазвану овим вирусом брзо претворили у здравствени и друштвени проблем највишег приоритета на глобалном нивоу. Иако су највеће биотехнолошке компаније убрзано почеле са развојем и масовном производњом дијагностичких тестова и вакцина, њихова доступност у тренуцима највеће потражње је и даље недовољна, а цене истих су лимитирајући фактор за бољу контролу болести и ширења пандемије [2]. Развој сопствених и одржива производња тестова и вакцина за COVID-19 су од великог друштвеног значаја. Важан предуслов за одрживу производњу тестова је доступност рекомбинантних антигена вируса и могућност производње истих на великој скали за потребе производње домаћих тестова. Овим техничким решењем се описује добијање два кључна антигена новог корона вируса рекомбинантном технологијом и њихова примена у серолошком ЕЛИСА тесту који производи Институт за примену нуклеарне енергије, ИНЕП, као и за добијање реагенаса за детекцију антигена новог корона вируса (специфичних антитела). У првој фази, оптимизоване су секвенце протеина које су подигле осетљивост постојећих серолошких тестова. Иновативност нашег приступа се огледа и у разрађеним експерименталним протоколима за добијање рекомбинантних протеина нуклеокапсида на великој скали, као и у солубилној форми, што олакшава поступак пречишћавања. Избор фрагмента нуклеокапсида који се хетеролого експримира у солубилној форми, а специфично детектује антитела и генерише јак имуни одговор током имунизације животиња (имуногеност) на основу прегледа познатих епитопских секвенци је кључна иновација овог техничког решења. Ово је први пример успешно примењеног рекомбинатног протеина произведеног у Србији у дијагностичком тесту који је регистрован
код Агенције за лекове и медицинска средства Републике Србије (број решења 515-02-02370-21-002), а који је примену нашао и на међународном нивоу.
T1  - Dobijanje rekombinantnog imunogenog fragmenta proteina nukleokapsida SARS-CoV-2 virusa za proizvodnju reagenasa i dijagnostičkih testova na novi korona virus
UR  - https://hdl.handle.net/21.15107/rcub_cherry_6014
ER  - 

@misc{
author = "Ćirković-Veličković, Tanja and Gnjatović, Marija and Ćujić, Danica and Todorović, Aleksandra and Stanić-Vučinić, Dragana and Đukić, Teodora and Mladenović, Maja and Vasović, Tamara and Stojadinović, Marija and Krstić-Ristivojević, Maja and Jovanović, Vesna and Simović, Ana and Radosavljević, Jelena and Aćimović, Jelena M. and Radomirović, Mirjana Ž. and Stojanović, Marijana",
year = "2023",
abstract = "Нови корона вирус (SARS CoV-2) који се појавио у Вухану 2019. године припада групи једноланчаних РНК вируса [1]. Представља нови инфективни агенс за хуману популацију и веома је брзо детектован у великом броју земаља. Узрочник је респираторних инфекција које могу да буду праћене и веома тешком клиничком сликом. Брзо ширење, одсуство имунитета на овај вирус и одсуство поузданих тестова за детекцију вируса у тренутку избијања пандемије су болест изазвану овим вирусом брзо претворили у здравствени и друштвени проблем највишег приоритета на глобалном нивоу. Иако су највеће биотехнолошке компаније убрзано почеле са развојем и масовном производњом дијагностичких тестова и вакцина, њихова доступност у тренуцима највеће потражње је и даље недовољна, а цене истих су лимитирајући фактор за бољу контролу болести и ширења пандемије [2]. Развој сопствених и одржива производња тестова и вакцина за COVID-19 су од великог друштвеног значаја. Важан предуслов за одрживу производњу тестова је доступност рекомбинантних антигена вируса и могућност производње истих на великој скали за потребе производње домаћих тестова. Овим техничким решењем се описује добијање два кључна антигена новог корона вируса рекомбинантном технологијом и њихова примена у серолошком ЕЛИСА тесту који производи Институт за примену нуклеарне енергије, ИНЕП, као и за добијање реагенаса за детекцију антигена новог корона вируса (специфичних антитела). У првој фази, оптимизоване су секвенце протеина које су подигле осетљивост постојећих серолошких тестова. Иновативност нашег приступа се огледа и у разрађеним експерименталним протоколима за добијање рекомбинантних протеина нуклеокапсида на великој скали, као и у солубилној форми, што олакшава поступак пречишћавања. Избор фрагмента нуклеокапсида који се хетеролого експримира у солубилној форми, а специфично детектује антитела и генерише јак имуни одговор током имунизације животиња (имуногеност) на основу прегледа познатих епитопских секвенци је кључна иновација овог техничког решења. Ово је први пример успешно примењеног рекомбинатног протеина произведеног у Србији у дијагностичком тесту који је регистрован
код Агенције за лекове и медицинска средства Републике Србије (број решења 515-02-02370-21-002), а који је примену нашао и на међународном нивоу.",
title = "Dobijanje rekombinantnog imunogenog fragmenta proteina nukleokapsida SARS-CoV-2 virusa za proizvodnju reagenasa i dijagnostičkih testova na novi korona virus",
url = "https://hdl.handle.net/21.15107/rcub_cherry_6014"
}

Ćirković-Veličković, T., Gnjatović, M., Ćujić, D., Todorović, A., Stanić-Vučinić, D., Đukić, T., Mladenović, M., Vasović, T., Stojadinović, M., Krstić-Ristivojević, M., Jovanović, V., Simović, A., Radosavljević, J., Aćimović, J. M., Radomirović, M. Ž.,& Stojanović, M.. (2023). Dobijanje rekombinantnog imunogenog fragmenta proteina nukleokapsida SARS-CoV-2 virusa za proizvodnju reagenasa i dijagnostičkih testova na novi korona virus. .
https://hdl.handle.net/21.15107/rcub_cherry_6014

Ćirković-Veličković T, Gnjatović M, Ćujić D, Todorović A, Stanić-Vučinić D, Đukić T, Mladenović M, Vasović T, Stojadinović M, Krstić-Ristivojević M, Jovanović V, Simović A, Radosavljević J, Aćimović JM, Radomirović MŽ, Stojanović M. Dobijanje rekombinantnog imunogenog fragmenta proteina nukleokapsida SARS-CoV-2 virusa za proizvodnju reagenasa i dijagnostičkih testova na novi korona virus. 2023;.
https://hdl.handle.net/21.15107/rcub_cherry_6014 .

Ćirković-Veličković, Tanja, Gnjatović, Marija, Ćujić, Danica, Todorović, Aleksandra, Stanić-Vučinić, Dragana, Đukić, Teodora, Mladenović, Maja, Vasović, Tamara, Stojadinović, Marija, Krstić-Ristivojević, Maja, Jovanović, Vesna, Simović, Ana, Radosavljević, Jelena, Aćimović, Jelena M., Radomirović, Mirjana Ž., Stojanović, Marijana, "Dobijanje rekombinantnog imunogenog fragmenta proteina nukleokapsida SARS-CoV-2 virusa za proizvodnju reagenasa i dijagnostičkih testova na novi korona virus" (2023),
https://hdl.handle.net/21.15107/rcub_cherry_6014 .

