TY  - CONF
AU  - Jovanović, Vesna
AU  - Radomirović, Mirjana Ž.
AU  - Krstić-Ristivojević, Maja
AU  - Stanić-Vučinić, Dragana
AU  - Ćirković-Veličković, Tanja
PY  - 2023
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/6023
AB  - In the last several decades, the trend of seafood consumption has significantly increased not only in the countries with a tradition of seafood consumption, but also in other ones [1]. The increase in the world's population and the awareness of healthy food, the globalization of markets, and the development of aquaculture are some of the factors that have led to this trend. The aquaculture of shellfish like clams, mussels, oysters and scallops has been very developed all around the world and the food products based on them have become part of the daily diet for many consumers. In addition, these food products are considered healthy food because of the high content of proteins and essential fatty acids, but their consumption may carry some risks of food allergy. Tropomyosin from shellfish (TPM) is the major allergen responsible for the development of anaphylaxis in persons with food allergy. The content of TPM in shellfish and its bioavailability from food products can have potential influence on the sensitization of consumers to TPM. It is known that food processing can change the bioavailability of food allergens [2]. The main goal of this study was the investigation of how processing and packaging of shellfish samples can affect the content of TPM in them.
For this study, clam Venerupis philippinarum was chosen as the species with the highest world aquaculture production [1]. After the purchasing of live clams, the animals were separated into 5 groups for the next treatments: fresh live (control group), freshly removed inner content was kept at +4°C for 3 days (three days` shelf-life), frozen in a plastic bag and kept at -20 °C during 7 days, marinated and kept in a glass jar at room temperature during 8 days and freshly boiled. After processing and packaging of samples, the total protein extracts were prepared in 10 mM sodium phosphate buffer pH 7.4 1M NaCl, 1 mM PMSF and the concentration of total proteins was determined by BCA method. The concentration of TPM in the total protein extracts was determined using a sandwich Enzyme-Linked Immunosorbent Assay (ELISA) using in-house prepared clams` TPM standard. The content of TPM (μg) in the samples was expressed per mg of extracted soluble proteins, individual animal and grams of soft wet tissue.
The cooked samples have significantly higher TPM content expressed per gram of soft wet tissue compared to all other treatments. Food processing such as freezing, marinating, or extending the shelf-life at 4°C by 3 days has very little effect on the change in TPM content per gram of soft wet tissue compared to the fresh samples. The processing of clams, like cooking or marinating, caused the content of total soluble extracted proteins to be three to four times lower compared to the other three treatments. In these samples the obtained ratio of the total TPM/ total soluble extracted proteins ratio was the highest. This result can be explained by the fact that TPM is thermostable and stays soluble after cooking, while other proteins become insoluble because of denaturation. The lower ratio of TPM/ total soluble extracted proteins was found in marinated samples compared to the cooked samples. The lowest total tropomyosin/ total soluble extracted proteins ratio was found in 3 days’ shelf-life. Treatments like cooking, marinating and keeping the inner content of the shell at +4°C can significantly affect extractability of proteins, particularly affecting the ratio of major allergen TPM in the total protein extracts. Further studies are needed to examine bioaccessibility of TPM in different biologically relevant fluids (gastric/intestinal) and during digestion in relation to the processing conditions.
PB  - Beograd : Srpsko hemijsko društvo
C3  - XXII EuroFoodChem conference, Book of Abstracts, 14th-16th June, 2023.
T1  - The effect of food processing and packaging of clams on the content of tropomyosin
SP  - 240
EP  - 240
UR  - https://hdl.handle.net/21.15107/rcub_cherry_6023
ER  - 

@conference{
author = "Jovanović, Vesna and Radomirović, Mirjana Ž. and Krstić-Ristivojević, Maja and Stanić-Vučinić, Dragana and Ćirković-Veličković, Tanja",
year = "2023",
abstract = "In the last several decades, the trend of seafood consumption has significantly increased not only in the countries with a tradition of seafood consumption, but also in other ones [1]. The increase in the world's population and the awareness of healthy food, the globalization of markets, and the development of aquaculture are some of the factors that have led to this trend. The aquaculture of shellfish like clams, mussels, oysters and scallops has been very developed all around the world and the food products based on them have become part of the daily diet for many consumers. In addition, these food products are considered healthy food because of the high content of proteins and essential fatty acids, but their consumption may carry some risks of food allergy. Tropomyosin from shellfish (TPM) is the major allergen responsible for the development of anaphylaxis in persons with food allergy. The content of TPM in shellfish and its bioavailability from food products can have potential influence on the sensitization of consumers to TPM. It is known that food processing can change the bioavailability of food allergens [2]. The main goal of this study was the investigation of how processing and packaging of shellfish samples can affect the content of TPM in them.
For this study, clam Venerupis philippinarum was chosen as the species with the highest world aquaculture production [1]. After the purchasing of live clams, the animals were separated into 5 groups for the next treatments: fresh live (control group), freshly removed inner content was kept at +4°C for 3 days (three days` shelf-life), frozen in a plastic bag and kept at -20 °C during 7 days, marinated and kept in a glass jar at room temperature during 8 days and freshly boiled. After processing and packaging of samples, the total protein extracts were prepared in 10 mM sodium phosphate buffer pH 7.4 1M NaCl, 1 mM PMSF and the concentration of total proteins was determined by BCA method. The concentration of TPM in the total protein extracts was determined using a sandwich Enzyme-Linked Immunosorbent Assay (ELISA) using in-house prepared clams` TPM standard. The content of TPM (μg) in the samples was expressed per mg of extracted soluble proteins, individual animal and grams of soft wet tissue.
The cooked samples have significantly higher TPM content expressed per gram of soft wet tissue compared to all other treatments. Food processing such as freezing, marinating, or extending the shelf-life at 4°C by 3 days has very little effect on the change in TPM content per gram of soft wet tissue compared to the fresh samples. The processing of clams, like cooking or marinating, caused the content of total soluble extracted proteins to be three to four times lower compared to the other three treatments. In these samples the obtained ratio of the total TPM/ total soluble extracted proteins ratio was the highest. This result can be explained by the fact that TPM is thermostable and stays soluble after cooking, while other proteins become insoluble because of denaturation. The lower ratio of TPM/ total soluble extracted proteins was found in marinated samples compared to the cooked samples. The lowest total tropomyosin/ total soluble extracted proteins ratio was found in 3 days’ shelf-life. Treatments like cooking, marinating and keeping the inner content of the shell at +4°C can significantly affect extractability of proteins, particularly affecting the ratio of major allergen TPM in the total protein extracts. Further studies are needed to examine bioaccessibility of TPM in different biologically relevant fluids (gastric/intestinal) and during digestion in relation to the processing conditions.",
publisher = "Beograd : Srpsko hemijsko društvo",
journal = "XXII EuroFoodChem conference, Book of Abstracts, 14th-16th June, 2023.",
title = "The effect of food processing and packaging of clams on the content of tropomyosin",
pages = "240-240",
url = "https://hdl.handle.net/21.15107/rcub_cherry_6023"
}

Jovanović, V., Radomirović, M. Ž., Krstić-Ristivojević, M., Stanić-Vučinić, D.,& Ćirković-Veličković, T.. (2023). The effect of food processing and packaging of clams on the content of tropomyosin. in XXII EuroFoodChem conference, Book of Abstracts, 14th-16th June, 2023.
Beograd : Srpsko hemijsko društvo., 240-240.
https://hdl.handle.net/21.15107/rcub_cherry_6023

Jovanović V, Radomirović MŽ, Krstić-Ristivojević M, Stanić-Vučinić D, Ćirković-Veličković T. The effect of food processing and packaging of clams on the content of tropomyosin. in XXII EuroFoodChem conference, Book of Abstracts, 14th-16th June, 2023.. 2023;:240-240.
https://hdl.handle.net/21.15107/rcub_cherry_6023 .

Jovanović, Vesna, Radomirović, Mirjana Ž., Krstić-Ristivojević, Maja, Stanić-Vučinić, Dragana, Ćirković-Veličković, Tanja, "The effect of food processing and packaging of clams on the content of tropomyosin" in XXII EuroFoodChem conference, Book of Abstracts, 14th-16th June, 2023. (2023):240-240,
https://hdl.handle.net/21.15107/rcub_cherry_6023 .

TY  - CONF
AU  - Krstić-Ristivojević, Maja
AU  - Jovanović, Vesna
AU  - Radomirović, Mirjana Ž.
AU  - Trifunović, Olga
AU  - Stanić-Vučinić, Dragana
AU  - Ćirković-Veličković, Tanja
PY  - 2023
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/6024
AB  - In the last 50 years, the annual per capita consumption of seafood products worldwide has more than doubled, from almost 10 kg in 1960 to over 20 kg in 2014. Seafood protein is an essential part of the diet in many countries, particularly where total protein intake is low [1]. However, as defined by the European Community, fish, and shellfish tropomyosins (TPM) are major allergens and major causes of anaphylaxis [2]. The increasing prevalence of food allergies is consistent with the increasing pollution of soil and water with plastic particles. To investigate the potential link between increasing plastic pollution and increasing food allergy prevalence, we aim to develop methods for precise and accurate monitoring of allergens and plastic in real seafood samples.
TPM was isolated from shrimp (Litopenaeus vannamei), clams (Venerupis philippinarum), and mussels (Mytilus galloprovincialis). The obtained in-house TPM proteins from three different sources were resolved using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The concentration of TPM in seashell samples from different geographical origin was determined using a sandwich Enzyme-Linked Immunosorbent Assay (ELISA) with prior optimization of adequate TPM standard curve using commercial and non-commercial in-house prepared TPM standards.
TPM standards resolved via 2D-PAGE revealed the presence of two isoforms of shrimp and mussels TPM standard, one dominant and one less abundant isoform. Two isoforms from both seafood sources, shrimp and mussels, are slightly different in molecular weight and pI value. As for the TPM standard obtained from clams, the 2D electrophoregram showed possibly eight isoforms with small differences in mass and pI values. Furthermore, the presence of three dominant isoforms can be observed that differ slightly in molecular mass, while other isoforms also differ in pI value. The ELISA results, regarding TPM standard curve optimization, showed that in both the commercial shrimp TPM and in-house shrimp TPM standards, sigmoidal concentration dependence is present in a range of 50 to 0.05 ng/ml, using serial double dilutions. On the other hand, TPM standards isolated from mussels and clams show sigmoidal concentration dependence in the range of 45 to 0.044 μg/ml with using the identical combination of capture and detection antibodies and serial double dilutions. TPM concentrations in clams and mussel samples extrapolated from standard curves of commercial shrimp TPM standard and corresponding in-house TPM standards are presented in Table 1.
Differences in TPM concentration of the same sample using different TPM standards differ from 40 to 600 times, which strongly indicates that the right choice of TPM standard is a critical step for accurate and precise determination of TPM concentration in seafood samples.
PB  - Beograd : Srpsko hemijsko društvo
C3  - XXII EuroFoodChem conference, 14th-16th June, 2023. In: Book of Abstracts
T1  - Tropomyosin quantification in seafood samples-right choice of standard makes a difference
SP  - 132
EP  - 132
UR  - https://hdl.handle.net/21.15107/rcub_cherry_6024
ER  - 

@conference{
author = "Krstić-Ristivojević, Maja and Jovanović, Vesna and Radomirović, Mirjana Ž. and Trifunović, Olga and Stanić-Vučinić, Dragana and Ćirković-Veličković, Tanja",
year = "2023",
abstract = "In the last 50 years, the annual per capita consumption of seafood products worldwide has more than doubled, from almost 10 kg in 1960 to over 20 kg in 2014. Seafood protein is an essential part of the diet in many countries, particularly where total protein intake is low [1]. However, as defined by the European Community, fish, and shellfish tropomyosins (TPM) are major allergens and major causes of anaphylaxis [2]. The increasing prevalence of food allergies is consistent with the increasing pollution of soil and water with plastic particles. To investigate the potential link between increasing plastic pollution and increasing food allergy prevalence, we aim to develop methods for precise and accurate monitoring of allergens and plastic in real seafood samples.
TPM was isolated from shrimp (Litopenaeus vannamei), clams (Venerupis philippinarum), and mussels (Mytilus galloprovincialis). The obtained in-house TPM proteins from three different sources were resolved using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The concentration of TPM in seashell samples from different geographical origin was determined using a sandwich Enzyme-Linked Immunosorbent Assay (ELISA) with prior optimization of adequate TPM standard curve using commercial and non-commercial in-house prepared TPM standards.
TPM standards resolved via 2D-PAGE revealed the presence of two isoforms of shrimp and mussels TPM standard, one dominant and one less abundant isoform. Two isoforms from both seafood sources, shrimp and mussels, are slightly different in molecular weight and pI value. As for the TPM standard obtained from clams, the 2D electrophoregram showed possibly eight isoforms with small differences in mass and pI values. Furthermore, the presence of three dominant isoforms can be observed that differ slightly in molecular mass, while other isoforms also differ in pI value. The ELISA results, regarding TPM standard curve optimization, showed that in both the commercial shrimp TPM and in-house shrimp TPM standards, sigmoidal concentration dependence is present in a range of 50 to 0.05 ng/ml, using serial double dilutions. On the other hand, TPM standards isolated from mussels and clams show sigmoidal concentration dependence in the range of 45 to 0.044 μg/ml with using the identical combination of capture and detection antibodies and serial double dilutions. TPM concentrations in clams and mussel samples extrapolated from standard curves of commercial shrimp TPM standard and corresponding in-house TPM standards are presented in Table 1.
Differences in TPM concentration of the same sample using different TPM standards differ from 40 to 600 times, which strongly indicates that the right choice of TPM standard is a critical step for accurate and precise determination of TPM concentration in seafood samples.",
publisher = "Beograd : Srpsko hemijsko društvo",
journal = "XXII EuroFoodChem conference, 14th-16th June, 2023. In: Book of Abstracts",
title = "Tropomyosin quantification in seafood samples-right choice of standard makes a difference",
pages = "132-132",
url = "https://hdl.handle.net/21.15107/rcub_cherry_6024"
}

Krstić-Ristivojević, M., Jovanović, V., Radomirović, M. Ž., Trifunović, O., Stanić-Vučinić, D.,& Ćirković-Veličković, T.. (2023). Tropomyosin quantification in seafood samples-right choice of standard makes a difference. in XXII EuroFoodChem conference, 14th-16th June, 2023. In: Book of Abstracts
Beograd : Srpsko hemijsko društvo., 132-132.
https://hdl.handle.net/21.15107/rcub_cherry_6024

Krstić-Ristivojević M, Jovanović V, Radomirović MŽ, Trifunović O, Stanić-Vučinić D, Ćirković-Veličković T. Tropomyosin quantification in seafood samples-right choice of standard makes a difference. in XXII EuroFoodChem conference, 14th-16th June, 2023. In: Book of Abstracts. 2023;:132-132.
https://hdl.handle.net/21.15107/rcub_cherry_6024 .

