TY  - CONF
AU  - Radomirović, Mirjana
AU  - Gligorijević, Nikola
AU  - Stanić-Vučinić, Dragana
AU  - Rajković, Andreja
AU  - Ćirković Veličković, Tanja
PY  - 2023
UR  - https://cer.ihtm.bg.ac.rs/handle/123456789/6668
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 a ccuracy a s l ow a s 1 9.8 p g/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  - Serbian Biochemical Society
C3  - Serbian Biochemical Society Twelfth Conference, International scientific meeting, “Biochemistry in Biotechnology,” 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_cer_6668
ER  - 

@conference{
author = "Radomirović, Mirjana 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 a ccuracy a s l ow a s 1 9.8 p g/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 = "Serbian Biochemical Society",
journal = "Serbian Biochemical Society Twelfth Conference, International scientific meeting, “Biochemistry in Biotechnology,” September 21-23, 2023, Belgrade, Serbia",
title = "Immuno-PCR for crustacean tropomyosin quantification",
pages = "130-130",
url = "https://hdl.handle.net/21.15107/rcub_cer_6668"
}

Radomirović, M., Gligorijević, N., Stanić-Vučinić, D., Rajković, A.,& Ćirković Veličković, T.. (2023). Immuno-PCR for crustacean tropomyosin quantification. in Serbian Biochemical Society Twelfth Conference, International scientific meeting, “Biochemistry in Biotechnology,” September 21-23, 2023, Belgrade, Serbia
Serbian Biochemical Society., 130-130.
https://hdl.handle.net/21.15107/rcub_cer_6668

Radomirović M, Gligorijević N, Stanić-Vučinić D, Rajković A, Ćirković Veličković T. Immuno-PCR for crustacean tropomyosin quantification. in Serbian Biochemical Society Twelfth Conference, International scientific meeting, “Biochemistry in Biotechnology,” September 21-23, 2023, Belgrade, Serbia. 2023;:130-130.
https://hdl.handle.net/21.15107/rcub_cer_6668 .

Radomirović, Mirjana, Gligorijević, Nikola, Stanić-Vučinić, Dragana, Rajković, Andreja, Ćirković Veličković, Tanja, "Immuno-PCR for crustacean tropomyosin quantification" in Serbian Biochemical Society Twelfth Conference, International scientific meeting, “Biochemistry in Biotechnology,” September 21-23, 2023, Belgrade, Serbia (2023):130-130,
https://hdl.handle.net/21.15107/rcub_cer_6668 .

TY  - JOUR
AU  - Radomirović, Mirjana
AU  - Gligorijević, Nikola
AU  - Stanić-Vučinić, Dragana
AU  - Rajković, Andreja
AU  - Ćirković Veličković, Tanja
PY  - 2023
UR  - https://cer.ihtm.bg.ac.rs/handle/123456789/6782
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  - 10.3390/ijms242015410
ER  - 

@article{
author = "Radomirović, Mirjana 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 = "10.3390/ijms242015410"
}

Radomirović, M., 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/10.3390/ijms242015410

Radomirović M, 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:10.3390/ijms242015410 .

Radomirović, Mirjana, 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/10.3390/ijms242015410 . .

TY  - CONF
AU  - Radomirović, Mirjana
AU  - Gligorijević, Nikola
AU  - Rajković, Andreja
AU  - Ćirković Veličković, Tanja
PY  - 2022
UR  - https://cer.ihtm.bg.ac.rs/handle/123456789/6799
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  - Serbian Biochemical Society
PB  - University of Belgrade - Faculty of Chemistry
C3  - Proceedings - XI Conference of Serbian Biochemical Society "Amazing Biochemistry", 22.09.2022. Novi Sad, Serbia, 2022, 2022, 141- Publisher:
T1  - Development and comparison of Western blot, dot blot and ELISA for mussel tropomyosin quantification
SP  - 125
EP  - 125
UR  - https://hdl.handle.net/21.15107/rcub_cer_6799
ER  - 

@conference{
author = "Radomirović, Mirjana 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 = "Serbian Biochemical Society, University of Belgrade - Faculty of Chemistry",
journal = "Proceedings - XI Conference of Serbian Biochemical Society "Amazing Biochemistry", 22.09.2022. Novi Sad, Serbia, 2022, 2022, 141- Publisher:",
title = "Development and comparison of Western blot, dot blot and ELISA for mussel tropomyosin quantification",
pages = "125-125",
url = "https://hdl.handle.net/21.15107/rcub_cer_6799"
}

Radomirović, M., Gligorijević, N., Rajković, A.,& Ćirković Veličković, T.. (2022). Development and comparison of Western blot, dot blot and ELISA for mussel tropomyosin quantification. in Proceedings - XI Conference of Serbian Biochemical Society "Amazing Biochemistry", 22.09.2022. Novi Sad, Serbia, 2022, 2022, 141- Publisher:
Serbian Biochemical Society., 125-125.
https://hdl.handle.net/21.15107/rcub_cer_6799

Radomirović M, Gligorijević N, Rajković A, Ćirković Veličković T. Development and comparison of Western blot, dot blot and ELISA for mussel tropomyosin quantification. in Proceedings - XI Conference of Serbian Biochemical Society "Amazing Biochemistry", 22.09.2022. Novi Sad, Serbia, 2022, 2022, 141- Publisher:. 2022;:125-125.
https://hdl.handle.net/21.15107/rcub_cer_6799 .