TY  - CONF
AU  - Ćirković-Veličković, Tanja
PY  - 2022
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/5778
AB  - Нови корона вирус (SARS CoV-2) је нови инфективни агенс за хуману популацију и веома је брзо детектован у великом броју земаља. Брзо ширење, одсуство имунитета на овај вирус и одсуство поузданих тестова за детекцију вируса у тренутку избијања пандемије су болест изазвану овим вирусом брзо претворили у здравствени и друштвени проблем највишег приоритета на глобалном нивоу. Иако су највеће биотехнолошке компаније убрзано почеле са развојем и масовном производњом дијагностичких тестова и вакцина, њихова доступност у тренуцима највеће потражње је и даље недовољна, а цене истих су лимитирајући фактор за бољу контролу болести и ширења пандемије. Тестови имају и своја ограничења, посебно када су у питању антигенски тестови, тако да се и даље ради на унапређењу осетљивости истих. У фокусу наших истраживања је развој дијагностичких и серолошких тестова за COVID-19. Дијагностички тестови детектују делове вируса: генетског материјала вируса , односно протеинске компоненте вируса. Серолошки тестови који детектују присуство антитела на протеине вируса. Важна компенента ових тестова је рекомбинанто произведен протеин. Истраживачки тим Хемијског факултета је до сада произвео рекомбинантне протеине шиљка и нуклеокапсида за потребе развоја серолошког теста и N протеин високе чистоће на великој скали за потребе добијања специфичких антитела и развоја антигенског теста. Добијена високо специфична поликлонска антитела на N протеин вируса из имунизованих животиња, биће употребљена за дизајн основног сендвич ЕЛИСА теста за детекцију SARS CoV-2 вируса на бази препознавања N-протеина вируса.  Oчекује се даље унапређење осетљивости теста применом различитих техника, као што су имобилисана специфична антитела на принципу имуноафинитетног ЕЛИСА теста што ће омогућити хватање вирусних антигена у хуманим телесним течностима попут крви, урина и пљувачке, и њихово даље концентрисање, што са једне стране повећава могућност за тачан резултат а са друге стране шири репертоар врсте узорака.
PB  - Београд : Српска академија наука и уметности
C3  - Симпозијум COVID-19 пандемије: поруке, нова сазнања и дилеме, САНУ, 4. јун 2021. године
T1  - Производња рекомбинантних антигена и развој одрживих тестова за Covid-19
UR  - https://hdl.handle.net/21.15107/rcub_cherry_5778
ER  - 

@conference{
author = "Ćirković-Veličković, Tanja",
year = "2022",
abstract = "Нови корона вирус (SARS CoV-2) је нови инфективни агенс за хуману популацију и веома је брзо детектован у великом броју земаља. Брзо ширење, одсуство имунитета на овај вирус и одсуство поузданих тестова за детекцију вируса у тренутку избијања пандемије су болест изазвану овим вирусом брзо претворили у здравствени и друштвени проблем највишег приоритета на глобалном нивоу. Иако су највеће биотехнолошке компаније убрзано почеле са развојем и масовном производњом дијагностичких тестова и вакцина, њихова доступност у тренуцима највеће потражње је и даље недовољна, а цене истих су лимитирајући фактор за бољу контролу болести и ширења пандемије. Тестови имају и своја ограничења, посебно када су у питању антигенски тестови, тако да се и даље ради на унапређењу осетљивости истих. У фокусу наших истраживања је развој дијагностичких и серолошких тестова за COVID-19. Дијагностички тестови детектују делове вируса: генетског материјала вируса , односно протеинске компоненте вируса. Серолошки тестови који детектују присуство антитела на протеине вируса. Важна компенента ових тестова је рекомбинанто произведен протеин. Истраживачки тим Хемијског факултета је до сада произвео рекомбинантне протеине шиљка и нуклеокапсида за потребе развоја серолошког теста и N протеин високе чистоће на великој скали за потребе добијања специфичких антитела и развоја антигенског теста. Добијена високо специфична поликлонска антитела на N протеин вируса из имунизованих животиња, биће употребљена за дизајн основног сендвич ЕЛИСА теста за детекцију SARS CoV-2 вируса на бази препознавања N-протеина вируса.  Oчекује се даље унапређење осетљивости теста применом различитих техника, као што су имобилисана специфична антитела на принципу имуноафинитетног ЕЛИСА теста што ће омогућити хватање вирусних антигена у хуманим телесним течностима попут крви, урина и пљувачке, и њихово даље концентрисање, што са једне стране повећава могућност за тачан резултат а са друге стране шири репертоар врсте узорака.",
publisher = "Београд : Српска академија наука и уметности",
journal = "Симпозијум COVID-19 пандемије: поруке, нова сазнања и дилеме, САНУ, 4. јун 2021. године",
title = "Производња рекомбинантних антигена и развој одрживих тестова за Covid-19",
url = "https://hdl.handle.net/21.15107/rcub_cherry_5778"
}

Ćirković-Veličković, T.. (2022). Производња рекомбинантних антигена и развој одрживих тестова за Covid-19. in Симпозијум COVID-19 пандемије: поруке, нова сазнања и дилеме, САНУ, 4. јун 2021. године
Београд : Српска академија наука и уметности..
https://hdl.handle.net/21.15107/rcub_cherry_5778

Ćirković-Veličković T. Производња рекомбинантних антигена и развој одрживих тестова за Covid-19. in Симпозијум COVID-19 пандемије: поруке, нова сазнања и дилеме, САНУ, 4. јун 2021. године. 2022;.
https://hdl.handle.net/21.15107/rcub_cherry_5778 .

Ćirković-Veličković, Tanja, "Производња рекомбинантних антигена и развој одрживих тестова за Covid-19" in Симпозијум COVID-19 пандемије: поруке, нова сазнања и дилеме, САНУ, 4. јун 2021. године (2022),
https://hdl.handle.net/21.15107/rcub_cherry_5778 .

TY  - CONF
AU  - Ćirković-Veličković, Tanja
AU  - Radomirović, Mirjana Ž.
AU  - Simović, Ana
AU  - Jovanović, Vesna B.
AU  - Ćujić, Danica R.
AU  - Gnjatović, Marija Lj.
AU  - Stojanović, Marijana
PY  - 2022
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/5777
AB  - RT-PCR remains the primary method of diagnosing SARS-CoV-2. Serological diagnosis of COVID-19 is simple and does not require complex techniques and equipment, rendering it suitable for rapid detection and massive screening. However, serological tests cannot confirm SARS-CoV-2, and results will be false negative when antibody concentrations fall below detection limit. Serological testing is important method for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) infection. Antigen-based tests show high specificity, but sensitivity is relatively low. For development and further improvement of both serological and antigen-based tests, availability of recombinantly producted SARS CoV-2 antigens is needed and during the periods of high demend in the pandemic, it was a limited factor both for research and diagnostic tests production by national producers of tests. Nucleocapsid (N) protein is the most abundant virus derived protein and strong immunogen. We aimed to find its efficient, low-cost production. SARS-CoV-2 recombinant fragment of nucleocapsid protein (rfNP; 58-419 aa) was expressed in E. coli in soluble form, purified 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 beta-sheet (40.7%). rfNP was tested in immunoblot using sera of COVID-19 convalescent patients. Cost-effective approach for soluble recombinant N protein fragment production was developed, with reliable IgG and IgM antibodies detection of SARS-CoV-2 infection. 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). Application of serological ELISA for seropositivity detection in 200 allergic children in comparison to non-allergic did not show a bias towards any of the tested groups.  Furthermore, we have optimized production of rfNP at a large scale to raise specific antibodies to N-protein in rabbit and mice and develop an in house ELISA for quantification of N protein in biological fluids. Test was clinically validated in 200 PCR positive patients and showed a sensitivity of around 50%.
C3  - International Congress on Molecular Immunology and Allergology (IMAC-2022), Moscow, December 1-3, 2022
T1  - SARS CoV-2 nucleocapsid-based diagnostic tests and serological response in allergic children
UR  - https://hdl.handle.net/21.15107/rcub_cherry_5777
ER  - 