Krstić-Ristivojević, Maja, Jovanović, Vesna, Radomirović, Mirjana Ž., Trifunović, Olga, Stanić-Vučinić, Dragana, Ćirković-Veličković, Tanja, "Tropomyosin quantification in seafood samples-right choice of standard makes a difference" in XXII EuroFoodChem conference, 14th-16th June, 2023. In: Book of Abstracts (2023):132-132,
https://hdl.handle.net/21.15107/rcub_cherry_6024 .

TY  - CONF
AU  - Simović, Ana
AU  - Bićanin, Maša
AU  - Radomirović, Mirjana Ž.
AU  - Aćimovič, Jelena
AU  - Mitić, Dragana
AU  - Stanić-Vučinić, Dragana
AU  - Ćirković-Veličković, Tanja
PY  - 2023
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/6418
AB  - The emergence of the novel coronavirus SARS-CoV-2 has attracted the attention of the whole scientific community. However, as there are significant concerns regarding the effectiveness of vaccines and drugs against novel SARS-CoV-2 variants, naturally derived broad-spectrum of antivirals seems to be precious adjuvant agents to assist in combat against this disease. Phycocyanobilin (PCB) is an open-chain tetrapyrrole chromophore of phycocyanin (PC), chromoprotein derived from Spirullina, with strong anti-oxidative action. The role of disulfide bonds and thiol-disulfide balance in RBD is considered to play a significant role in the binding of S protein to ACE2 receptor. In RBD, in contrast to C480–C488 disulfide, which is thermodynamically stable, C379–C432 and C391–C525 disulfides are in dynamic equilibrium with their thiol states and, thus these two pairs of disulfides are more sensitive to changes in redox poise. Our study aimed to investigate impact of PCB on disulfide balance of RBD by redox proteomics and to investigate structural changes in the protein exposed to PCB. The effect of PCB on RBD secondary structures was examined by far-UV CD spectroscopy after titration of RBD with increasing concentrations of PCB. The presence of PCB had a pronounced effect on the spectral shape. RBD is dominantly composed of random coils and β-sheets. In the presence of PCB a slight increase of α-helical and random coils content, while the content of β-sheets and β-turns is decreased. Mapping redox-active disulfides and reactive cysteines in recombinant SARS-CoV-2 RBD was done using redox proteomics on both recombinant RBD and PCB-exposed RBD. A mass shift caused by alkylation of free Cys residues was detected on three Cys residues demonstrating disulfides C379–C432 and 432-391 to be semi-stable in both RBD and PCB-exposed RBD. Our results demonstrate that RBD exposed to PCB undergo structural changes but does not change the redox state of its critical semi-stable disulfides.
Acknowledgment: The authors acknowledge support of the special Research Fund (BOF) of Ghent University (grant number 01N01718) and the Ministry of Science, Innovation and technological development of the Republic of Serbia (Contract number: 451-03-68/2023-14/200168).
PB  - Italian Proteomics Association
C3  - ItPA HPS and SePA XVII International Congress: Proteomics and Metabolomics towards Global Health, November 29th – December 1st, 2023, Roma, Italy
T1  - Redox status of critical disulfides of SARS-CoV-2 receptor-binding-domain exposed to bioactive chomophore phycocyanobilin
SP  - 63
EP  - 63
UR  - https://hdl.handle.net/21.15107/rcub_cherry_6418
ER  - 

@conference{
author = "Simović, Ana and Bićanin, Maša and Radomirović, Mirjana Ž. and Aćimovič, Jelena and Mitić, Dragana and Stanić-Vučinić, Dragana and Ćirković-Veličković, Tanja",
year = "2023",
abstract = "The emergence of the novel coronavirus SARS-CoV-2 has attracted the attention of the whole scientific community. However, as there are significant concerns regarding the effectiveness of vaccines and drugs against novel SARS-CoV-2 variants, naturally derived broad-spectrum of antivirals seems to be precious adjuvant agents to assist in combat against this disease. Phycocyanobilin (PCB) is an open-chain tetrapyrrole chromophore of phycocyanin (PC), chromoprotein derived from Spirullina, with strong anti-oxidative action. The role of disulfide bonds and thiol-disulfide balance in RBD is considered to play a significant role in the binding of S protein to ACE2 receptor. In RBD, in contrast to C480–C488 disulfide, which is thermodynamically stable, C379–C432 and C391–C525 disulfides are in dynamic equilibrium with their thiol states and, thus these two pairs of disulfides are more sensitive to changes in redox poise. Our study aimed to investigate impact of PCB on disulfide balance of RBD by redox proteomics and to investigate structural changes in the protein exposed to PCB. The effect of PCB on RBD secondary structures was examined by far-UV CD spectroscopy after titration of RBD with increasing concentrations of PCB. The presence of PCB had a pronounced effect on the spectral shape. RBD is dominantly composed of random coils and β-sheets. In the presence of PCB a slight increase of α-helical and random coils content, while the content of β-sheets and β-turns is decreased. Mapping redox-active disulfides and reactive cysteines in recombinant SARS-CoV-2 RBD was done using redox proteomics on both recombinant RBD and PCB-exposed RBD. A mass shift caused by alkylation of free Cys residues was detected on three Cys residues demonstrating disulfides C379–C432 and 432-391 to be semi-stable in both RBD and PCB-exposed RBD. Our results demonstrate that RBD exposed to PCB undergo structural changes but does not change the redox state of its critical semi-stable disulfides.
Acknowledgment: The authors acknowledge support of the special Research Fund (BOF) of Ghent University (grant number 01N01718) and the Ministry of Science, Innovation and technological development of the Republic of Serbia (Contract number: 451-03-68/2023-14/200168).",
publisher = "Italian Proteomics Association",
journal = "ItPA HPS and SePA XVII International Congress: Proteomics and Metabolomics towards Global Health, November 29th – December 1st, 2023, Roma, Italy",
title = "Redox status of critical disulfides of SARS-CoV-2 receptor-binding-domain exposed to bioactive chomophore phycocyanobilin",
pages = "63-63",
url = "https://hdl.handle.net/21.15107/rcub_cherry_6418"
}

Simović, A., Bićanin, M., Radomirović, M. Ž., Aćimovič, J., Mitić, D., Stanić-Vučinić, D.,& Ćirković-Veličković, T.. (2023). Redox status of critical disulfides of SARS-CoV-2 receptor-binding-domain exposed to bioactive chomophore phycocyanobilin. in ItPA HPS and SePA XVII International Congress: Proteomics and Metabolomics towards Global Health, November 29th – December 1st, 2023, Roma, Italy
Italian Proteomics Association., 63-63.
https://hdl.handle.net/21.15107/rcub_cherry_6418

Simović A, Bićanin M, Radomirović MŽ, Aćimovič J, Mitić D, Stanić-Vučinić D, Ćirković-Veličković T. Redox status of critical disulfides of SARS-CoV-2 receptor-binding-domain exposed to bioactive chomophore phycocyanobilin. in ItPA HPS and SePA XVII International Congress: Proteomics and Metabolomics towards Global Health, November 29th – December 1st, 2023, Roma, Italy. 2023;:63-63.
https://hdl.handle.net/21.15107/rcub_cherry_6418 .

Simović, Ana, Bićanin, Maša, Radomirović, Mirjana Ž., Aćimovič, Jelena, Mitić, Dragana, Stanić-Vučinić, Dragana, Ćirković-Veličković, Tanja, "Redox status of critical disulfides of SARS-CoV-2 receptor-binding-domain exposed to bioactive chomophore phycocyanobilin" in ItPA HPS and SePA XVII International Congress: Proteomics and Metabolomics towards Global Health, November 29th – December 1st, 2023, Roma, Italy (2023):63-63,
https://hdl.handle.net/21.15107/rcub_cherry_6418 .

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  - DATA
AU  - Radomirović, Mirjana
AU  - Gligorijević, Nikola
AU  - Stanić-Vučinić, Dragana
AU  - Rajković, Andreja
AU  - Ćirković-Veličković, Tanja
PY  - 2023
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/6466
AB  - Experimental data for the results shown in the manuscript Ultrasensitive Quantification of Crustacean Tropomyosin by Immuno-PCR are given in the attachment.
PB  - MDPI
T2  - International Journal of Molecular Sciences
T1  - Research data for: Radomirović, M. Ž., Gligorijević, N., Stanić-Vučinić, D., Rajković, A.,& Ćirković-Veličković, T.. (2023). Ultrasensitive Quantification of Crustacean Tropomyosin by Immuno-PCR. in International Journal of Molecular Sciences MDPI., 24(20), 15410. https://doi.org/doi.org/10.3390/ ijms242015410
VL  - 24
IS  - 20
UR  - https://hdl.handle.net/21.15107/rcub_cherry_6466
ER  - 

@misc{
author = "Radomirović, Mirjana and Gligorijević, Nikola and Stanić-Vučinić, Dragana and Rajković, Andreja and Ćirković-Veličković, Tanja",
year = "2023",
abstract = "Experimental data for the results shown in the manuscript Ultrasensitive Quantification of Crustacean Tropomyosin by Immuno-PCR are given in the attachment.",
publisher = "MDPI",
journal = "International Journal of Molecular Sciences",
title = "Research data for: Radomirović, M. Ž., Gligorijević, N., Stanić-Vučinić, D., Rajković, A.,& Ćirković-Veličković, T.. (2023). Ultrasensitive Quantification of Crustacean Tropomyosin by Immuno-PCR. in International Journal of Molecular Sciences MDPI., 24(20), 15410. https://doi.org/doi.org/10.3390/ ijms242015410",
volume = "24",
number = "20",
url = "https://hdl.handle.net/21.15107/rcub_cherry_6466"
}

Radomirović, M., Gligorijević, N., Stanić-Vučinić, D., Rajković, A.,& Ćirković-Veličković, T.. (2023). Research data for: Radomirović, M. Ž., Gligorijević, N., Stanić-Vučinić, D., Rajković, A.,& Ćirković-Veličković, T.. (2023). Ultrasensitive Quantification of Crustacean Tropomyosin by Immuno-PCR. in International Journal of Molecular Sciences MDPI., 24(20), 15410. https://doi.org/doi.org/10.3390/ ijms242015410. in International Journal of Molecular Sciences
MDPI., 24(20).
https://hdl.handle.net/21.15107/rcub_cherry_6466

Radomirović M, Gligorijević N, Stanić-Vučinić D, Rajković A, Ćirković-Veličković T. Research data for: Radomirović, M. Ž., Gligorijević, N., Stanić-Vučinić, D., Rajković, A.,& Ćirković-Veličković, T.. (2023). Ultrasensitive Quantification of Crustacean Tropomyosin by Immuno-PCR. in International Journal of Molecular Sciences MDPI., 24(20), 15410. https://doi.org/doi.org/10.3390/ ijms242015410. in International Journal of Molecular Sciences. 2023;24(20).
https://hdl.handle.net/21.15107/rcub_cherry_6466 .

Radomirović, Mirjana, Gligorijević, Nikola, Stanić-Vučinić, Dragana, Rajković, Andreja, Ćirković-Veličković, Tanja, "Research data for: Radomirović, M. Ž., Gligorijević, N., Stanić-Vučinić, D., Rajković, A.,& Ćirković-Veličković, T.. (2023). Ultrasensitive Quantification of Crustacean Tropomyosin by Immuno-PCR. in International Journal of Molecular Sciences MDPI., 24(20), 15410. https://doi.org/doi.org/10.3390/ ijms242015410" in International Journal of Molecular Sciences, 24, no. 20 (2023),
https://hdl.handle.net/21.15107/rcub_cherry_6466 .