Radomirović, Mirjana, Gligorijević, Nikola, Rajković, Andreja, Ćirković Veličković, Tanja, "Development and comparison of Western blot, dot blot and ELISA for mussel tropomyosin quantification" in Proceedings - XI Conference of Serbian Biochemical Society "Amazing Biochemistry", 22.09.2022. Novi Sad, Serbia, 2022, 2022, 141- Publisher: (2022):125-125,
https://hdl.handle.net/21.15107/rcub_cer_6799 .

TY  - CONF
AU  - Radomirović, Mirjana
AU  - Čolaković, Maša
AU  - Pismestrović, Marina
AU  - Gligorijević, Nikola
AU  - Stanić-Vučinić, Dragana
AU  - Rajković, Andreja
AU  - Ćirković Veličković, Tanja
PY  - 2022
UR  - https://cer.ihtm.bg.ac.rs/handle/123456789/6823
AB  - Tropomyosin (TPM) is considered as a major allergen among different shellfish species.
Therefore, the development of methods for quantifying TPM in food products iscrucial 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. SDSPAGE
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.
AB  - Tropomiozin (TPM) se smatra glavnim alergenom morskih plodova. Razvoj metoda za kvantifikaciju TPM-a u prehrambenim proizvodima je ključan za alergične osobe. Nekoliko pufera za ekstrakciju je upoređeno u pogledu efikasnosti ekstrakcije protein iz sveže zamrznutih i kuvanih gambora, tokom 2 i 24 časa. Sadržaj proteina u solubilnim ekstraktima je određen Bredfordovom metodom. Proteinski profil ekstrakata je analiziran SDS-PAGE metodom. Za kvantifikaciju TPM-a je razvijena sendvič ELISA metoda. Nijedan puffer nije pokazao značajnu razliku u količini ekstrahovanih protein nakon 2 i 24 časa ekstrakcije. U poređenju sa sirovim, značajno manje proteina je ekstrahovano iz kuvanih gambora. Kvantifikacija TPM-a ELISA metodom je pokazala da se fosfatom puferisanim fiziološkim rastvorom (PBS) koji sadrži 1 M NaCl (PBSN) i karbonatnim 
puferom, pH 10, ekstrahuje oko 6 puta više TPM-a u poređenju sa PBS-om. Ovo ukazuje 
na značaj odabira adekvatnog pufera za ekstrakciju TPM-a, jer se upotrebom tradicionalno
korišćenih pufera može potceniti koncentracija TPM-a u gamborima.
PB  - Belgrade : Serbian Chemical Society
C3  - 58th Meeting of  the Serbian Chemical Society - Book of abstracts, Proceedings,  June 9-10, 2022 Belgrade / 58. Savetovanje Srpskog hemijskog društva, Kratki izvodi radova, Knjiga radova, 9. i 10. jun 2022. godine, Beograd
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_cer_6823
ER  - 

@conference{
author = "Radomirović, Mirjana 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 as a major allergen among different shellfish species.
Therefore, the development of methods for quantifying TPM in food products iscrucial 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. SDSPAGE
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., Tropomiozin (TPM) se smatra glavnim alergenom morskih plodova. Razvoj metoda za kvantifikaciju TPM-a u prehrambenim proizvodima je ključan za alergične osobe. Nekoliko pufera za ekstrakciju je upoređeno u pogledu efikasnosti ekstrakcije protein iz sveže zamrznutih i kuvanih gambora, tokom 2 i 24 časa. Sadržaj proteina u solubilnim ekstraktima je određen Bredfordovom metodom. Proteinski profil ekstrakata je analiziran SDS-PAGE metodom. Za kvantifikaciju TPM-a je razvijena sendvič ELISA metoda. Nijedan puffer nije pokazao značajnu razliku u količini ekstrahovanih protein nakon 2 i 24 časa ekstrakcije. U poređenju sa sirovim, značajno manje proteina je ekstrahovano iz kuvanih gambora. Kvantifikacija TPM-a ELISA metodom je pokazala da se fosfatom puferisanim fiziološkim rastvorom (PBS) koji sadrži 1 M NaCl (PBSN) i karbonatnim 
puferom, pH 10, ekstrahuje oko 6 puta više TPM-a u poređenju sa PBS-om. Ovo ukazuje 
na značaj odabira adekvatnog pufera za ekstrakciju TPM-a, jer se upotrebom tradicionalno
korišćenih pufera može potceniti koncentracija TPM-a u gamborima.",
publisher = "Belgrade : Serbian Chemical Society",
journal = "58th Meeting of  the Serbian Chemical Society - Book of abstracts, Proceedings,  June 9-10, 2022 Belgrade / 58. Savetovanje Srpskog hemijskog društva, Kratki izvodi radova, Knjiga radova, 9. i 10. jun 2022. godine, Beograd",
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_cer_6823"
}