@conference{
author = "Ćirković-Veličković, Tanja and Radomirović, Mirjana Ž. and Simović, Ana and Jovanović, Vesna B. and Ćujić, Danica R. and Gnjatović, Marija Lj. and Stojanović, Marijana",
year = "2022",
abstract = "RT-PCR remains the primary method of diagnosing SARS-CoV-2. Serological diagnosis of COVID-19 is simple and does not require complex techniques and equipment, rendering it suitable for rapid detection and massive screening. However, serological tests cannot confirm SARS-CoV-2, and results will be false negative when antibody concentrations fall below detection limit. Serological testing is important method for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) infection. Antigen-based tests show high specificity, but sensitivity is relatively low. For development and further improvement of both serological and antigen-based tests, availability of recombinantly producted SARS CoV-2 antigens is needed and during the periods of high demend in the pandemic, it was a limited factor both for research and diagnostic tests production by national producers of tests. Nucleocapsid (N) protein is the most abundant virus derived protein and strong immunogen. We aimed to find its efficient, low-cost production. SARS-CoV-2 recombinant fragment of nucleocapsid protein (rfNP; 58-419 aa) was expressed in E. coli in soluble form, purified 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 beta-sheet (40.7%). rfNP was tested in immunoblot using sera of COVID-19 convalescent patients. Cost-effective approach for soluble recombinant N protein fragment production was developed, with reliable IgG and IgM antibodies detection of SARS-CoV-2 infection. 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). Application of serological ELISA for seropositivity detection in 200 allergic children in comparison to non-allergic did not show a bias towards any of the tested groups.  Furthermore, we have optimized production of rfNP at a large scale to raise specific antibodies to N-protein in rabbit and mice and develop an in house ELISA for quantification of N protein in biological fluids. Test was clinically validated in 200 PCR positive patients and showed a sensitivity of around 50%.",
journal = "International Congress on Molecular Immunology and Allergology (IMAC-2022), Moscow, December 1-3, 2022",
title = "SARS CoV-2 nucleocapsid-based diagnostic tests and serological response in allergic children",
url = "https://hdl.handle.net/21.15107/rcub_cherry_5777"
}

Ćirković-Veličković, T., Radomirović, M. Ž., Simović, A., Jovanović, V. B., Ćujić, D. R., Gnjatović, M. Lj.,& Stojanović, M.. (2022). SARS CoV-2 nucleocapsid-based diagnostic tests and serological response in allergic children. in International Congress on Molecular Immunology and Allergology (IMAC-2022), Moscow, December 1-3, 2022.
https://hdl.handle.net/21.15107/rcub_cherry_5777

Ćirković-Veličković T, Radomirović MŽ, Simović A, Jovanović VB, Ćujić DR, Gnjatović ML, Stojanović M. SARS CoV-2 nucleocapsid-based diagnostic tests and serological response in allergic children. in International Congress on Molecular Immunology and Allergology (IMAC-2022), Moscow, December 1-3, 2022. 2022;.
https://hdl.handle.net/21.15107/rcub_cherry_5777 .

Ćirković-Veličković, Tanja, Radomirović, Mirjana Ž., Simović, Ana, Jovanović, Vesna B., Ćujić, Danica R., Gnjatović, Marija Lj., Stojanović, Marijana, "SARS CoV-2 nucleocapsid-based diagnostic tests and serological response in allergic children" in International Congress on Molecular Immunology and Allergology (IMAC-2022), Moscow, December 1-3, 2022 (2022),
https://hdl.handle.net/21.15107/rcub_cherry_5777 .

TY  - CONF
AU  - Radomirović, Mirjana Ž.
AU  - Simović, Ana
AU  - Udovički, Božidar D.
AU  - Krstić-Ristivojević, Maja
AU  - Sabljić, Ljiljana Z.
AU  - Lukić, Ivana D.
AU  - Glamočlija, Sofija Đ.
AU  - Ćujić, Danica R.
AU  - Gnjatović, Marija Lj.
AU  - Stojanović, Marijana M.
AU  - Stanić-Vučinić, Dragana
AU  - Radosavljević, Jelena
AU  - Ćirković-Veličković, Tanja
PY  - 2022
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/5361
AB  - Ta na dijagnoza ljudi sa sumnjom na infekciju SARS-CoV-2 je od suštinskog zna aja za
suzbijanje globalnog širenja COVID-19. Prisustvo SARS-CoV-2 može se otkriti RT-PCRom (otkriva RNK virusa) ili detekcijom prisustva virusnih antigena u biološkim te nostima
ELISA-om ili sli nom tehnikom koje koriste antitela razvijena u životinjama. Cilj studije
je bio uspostavljanje kvantitativnog testa koji se zasniva na koriš enju poliklonskih seruma
za rutinsko odre ivanje koncentracije SARS-CoV-2 nukleokapsidnog proteina merenjam
apsorbancije u standardnoj mikrotitarskoj plo ici sa 96 bunara. Za potrebe razvoja testa
proizveden je rekombinantni N-protein i koriš en za proizvodnju antiseruma u miševima i
ze evima. Proizvedeni antiserumi su pre iš eni i odre en im je titar. Poliklonskiantiserumi
visokog afiniteta specifi ni za N-protein koriš eni su za razvoj ELISA testa specifi nog za
ovaj protein. Test se zasniva na koriš enju poliklonskih seruma miševa koji su adherirani
na dno bunara mikrotitarske plo ice za hvatanje N-proteina iz uzorka. Razli ite
koncentracije rekombinantnog N-proteina su koriš ene za standardnu krivu za
kvantifikaciju proteina. N-protein vezan za antitela miševa je detektovan ze jim
poliklonskim serumom i anti-ze jim antitelom povezanim sa enzimom koji obezbe uje
spektrofotometrijsko merenje. Uspešno smo razvili prototip ELISA testa za kvantifikaciju
N-proteina sa granicom detekcije u opsegu od ng/mL. Prose na vrednost LOD za prototip
ELISA testa za detekciju N-proteina je 9,2 ng/mL, dok je prose na vrednost LOQ
10,2 ng/mL. Pokazali smo da su proizvedeni poliklonski antiserumi pogodni za detekciju
N-proteina sa sli nim ili boljim afinitetom i specifi noš u od komercijalnih antitela.
Štaviše, prototip ELISA testa se može koristiti sa zadovoljavaju om pouzdanoš u za
kvantifikaciju N-proteina u uzorcima bogatim proteinima, poput ljudskih seruma.
AB  - The accurate diagnosis of people with suspected infection with the SARS-CoV-2 is
essential to curb the global spread of COVID-19. The presence of SARS-CoV-2 can be
detected by RT-PCR (it detects RNA of the virus) or by the presence of viral antigens in
biological fluids in ELISA or similar techniques using antibodies developed in animals.
The aim of the study was the establishment of a quantitative polyclonal sera-based test for
routine measurement of the concentration of SARS CoV-2 nucleocapsid protein using
absorbance measurement in a standard 96-well microtiter plate. For the purposes of the test
development, recombinant N protein was produced and used for the production of mice
and rabbit antisera. Produced antisera were purified and titer was determined. High-affinity
polyclonal N-protein specific antisera were used for N-protein specific ELISA test
development. The test is based on mice polyclonal sera adhered to microtiter plate bottom
for the capture of the N protein from the specimen. Various concentrations of the
recombinant N-protein were used to generate a standard curve for protein quantification.
The N-protein bound to the mice antibodies was detected with rabbit polyclonal sera and
anti-rabbit antibody coupled to an enzyme that provides spectrophotometric measurement.
We have successfully developed the prototype ELISA for the quantification of N-protein
with the detection limit being in the range of ng/mL. The average LOD value for the
prototype ELISA was determined to be 9.2 ng/mL, while the average LOQ value was
10.2 ng/mL. We have demonstrated that produced polyclonal antisera are suitable for the
detection of N-protein with affinity and specificity similar to, or better than commercial
antibodies. Furthermore, the prototype ELISA can be used with satisfactory confidence for
quantification of the N-protein in protein-rich samples, similar to human sera.
PB  - Belgrade : Serbian Chemical Society
C3  - 58th Meeting of the Serbian Chemical Society, Belgrade, Serbia, 9th-10th June, 2022. In: Book of Abstracts and Proceedings
T1  - Razvoj sendvič ELISA testa specifičnog za SARS-CoV-2 N-protein
T1  - Development of SARS-CoV-2 N-protein specific capture ELISA
SP  - 65
EP  - 66
UR  - https://hdl.handle.net/21.15107/rcub_cherry_5361
ER  - 