TY  - JOUR
AU  - Radomirović, Mirjana Ž.
AU  - Gligorijević, Nikola
AU  - Stanić-Vučinić, Dragana
AU  - Rajković, Andreja
AU  - Ćirković-Veličković, Tanja
PY  - 2023
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/6019
AB  - Tropomyosin is the major and predominant allergen among shellfish. This study developed
an ultrasensitive immuno-PCR method for the quantification of crustacean tropomyosin in foods.
The method couples sandwich ELISA with the real-time PCR (rtPCR) amplification of marker DNAs. Monoclonal anti-TPM antibody was the capture antibody, polyclonal rabbit anti-shrimp tropomyosin antibody was the detection antibody, while natural shrimp tropomyosin served as the standard. A double-stranded amino-DNA was covalently conjugated to a secondary anti-rabbit antibody and subsequently amplified and quantified via rtPCR. The quantification sensitivity of immuno-PCR was 20-fold higher than analogous ELISA, with LOQ 19.8 pg/mL. The developed immuno-PCR method is highly specific for the detection of crustacean tropomyosin and is highly precise in a broad concentration range. Tropomyosin recovery in the spiked vegetable soup was 87.7–115.6%. Crustacean tropomyosin was also quantified in commercial food products. The reported immuno-PCR assay is the most sensitive method for the quantification of crustacean tropomyosin and is the first immuno-PCR-based assay for the quantification of food allergen and food protein in general. The described method could be easily adapted for the specific and ultrasensitive immuno-PCR-based detection of traces of any food allergen that is currently being quantified with ELISA, which is of critical importance for people with food allergies.
PB  - MDPI
T2  - International Journal of Molecular Sciences
T1  - Ultrasensitive Quantification of Crustacean Tropomyosin by Immuno-PCR
VL  - 24
IS  - 20
SP  - 15410
DO  - doi.org/10.3390/ ijms242015410
ER  - 

@article{
author = "Radomirović, Mirjana Ž. and Gligorijević, Nikola and Stanić-Vučinić, Dragana and Rajković, Andreja and Ćirković-Veličković, Tanja",
year = "2023",
abstract = "Tropomyosin is the major and predominant allergen among shellfish. This study developed
an ultrasensitive immuno-PCR method for the quantification of crustacean tropomyosin in foods.
The method couples sandwich ELISA with the real-time PCR (rtPCR) amplification of marker DNAs. Monoclonal anti-TPM antibody was the capture antibody, polyclonal rabbit anti-shrimp tropomyosin antibody was the detection antibody, while natural shrimp tropomyosin served as the standard. A double-stranded amino-DNA was covalently conjugated to a secondary anti-rabbit antibody and subsequently amplified and quantified via rtPCR. The quantification sensitivity of immuno-PCR was 20-fold higher than analogous ELISA, with LOQ 19.8 pg/mL. The developed immuno-PCR method is highly specific for the detection of crustacean tropomyosin and is highly precise in a broad concentration range. Tropomyosin recovery in the spiked vegetable soup was 87.7–115.6%. Crustacean tropomyosin was also quantified in commercial food products. The reported immuno-PCR assay is the most sensitive method for the quantification of crustacean tropomyosin and is the first immuno-PCR-based assay for the quantification of food allergen and food protein in general. The described method could be easily adapted for the specific and ultrasensitive immuno-PCR-based detection of traces of any food allergen that is currently being quantified with ELISA, which is of critical importance for people with food allergies.",
publisher = "MDPI",
journal = "International Journal of Molecular Sciences",
title = "Ultrasensitive Quantification of Crustacean Tropomyosin by Immuno-PCR",
volume = "24",
number = "20",
pages = "15410",
doi = "doi.org/10.3390/ ijms242015410"
}

Radomirović, M. Ž., Gligorijević, N., Stanić-Vučinić, D., Rajković, A.,& Ćirković-Veličković, T.. (2023). Ultrasensitive Quantification of Crustacean Tropomyosin by Immuno-PCR. in International Journal of Molecular Sciences
MDPI., 24(20), 15410.
https://doi.org/doi.org/10.3390/ ijms242015410

Radomirović MŽ, Gligorijević N, Stanić-Vučinić D, Rajković A, Ćirković-Veličković T. Ultrasensitive Quantification of Crustacean Tropomyosin by Immuno-PCR. in International Journal of Molecular Sciences. 2023;24(20):15410.
doi:doi.org/10.3390/ ijms242015410 .

Radomirović, Mirjana Ž., Gligorijević, Nikola, Stanić-Vučinić, Dragana, Rajković, Andreja, Ćirković-Veličković, Tanja, "Ultrasensitive Quantification of Crustacean Tropomyosin by Immuno-PCR" in International Journal of Molecular Sciences, 24, no. 20 (2023):15410,
https://doi.org/doi.org/10.3390/ ijms242015410 . .

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  - Radomirović, Mirjana Ž.
AU  - Bićanin, Maša
AU  - Udovički, Božidar
AU  - Krstić-Ristivojević, Maja
AU  - Đukić, Teodora
AU  - Vasović, Tamara
AU  - Jovanović, Vesna
AU  - Stanić-Vučinić, Dragana
AU  - Rajković, Andreja
AU  - Ćirković-Veličković, Tanja
PY  - 2023
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/6021
AB  - Accurately diagnosing people with suspected SARS-CoV-2 infection is essential to help manage COVID-19. Currently available SARS-CoV-2 diagnostics detect either RNA of the virus by RT-PCR or the presence of viral antigens in biological fluids by ELISA or similar techniques. Low sensitivity of antigen tests could lead to the risk of false negative results. Therefore, this study aimed to develop a highly sensitive immuno-PCR method for quantifying SARS-CoV-2 nucleocapsid (N) protein that combines the specificity of sandwich ELISA with the sensitivity of PCR. Recombinant N protein fragment was produced in E. coli as an expression system and purified using immobilized metal ion affinity chromatography. The antibodies against the N protein were raised in rabbits and mice. High-affinity polyclonal mice and rabbit N protein-specific antisera were purified using ammonium sulfate precipitation and used to develop sandwich ELISA for the quantification of N protein. Mice polyclonal serum was used as a capture for N protein. N
protein bound to mice antibodies was detected with rabbit polyclonal sera. A double-stranded amino-DNA molecule of 77 base pairs was PCR-synthesized, covalently conjugated to a secondary goat anti-rabbit antibody and subsequently amplified and quantified by real-time PCR. The results were compared to analogous sandwich ELISA consisting of alkaline phosphatase-labeled goat anti-rabbit antibody. The sensitivity of immuno-PCR for quantification of N protein was increased by up to 7-fold compared to analogous ELISA, having a limit of detection of 92 pg/mL and a limit of quantification of 840 pg/mL. The developed immuno-PCR method thus has the potential to be used as a new antigen test for COVID-19 and beyond.
PB  - Federation of European Biochemical Societies, Wiley
C3  - The 47th FEBS Congress, 8th-12th July, 2023. In: FEBS Open Bio, 13: Suppl. 2
T1  - Development of immuno-PCR for sensitive quantification of SARS-CoV-2 nucleocapsid protein
SP  - 44
EP  - 44
UR  - https://hdl.handle.net/21.15107/rcub_cherry_6021
ER  - 

@conference{
author = "Radomirović, Mirjana Ž. and Bićanin, Maša and Udovički, Božidar and Krstić-Ristivojević, Maja and Đukić, Teodora and Vasović, Tamara and Jovanović, Vesna and Stanić-Vučinić, Dragana and Rajković, Andreja and Ćirković-Veličković, Tanja",
year = "2023",
abstract = "Accurately diagnosing people with suspected SARS-CoV-2 infection is essential to help manage COVID-19. Currently available SARS-CoV-2 diagnostics detect either RNA of the virus by RT-PCR or the presence of viral antigens in biological fluids by ELISA or similar techniques. Low sensitivity of antigen tests could lead to the risk of false negative results. Therefore, this study aimed to develop a highly sensitive immuno-PCR method for quantifying SARS-CoV-2 nucleocapsid (N) protein that combines the specificity of sandwich ELISA with the sensitivity of PCR. Recombinant N protein fragment was produced in E. coli as an expression system and purified using immobilized metal ion affinity chromatography. The antibodies against the N protein were raised in rabbits and mice. High-affinity polyclonal mice and rabbit N protein-specific antisera were purified using ammonium sulfate precipitation and used to develop sandwich ELISA for the quantification of N protein. Mice polyclonal serum was used as a capture for N protein. N
protein bound to mice antibodies was detected with rabbit polyclonal sera. A double-stranded amino-DNA molecule of 77 base pairs was PCR-synthesized, covalently conjugated to a secondary goat anti-rabbit antibody and subsequently amplified and quantified by real-time PCR. The results were compared to analogous sandwich ELISA consisting of alkaline phosphatase-labeled goat anti-rabbit antibody. The sensitivity of immuno-PCR for quantification of N protein was increased by up to 7-fold compared to analogous ELISA, having a limit of detection of 92 pg/mL and a limit of quantification of 840 pg/mL. The developed immuno-PCR method thus has the potential to be used as a new antigen test for COVID-19 and beyond.",
publisher = "Federation of European Biochemical Societies, Wiley",
journal = "The 47th FEBS Congress, 8th-12th July, 2023. In: FEBS Open Bio, 13: Suppl. 2",
title = "Development of immuno-PCR for sensitive quantification of SARS-CoV-2 nucleocapsid protein",
pages = "44-44",
url = "https://hdl.handle.net/21.15107/rcub_cherry_6021"
}

Radomirović, M. Ž., Bićanin, M., Udovički, B., Krstić-Ristivojević, M., Đukić, T., Vasović, T., Jovanović, V., Stanić-Vučinić, D., Rajković, A.,& Ćirković-Veličković, T.. (2023). Development of immuno-PCR for sensitive quantification of SARS-CoV-2 nucleocapsid protein. in The 47th FEBS Congress, 8th-12th July, 2023. In: FEBS Open Bio, 13: Suppl. 2
Federation of European Biochemical Societies, Wiley., 44-44.
https://hdl.handle.net/21.15107/rcub_cherry_6021

Radomirović MŽ, Bićanin M, Udovički B, Krstić-Ristivojević M, Đukić T, Vasović T, Jovanović V, Stanić-Vučinić D, Rajković A, Ćirković-Veličković T. Development of immuno-PCR for sensitive quantification of SARS-CoV-2 nucleocapsid protein. in The 47th FEBS Congress, 8th-12th July, 2023. In: FEBS Open Bio, 13: Suppl. 2. 2023;:44-44.
https://hdl.handle.net/21.15107/rcub_cherry_6021 .

Radomirović, Mirjana Ž., Bićanin, Maša, Udovički, Božidar, Krstić-Ristivojević, Maja, Đukić, Teodora, Vasović, Tamara, Jovanović, Vesna, Stanić-Vučinić, Dragana, Rajković, Andreja, Ćirković-Veličković, Tanja, "Development of immuno-PCR for sensitive quantification of SARS-CoV-2 nucleocapsid protein" in The 47th FEBS Congress, 8th-12th July, 2023. In: FEBS Open Bio, 13: Suppl. 2 (2023):44-44,
https://hdl.handle.net/21.15107/rcub_cherry_6021 .

TY  - JOUR
AU  - Nedić, Olgica
AU  - Penezić, Ana
AU  - Minić, Simeon
AU  - Radomirović, Mirjana Ž.
AU  - Nikolić, Milan
AU  - Ćirković-Veličković, Tanja
AU  - Gligorijević, Nikola
PY  - 2023
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/6028
AB  - Common to all biological systems and living organisms are molecular interactions, which
may lead to specific physiological events. Most often, a cascade of events occurs, establishing an equilibrium between possibly competing and/or synergistic processes. Biochemical pathways that sustain life depend on multiple intrinsic and extrinsic factors contributing to aging and/or diseases. This article deals with food antioxidants and human proteins from the circulation, their interaction, their effect on the structure, properties, and function of antioxidant-bound proteins, and the possible impact of complex formation on antioxidants. An overview of studies examining interactions between individual antioxidant compounds and major blood proteins is presented with findings. Investigating antioxidant/protein interactions at the level of the human organism and determining antioxidant distribution between proteins and involvement in the particular physiological role is a very complex and challenging task. However, by knowing the role of a particular protein in certain pathology or aging, and the effect exerted by a particular antioxidant bound to it, it is possible to recommend specific food intake or resistance to it to improve the condition or slow down the process.
PB  - MDPI
T2  - Antioxidants
T1  - Food Antioxidants and Their Interaction with Human Proteins
VL  - 12
IS  - 4
SP  - 815
DO  - 10.3390/antiox12040815
ER  - 

@article{
author = "Nedić, Olgica and Penezić, Ana and Minić, Simeon and Radomirović, Mirjana Ž. and Nikolić, Milan and Ćirković-Veličković, Tanja and Gligorijević, Nikola",
year = "2023",
abstract = "Common to all biological systems and living organisms are molecular interactions, which
may lead to specific physiological events. Most often, a cascade of events occurs, establishing an equilibrium between possibly competing and/or synergistic processes. Biochemical pathways that sustain life depend on multiple intrinsic and extrinsic factors contributing to aging and/or diseases. This article deals with food antioxidants and human proteins from the circulation, their interaction, their effect on the structure, properties, and function of antioxidant-bound proteins, and the possible impact of complex formation on antioxidants. An overview of studies examining interactions between individual antioxidant compounds and major blood proteins is presented with findings. Investigating antioxidant/protein interactions at the level of the human organism and determining antioxidant distribution between proteins and involvement in the particular physiological role is a very complex and challenging task. However, by knowing the role of a particular protein in certain pathology or aging, and the effect exerted by a particular antioxidant bound to it, it is possible to recommend specific food intake or resistance to it to improve the condition or slow down the process.",
publisher = "MDPI",
journal = "Antioxidants",
title = "Food Antioxidants and Their Interaction with Human Proteins",
volume = "12",
number = "4",
pages = "815",
doi = "10.3390/antiox12040815"
}

Nedić, O., Penezić, A., Minić, S., Radomirović, M. Ž., Nikolić, M., Ćirković-Veličković, T.,& Gligorijević, N.. (2023). Food Antioxidants and Their Interaction with Human Proteins. in Antioxidants
MDPI., 12(4), 815.
https://doi.org/10.3390/antiox12040815

Nedić O, Penezić A, Minić S, Radomirović MŽ, Nikolić M, Ćirković-Veličković T, Gligorijević N. Food Antioxidants and Their Interaction with Human Proteins. in Antioxidants. 2023;12(4):815.
doi:10.3390/antiox12040815 .

Nedić, Olgica, Penezić, Ana, Minić, Simeon, Radomirović, Mirjana Ž., Nikolić, Milan, Ćirković-Veličković, Tanja, Gligorijević, Nikola, "Food Antioxidants and Their Interaction with Human Proteins" in Antioxidants, 12, no. 4 (2023):815,
https://doi.org/10.3390/antiox12040815 . .