@conference{
author = "Radomirović, Mirjana Ž. and Simović, Ana and Udovički, Božidar D. and Krstić-Ristivojević, Maja and Sabljić, Ljiljana Z. and Lukić, Ivana D. and Glamočlija, Sofija Đ. and Ćujić, Danica R. and Gnjatović, Marija Lj. and Stojanović, Marijana M. and Stanić-Vučinić, Dragana and Radosavljević, Jelena and Ćirković-Veličković, Tanja",
year = "2022",
abstract = "Ta na dijagnoza ljudi sa sumnjom na infekciju SARS-CoV-2 je od suštinskog zna aja za
suzbijanje globalnog širenja COVID-19. Prisustvo SARS-CoV-2 može se otkriti RT-PCRom (otkriva RNK virusa) ili detekcijom prisustva virusnih antigena u biološkim te nostima
ELISA-om ili sli nom tehnikom koje koriste antitela razvijena u životinjama. Cilj studije
je bio uspostavljanje kvantitativnog testa koji se zasniva na koriš enju poliklonskih seruma
za rutinsko odre ivanje koncentracije SARS-CoV-2 nukleokapsidnog proteina merenjam
apsorbancije u standardnoj mikrotitarskoj plo ici sa 96 bunara. Za potrebe razvoja testa
proizveden je rekombinantni N-protein i koriš en za proizvodnju antiseruma u miševima i
ze evima. Proizvedeni antiserumi su pre iš eni i odre en im je titar. Poliklonskiantiserumi
visokog afiniteta specifi ni za N-protein koriš eni su za razvoj ELISA testa specifi nog za
ovaj protein. Test se zasniva na koriš enju poliklonskih seruma miševa koji su adherirani
na dno bunara mikrotitarske plo ice za hvatanje N-proteina iz uzorka. Razli ite
koncentracije rekombinantnog N-proteina su koriš ene za standardnu krivu za
kvantifikaciju proteina. N-protein vezan za antitela miševa je detektovan ze jim
poliklonskim serumom i anti-ze jim antitelom povezanim sa enzimom koji obezbe uje
spektrofotometrijsko merenje. Uspešno smo razvili prototip ELISA testa za kvantifikaciju
N-proteina sa granicom detekcije u opsegu od ng/mL. Prose na vrednost LOD za prototip
ELISA testa za detekciju N-proteina je 9,2 ng/mL, dok je prose na vrednost LOQ
10,2 ng/mL. Pokazali smo da su proizvedeni poliklonski antiserumi pogodni za detekciju
N-proteina sa sli nim ili boljim afinitetom i specifi noš u od komercijalnih antitela.
Štaviše, prototip ELISA testa se može koristiti sa zadovoljavaju om pouzdanoš u za
kvantifikaciju N-proteina u uzorcima bogatim proteinima, poput ljudskih seruma., The accurate diagnosis of people with suspected infection with the SARS-CoV-2 is
essential to curb the global spread of COVID-19. The presence of SARS-CoV-2 can be
detected by RT-PCR (it detects RNA of the virus) or by the presence of viral antigens in
biological fluids in ELISA or similar techniques using antibodies developed in animals.
The aim of the study was the establishment of a quantitative polyclonal sera-based test for
routine measurement of the concentration of SARS CoV-2 nucleocapsid protein using
absorbance measurement in a standard 96-well microtiter plate. For the purposes of the test
development, recombinant N protein was produced and used for the production of mice
and rabbit antisera. Produced antisera were purified and titer was determined. High-affinity
polyclonal N-protein specific antisera were used for N-protein specific ELISA test
development. The test is based on mice polyclonal sera adhered to microtiter plate bottom
for the capture of the N protein from the specimen. Various concentrations of the
recombinant N-protein were used to generate a standard curve for protein quantification.
The N-protein bound to the mice antibodies was detected with rabbit polyclonal sera and
anti-rabbit antibody coupled to an enzyme that provides spectrophotometric measurement.
We have successfully developed the prototype ELISA for the quantification of N-protein
with the detection limit being in the range of ng/mL. The average LOD value for the
prototype ELISA was determined to be 9.2 ng/mL, while the average LOQ value was
10.2 ng/mL. We have demonstrated that produced polyclonal antisera are suitable for the
detection of N-protein with affinity and specificity similar to, or better than commercial
antibodies. Furthermore, the prototype ELISA can be used with satisfactory confidence for
quantification of the N-protein in protein-rich samples, similar to human sera.",
publisher = "Belgrade : Serbian Chemical Society",
journal = "58th Meeting of the Serbian Chemical Society, Belgrade, Serbia, 9th-10th June, 2022. In: Book of Abstracts and Proceedings",
title = "Razvoj sendvič ELISA testa specifičnog za SARS-CoV-2 N-protein, Development of SARS-CoV-2 N-protein specific capture ELISA",
pages = "65-66",
url = "https://hdl.handle.net/21.15107/rcub_cherry_5361"
}

Radomirović, M. Ž., Simović, A., Udovički, B. D., Krstić-Ristivojević, M., Sabljić, L. Z., Lukić, I. D., Glamočlija, S. Đ., Ćujić, D. R., Gnjatović, M. Lj., Stojanović, M. M., Stanić-Vučinić, D., Radosavljević, J.,& Ćirković-Veličković, T.. (2022). Razvoj sendvič ELISA testa specifičnog za SARS-CoV-2 N-protein. in 58th Meeting of the Serbian Chemical Society, Belgrade, Serbia, 9th-10th June, 2022. In: Book of Abstracts and Proceedings
Belgrade : Serbian Chemical Society., 65-66.
https://hdl.handle.net/21.15107/rcub_cherry_5361

Radomirović MŽ, Simović A, Udovički BD, Krstić-Ristivojević M, Sabljić LZ, Lukić ID, Glamočlija SĐ, Ćujić DR, Gnjatović ML, Stojanović MM, Stanić-Vučinić D, Radosavljević J, Ćirković-Veličković T. Razvoj sendvič ELISA testa specifičnog za SARS-CoV-2 N-protein. in 58th Meeting of the Serbian Chemical Society, Belgrade, Serbia, 9th-10th June, 2022. In: Book of Abstracts and Proceedings. 2022;:65-66.
https://hdl.handle.net/21.15107/rcub_cherry_5361 .

Radomirović, Mirjana Ž., Simović, Ana, Udovički, Božidar D., Krstić-Ristivojević, Maja, Sabljić, Ljiljana Z., Lukić, Ivana D., Glamočlija, Sofija Đ., Ćujić, Danica R., Gnjatović, Marija Lj., Stojanović, Marijana M., Stanić-Vučinić, Dragana, Radosavljević, Jelena, Ćirković-Veličković, Tanja, "Razvoj sendvič ELISA testa specifičnog za SARS-CoV-2 N-protein" in 58th Meeting of the Serbian Chemical Society, Belgrade, Serbia, 9th-10th June, 2022. In: Book of Abstracts and Proceedings (2022):65-66,
https://hdl.handle.net/21.15107/rcub_cherry_5361 .

TY  - CONF
AU  - Radomirović, Mirjana Ž.
AU  - Simović, Ana
AU  - Udovički, Božidar D.
AU  - Krstić-Ristivojević, Maja
AU  - Sabljić, Ljiljana Z.
AU  - Lukić, Ivana D.
AU  - Glamočlija, Sofija Đ.
AU  - Ćujić, Danica R.
AU  - Gnjatović, Marija Lj.
AU  - Stojanović, Marijana M.
AU  - Stanić-Vučinić, Dragana
AU  - Radosavljević, Jelena
AU  - Ćirković-Veličković, Tanja
PY  - 2022
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/5362
AB  - Ta na dijagnoza ljudi sa sumnjom na infekciju SARS-CoV-2 je od suštinskog zna aja zasuzbijanje globalnog širenja COVID-19. Prisustvo SARS-CoV-2 može se otkriti RT-PCRom (otkriva RNK virusa) ili detekcijom prisustva virusnih antigena u biološkim te nostimaELISA-om ili sli nom tehnikom koje koriste antitela razvijena u životinjama. Cilj studijeje bio uspostavljanje kvantitativnog testa koji se zasniva na koriš enju poliklonskih serumaza rutinsko odre ivanje koncentracije SARS-CoV-2 nukleokapsidnog proteina merenjamapsorbancije u standardnoj mikrotitarskoj plo ici sa 96 bunara. Za potrebe razvoja testaproizveden je rekombinantni N-protein i koriš en za proizvodnju antiseruma u miševima ize evima. Proizvedeni antiserumi su pre iš eni i odre en im je titar. Poliklonskiantiserumivisokog afiniteta specifi ni za N-protein koriš eni su za razvoj ELISA testa specifi nog zaovaj protein. Test se zasniva na koriš enju poliklonskih seruma miševa koji su adheriranina dno bunara mikrotitarske plo ice za hvatanje N-proteina iz uzorka. Razli itekoncentracije rekombinantnog N-proteina su koriš ene za standardnu krivu zakvantifikaciju proteina. N-protein vezan za antitela miševa je detektovan ze jimpoliklonskim serumom i anti-ze jim antitelom povezanim sa enzimom koji obezbe ujespektrofotometrijsko merenje. Uspešno smo razvili prototip ELISA testa za kvantifikacijuN-proteina sa granicom detekcije u opsegu od ng/mL. Prose na vrednost LOD za prototipELISA testa za detekciju N-proteina je 9,2 ng/mL, dok je prose na vrednost LOQ10,2 ng/mL. Pokazali smo da su proizvedeni poliklonski antiserumi pogodni za detekcijuN-proteina sa sli nim ili boljim afinitetom i specifi noš u od komercijalnih antitela.Štaviše, prototip ELISA testa se može koristiti sa zadovoljavaju om pouzdanoš u zakvantifikaciju N-proteina u uzorcima bogatim proteinima, poput ljudskih seruma.
AB  - The accurate diagnosis of people with suspected infection with the SARS-CoV-2 isessential to curb the global spread of COVID-19. The presence of SARS-CoV-2 can bedetected by RT-PCR (it detects RNA of the virus) or by the presence of viral antigens inbiological fluids in ELISA or similar techniques using antibodies developed in animals.The aim of the study was the establishment of a quantitative polyclonal sera-based test forroutine measurement of the concentration of SARS CoV-2 nucleocapsid protein usingabsorbance measurement in a standard 96-well microtiter plate. For the purposes of the testdevelopment, recombinant N protein was produced and used for the production of miceand rabbit antisera. Produced antisera were purified and titer was determined. High-affinitypolyclonal N-protein specific antisera were used for N-protein specific ELISA testdevelopment. The test is based on mice polyclonal sera adhered to microtiter plate bottomfor the capture of the N protein from the specimen. Various concentrations of therecombinant N-protein were used to generate a standard curve for protein quantification.The N-protein bound to the mice antibodies was detected with rabbit polyclonal sera andanti-rabbit antibody coupled to an enzyme that provides spectrophotometric measurement.We have successfully developed the prototype ELISA for the quantification of N-proteinwith the detection limit being in the range of ng/mL. The average LOD value for theprototype ELISA was determined to be 9.2 ng/mL, while the average LOQ value was10.2 ng/mL. We have demonstrated that produced polyclonal antisera are suitable for thedetection of N-protein with affinity and specificity similar to, or better than commercialantibodies. Furthermore, the prototype ELISA can be used with satisfactory confidence forquantification of the N-protein in protein-rich samples, similar to human sera.
PB  - Belgrade : Serbian Chemical Society
C3  - 58th Meeting of the Serbian Chemical Society, Belgrade, Serbia, 9th-10th June, 2022. In: Book of Abstracts and Proceedings
T1  - Razvoj sendvič ELISA testa specifičnog za SARS-CoV-2 N-protein
T1  - Development of SARS-CoV-2 N-protein specific capture ELISA
UR  - https://hdl.handle.net/21.15107/rcub_cherry_5362
ER  - 