TY  - CONF
AU  - Radomirović, Mirjana Ž.
AU  - Gligorijević, Nikola
AU  - Stanić-Vučinić, Dragana
AU  - Rajković, Andreja
AU  - Ćirković-Veličković, Tanja
PY  - 2023
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/6020
AB  - Tropomyosin has been recognized as one of the most common allergens among shellfish allergens. Sensitive and specific quantification of traces of allergens present in food samples is of critical importance for people with food allergies. This study thus aimed to develop a highly sensitive immuno-PCR method for detecting crustacean tropomyosin in foods. Method couples conventional sandwich ELISA assay with real-time PCR amplification of marker DNA. Monoclonal mouse anti-tropomyosin antibody was used as a capture antibody, while polyclonal rabbit anti-tropomyosin antibody served as a detection antibody in sandwich ELISA. A double-stranded amino-DNA molecule of 77 base pairs was covalently conjugated to a secondary goat anti-rabbit antibody and subsequently amplified and quantified by real-time PCR. Tropomyosin was quantified using highly purified natural shrimp tropomyosin as standard. The sensitivity of immuno-PCR for quantification of tropomyosin was increased by up to 20-fold compared to ELISA, demonstrating accuracy as low as 19.8 pg/mL. Recovery of tropomyosin in vegetable soup as a food matrix was in the 87.7–115.6% range, with relative standard deviations in the 5–24.5% range. Tropomyosin was also quantified in the commercially available food products. Developed immuno-PCR technique thus shows the potential to be a method of choice for specific and ultrasensitive detection of tropomyosin in food samples, with the final aim of reducing risks of accidental food contamination.
PB  - Belgrade : Faculty of Chemistry
PB  - Belgrade : Serbian Biochemical Society
C3  - "Biochemistry in Biotechnology", Twelfth Conference, International scientific meeting, September 21-23, 2023, Belgrade, Serbia
T1  - Immuno-PCR for crustacean tropomyosin quantification
SP  - 130
EP  - 130
UR  - https://hdl.handle.net/21.15107/rcub_cherry_6020
ER  - 

@conference{
author = "Radomirović, Mirjana Ž. and Gligorijević, Nikola and Stanić-Vučinić, Dragana and Rajković, Andreja and Ćirković-Veličković, Tanja",
year = "2023",
abstract = "Tropomyosin has been recognized as one of the most common allergens among shellfish allergens. Sensitive and specific quantification of traces of allergens present in food samples is of critical importance for people with food allergies. This study thus aimed to develop a highly sensitive immuno-PCR method for detecting crustacean tropomyosin in foods. Method couples conventional sandwich ELISA assay with real-time PCR amplification of marker DNA. Monoclonal mouse anti-tropomyosin antibody was used as a capture antibody, while polyclonal rabbit anti-tropomyosin antibody served as a detection antibody in sandwich ELISA. A double-stranded amino-DNA molecule of 77 base pairs was covalently conjugated to a secondary goat anti-rabbit antibody and subsequently amplified and quantified by real-time PCR. Tropomyosin was quantified using highly purified natural shrimp tropomyosin as standard. The sensitivity of immuno-PCR for quantification of tropomyosin was increased by up to 20-fold compared to ELISA, demonstrating accuracy as low as 19.8 pg/mL. Recovery of tropomyosin in vegetable soup as a food matrix was in the 87.7–115.6% range, with relative standard deviations in the 5–24.5% range. Tropomyosin was also quantified in the commercially available food products. Developed immuno-PCR technique thus shows the potential to be a method of choice for specific and ultrasensitive detection of tropomyosin in food samples, with the final aim of reducing risks of accidental food contamination.",
publisher = "Belgrade : Faculty of Chemistry, Belgrade : Serbian Biochemical Society",
journal = ""Biochemistry in Biotechnology", Twelfth Conference, International scientific meeting, September 21-23, 2023, Belgrade, Serbia",
title = "Immuno-PCR for crustacean tropomyosin quantification",
pages = "130-130",
url = "https://hdl.handle.net/21.15107/rcub_cherry_6020"
}

Radomirović, M. Ž., Gligorijević, N., Stanić-Vučinić, D., Rajković, A.,& Ćirković-Veličković, T.. (2023). Immuno-PCR for crustacean tropomyosin quantification. in "Biochemistry in Biotechnology", Twelfth Conference, International scientific meeting, September 21-23, 2023, Belgrade, Serbia
Belgrade : Faculty of Chemistry., 130-130.
https://hdl.handle.net/21.15107/rcub_cherry_6020

Radomirović MŽ, Gligorijević N, Stanić-Vučinić D, Rajković A, Ćirković-Veličković T. Immuno-PCR for crustacean tropomyosin quantification. in "Biochemistry in Biotechnology", Twelfth Conference, International scientific meeting, September 21-23, 2023, Belgrade, Serbia. 2023;:130-130.
https://hdl.handle.net/21.15107/rcub_cherry_6020 .

Radomirović, Mirjana Ž., Gligorijević, Nikola, Stanić-Vučinić, Dragana, Rajković, Andreja, Ćirković-Veličković, Tanja, "Immuno-PCR for crustacean tropomyosin quantification" in "Biochemistry in Biotechnology", Twelfth Conference, International scientific meeting, September 21-23, 2023, Belgrade, Serbia (2023):130-130,
https://hdl.handle.net/21.15107/rcub_cherry_6020 .

TY  - PAT
AU  - Ćirković-Veličković, Tanja
AU  - Radomirović, Mirjana Ž.
AU  - Stanić-Vučinić, Dragana
PY  - 2023
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/6007
AB  - Предметни проналазак се односи на област биоаналитике и представља универзални кит за амплификацију сигнала секундарних антитела у ЕЛИСА есејима имуно-ПЦР-ом. Описане су компоненте кита, укључујући маркер нуклеинске киселине који садржи унапред одређену нуклеотидну секвенцу, као и везни молекул способан да са једне стране специфично веже рецептор који није нуклеинска киселина, а који је способан да специфично веже аналит имобилисан на чврстом носачу везивањем за други рецептор који није нуклеинска киселина, и са друге стране да специфично веже маркер нуклеинске киселине. Поступци за специфично откривање присуства маркера нуклеинске киселине у комплексу везног молекула са аналитом, који укључују амплификацију нуклеинске киселине у ПЦР реакцији у реалном времену, и указују на присуство аналита у поменутом узорку, су такође обезбеђени. Предметни проналазак може да буде коришћен за изузетно осетљиву и специфичну детекцију широког спектра анaлита, за који могу да се произведу специфична антитела, у области медицине, прехрамбене индустрије и биотехнологије.
AB  - The present invention belongs to the field of bioanalytics and relates to universal kit for amplification of secondary antibodies signal in ELISA assays by immuno-PCR. Kit komponents include nucleic acid marker comprising of predetermined nucleotide sequence, as well as linker molecule, which is on one hand capable of specifically binding non-nucleic acid receptor that is capable of binding to solid surface-immobilized analyte, and on other hand is capable to specifically bind nucleic acid marker. Methods for specific detection of presence of nucleic acid marker in complex with analyte through linker molecule, that include amplification of nucleic acid marker in real-time PCR, wherein the presence of nucleic acid markers indicates presence of analyte in said sample, are also provided. The present invention can be used for sensitive and specific detection of wide range of analytes, for which specific antibodies can be produced, in the field of medicine, food industry and biotechnology.
PB  - Zavod za intelektualnu svojinu Republike Srbije
T2  - Glasnik intelektualne svojine
T1  - Kit za amplifikaciju signala sekundarnih antitela u indirektnim ELISA esejima imuno-PCR-om
IS  - 9
UR  - https://hdl.handle.net/21.15107/rcub_cherry_6007
ER  - 

@misc{
author = "Ćirković-Veličković, Tanja and Radomirović, Mirjana Ž. and Stanić-Vučinić, Dragana",
year = "2023",
abstract = "Предметни проналазак се односи на област биоаналитике и представља универзални кит за амплификацију сигнала секундарних антитела у ЕЛИСА есејима имуно-ПЦР-ом. Описане су компоненте кита, укључујући маркер нуклеинске киселине који садржи унапред одређену нуклеотидну секвенцу, као и везни молекул способан да са једне стране специфично веже рецептор који није нуклеинска киселина, а који је способан да специфично веже аналит имобилисан на чврстом носачу везивањем за други рецептор који није нуклеинска киселина, и са друге стране да специфично веже маркер нуклеинске киселине. Поступци за специфично откривање присуства маркера нуклеинске киселине у комплексу везног молекула са аналитом, који укључују амплификацију нуклеинске киселине у ПЦР реакцији у реалном времену, и указују на присуство аналита у поменутом узорку, су такође обезбеђени. Предметни проналазак може да буде коришћен за изузетно осетљиву и специфичну детекцију широког спектра анaлита, за који могу да се произведу специфична антитела, у области медицине, прехрамбене индустрије и биотехнологије., The present invention belongs to the field of bioanalytics and relates to universal kit for amplification of secondary antibodies signal in ELISA assays by immuno-PCR. Kit komponents include nucleic acid marker comprising of predetermined nucleotide sequence, as well as linker molecule, which is on one hand capable of specifically binding non-nucleic acid receptor that is capable of binding to solid surface-immobilized analyte, and on other hand is capable to specifically bind nucleic acid marker. Methods for specific detection of presence of nucleic acid marker in complex with analyte through linker molecule, that include amplification of nucleic acid marker in real-time PCR, wherein the presence of nucleic acid markers indicates presence of analyte in said sample, are also provided. The present invention can be used for sensitive and specific detection of wide range of analytes, for which specific antibodies can be produced, in the field of medicine, food industry and biotechnology.",
publisher = "Zavod za intelektualnu svojinu Republike Srbije",
journal = "Glasnik intelektualne svojine",
title = "Kit za amplifikaciju signala sekundarnih antitela u indirektnim ELISA esejima imuno-PCR-om",
number = "9",
url = "https://hdl.handle.net/21.15107/rcub_cherry_6007"
}

Ćirković-Veličković, T., Radomirović, M. Ž.,& Stanić-Vučinić, D.. (2023). Kit za amplifikaciju signala sekundarnih antitela u indirektnim ELISA esejima imuno-PCR-om. in Glasnik intelektualne svojine
Zavod za intelektualnu svojinu Republike Srbije.(9).
https://hdl.handle.net/21.15107/rcub_cherry_6007

Ćirković-Veličković T, Radomirović MŽ, Stanić-Vučinić D. Kit za amplifikaciju signala sekundarnih antitela u indirektnim ELISA esejima imuno-PCR-om. in Glasnik intelektualne svojine. 2023;(9).
https://hdl.handle.net/21.15107/rcub_cherry_6007 .

Ćirković-Veličković, Tanja, Radomirović, Mirjana Ž., Stanić-Vučinić, Dragana, "Kit za amplifikaciju signala sekundarnih antitela u indirektnim ELISA esejima imuno-PCR-om" in Glasnik intelektualne svojine, no. 9 (2023),
https://hdl.handle.net/21.15107/rcub_cherry_6007 .

TY  - CONF
AU  - Nedić, Olgica
AU  - Gligorijević, Nikola
AU  - Penezić, Ana
AU  - Minić, Simeon
AU  - Radomirović, Mirjana Ž.
AU  - Nikolić, Milan
AU  - Ćirković-Veličković, Tanja
PY  - 2022
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/6031
AB  - Our research work was focused on interactions between resveratrol (R) and fibrinogen (I), (dihydro)alpha-lipoic acid (ALA) and fibrinogen or albumin, and phycocyanobilin (PCB) and catalase. Resveratrol is found in grapes and berries, leafy greens are a source of ALA and alga Spirulina is a source of PCB. L-P interactions were investigated by following-up structural changes of proteins and/or ligands using spectrometric methods (spectrofluorimetry, CD, FTIR) and by examining the primary role of individual proteins upon ligand binding.
PB  - Belgrade : Faculty of Agriculture
C3  - 1st European Symposium on Phytochemicals in Medicine and Food, 7th-9th September, 2022. In: Book of Abstracts
T1  - Food antioxidants and their interaction with human proteins
SP  - 13
EP  - 13
UR  - https://hdl.handle.net/21.15107/rcub_cherry_6031
ER  - 

@conference{
author = "Nedić, Olgica and Gligorijević, Nikola and Penezić, Ana and Minić, Simeon and Radomirović, Mirjana Ž. and Nikolić, Milan and Ćirković-Veličković, Tanja",
year = "2022",
abstract = "Our research work was focused on interactions between resveratrol (R) and fibrinogen (I), (dihydro)alpha-lipoic acid (ALA) and fibrinogen or albumin, and phycocyanobilin (PCB) and catalase. Resveratrol is found in grapes and berries, leafy greens are a source of ALA and alga Spirulina is a source of PCB. L-P interactions were investigated by following-up structural changes of proteins and/or ligands using spectrometric methods (spectrofluorimetry, CD, FTIR) and by examining the primary role of individual proteins upon ligand binding.",
publisher = "Belgrade : Faculty of Agriculture",
journal = "1st European Symposium on Phytochemicals in Medicine and Food, 7th-9th September, 2022. In: Book of Abstracts",
title = "Food antioxidants and their interaction with human proteins",
pages = "13-13",
url = "https://hdl.handle.net/21.15107/rcub_cherry_6031"
}

Nedić, O., Gligorijević, N., Penezić, A., Minić, S., Radomirović, M. Ž., Nikolić, M.,& Ćirković-Veličković, T.. (2022). Food antioxidants and their interaction with human proteins. in 1st European Symposium on Phytochemicals in Medicine and Food, 7th-9th September, 2022. In: Book of Abstracts
Belgrade : Faculty of Agriculture., 13-13.
https://hdl.handle.net/21.15107/rcub_cherry_6031

Nedić O, Gligorijević N, Penezić A, Minić S, Radomirović MŽ, Nikolić M, Ćirković-Veličković T. Food antioxidants and their interaction with human proteins. in 1st European Symposium on Phytochemicals in Medicine and Food, 7th-9th September, 2022. In: Book of Abstracts. 2022;:13-13.
https://hdl.handle.net/21.15107/rcub_cherry_6031 .

Nedić, Olgica, Gligorijević, Nikola, Penezić, Ana, Minić, Simeon, Radomirović, Mirjana Ž., Nikolić, Milan, Ćirković-Veličković, Tanja, "Food antioxidants and their interaction with human proteins" in 1st European Symposium on Phytochemicals in Medicine and Food, 7th-9th September, 2022. In: Book of Abstracts (2022):13-13,
https://hdl.handle.net/21.15107/rcub_cherry_6031 .