@conference{
author = "Radomirović, Mirjana Ž. and Simović, Ana and Udovički, Božidar D. and Krstić-Ristivojević, Maja and Sabljić, Ljiljana Z. and Lukić, Ivana D. and Glamočlija, Sofija Đ. and Ćujić, Danica R. and Gnjatović, Marija Lj. and Stojanović, Marijana M. and Stanić-Vučinić, Dragana and Radosavljević, Jelena and Ćirković-Veličković, Tanja",
year = "2022",
abstract = "Ta na dijagnoza ljudi sa sumnjom na infekciju SARS-CoV-2 je od suštinskog zna aja zasuzbijanje globalnog širenja COVID-19. Prisustvo SARS-CoV-2 može se otkriti RT-PCRom (otkriva RNK virusa) ili detekcijom prisustva virusnih antigena u biološkim te nostimaELISA-om ili sli nom tehnikom koje koriste antitela razvijena u životinjama. Cilj studijeje bio uspostavljanje kvantitativnog testa koji se zasniva na koriš enju poliklonskih serumaza rutinsko odre ivanje koncentracije SARS-CoV-2 nukleokapsidnog proteina merenjamapsorbancije u standardnoj mikrotitarskoj plo ici sa 96 bunara. Za potrebe razvoja testaproizveden je rekombinantni N-protein i koriš en za proizvodnju antiseruma u miševima ize evima. Proizvedeni antiserumi su pre iš eni i odre en im je titar. Poliklonskiantiserumivisokog afiniteta specifi ni za N-protein koriš eni su za razvoj ELISA testa specifi nog zaovaj protein. Test se zasniva na koriš enju poliklonskih seruma miševa koji su adheriranina dno bunara mikrotitarske plo ice za hvatanje N-proteina iz uzorka. Razli itekoncentracije rekombinantnog N-proteina su koriš ene za standardnu krivu zakvantifikaciju proteina. N-protein vezan za antitela miševa je detektovan ze jimpoliklonskim serumom i anti-ze jim antitelom povezanim sa enzimom koji obezbe ujespektrofotometrijsko merenje. Uspešno smo razvili prototip ELISA testa za kvantifikacijuN-proteina sa granicom detekcije u opsegu od ng/mL. Prose na vrednost LOD za prototipELISA testa za detekciju N-proteina je 9,2 ng/mL, dok je prose na vrednost LOQ10,2 ng/mL. Pokazali smo da su proizvedeni poliklonski antiserumi pogodni za detekcijuN-proteina sa sli nim ili boljim afinitetom i specifi noš u od komercijalnih antitela.Štaviše, prototip ELISA testa se može koristiti sa zadovoljavaju om pouzdanoš u zakvantifikaciju N-proteina u uzorcima bogatim proteinima, poput ljudskih seruma., The accurate diagnosis of people with suspected infection with the SARS-CoV-2 isessential to curb the global spread of COVID-19. The presence of SARS-CoV-2 can bedetected by RT-PCR (it detects RNA of the virus) or by the presence of viral antigens inbiological fluids in ELISA or similar techniques using antibodies developed in animals.The aim of the study was the establishment of a quantitative polyclonal sera-based test forroutine measurement of the concentration of SARS CoV-2 nucleocapsid protein usingabsorbance measurement in a standard 96-well microtiter plate. For the purposes of the testdevelopment, recombinant N protein was produced and used for the production of miceand rabbit antisera. Produced antisera were purified and titer was determined. High-affinitypolyclonal N-protein specific antisera were used for N-protein specific ELISA testdevelopment. The test is based on mice polyclonal sera adhered to microtiter plate bottomfor the capture of the N protein from the specimen. Various concentrations of therecombinant N-protein were used to generate a standard curve for protein quantification.The N-protein bound to the mice antibodies was detected with rabbit polyclonal sera andanti-rabbit antibody coupled to an enzyme that provides spectrophotometric measurement.We have successfully developed the prototype ELISA for the quantification of N-proteinwith the detection limit being in the range of ng/mL. The average LOD value for theprototype ELISA was determined to be 9.2 ng/mL, while the average LOQ value was10.2 ng/mL. We have demonstrated that produced polyclonal antisera are suitable for thedetection of N-protein with affinity and specificity similar to, or better than commercialantibodies. Furthermore, the prototype ELISA can be used with satisfactory confidence forquantification of the N-protein in protein-rich samples, similar to human sera.",
publisher = "Belgrade : Serbian Chemical Society",
journal = "58th Meeting of the Serbian Chemical Society, Belgrade, Serbia, 9th-10th June, 2022. In: Book of Abstracts and Proceedings",
title = "Razvoj sendvič ELISA testa specifičnog za SARS-CoV-2 N-protein, Development of SARS-CoV-2 N-protein specific capture ELISA",
url = "https://hdl.handle.net/21.15107/rcub_cherry_5362"
}

Radomirović, M. Ž., Simović, A., Udovički, B. D., Krstić-Ristivojević, M., Sabljić, L. Z., Lukić, I. D., Glamočlija, S. Đ., Ćujić, D. R., Gnjatović, M. Lj., Stojanović, M. M., Stanić-Vučinić, D., Radosavljević, J.,& Ćirković-Veličković, T.. (2022). Razvoj sendvič ELISA testa specifičnog za SARS-CoV-2 N-protein. in 58th Meeting of the Serbian Chemical Society, Belgrade, Serbia, 9th-10th June, 2022. In: Book of Abstracts and Proceedings
Belgrade : Serbian Chemical Society..
https://hdl.handle.net/21.15107/rcub_cherry_5362

Radomirović MŽ, Simović A, Udovički BD, Krstić-Ristivojević M, Sabljić LZ, Lukić ID, Glamočlija SĐ, Ćujić DR, Gnjatović ML, Stojanović MM, Stanić-Vučinić D, Radosavljević J, Ćirković-Veličković T. Razvoj sendvič ELISA testa specifičnog za SARS-CoV-2 N-protein. in 58th Meeting of the Serbian Chemical Society, Belgrade, Serbia, 9th-10th June, 2022. In: Book of Abstracts and Proceedings. 2022;.
https://hdl.handle.net/21.15107/rcub_cherry_5362 .

Radomirović, Mirjana Ž., Simović, Ana, Udovički, Božidar D., Krstić-Ristivojević, Maja, Sabljić, Ljiljana Z., Lukić, Ivana D., Glamočlija, Sofija Đ., Ćujić, Danica R., Gnjatović, Marija Lj., Stojanović, Marijana M., Stanić-Vučinić, Dragana, Radosavljević, Jelena, Ćirković-Veličković, Tanja, "Razvoj sendvič ELISA testa specifičnog za SARS-CoV-2 N-protein" in 58th Meeting of the Serbian Chemical Society, Belgrade, Serbia, 9th-10th June, 2022. In: Book of Abstracts and Proceedings (2022),
https://hdl.handle.net/21.15107/rcub_cherry_5362 .

TY  - THES
AU  - Jošić, Ljiljana
PY  - 2022
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/5588
AB  - SARS-CoV-2 je virus koji je uzročnik pandemije kovid-19. Virus ima visoku stopu smrtnosti, pogotovo kod osetljivih grupa, i veoma je zarazan. Lako se širi te je detekcija virusa , a potom i izolacija obolelih ključan korak u borbi protiv istog i sprečavanju širenja pandemije. Detekcija virusa može da se radi indirektno, detekcijom proizvedenih antitela od strane imunog sistema čoveka ili direktnim putem, PCR ili antigenskim testovima koji identifikuju delove virusa. Iako su PCR testovi pouzdani, jer detektuju genetski materijal virusa, oni su skupi, zahtevaju vremena i u kriznim momentima ne predstavljaju najbolji izbor za identifikaciju obolelih. Antigenski testovi detektuju ključne proteine virusa, brzi su, pristupačni i laki za upotrebu. U ovom radu je razvijan novi antigenski test za detekciju SARS-CoV-2 virusa u biološkim fluidima pacijenata. Test na prisutnost SARS-CoV-2 virusa opisan u ovom radu se zasniva na specifičnom vezivanju N proteina, sastavnog dela virusa, pomoću streptavidinskih magnetnih kuglica, a potom njegova detekcija ELISA testom. U radu je prikazana stabilnost i uspešna reciklaža magnetnih kuglica, koji daju osnovu za potencijalnu ponovnu upotrebu kuglica za nekoliko uzoraka. Rezultati opisanih eksperimenata u ovom radu ukazuju na visok potencijal za razvijanja standardizovanog antigenskog testa za detekciju SARS-CoV-2 virusa u oralnim i nazalnim brisevima pacijenata. Predložena metoda može imati primenu i u medicinskim istraživanjima jer omogućava kvantitativno vezivanje i koncentrovanje N proteina iz razblaženih uzoraka poput urina i seruma pacijenata.
T1  - Razvoj Antigenskog Testa za Detekciju Nukleokapsidnog Proteina SARS-CoV-2
SP  - 1
EP  - 50
UR  - https://hdl.handle.net/21.15107/rcub_cherry_5588
ER  - 