TY  - CONF
AU  - Pismestrović, Marina
AU  - Radomirović, Mirjana Ž.
AU  - Čolaković, Maša
AU  - Ćirković-Veličković, Tanja
PY  - 2022
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/6026
AB  - Tropomyosin (TPM) is a major allergen among different shellfish species. Developing sensitive, specific, and reliable methods for quantifying TPM in food products is crucial for persons allergic to shellfish. Commonly used extraction buffers often show shortcomings in their extraction efficiency, which is why sometimes the presence of some allergens can be overlooked in the biological material. Therefore, this work aimed to optimize Mediterranean mussel TPM extraction conditions and develop a sandwich ELISA method for TPM quantification. Several extraction buffers were tested for their efficiency in recovering proteins from fresh frozen and cooked mussels during 2 and 24 hours of extraction. The protein content was quantified using the Bradford protein assay. Protein components of soluble extracts were profiled using SDS-PAGE. TPM presence in soluble extracts was confirmed by Western blot using both monoclonal and polyclonal anti-TPM antibodies. Sandwich ELISA was developed and used to quantify TPM content. None of the extraction buffers showed a significant difference in total protein content between 2 and 24 hours of extraction, indicating that 2 hours is sufficient for protein recovery in both raw and cooked mussels. Significantly fewer proteins were extracted from cooked mussels compared to raw mussels. Densitometrically estimated TPM concentrations indicate that PBS containing 1M NaCl (PBSN) extracts around 40% more TPM than PBS. Carbonate buffers extract even three times higher amounts of TPM than traditionally used extraction buffer PBS. Developed sandwich ELISA has shown not to be reliable for quantifying TPM from mussels, significantly underestimating its concentration, as concluded by comparing TPM concentrations obtained by ELISA with those obtained densitometrically. Therefore, Western blot has been used as an alternative method for mussel TPM quantification. The linear range for TPM quantification using Western blot was between 1.25 and 10 µg/ml. TPM concentrations in mussel extracts estimated using Western blot correlated well with those calculated by densitometric gel analysis. Further work will be aimed at improving the sensitivity of the presented methods and developing new methods for TPM quantification.
PB  - Beograd : Srpsko hemijsko društvo
PB  - Beograd : Klub mladih hemičara Srbije
C3  - 8th Conference of Young Chemists of Serbia, Belgrade, Serbia, 29th October, 2022. In: Book of Abstracts
T1  - Optimization of extraction conditions of tropomyosin from Mediterranean mussel and its quantification by developed ELISA
SP  - 54
EP  - 54
UR  - https://hdl.handle.net/21.15107/rcub_cherry_6026
ER  - 

@conference{
author = "Pismestrović, Marina and Radomirović, Mirjana Ž. and Čolaković, Maša and Ćirković-Veličković, Tanja",
year = "2022",
abstract = "Tropomyosin (TPM) is a major allergen among different shellfish species. Developing sensitive, specific, and reliable methods for quantifying TPM in food products is crucial for persons allergic to shellfish. Commonly used extraction buffers often show shortcomings in their extraction efficiency, which is why sometimes the presence of some allergens can be overlooked in the biological material. Therefore, this work aimed to optimize Mediterranean mussel TPM extraction conditions and develop a sandwich ELISA method for TPM quantification. Several extraction buffers were tested for their efficiency in recovering proteins from fresh frozen and cooked mussels during 2 and 24 hours of extraction. The protein content was quantified using the Bradford protein assay. Protein components of soluble extracts were profiled using SDS-PAGE. TPM presence in soluble extracts was confirmed by Western blot using both monoclonal and polyclonal anti-TPM antibodies. Sandwich ELISA was developed and used to quantify TPM content. None of the extraction buffers showed a significant difference in total protein content between 2 and 24 hours of extraction, indicating that 2 hours is sufficient for protein recovery in both raw and cooked mussels. Significantly fewer proteins were extracted from cooked mussels compared to raw mussels. Densitometrically estimated TPM concentrations indicate that PBS containing 1M NaCl (PBSN) extracts around 40% more TPM than PBS. Carbonate buffers extract even three times higher amounts of TPM than traditionally used extraction buffer PBS. Developed sandwich ELISA has shown not to be reliable for quantifying TPM from mussels, significantly underestimating its concentration, as concluded by comparing TPM concentrations obtained by ELISA with those obtained densitometrically. Therefore, Western blot has been used as an alternative method for mussel TPM quantification. The linear range for TPM quantification using Western blot was between 1.25 and 10 µg/ml. TPM concentrations in mussel extracts estimated using Western blot correlated well with those calculated by densitometric gel analysis. Further work will be aimed at improving the sensitivity of the presented methods and developing new methods for TPM quantification.",
publisher = "Beograd : Srpsko hemijsko društvo, Beograd : Klub mladih hemičara Srbije",
journal = "8th Conference of Young Chemists of Serbia, Belgrade, Serbia, 29th October, 2022. In: Book of Abstracts",
title = "Optimization of extraction conditions of tropomyosin from Mediterranean mussel and its quantification by developed ELISA",
pages = "54-54",
url = "https://hdl.handle.net/21.15107/rcub_cherry_6026"
}

Pismestrović, M., Radomirović, M. Ž., Čolaković, M.,& Ćirković-Veličković, T.. (2022). Optimization of extraction conditions of tropomyosin from Mediterranean mussel and its quantification by developed ELISA. in 8th Conference of Young Chemists of Serbia, Belgrade, Serbia, 29th October, 2022. In: Book of Abstracts
Beograd : Srpsko hemijsko društvo., 54-54.
https://hdl.handle.net/21.15107/rcub_cherry_6026

Pismestrović M, Radomirović MŽ, Čolaković M, Ćirković-Veličković T. Optimization of extraction conditions of tropomyosin from Mediterranean mussel and its quantification by developed ELISA. in 8th Conference of Young Chemists of Serbia, Belgrade, Serbia, 29th October, 2022. In: Book of Abstracts. 2022;:54-54.
https://hdl.handle.net/21.15107/rcub_cherry_6026 .

Pismestrović, Marina, Radomirović, Mirjana Ž., Čolaković, Maša, Ćirković-Veličković, Tanja, "Optimization of extraction conditions of tropomyosin from Mediterranean mussel and its quantification by developed ELISA" in 8th Conference of Young Chemists of Serbia, Belgrade, Serbia, 29th October, 2022. In: Book of Abstracts (2022):54-54,
https://hdl.handle.net/21.15107/rcub_cherry_6026 .

TY  - CONF
AU  - Radomirović, Mirjana Ž.
AU  - Čolaković, Maša
AU  - Pismestrović, Marina
AU  - Gligorijević, Nikola
AU  - Stanić-Vučinić, Dragana
AU  - Rajković, Andreja
AU  - Ćirković-Veličković, Tanja
PY  - 2022
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/6027
AB  - Tropomyosin (TPM) is considered a major allergen among different shellfish species. Therefore, the development of methods for quantifying TPM in food products is crucial for allergic persons. Several extraction buffers were tested for their efficiency in recovering proteins from fresh frozen and cooked razor mud shrimp during 2 and 24 hours of extraction. The protein content was quantified using the Bradford protein assay. SDS-PAGE was used for protein profiling of soluble extracts. Sandwich ELISA was developed and used to quantify TPM content. None of the extraction buffers showed a significant difference in total protein content between 2 and 24 hours of extraction. Significantly fewer proteins were extracted from cooked shrimp compared to the raw shrimp. ELISA quantification showed that phosphate-buffered saline (PBS) containing 1 M NaCl (PBSN) and carbonate buffer, pH 10, extracted approximately 6 times higher amount of tropomyosin in comparison to PBS, highlighting the importance of choosing the appropriate extraction buffer for the precise quantification of TPM. Traditionally used extraction buffer PBS could significantly underestimate shrimp TPM content.
PB  - Beograd : Srpsko hemijsko društvo
C3  - 58th Meeting of the Serbian Chemical Society, Belgrade, Serbia, 9th-10th June, 2022. In: Book of Abstracts and Proceedings
T1  - Optimization of extraction conditions of tropomyosin from razor mud shrimp and its quantification by developed ELISA
SP  - 64
EP  - 64
UR  - https://hdl.handle.net/21.15107/rcub_cherry_6027
ER  - 

@conference{
author = "Radomirović, Mirjana Ž. and Čolaković, Maša and Pismestrović, Marina and Gligorijević, Nikola and Stanić-Vučinić, Dragana and Rajković, Andreja and Ćirković-Veličković, Tanja",
year = "2022",
abstract = "Tropomyosin (TPM) is considered a major allergen among different shellfish species. Therefore, the development of methods for quantifying TPM in food products is crucial for allergic persons. Several extraction buffers were tested for their efficiency in recovering proteins from fresh frozen and cooked razor mud shrimp during 2 and 24 hours of extraction. The protein content was quantified using the Bradford protein assay. SDS-PAGE was used for protein profiling of soluble extracts. Sandwich ELISA was developed and used to quantify TPM content. None of the extraction buffers showed a significant difference in total protein content between 2 and 24 hours of extraction. Significantly fewer proteins were extracted from cooked shrimp compared to the raw shrimp. ELISA quantification showed that phosphate-buffered saline (PBS) containing 1 M NaCl (PBSN) and carbonate buffer, pH 10, extracted approximately 6 times higher amount of tropomyosin in comparison to PBS, highlighting the importance of choosing the appropriate extraction buffer for the precise quantification of TPM. Traditionally used extraction buffer PBS could significantly underestimate shrimp TPM content.",
publisher = "Beograd : Srpsko hemijsko društvo",
journal = "58th Meeting of the Serbian Chemical Society, Belgrade, Serbia, 9th-10th June, 2022. In: Book of Abstracts and Proceedings",
title = "Optimization of extraction conditions of tropomyosin from razor mud shrimp and its quantification by developed ELISA",
pages = "64-64",
url = "https://hdl.handle.net/21.15107/rcub_cherry_6027"
}

Radomirović, M. Ž., Čolaković, M., Pismestrović, M., Gligorijević, N., Stanić-Vučinić, D., Rajković, A.,& Ćirković-Veličković, T.. (2022). Optimization of extraction conditions of tropomyosin from razor mud shrimp and its quantification by developed ELISA. in 58th Meeting of the Serbian Chemical Society, Belgrade, Serbia, 9th-10th June, 2022. In: Book of Abstracts and Proceedings
Beograd : Srpsko hemijsko društvo., 64-64.
https://hdl.handle.net/21.15107/rcub_cherry_6027

Radomirović MŽ, Čolaković M, Pismestrović M, Gligorijević N, Stanić-Vučinić D, Rajković A, Ćirković-Veličković T. Optimization of extraction conditions of tropomyosin from razor mud shrimp and its quantification by developed ELISA. in 58th Meeting of the Serbian Chemical Society, Belgrade, Serbia, 9th-10th June, 2022. In: Book of Abstracts and Proceedings. 2022;:64-64.
https://hdl.handle.net/21.15107/rcub_cherry_6027 .

Radomirović, Mirjana Ž., Čolaković, Maša, Pismestrović, Marina, Gligorijević, Nikola, Stanić-Vučinić, Dragana, Rajković, Andreja, Ćirković-Veličković, Tanja, "Optimization of extraction conditions of tropomyosin from razor mud shrimp and its quantification by developed ELISA" in 58th Meeting of the Serbian Chemical Society, Belgrade, Serbia, 9th-10th June, 2022. In: Book of Abstracts and Proceedings (2022):64-64,
https://hdl.handle.net/21.15107/rcub_cherry_6027 .

TY  - CONF
AU  - Simović, Ana
AU  - Radomirović, Mirjana Ž.
AU  - Gligorijević, Nikola
AU  - Stanić-Vučinić, Dragana
AU  - Minić, Simeon L.
AU  - Nikolić, Milan
AU  - Ćirković-Veličković, Tanja
PY  - 2022
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/5520
AB  - The emergence of the coronavirus SARS-CoV-2 has attracted attention of the whole scientific community. The SARS-CoV-2 spike (S) protein plays the most important role in viral attachment to host receptor angiotensin-converting enzyme 2 (ACE2), via the receptor-binding domain (RBD), fusion and entry into the host, and it serves as a target for the development of antibodies, entry inhibitors and vaccines. It has been demonstrated that phycocyanobilin (PCB), a bioactive open-chain tetrapyrrole chromophore of phycocyanin (PC), chromoprotein derived from the cyanobacterium Arthrospira platensis, can bind a plethora of different proteins, both in a noncovalent and covalent manner. This study aimed to investigate interactions of PCB with S protein and RBD respectively. Electrophoretic techniques, fluorescence spectroscopy, and inhibition of S–PCB and RBD–PCB covalent adduct formation using iodoacetamide and N-ethylmaleimide, were employed to examine interactions of PCB with S protein and RBD, while the effects of PCB binding on RBD structure were studied by CD spectroscopy. SDS-PAGE with Zn2+ staining has revealed that PCB covalently binds to both S protein and RBD, via free cysteine residues. Binding constants determined by the fluorescence quenching method were: 2.1×107 M–1 for PCB and S protein and 8.4×104 M–1 for PCB and RBD. Far-UV circular dichroism spectra showed that the binding of PCB influences RBD structure by decreasing the disordered structure content. Due to moderately strong noncovalent interactions of PCB with S protein and RBD, as well as covalent adducts formation, it may exert one of its many bioactive effects via impact on S protein binding to ACE2 receptor.
PB  - Faculty of Chemistry, Serbian Biochemical Society
C3  - Serbian Biochemical Society Eleventh Conference, Scientific meeting of an international character, September 22nd and 23rd, 2022, Novi Sad, Serbia
T1  - Noncovalent and covalent binding of phycocyanobilin to S protein of SARS-CoV-2 and its receptor-binding domain
SP  - 130
EP  - 131
UR  - https://hdl.handle.net/21.15107/rcub_cherry_5520
ER  - 

@conference{
author = "Simović, Ana and Radomirović, Mirjana Ž. and Gligorijević, Nikola and Stanić-Vučinić, Dragana and Minić, Simeon L. and Nikolić, Milan and Ćirković-Veličković, Tanja",
year = "2022",
abstract = "The emergence of the coronavirus SARS-CoV-2 has attracted attention of the whole scientific community. The SARS-CoV-2 spike (S) protein plays the most important role in viral attachment to host receptor angiotensin-converting enzyme 2 (ACE2), via the receptor-binding domain (RBD), fusion and entry into the host, and it serves as a target for the development of antibodies, entry inhibitors and vaccines. It has been demonstrated that phycocyanobilin (PCB), a bioactive open-chain tetrapyrrole chromophore of phycocyanin (PC), chromoprotein derived from the cyanobacterium Arthrospira platensis, can bind a plethora of different proteins, both in a noncovalent and covalent manner. This study aimed to investigate interactions of PCB with S protein and RBD respectively. Electrophoretic techniques, fluorescence spectroscopy, and inhibition of S–PCB and RBD–PCB covalent adduct formation using iodoacetamide and N-ethylmaleimide, were employed to examine interactions of PCB with S protein and RBD, while the effects of PCB binding on RBD structure were studied by CD spectroscopy. SDS-PAGE with Zn2+ staining has revealed that PCB covalently binds to both S protein and RBD, via free cysteine residues. Binding constants determined by the fluorescence quenching method were: 2.1×107 M–1 for PCB and S protein and 8.4×104 M–1 for PCB and RBD. Far-UV circular dichroism spectra showed that the binding of PCB influences RBD structure by decreasing the disordered structure content. Due to moderately strong noncovalent interactions of PCB with S protein and RBD, as well as covalent adducts formation, it may exert one of its many bioactive effects via impact on S protein binding to ACE2 receptor.",
publisher = "Faculty of Chemistry, Serbian Biochemical Society",
journal = "Serbian Biochemical Society Eleventh Conference, Scientific meeting of an international character, September 22nd and 23rd, 2022, Novi Sad, Serbia",
title = "Noncovalent and covalent binding of phycocyanobilin to S protein of SARS-CoV-2 and its receptor-binding domain",
pages = "130-131",
url = "https://hdl.handle.net/21.15107/rcub_cherry_5520"
}

Simović, A., Radomirović, M. Ž., Gligorijević, N., Stanić-Vučinić, D., Minić, S. L., Nikolić, M.,& Ćirković-Veličković, T.. (2022). Noncovalent and covalent binding of phycocyanobilin to S protein of SARS-CoV-2 and its receptor-binding domain. in Serbian Biochemical Society Eleventh Conference, Scientific meeting of an international character, September 22nd and 23rd, 2022, Novi Sad, Serbia
Faculty of Chemistry, Serbian Biochemical Society., 130-131.
https://hdl.handle.net/21.15107/rcub_cherry_5520

Simović A, Radomirović MŽ, Gligorijević N, Stanić-Vučinić D, Minić SL, Nikolić M, Ćirković-Veličković T. Noncovalent and covalent binding of phycocyanobilin to S protein of SARS-CoV-2 and its receptor-binding domain. in Serbian Biochemical Society Eleventh Conference, Scientific meeting of an international character, September 22nd and 23rd, 2022, Novi Sad, Serbia. 2022;:130-131.
https://hdl.handle.net/21.15107/rcub_cherry_5520 .

Simović, Ana, Radomirović, Mirjana Ž., Gligorijević, Nikola, Stanić-Vučinić, Dragana, Minić, Simeon L., Nikolić, Milan, Ćirković-Veličković, Tanja, "Noncovalent and covalent binding of phycocyanobilin to S protein of SARS-CoV-2 and its receptor-binding domain" in Serbian Biochemical Society Eleventh Conference, Scientific meeting of an international character, September 22nd and 23rd, 2022, Novi Sad, Serbia (2022):130-131,
https://hdl.handle.net/21.15107/rcub_cherry_5520 .

TY  - CONF
AU  - Radomirović, Mirjana Ž.
AU  - Gligorijević, Nikola
AU  - Rajković, Andreja
AU  - Ćirković-Veličković, Tanja
PY  - 2022
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/6022
AB  - Tropomyosin (TPM) is considered a major allergen among different shellfish species. Developing sensitive, specific, and reliable methods for quantifying TPM in food products is crucial for persons allergic to shellfish. We have previously developed a highly sensitive sandwich ELISA method for quantifying shrimp TPM. Despite high amino acid sequence homology between shrimp and mussels TPM, the method has not been reliable for quantifying TPM from mussels, underestimating its concentration up to three orders of magnitude. Therefore, this work aimed to develop alternative immunological methods for mussel TPM quantification. Western blot, dot blot, and indirect ELISA using monoclonal anti-TPM antibody and alkaline phosphatase-labeled secondary antibody were developed and compared in terms of their sensitivity. Tropomyosin in mussels extracts was quantified using highly purified natural shrimp tropomyosin as standard. The linear range for TPM quantification using dot blot was between 5 and 50 µg/ml, while Western blot has slightly increased sensitivity, with a linear range between 1.25 and 12.5 µg/ml. Indirect ELISA has further improved the sensitivity of TPM quantification, with a 0.04-0.4 µg/ml linear range. Additional work will be performed to enhance the sensitivity of the presented methods, with the final aim of reducing risks of inadvertent food contamination.
PB  - Belgrade : Faculty of Chemistry
PB  - Belgrade : Serbian Biochemical Society
C3  - Serbian Biochemical Society Eleventh Conference, 22nd-23rd September, 2022. In: Conference Proceedings
T1  - Development and comparison of Western blot, dot blot and ELISA for mussels tropomyosin quantification
SP  - 125
EP  - 125
UR  - https://hdl.handle.net/21.15107/rcub_cherry_6022
ER  - 

@conference{
author = "Radomirović, Mirjana Ž. and Gligorijević, Nikola and Rajković, Andreja and Ćirković-Veličković, Tanja",
year = "2022",
abstract = "Tropomyosin (TPM) is considered a major allergen among different shellfish species. Developing sensitive, specific, and reliable methods for quantifying TPM in food products is crucial for persons allergic to shellfish. We have previously developed a highly sensitive sandwich ELISA method for quantifying shrimp TPM. Despite high amino acid sequence homology between shrimp and mussels TPM, the method has not been reliable for quantifying TPM from mussels, underestimating its concentration up to three orders of magnitude. Therefore, this work aimed to develop alternative immunological methods for mussel TPM quantification. Western blot, dot blot, and indirect ELISA using monoclonal anti-TPM antibody and alkaline phosphatase-labeled secondary antibody were developed and compared in terms of their sensitivity. Tropomyosin in mussels extracts was quantified using highly purified natural shrimp tropomyosin as standard. The linear range for TPM quantification using dot blot was between 5 and 50 µg/ml, while Western blot has slightly increased sensitivity, with a linear range between 1.25 and 12.5 µg/ml. Indirect ELISA has further improved the sensitivity of TPM quantification, with a 0.04-0.4 µg/ml linear range. Additional work will be performed to enhance the sensitivity of the presented methods, with the final aim of reducing risks of inadvertent food contamination.",
publisher = "Belgrade : Faculty of Chemistry, Belgrade : Serbian Biochemical Society",
journal = "Serbian Biochemical Society Eleventh Conference, 22nd-23rd September, 2022. In: Conference Proceedings",
title = "Development and comparison of Western blot, dot blot and ELISA for mussels tropomyosin quantification",
pages = "125-125",
url = "https://hdl.handle.net/21.15107/rcub_cherry_6022"
}

Radomirović, M. Ž., Gligorijević, N., Rajković, A.,& Ćirković-Veličković, T.. (2022). Development and comparison of Western blot, dot blot and ELISA for mussels tropomyosin quantification. in Serbian Biochemical Society Eleventh Conference, 22nd-23rd September, 2022. In: Conference Proceedings
Belgrade : Faculty of Chemistry., 125-125.
https://hdl.handle.net/21.15107/rcub_cherry_6022

Radomirović MŽ, Gligorijević N, Rajković A, Ćirković-Veličković T. Development and comparison of Western blot, dot blot and ELISA for mussels tropomyosin quantification. in Serbian Biochemical Society Eleventh Conference, 22nd-23rd September, 2022. In: Conference Proceedings. 2022;:125-125.
https://hdl.handle.net/21.15107/rcub_cherry_6022 .

Radomirović, Mirjana Ž., Gligorijević, Nikola, Rajković, Andreja, Ćirković-Veličković, Tanja, "Development and comparison of Western blot, dot blot and ELISA for mussels tropomyosin quantification" in Serbian Biochemical Society Eleventh Conference, 22nd-23rd September, 2022. In: Conference Proceedings (2022):125-125,
https://hdl.handle.net/21.15107/rcub_cherry_6022 .

TY  - CONF
AU  - Radomirović, Mirjana Ž.
AU  - Gligorijević, Nikola
AU  - Minić, Simeon
AU  - Nikolić, Milan
AU  - Stanić-Vučinić, Dragana
AU  - Ćirković-Veličković, Tanja
PY  - 2022
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/6029
AB  - Phycocyanobilin (PCB) is an open-chain tetrapyrrole chromophore of phycocyanin (PC), chromoprotein derived from the cyanobacterium Arthrospira platensis. Our group has previously demonstrated the potential of PCB to covalently modify free cysteine residue of proteins using food protein β-lactoglobulin as a model protein. Relying on the proven ability of PCB to be attached to sulfhydryl groups of proteins, we propose a new method for covalent attachment of PCB to potentially any protein. We used Traut’s reagent (TR, 2-iminothiolane) to introduce free sulfhydryl groups in the model protein, bovine serum albumin (BSA), by modifying its lysine residues. All tested molar ratios of TR per mole of protein successfully modified BSA. A higher degree of modification by TR induced more profound alterations of BSA structure, as evidenced by near-UV and far-UV circular dichroism spectroscopy. At the same time, minor changes in BSA oligomerization and aggregation profile occurred with increasing TR molar ratio. PCB was covalently attached to introduced sulfhydryl groups at pH 9 at 20–fold molar ratio of PCB per mole of protein. An increase in the molar ratio of TR per mole of BSA leads to amplification of fluorescent signal of PCB-modified BSA, most significantly observed starting from 50-fold and higher TR ratios. Using BSA as a model protein, a 50-fold molar excess of TR seems to be the optimal choice for balancing a satisfactory signal amplification level and the negative effect on protein structure. BSA covalently modified with PCB has higher antioxidative activity than free BSA. The proposed method thus serves as a proof of concept for labeling virtually any protein with PCB as means of either functionalization through covalent attachment of bioactive PCB or obtaining fluorescent probes for application in fluorescence-based techniques.
PB  - Federation of European Biochemical Societies
PB  - Wiley
C3  - The Biochemistry Global Summit, 9th-14th July, 2022. In: FEBS Open Bio
T1  - Covalent modification of bovine serum albumin with phycocyanobilin using Traut’s reagent
VL  - 12
IS  - Suppl. 1
SP  - 302
EP  - 303
UR  - https://hdl.handle.net/21.15107/rcub_cherry_6029
ER  - 

@conference{
author = "Radomirović, Mirjana Ž. and Gligorijević, Nikola and Minić, Simeon and Nikolić, Milan and Stanić-Vučinić, Dragana and Ćirković-Veličković, Tanja",
year = "2022",
abstract = "Phycocyanobilin (PCB) is an open-chain tetrapyrrole chromophore of phycocyanin (PC), chromoprotein derived from the cyanobacterium Arthrospira platensis. Our group has previously demonstrated the potential of PCB to covalently modify free cysteine residue of proteins using food protein β-lactoglobulin as a model protein. Relying on the proven ability of PCB to be attached to sulfhydryl groups of proteins, we propose a new method for covalent attachment of PCB to potentially any protein. We used Traut’s reagent (TR, 2-iminothiolane) to introduce free sulfhydryl groups in the model protein, bovine serum albumin (BSA), by modifying its lysine residues. All tested molar ratios of TR per mole of protein successfully modified BSA. A higher degree of modification by TR induced more profound alterations of BSA structure, as evidenced by near-UV and far-UV circular dichroism spectroscopy. At the same time, minor changes in BSA oligomerization and aggregation profile occurred with increasing TR molar ratio. PCB was covalently attached to introduced sulfhydryl groups at pH 9 at 20–fold molar ratio of PCB per mole of protein. An increase in the molar ratio of TR per mole of BSA leads to amplification of fluorescent signal of PCB-modified BSA, most significantly observed starting from 50-fold and higher TR ratios. Using BSA as a model protein, a 50-fold molar excess of TR seems to be the optimal choice for balancing a satisfactory signal amplification level and the negative effect on protein structure. BSA covalently modified with PCB has higher antioxidative activity than free BSA. The proposed method thus serves as a proof of concept for labeling virtually any protein with PCB as means of either functionalization through covalent attachment of bioactive PCB or obtaining fluorescent probes for application in fluorescence-based techniques.",
publisher = "Federation of European Biochemical Societies, Wiley",
journal = "The Biochemistry Global Summit, 9th-14th July, 2022. In: FEBS Open Bio",
title = "Covalent modification of bovine serum albumin with phycocyanobilin using Traut’s reagent",
volume = "12",
number = "Suppl. 1",
pages = "302-303",
url = "https://hdl.handle.net/21.15107/rcub_cherry_6029"
}

Radomirović, M. Ž., Gligorijević, N., Minić, S., Nikolić, M., Stanić-Vučinić, D.,& Ćirković-Veličković, T.. (2022). Covalent modification of bovine serum albumin with phycocyanobilin using Traut’s reagent. in The Biochemistry Global Summit, 9th-14th July, 2022. In: FEBS Open Bio
Federation of European Biochemical Societies., 12(Suppl. 1), 302-303.
https://hdl.handle.net/21.15107/rcub_cherry_6029

Radomirović MŽ, Gligorijević N, Minić S, Nikolić M, Stanić-Vučinić D, Ćirković-Veličković T. Covalent modification of bovine serum albumin with phycocyanobilin using Traut’s reagent. in The Biochemistry Global Summit, 9th-14th July, 2022. In: FEBS Open Bio. 2022;12(Suppl. 1):302-303.
https://hdl.handle.net/21.15107/rcub_cherry_6029 .

Radomirović, Mirjana Ž., Gligorijević, Nikola, Minić, Simeon, Nikolić, Milan, Stanić-Vučinić, Dragana, Ćirković-Veličković, Tanja, "Covalent modification of bovine serum albumin with phycocyanobilin using Traut’s reagent" in The Biochemistry Global Summit, 9th-14th July, 2022. In: FEBS Open Bio, 12, no. Suppl. 1 (2022):302-303,
https://hdl.handle.net/21.15107/rcub_cherry_6029 .