@mastersthesis{
author = "Jošić, Ljiljana",
year = "2022",
abstract = "SARS-CoV-2 je virus koji je uzročnik pandemije kovid-19. Virus ima visoku stopu smrtnosti, pogotovo kod osetljivih grupa, i veoma je zarazan. Lako se širi te je detekcija virusa , a potom i izolacija obolelih ključan korak u borbi protiv istog i sprečavanju širenja pandemije. Detekcija virusa može da se radi indirektno, detekcijom proizvedenih antitela od strane imunog sistema čoveka ili direktnim putem, PCR ili antigenskim testovima koji identifikuju delove virusa. Iako su PCR testovi pouzdani, jer detektuju genetski materijal virusa, oni su skupi, zahtevaju vremena i u kriznim momentima ne predstavljaju najbolji izbor za identifikaciju obolelih. Antigenski testovi detektuju ključne proteine virusa, brzi su, pristupačni i laki za upotrebu. U ovom radu je razvijan novi antigenski test za detekciju SARS-CoV-2 virusa u biološkim fluidima pacijenata. Test na prisutnost SARS-CoV-2 virusa opisan u ovom radu se zasniva na specifičnom vezivanju N proteina, sastavnog dela virusa, pomoću streptavidinskih magnetnih kuglica, a potom njegova detekcija ELISA testom. U radu je prikazana stabilnost i uspešna reciklaža magnetnih kuglica, koji daju osnovu za potencijalnu ponovnu upotrebu kuglica za nekoliko uzoraka. Rezultati opisanih eksperimenata u ovom radu ukazuju na visok potencijal za razvijanja standardizovanog antigenskog testa za detekciju SARS-CoV-2 virusa u oralnim i nazalnim brisevima pacijenata. Predložena metoda može imati primenu i u medicinskim istraživanjima jer omogućava kvantitativno vezivanje i koncentrovanje N proteina iz razblaženih uzoraka poput urina i seruma pacijenata.",
title = "Razvoj Antigenskog Testa za Detekciju Nukleokapsidnog Proteina SARS-CoV-2",
pages = "1-50",
url = "https://hdl.handle.net/21.15107/rcub_cherry_5588"
}

Jošić, L.. (2022). Razvoj Antigenskog Testa za Detekciju Nukleokapsidnog Proteina SARS-CoV-2. , 1-50.
https://hdl.handle.net/21.15107/rcub_cherry_5588

Jošić L. Razvoj Antigenskog Testa za Detekciju Nukleokapsidnog Proteina SARS-CoV-2. 2022;:1-50.
https://hdl.handle.net/21.15107/rcub_cherry_5588 .

Jošić, Ljiljana, "Razvoj Antigenskog Testa za Detekciju Nukleokapsidnog Proteina SARS-CoV-2" (2022):1-50,
https://hdl.handle.net/21.15107/rcub_cherry_5588 .

TY  - JOUR
AU  - Đukić, Teodora
AU  - Mladenović, Maja
AU  - Stanić-Vučinić, Dragana
AU  - Radosavljević, Jelena
AU  - Smiljanić, Katarina
AU  - Sabljić, Ljiljana
AU  - Dević, Marija
AU  - Ćujić, Danica R.
AU  - Vasović, Tamara
AU  - Simović, Ana
AU  - Radomirović, Mirjana Ž.
AU  - Ćirković-Veličković, Tanja
PY  - 2021
UR  - https://cherry.chem.bg.ac.rs/handle/123456789/4330
AB  - Serological testing is important method for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Nucleocapsid (N) protein is the most abundant virus derived protein and strong immunogen. We aimed to find its efficient, low-cost production.

SARS-CoV-2 recombinant fragment of nucleocapsid protein (rfNP; 58–419 aa) was expressed in E. coli in soluble form, purified 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.
PB  - Elsevier
T2  - Virology journal
T1  - Expression, purification and immunological characterization of recombinant nucleocapsid protein fragment from SARS-CoV-2
VL  - 557
SP  - 15
EP  - 22
DO  - 10.1016/j.virol.2021.01.004
ER  - 

@article{
author = "Đukić, Teodora and Mladenović, Maja and Stanić-Vučinić, Dragana and Radosavljević, Jelena and Smiljanić, Katarina and Sabljić, Ljiljana and Dević, Marija and Ćujić, Danica R. 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 (SARS-CoV-2) infection. Nucleocapsid (N) protein is the most abundant virus derived protein and strong immunogen. We aimed to find its efficient, low-cost production.

SARS-CoV-2 recombinant fragment of nucleocapsid protein (rfNP; 58–419 aa) was expressed in E. coli in soluble form, purified 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.",
publisher = "Elsevier",
journal = "Virology journal",
title = "Expression, purification and immunological characterization of recombinant nucleocapsid protein fragment from SARS-CoV-2",
volume = "557",
pages = "15-22",
doi = "10.1016/j.virol.2021.01.004"
}

Đukić, T., Mladenović, M., Stanić-Vučinić, D., Radosavljević, J., Smiljanić, K., Sabljić, L., Dević, M., Ćujić, D. R., Vasović, T., Simović, A., Radomirović, M. Ž.,& Ćirković-Veličković, T.. (2021). Expression, purification and immunological characterization of recombinant nucleocapsid protein fragment from SARS-CoV-2. in Virology journal
Elsevier., 557, 15-22.
https://doi.org/10.1016/j.virol.2021.01.004

Đukić T, Mladenović M, Stanić-Vučinić D, Radosavljević J, Smiljanić K, Sabljić L, Dević M, Ćujić DR, Vasović T, Simović A, Radomirović MŽ, Ćirković-Veličković T. Expression, purification and immunological characterization of recombinant nucleocapsid protein fragment from SARS-CoV-2. in Virology journal. 2021;557:15-22.
doi:10.1016/j.virol.2021.01.004 .

Đukić, Teodora, Mladenović, Maja, Stanić-Vučinić, Dragana, Radosavljević, Jelena, Smiljanić, Katarina, Sabljić, Ljiljana, Dević, Marija, Ćujić, Danica R., Vasović, Tamara, Simović, Ana, Radomirović, Mirjana Ž., Ćirković-Veličković, Tanja, "Expression, purification and immunological characterization of recombinant nucleocapsid protein fragment from SARS-CoV-2" in Virology journal, 557 (2021):15-22,
https://doi.org/10.1016/j.virol.2021.01.004 . .

TY  - JOUR
AU  - Đukić, Teodora
AU  - Mladenović, Maja
AU  - Stanić-Vučinić, Dragana
AU  - Radosavljević, Jelena
AU  - Smiljanić, Katarina
AU  - Sabljić, Ljiljana
AU  - Dević, Marija
AU  - Ćujić, Danica R.
AU  - Vasović, Tamara
AU  - Simović, Ana
AU  - Radomirović, Mirjana Ž.
AU  - Ćirković-Veličković, Tanja
PY  - 2021
UR  - https://cherry.chem.bg.ac.rs/handle/123456789/4331
AB  - Serological testing is important method for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Nucleocapsid (N) protein is the most abundant virus derived protein and strong immunogen. We aimed to find its efficient, low-cost production.SARS-CoV-2 recombinant fragment of nucleocapsid protein (rfNP; 58–419 aa) was expressed in E. coli in soluble form, purified 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.
PB  - Elsevier
T2  - Virology journal
T1  - Expression, purification and immunological characterization of recombinant nucleocapsid protein fragment from SARS-CoV-2
VL  - 557
SP  - 15
EP  - 22
DO  - 10.1016/j.virol.2021.01.004
ER  - 