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  - JOUR
AU  - Stanić-Vučinić, Dragana
AU  - Stojadinović, Marija M.
AU  - Radomirović, Mirjana Ž.
AU  - Simović, Ana
AU  - Radibratović, Milica
AU  - Ćirković-Veličković, Tanja
PY  - 2022
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/4884
AB  - Background: The world’s production of whey is estimated to be more than 200 milliontons per year. Although whey is an important source of proteins with high nutritional value andbiotechnological importance, it is still considered as a by-product of the dairy industry with loweconomic value due to low industrial exploitation. There are several challenges in the separation ofwhey proteins: low concentration, the complexity of the material and similar properties (pI, molecularmass) of some proteins.Methods: A narrative review of all the relevant papers on the present methodologies based on ionexchange and adsorption principles for isolation of whey proteins, known to the authors, was conductedResults: Traditional ion exchange techniques are widely used for the separation and purification ofthe bovine whey proteins. These methodologies, based on the anion or cation chromatographicprocedures, as well as the combination of aforementioned techniques are still preferential methodsfor the isolation of the whey proteins on the laboratory scale. However, more recent research on ionexchange membranes for this purpose has been introduced, with promising potential to be appliedon the pilot industrial scale. Newly developed methodologies based either on the ion exchangeseparation (for example: simulated moving bed chromatography, expanded bed adsorption, magneticion exchangers, etc.) or adsorption (for example: adsorption on hydroxyapatite or activatedcarbon, or molecular imprinting) are promising approaches for scaling up of the whey proteins’purification processes.Conclusion: Many procedures based on ion exchange are successfully implemented for the separationand purification of whey proteins, providing protein preparations of moderate-to-high yieldand satisfactory purity. However, the authors anticipate further development of adsorption-basedmethodologies for the separation of whey proteins by targeting the differences in proteins’ structuresrather than targeting the differences in molecular masses and pI. The complex composite multilayeredmatrices, including also inorganic components, are promising materials for simultaneousexploiting of the differences in the masses, pI and structures of whey proteins for the separation.
PB  - Bentham Science
T2  - Current Analytical Chemistry
T1  - Application of Ion Exchange and Adsorption Techniques for Separation of Whey Proteins from Bovine Milk
VL  - 18
IS  - 3
SP  - 341
EP  - 359
DO  - 10.2174/1573411017666210108092338
UR  - https://hdl.handle.net/21.15107/rcub_cherry_4884
ER  - 

@article{
author = "Stanić-Vučinić, Dragana and Stojadinović, Marija M. and Radomirović, Mirjana Ž. and Simović, Ana and Radibratović, Milica and Ćirković-Veličković, Tanja",
year = "2022",
abstract = "Background: The world’s production of whey is estimated to be more than 200 milliontons per year. Although whey is an important source of proteins with high nutritional value andbiotechnological importance, it is still considered as a by-product of the dairy industry with loweconomic value due to low industrial exploitation. There are several challenges in the separation ofwhey proteins: low concentration, the complexity of the material and similar properties (pI, molecularmass) of some proteins.Methods: A narrative review of all the relevant papers on the present methodologies based on ionexchange and adsorption principles for isolation of whey proteins, known to the authors, was conductedResults: Traditional ion exchange techniques are widely used for the separation and purification ofthe bovine whey proteins. These methodologies, based on the anion or cation chromatographicprocedures, as well as the combination of aforementioned techniques are still preferential methodsfor the isolation of the whey proteins on the laboratory scale. However, more recent research on ionexchange membranes for this purpose has been introduced, with promising potential to be appliedon the pilot industrial scale. Newly developed methodologies based either on the ion exchangeseparation (for example: simulated moving bed chromatography, expanded bed adsorption, magneticion exchangers, etc.) or adsorption (for example: adsorption on hydroxyapatite or activatedcarbon, or molecular imprinting) are promising approaches for scaling up of the whey proteins’purification processes.Conclusion: Many procedures based on ion exchange are successfully implemented for the separationand purification of whey proteins, providing protein preparations of moderate-to-high yieldand satisfactory purity. However, the authors anticipate further development of adsorption-basedmethodologies for the separation of whey proteins by targeting the differences in proteins’ structuresrather than targeting the differences in molecular masses and pI. The complex composite multilayeredmatrices, including also inorganic components, are promising materials for simultaneousexploiting of the differences in the masses, pI and structures of whey proteins for the separation.",
publisher = "Bentham Science",
journal = "Current Analytical Chemistry",
title = "Application of Ion Exchange and Adsorption Techniques for Separation of Whey Proteins from Bovine Milk",
volume = "18",
number = "3",
pages = "341-359",
doi = "10.2174/1573411017666210108092338",
url = "https://hdl.handle.net/21.15107/rcub_cherry_4884"
}

Stanić-Vučinić, D., Stojadinović, M. M., Radomirović, M. Ž., Simović, A., Radibratović, M.,& Ćirković-Veličković, T.. (2022). Application of Ion Exchange and Adsorption Techniques for Separation of Whey Proteins from Bovine Milk. in Current Analytical Chemistry
Bentham Science., 18(3), 341-359.
https://doi.org/10.2174/1573411017666210108092338
https://hdl.handle.net/21.15107/rcub_cherry_4884

Stanić-Vučinić D, Stojadinović MM, Radomirović MŽ, Simović A, Radibratović M, Ćirković-Veličković T. Application of Ion Exchange and Adsorption Techniques for Separation of Whey Proteins from Bovine Milk. in Current Analytical Chemistry. 2022;18(3):341-359.
doi:10.2174/1573411017666210108092338
https://hdl.handle.net/21.15107/rcub_cherry_4884 .

Stanić-Vučinić, Dragana, Stojadinović, Marija M., Radomirović, Mirjana Ž., Simović, Ana, Radibratović, Milica, Ćirković-Veličković, Tanja, "Application of Ion Exchange and Adsorption Techniques for Separation of Whey Proteins from Bovine Milk" in Current Analytical Chemistry, 18, no. 3 (2022):341-359,
https://doi.org/10.2174/1573411017666210108092338 .,
https://hdl.handle.net/21.15107/rcub_cherry_4884 .

TY  - JOUR
AU  - Radosavljević, Jelena
AU  - Stanić-Vučinić, Dragana
AU  - Stojadinović, Marija M.
AU  - Radomirović, Mirjana Ž.
AU  - Simović, Ana
AU  - Radibratović, Milica
AU  - Ćirković-Veličković, Tanja
PY  - 2022
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/4883
AB  - Background: The world’s production of whey is estimated to be more than 200 milliontons per year. Although whey is an important source of proteins with high nutritional value andbiotechnological importance, it is still considered as a by-product of the dairy industry with loweconomic value due to low industrial exploitation. There are several challenges in the separation ofwhey proteins: low concentration, the complexity of the material and similar properties (pI, molecularmass) of some proteins.Methods: A narrative review of all the relevant papers on the present methodologies based on ionexchange and adsorption principles for isolation of whey proteins, known to the authors, was conductedResults: Traditional ion exchange techniques are widely used for the separation and purification ofthe bovine whey proteins. These methodologies, based on the anion or cation chromatographicprocedures, as well as the combination of aforementioned techniques are still preferential methodsfor the isolation of the whey proteins on the laboratory scale. However, more recent research on ionexchange membranes for this purpose has been introduced, with promising potential to be appliedon the pilot industrial scale. Newly developed methodologies based either on the ion exchangeseparation (for example: simulated moving bed chromatography, expanded bed adsorption, magneticion exchangers, etc.) or adsorption (for example: adsorption on hydroxyapatite or activatedcarbon, or molecular imprinting) are promising approaches for scaling up of the whey proteins’purification processes.Conclusion: Many procedures based on ion exchange are successfully implemented for the separationand purification of whey proteins, providing protein preparations of moderate-to-high yieldand satisfactory purity. However, the authors anticipate further development of adsorption-basedmethodologies for the separation of whey proteins by targeting the differences in proteins’ structuresrather than targeting the differences in molecular masses and pI. The complex composite multilayeredmatrices, including also inorganic components, are promising materials for simultaneousexploiting of the differences in the masses, pI and structures of whey proteins for the separation.
PB  - Bentham Science
T2  - Current Analytical Chemistry
T1  - Application of Ion Exchange and Adsorption Techniques for Separation of Whey Proteins from Bovine Milk
VL  - 18
IS  - 3
SP  - 341
EP  - 359
DO  - 10.2174/1573411017666210108092338
UR  - https://hdl.handle.net/21.15107/rcub_cherry_4883
ER  - 

@article{
author = "Radosavljević, Jelena and Stanić-Vučinić, Dragana and Stojadinović, Marija M. and Radomirović, Mirjana Ž. and Simović, Ana and Radibratović, Milica and Ćirković-Veličković, Tanja",
year = "2022",
abstract = "Background: The world’s production of whey is estimated to be more than 200 milliontons per year. Although whey is an important source of proteins with high nutritional value andbiotechnological importance, it is still considered as a by-product of the dairy industry with loweconomic value due to low industrial exploitation. There are several challenges in the separation ofwhey proteins: low concentration, the complexity of the material and similar properties (pI, molecularmass) of some proteins.Methods: A narrative review of all the relevant papers on the present methodologies based on ionexchange and adsorption principles for isolation of whey proteins, known to the authors, was conductedResults: Traditional ion exchange techniques are widely used for the separation and purification ofthe bovine whey proteins. These methodologies, based on the anion or cation chromatographicprocedures, as well as the combination of aforementioned techniques are still preferential methodsfor the isolation of the whey proteins on the laboratory scale. However, more recent research on ionexchange membranes for this purpose has been introduced, with promising potential to be appliedon the pilot industrial scale. Newly developed methodologies based either on the ion exchangeseparation (for example: simulated moving bed chromatography, expanded bed adsorption, magneticion exchangers, etc.) or adsorption (for example: adsorption on hydroxyapatite or activatedcarbon, or molecular imprinting) are promising approaches for scaling up of the whey proteins’purification processes.Conclusion: Many procedures based on ion exchange are successfully implemented for the separationand purification of whey proteins, providing protein preparations of moderate-to-high yieldand satisfactory purity. However, the authors anticipate further development of adsorption-basedmethodologies for the separation of whey proteins by targeting the differences in proteins’ structuresrather than targeting the differences in molecular masses and pI. The complex composite multilayeredmatrices, including also inorganic components, are promising materials for simultaneousexploiting of the differences in the masses, pI and structures of whey proteins for the separation.",
publisher = "Bentham Science",
journal = "Current Analytical Chemistry",
title = "Application of Ion Exchange and Adsorption Techniques for Separation of Whey Proteins from Bovine Milk",
volume = "18",
number = "3",
pages = "341-359",
doi = "10.2174/1573411017666210108092338",
url = "https://hdl.handle.net/21.15107/rcub_cherry_4883"
}

Radosavljević, J., Stanić-Vučinić, D., Stojadinović, M. M., Radomirović, M. Ž., Simović, A., Radibratović, M.,& Ćirković-Veličković, T.. (2022). Application of Ion Exchange and Adsorption Techniques for Separation of Whey Proteins from Bovine Milk. in Current Analytical Chemistry
Bentham Science., 18(3), 341-359.
https://doi.org/10.2174/1573411017666210108092338
https://hdl.handle.net/21.15107/rcub_cherry_4883

Radosavljević J, Stanić-Vučinić D, Stojadinović MM, Radomirović MŽ, Simović A, Radibratović M, Ćirković-Veličković T. Application of Ion Exchange and Adsorption Techniques for Separation of Whey Proteins from Bovine Milk. in Current Analytical Chemistry. 2022;18(3):341-359.
doi:10.2174/1573411017666210108092338
https://hdl.handle.net/21.15107/rcub_cherry_4883 .

Radosavljević, Jelena, Stanić-Vučinić, Dragana, Stojadinović, Marija M., Radomirović, Mirjana Ž., Simović, Ana, Radibratović, Milica, Ćirković-Veličković, Tanja, "Application of Ion Exchange and Adsorption Techniques for Separation of Whey Proteins from Bovine Milk" in Current Analytical Chemistry, 18, no. 3 (2022):341-359,
https://doi.org/10.2174/1573411017666210108092338 .,
https://hdl.handle.net/21.15107/rcub_cherry_4883 .

TY  - JOUR
AU  - Radomirović, Mirjana Ž.
AU  - Minić, Simeon L.
AU  - Stanić-Vučinić, Dragana
AU  - Nikolić, Milan
AU  - Van Haute, Sam
AU  - Rajković, Andreja
AU  - Ćirković-Veličković, Tanja
PY  - 2022
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/4863
AB  - β-lactoglobulin (BLG) is a major whey protein with numerous techno-functional properties desirable for the food industry. Phycocyanobilin (PCB), a bioactive pigment of Arthrospira platensis with health-promoting effects, covalently binds to BLG at physiological pH. This study investigated the effects of this covalent modification on BLG functional properties. The BLG–PCB adduct possesses enhanced antioxidant properties, and bound PCB protects BLG against free radical-induced oxidation. Despite the similar thermal stabilities of BLG and BLG–PCB, BLG–PCB is less susceptible to covalent and noncovalent aggregation under moderate heat treatment (63 °C, 30 min). Blocked thiol group and reduced hydrophobicity due to hindering of hydrophobic residues by bound PCB, as well as the heat-induced transition of β-sheet to α-helix, contributed to the low susceptibility of BLG–PCB to aggregation. BLG–PCB has a higher resistance to pepsin and pancreatin digestion than BLG and unaltered IgE-binding properties. The improved functional properties of BLG–PCB make it a useful ingredient in the food industry.
PB  - Elsevier
T2  - Food Hydrocolloids
T1  - Phycocyanobilin-modified β-lactoglobulin exhibits increased antioxidant properties and stability to digestion and heating
VL  - 123
SP  - 107169
DO  - 10.1016/j.foodhyd.2021.107169
ER  - 

@article{
author = "Radomirović, Mirjana Ž. and Minić, Simeon L. and Stanić-Vučinić, Dragana and Nikolić, Milan and Van Haute, Sam and Rajković, Andreja and Ćirković-Veličković, Tanja",
year = "2022",
abstract = "β-lactoglobulin (BLG) is a major whey protein with numerous techno-functional properties desirable for the food industry. Phycocyanobilin (PCB), a bioactive pigment of Arthrospira platensis with health-promoting effects, covalently binds to BLG at physiological pH. This study investigated the effects of this covalent modification on BLG functional properties. The BLG–PCB adduct possesses enhanced antioxidant properties, and bound PCB protects BLG against free radical-induced oxidation. Despite the similar thermal stabilities of BLG and BLG–PCB, BLG–PCB is less susceptible to covalent and noncovalent aggregation under moderate heat treatment (63 °C, 30 min). Blocked thiol group and reduced hydrophobicity due to hindering of hydrophobic residues by bound PCB, as well as the heat-induced transition of β-sheet to α-helix, contributed to the low susceptibility of BLG–PCB to aggregation. BLG–PCB has a higher resistance to pepsin and pancreatin digestion than BLG and unaltered IgE-binding properties. The improved functional properties of BLG–PCB make it a useful ingredient in the food industry.",
publisher = "Elsevier",
journal = "Food Hydrocolloids",
title = "Phycocyanobilin-modified β-lactoglobulin exhibits increased antioxidant properties and stability to digestion and heating",
volume = "123",
pages = "107169",
doi = "10.1016/j.foodhyd.2021.107169"
}

Radomirović, M. Ž., Minić, S. L., Stanić-Vučinić, D., Nikolić, M., Van Haute, S., Rajković, A.,& Ćirković-Veličković, T.. (2022). Phycocyanobilin-modified β-lactoglobulin exhibits increased antioxidant properties and stability to digestion and heating. in Food Hydrocolloids
Elsevier., 123, 107169.
https://doi.org/10.1016/j.foodhyd.2021.107169

Radomirović MŽ, Minić SL, Stanić-Vučinić D, Nikolić M, Van Haute S, Rajković A, Ćirković-Veličković T. Phycocyanobilin-modified β-lactoglobulin exhibits increased antioxidant properties and stability to digestion and heating. in Food Hydrocolloids. 2022;123:107169.
doi:10.1016/j.foodhyd.2021.107169 .

Radomirović, Mirjana Ž., Minić, Simeon L., Stanić-Vučinić, Dragana, Nikolić, Milan, Van Haute, Sam, Rajković, Andreja, Ćirković-Veličković, Tanja, "Phycocyanobilin-modified β-lactoglobulin exhibits increased antioxidant properties and stability to digestion and heating" in Food Hydrocolloids, 123 (2022):107169,
https://doi.org/10.1016/j.foodhyd.2021.107169 . .

TY  - CONF
AU  - Radomirović, Mirjana Ž.
AU  - Gligorijević, Nikola
AU  - Stanić-Vučinić, Dragana
AU  - Rajković, Andreja
AU  - Ćirković-Veličković, Tanja
PY  - 2021
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/6025
AB  - Food allergies affect up to 10% of the general population and represent an important health problem in the field of food safety in industrialized countries. Hence, developing reliable, specific, and sensitive methods for detecting and quantifying allergens in food products is of high importance. Shellfish have been recognized as one of the eight most common sources of allergens, with tropomyosin (TPM) being considered a major heat-stable allergen, having a highly conserved amino acid sequence among different shellfish species. Allergenicity of TPM may change during food processing, such as cooking. The objective of this study was to develop an enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of shellfish tropomyosin in food samples. 
Two different extraction buffers - phosphate-buffered saline (PBS) and PBS containing 1 M sodium-chloride (PBSN), were compared for their ability to recover proteins from pre-cooked frozen Mediterranean mussel (Mytilus galloprovincialis) and fresh frozen razor mud shrimp (Solenocera melantho). The samples were additionally cooked according to the manufacturer's instruction and analyzed as such. The protein content was quantified using Bradford protein assay, and the protein components of soluble extracts were profiled using SDS-PAGE. TPM presence was confirmed using Western blot. Sandwich ELISA was developed using a monoclonal anti-TPM antibody as a capture antibody, while polyclonal anti-TPM antibody served as a detection antibody and was coupled to the biotinylated secondary antibody and streptavidin-alkaline phosphatase conjugate. Tropomyosin was quantified using highly purified natural shrimp tropomyosin as standard. 
The profile of extracted proteins was changed when using PBSN instead of PBS. A higher concentration of proteins was recovered from raw shrimp using PBSN instead of PBS. At the same time, the type of extraction buffer did not affect protein recovery either from heated shrimp or pre-cooked/heated mussels. Significantly fewer proteins were extracted from cooked shrimp sample compared to the raw shrimp, while cooking showed no effect on the extraction of proteins from mussels. Cooking did not affect TPM recognition in Western blot. TPM was quantified in shrimp samples in sandwich ELISA. However, developed ELISA could not quantify mussel's TPM, indicating that this approach may distinguish mussels and shrimp TPM.
PB  - Sociedade Portuguesa de Química, Av. Da República, 45 – 3º Esq, 1050-187 Lisboa – Portugal
C3  - XXI EuroFoodChem conference, Virtual Congress, 22nd-24th November, 2021. In: Book of Abstracts
T1  - Extraction and quantification of tropomyosin in selected samples of shellfish
SP  - 118
EP  - 118
UR  - https://hdl.handle.net/21.15107/rcub_cherry_6025
ER  - 

@conference{
author = "Radomirović, Mirjana Ž. and Gligorijević, Nikola and Stanić-Vučinić, Dragana and Rajković, Andreja and Ćirković-Veličković, Tanja",
year = "2021",
abstract = "Food allergies affect up to 10% of the general population and represent an important health problem in the field of food safety in industrialized countries. Hence, developing reliable, specific, and sensitive methods for detecting and quantifying allergens in food products is of high importance. Shellfish have been recognized as one of the eight most common sources of allergens, with tropomyosin (TPM) being considered a major heat-stable allergen, having a highly conserved amino acid sequence among different shellfish species. Allergenicity of TPM may change during food processing, such as cooking. The objective of this study was to develop an enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of shellfish tropomyosin in food samples. 
Two different extraction buffers - phosphate-buffered saline (PBS) and PBS containing 1 M sodium-chloride (PBSN), were compared for their ability to recover proteins from pre-cooked frozen Mediterranean mussel (Mytilus galloprovincialis) and fresh frozen razor mud shrimp (Solenocera melantho). The samples were additionally cooked according to the manufacturer's instruction and analyzed as such. The protein content was quantified using Bradford protein assay, and the protein components of soluble extracts were profiled using SDS-PAGE. TPM presence was confirmed using Western blot. Sandwich ELISA was developed using a monoclonal anti-TPM antibody as a capture antibody, while polyclonal anti-TPM antibody served as a detection antibody and was coupled to the biotinylated secondary antibody and streptavidin-alkaline phosphatase conjugate. Tropomyosin was quantified using highly purified natural shrimp tropomyosin as standard. 
The profile of extracted proteins was changed when using PBSN instead of PBS. A higher concentration of proteins was recovered from raw shrimp using PBSN instead of PBS. At the same time, the type of extraction buffer did not affect protein recovery either from heated shrimp or pre-cooked/heated mussels. Significantly fewer proteins were extracted from cooked shrimp sample compared to the raw shrimp, while cooking showed no effect on the extraction of proteins from mussels. Cooking did not affect TPM recognition in Western blot. TPM was quantified in shrimp samples in sandwich ELISA. However, developed ELISA could not quantify mussel's TPM, indicating that this approach may distinguish mussels and shrimp TPM.",
publisher = "Sociedade Portuguesa de Química, Av. Da República, 45 – 3º Esq, 1050-187 Lisboa – Portugal",
journal = "XXI EuroFoodChem conference, Virtual Congress, 22nd-24th November, 2021. In: Book of Abstracts",
title = "Extraction and quantification of tropomyosin in selected samples of shellfish",
pages = "118-118",
url = "https://hdl.handle.net/21.15107/rcub_cherry_6025"
}

Radomirović, M. Ž., Gligorijević, N., Stanić-Vučinić, D., Rajković, A.,& Ćirković-Veličković, T.. (2021). Extraction and quantification of tropomyosin in selected samples of shellfish. in XXI EuroFoodChem conference, Virtual Congress, 22nd-24th November, 2021. In: Book of Abstracts
Sociedade Portuguesa de Química, Av. Da República, 45 – 3º Esq, 1050-187 Lisboa – Portugal., 118-118.
https://hdl.handle.net/21.15107/rcub_cherry_6025

Radomirović MŽ, Gligorijević N, Stanić-Vučinić D, Rajković A, Ćirković-Veličković T. Extraction and quantification of tropomyosin in selected samples of shellfish. in XXI EuroFoodChem conference, Virtual Congress, 22nd-24th November, 2021. In: Book of Abstracts. 2021;:118-118.
https://hdl.handle.net/21.15107/rcub_cherry_6025 .

Radomirović, Mirjana Ž., Gligorijević, Nikola, Stanić-Vučinić, Dragana, Rajković, Andreja, Ćirković-Veličković, Tanja, "Extraction and quantification of tropomyosin in selected samples of shellfish" in XXI EuroFoodChem conference, Virtual Congress, 22nd-24th November, 2021. In: Book of Abstracts (2021):118-118,
https://hdl.handle.net/21.15107/rcub_cherry_6025 .

TY  - CONF
AU  - Radomirović, Mirjana Ž.
AU  - Gligorijević, Nikola
AU  - Minić, Simeon
AU  - Nikolić, Milan
AU  - Stanić-Vučinić, Dragana
AU  - Ćirković-Veličković, Tanja
PY  - 2021
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/6032
AB  - Phycobiliproteins (PBP) have been employed in numerous fluorescence-based techniques owing to highly fluorescent, covalently bound tetrapyrrole chromophores. So far, only entire PBPs have been utilized as fluorescent probes. A new method for covalent attachment of phycocyanin’s chromophore, phycocyanobilin (PCB), to potentially any protein, is proposed, relying on the ability of PCB to be attached to sulfhydryl groups of proteins. Traut’s reagent (TR, 2-iminothiolane) was exploited for introduction of sulfhydryl groups in the model protein, bovine serum albumin (BSA), by modifying its primary amines. Introduced sulfhydryl groups were then targeted for modification by PCB. All tested molar ratios of TR per mole of protein were successful in modification of BSA. Near-UV and far-UV circular dichroism spectroscopy revealed that a higher degree of modification by TR induces more profound alterations of BSA structure, leading at the same time to minor changes in BSA oligomerization and aggregation profile. PCB was covalently attached to introduced sulfhydryl groups at pH 9 at 20–fold ratio of TR. An increase in the molar ratio of TR per mole of BSA leads to amplification of fluorescent signal of PCB-modified BSA, most significantly observed starting from 50-fold and higher TR ratios. Using BSA as a model protein, a 50-fold molar excess of TR seems to be the optimal ratio for balancing between the effect on protein structure and the degree of labeling and thus fluorescent signal obtained. The proposed method could be used for labeling of virtually any protein, as means of either obtaining fluorescent probes for application in fluorescent techniques or functionalization of, for example, food proteins through covalent attachment of bioactive PCB.
PB  - Belgrade : Faculty of Chemistry
C3  - FoodEnTwin Symposium “Novel analytical approaches in food and environmental sciences”, Belgrade, Serbia, 16th-18th June, 2021. In: Book of Abstracts
T1  - Traut’s reagent application in fluorescent labeling of bovine serum albumin with phycocyanobilin
SP  - 37
EP  - 37
UR  - https://hdl.handle.net/21.15107/rcub_cherry_6032
ER  - 

@conference{
author = "Radomirović, Mirjana Ž. and Gligorijević, Nikola and Minić, Simeon and Nikolić, Milan and Stanić-Vučinić, Dragana and Ćirković-Veličković, Tanja",
year = "2021",
abstract = "Phycobiliproteins (PBP) have been employed in numerous fluorescence-based techniques owing to highly fluorescent, covalently bound tetrapyrrole chromophores. So far, only entire PBPs have been utilized as fluorescent probes. A new method for covalent attachment of phycocyanin’s chromophore, phycocyanobilin (PCB), to potentially any protein, is proposed, relying on the ability of PCB to be attached to sulfhydryl groups of proteins. Traut’s reagent (TR, 2-iminothiolane) was exploited for introduction of sulfhydryl groups in the model protein, bovine serum albumin (BSA), by modifying its primary amines. Introduced sulfhydryl groups were then targeted for modification by PCB. All tested molar ratios of TR per mole of protein were successful in modification of BSA. Near-UV and far-UV circular dichroism spectroscopy revealed that a higher degree of modification by TR induces more profound alterations of BSA structure, leading at the same time to minor changes in BSA oligomerization and aggregation profile. PCB was covalently attached to introduced sulfhydryl groups at pH 9 at 20–fold ratio of TR. An increase in the molar ratio of TR per mole of BSA leads to amplification of fluorescent signal of PCB-modified BSA, most significantly observed starting from 50-fold and higher TR ratios. Using BSA as a model protein, a 50-fold molar excess of TR seems to be the optimal ratio for balancing between the effect on protein structure and the degree of labeling and thus fluorescent signal obtained. The proposed method could be used for labeling of virtually any protein, as means of either obtaining fluorescent probes for application in fluorescent techniques or functionalization of, for example, food proteins through covalent attachment of bioactive PCB.",
publisher = "Belgrade : Faculty of Chemistry",
journal = "FoodEnTwin Symposium “Novel analytical approaches in food and environmental sciences”, Belgrade, Serbia, 16th-18th June, 2021. In: Book of Abstracts",
title = "Traut’s reagent application in fluorescent labeling of bovine serum albumin with phycocyanobilin",
pages = "37-37",
url = "https://hdl.handle.net/21.15107/rcub_cherry_6032"
}

Radomirović, M. Ž., Gligorijević, N., Minić, S., Nikolić, M., Stanić-Vučinić, D.,& Ćirković-Veličković, T.. (2021). Traut’s reagent application in fluorescent labeling of bovine serum albumin with phycocyanobilin. in FoodEnTwin Symposium “Novel analytical approaches in food and environmental sciences”, Belgrade, Serbia, 16th-18th June, 2021. In: Book of Abstracts
Belgrade : Faculty of Chemistry., 37-37.
https://hdl.handle.net/21.15107/rcub_cherry_6032

Radomirović MŽ, Gligorijević N, Minić S, Nikolić M, Stanić-Vučinić D, Ćirković-Veličković T. Traut’s reagent application in fluorescent labeling of bovine serum albumin with phycocyanobilin. in FoodEnTwin Symposium “Novel analytical approaches in food and environmental sciences”, Belgrade, Serbia, 16th-18th June, 2021. In: Book of Abstracts. 2021;:37-37.
https://hdl.handle.net/21.15107/rcub_cherry_6032 .

Radomirović, Mirjana Ž., Gligorijević, Nikola, Minić, Simeon, Nikolić, Milan, Stanić-Vučinić, Dragana, Ćirković-Veličković, Tanja, "Traut’s reagent application in fluorescent labeling of bovine serum albumin with phycocyanobilin" in FoodEnTwin Symposium “Novel analytical approaches in food and environmental sciences”, Belgrade, Serbia, 16th-18th June, 2021. In: Book of Abstracts (2021):37-37,
https://hdl.handle.net/21.15107/rcub_cherry_6032 .