@article{
author = "Đukić, Teodora and Mladenović, Maja and Stanić-Vučinić, Dragana and Radosavljević, Jelena and Smiljanić, Katarina and Sabljić, Ljiljana and Dević, Marija and Ćujić, Danica R. 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 (SARS-CoV-2) infection. Nucleocapsid (N) protein is the most abundant virus derived protein and strong immunogen. We aimed to find its efficient, low-cost production.SARS-CoV-2 recombinant fragment of nucleocapsid protein (rfNP; 58–419 aa) was expressed in E. coli in soluble form, purified 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.",
publisher = "Elsevier",
journal = "Virology journal",
title = "Expression, purification and immunological characterization of recombinant nucleocapsid protein fragment from SARS-CoV-2",
volume = "557",
pages = "15-22",
doi = "10.1016/j.virol.2021.01.004"
}

Đukić, T., Mladenović, M., Stanić-Vučinić, D., Radosavljević, J., Smiljanić, K., Sabljić, L., Dević, M., Ćujić, D. R., Vasović, T., Simović, A., Radomirović, M. Ž.,& Ćirković-Veličković, T.. (2021). Expression, purification and immunological characterization of recombinant nucleocapsid protein fragment from SARS-CoV-2. in Virology journal
Elsevier., 557, 15-22.
https://doi.org/10.1016/j.virol.2021.01.004

Đukić T, Mladenović M, Stanić-Vučinić D, Radosavljević J, Smiljanić K, Sabljić L, Dević M, Ćujić DR, Vasović T, Simović A, Radomirović MŽ, Ćirković-Veličković T. Expression, purification and immunological characterization of recombinant nucleocapsid protein fragment from SARS-CoV-2. in Virology journal. 2021;557:15-22.
doi:10.1016/j.virol.2021.01.004 .

Đukić, Teodora, Mladenović, Maja, Stanić-Vučinić, Dragana, Radosavljević, Jelena, Smiljanić, Katarina, Sabljić, Ljiljana, Dević, Marija, Ćujić, Danica R., Vasović, Tamara, Simović, Ana, Radomirović, Mirjana Ž., Ćirković-Veličković, Tanja, "Expression, purification and immunological characterization of recombinant nucleocapsid protein fragment from SARS-CoV-2" in Virology journal, 557 (2021):15-22,
https://doi.org/10.1016/j.virol.2021.01.004 . .

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 .

TY  - CONF
AU  - Đukić, Teodora
AU  - Vasović, Tamara
AU  - Mladenović, Maja
AU  - Jovanović, Vesna B.
AU  - Smiljanić, Katarina
AU  - Radosavljević, Jelena
AU  - Stanić-Vučinić, Dragana
AU  - Ćirković-Veličković, Tanja
PY  - 2021
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/5774
AB  - Nucleocapsid (N) protein is the most abundant SARS-CoV-2 virus derived protein and strong immunogen which can be used as a component of the immunological tests for the diagnosis of SARS-CoV-2 infection. Recombinant fragment of N-protein (58–419 aa) was expressed in E. coli in a soluble form using developed optimized protocol of expression (16-18h, 37 °C, 0.4 mM IPTG). After lysis of cells, N-protein from soluble fraction of lysate was purified using optimized protocol for purification by immobilized metal affinity chromatography on Ni-Sepharose in two repeated steps under different elution conditions. Obtained fraction of N-protein after the second chromatography was desalted and concentrated using phosphate buffer solution and ultrafiltration. The purity of isolated N-protein was deterrmined by SDS PAGE, while high resolution mass spectrometry was used for its characterization. Isolated N-protein was over 90% purity and identified as the most intense and abundant protein fragment, with PEAKS PTM score of 508 and sequence coverage of over 70%, including 173 unique peptides.
PB  - Belgrade : Serbian Biochemical Society
C3  - Serbian Biochemical Society Tenth Conference
with international participation “Biochemical Insights into Molecular Mechanisms”, Kragujevac, Serbia, 24th September 2021
T1  - Optimization of expression, purification and HRMS characterization of recombinant N-protein fragment from SARS-CoV-2
SP  - 108
EP  - 108
UR  - https://hdl.handle.net/21.15107/rcub_cherry_5774
ER  - 

@conference{
author = "Đukić, Teodora and Vasović, Tamara and Mladenović, Maja and Jovanović, Vesna B. and Smiljanić, Katarina and Radosavljević, Jelena and Stanić-Vučinić, Dragana and Ćirković-Veličković, Tanja",
year = "2021",
abstract = "Nucleocapsid (N) protein is the most abundant SARS-CoV-2 virus derived protein and strong immunogen which can be used as a component of the immunological tests for the diagnosis of SARS-CoV-2 infection. Recombinant fragment of N-protein (58–419 aa) was expressed in E. coli in a soluble form using developed optimized protocol of expression (16-18h, 37 °C, 0.4 mM IPTG). After lysis of cells, N-protein from soluble fraction of lysate was purified using optimized protocol for purification by immobilized metal affinity chromatography on Ni-Sepharose in two repeated steps under different elution conditions. Obtained fraction of N-protein after the second chromatography was desalted and concentrated using phosphate buffer solution and ultrafiltration. The purity of isolated N-protein was deterrmined by SDS PAGE, while high resolution mass spectrometry was used for its characterization. Isolated N-protein was over 90% purity and identified as the most intense and abundant protein fragment, with PEAKS PTM score of 508 and sequence coverage of over 70%, including 173 unique peptides.",
publisher = "Belgrade : Serbian Biochemical Society",
journal = "Serbian Biochemical Society Tenth Conference
with international participation “Biochemical Insights into Molecular Mechanisms”, Kragujevac, Serbia, 24th September 2021",
title = "Optimization of expression, purification and HRMS characterization of recombinant N-protein fragment from SARS-CoV-2",
pages = "108-108",
url = "https://hdl.handle.net/21.15107/rcub_cherry_5774"
}

Đukić, T., Vasović, T., Mladenović, M., Jovanović, V. B., Smiljanić, K., Radosavljević, J., Stanić-Vučinić, D.,& Ćirković-Veličković, T.. (2021). Optimization of expression, purification and HRMS characterization of recombinant N-protein fragment from SARS-CoV-2. in Serbian Biochemical Society Tenth Conference
with international participation “Biochemical Insights into Molecular Mechanisms”, Kragujevac, Serbia, 24th September 2021
Belgrade : Serbian Biochemical Society., 108-108.
https://hdl.handle.net/21.15107/rcub_cherry_5774

Đukić T, Vasović T, Mladenović M, Jovanović VB, Smiljanić K, Radosavljević J, Stanić-Vučinić D, Ćirković-Veličković T. Optimization of expression, purification and HRMS characterization of recombinant N-protein fragment from SARS-CoV-2. in Serbian Biochemical Society Tenth Conference
with international participation “Biochemical Insights into Molecular Mechanisms”, Kragujevac, Serbia, 24th September 2021. 2021;:108-108.
https://hdl.handle.net/21.15107/rcub_cherry_5774 .

Đukić, Teodora, Vasović, Tamara, Mladenović, Maja, Jovanović, Vesna B., Smiljanić, Katarina, Radosavljević, Jelena, Stanić-Vučinić, Dragana, Ćirković-Veličković, Tanja, "Optimization of expression, purification and HRMS characterization of recombinant N-protein fragment from SARS-CoV-2" in Serbian Biochemical Society Tenth Conference
with international participation “Biochemical Insights into Molecular Mechanisms”, Kragujevac, Serbia, 24th September 2021 (2021):108-108,
https://hdl.handle.net/21.15107/rcub_cherry_5774 .