Radomirović, M., 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 - Book of abstracts, Proceedings,  June 9-10, 2022 Belgrade / 58. Savetovanje Srpskog hemijskog društva, Kratki izvodi radova, Knjiga radova, 9. i 10. jun 2022. godine, Beograd
Belgrade : Serbian Chemical Society., 64-64.
https://hdl.handle.net/21.15107/rcub_cer_6823

Radomirović M, 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 - Book of abstracts, Proceedings,  June 9-10, 2022 Belgrade / 58. Savetovanje Srpskog hemijskog društva, Kratki izvodi radova, Knjiga radova, 9. i 10. jun 2022. godine, Beograd. 2022;:64-64.
https://hdl.handle.net/21.15107/rcub_cer_6823 .

Radomirović, Mirjana, 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 - Book of abstracts, Proceedings,  June 9-10, 2022 Belgrade / 58. Savetovanje Srpskog hemijskog društva, Kratki izvodi radova, Knjiga radova, 9. i 10. jun 2022. godine, Beograd (2022):64-64,
https://hdl.handle.net/21.15107/rcub_cer_6823 .

TY  - CONF
AU  - Radomirović, Mirjana
AU  - Gligorijević, Nikola
AU  - Stanić-Vučinić, Dragana
AU  - Rajković, Andreja
AU  - Ćirković Veličković, Tanja
PY  - 2021
UR  - https://cer.ihtm.bg.ac.rs/handle/123456789/7280
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
C3  - Book of Abstracts - XXI EuroFoodChem conference, 22nd-24th November, 2021, Virtual Congress
T1  - Extraction and quantification of tropomyosin in selected samples of shellfish
SP  - 118
EP  - 118
UR  - https://hdl.handle.net/21.15107/rcub_cer_7280
ER  - 

@conference{
author = "Radomirović, Mirjana 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",
journal = "Book of Abstracts - XXI EuroFoodChem conference, 22nd-24th November, 2021, Virtual Congress",
title = "Extraction and quantification of tropomyosin in selected samples of shellfish",
pages = "118-118",
url = "https://hdl.handle.net/21.15107/rcub_cer_7280"
}

Radomirović, M., Gligorijević, N., Stanić-Vučinić, D., Rajković, A.,& Ćirković Veličković, T.. (2021). Extraction and quantification of tropomyosin in selected samples of shellfish. in Book of Abstracts - XXI EuroFoodChem conference, 22nd-24th November, 2021, Virtual Congress
Sociedade Portuguesa de Química., 118-118.
https://hdl.handle.net/21.15107/rcub_cer_7280

Radomirović M, Gligorijević N, Stanić-Vučinić D, Rajković A, Ćirković Veličković T. Extraction and quantification of tropomyosin in selected samples of shellfish. in Book of Abstracts - XXI EuroFoodChem conference, 22nd-24th November, 2021, Virtual Congress. 2021;:118-118.
https://hdl.handle.net/21.15107/rcub_cer_7280 .

Radomirović, Mirjana, Gligorijević, Nikola, Stanić-Vučinić, Dragana, Rajković, Andreja, Ćirković Veličković, Tanja, "Extraction and quantification of tropomyosin in selected samples of shellfish" in Book of Abstracts - XXI EuroFoodChem conference, 22nd-24th November, 2021, Virtual Congress (2021):118-118,
https://hdl.handle.net/21.15107/rcub_cer_7280 .

TY  - CONF
AU  - Gligorijević, Nikola
AU  - Radomirović, Mirjana
AU  - Rajković, Andreja
AU  - Nedić, Olgica
AU  - Ćirković Veličković, Tanja
PY  - 2021
UR  - https://ec.europa.eu/research/participants/documents/downloadPublic?documentIds=080166e5deeb546e&appId=PPGMS
UR  - https://cer.ihtm.bg.ac.rs/handle/123456789/7284
AB  - The French paradox describes a lower incidence of cardiovascular problems despite a high intake of
saturated fats. This phenomenon was associated with higher consumption of red wine, only to be later discovered that the presence of several antioxidants, including resveratrol, are responsible for it. We investigated if resveratrol has a more direct role in protection from harmful oxidation and development of thrombosis, presumably through binding to important proteins of the blood coagulation process. Spectrofluorimetric analysis demonstrated binding of resveratrol to fibrinogen, the main protein in the coagulation process, which also has an important application as a food additive in making of fibrin gels. Various spectroscopic methods have demonstrated that binding of
resveratrol does not unfold or destabilize fibrinogen since both near and far-UV CD spectra as well as its melting temperature remained unchanged. A mutuallyprotective effect against the free radical-
induced oxidation of resveratrol and fibrinogen was found. The presence of fibrinogen caused a very
small masking effect of the antioxidative potential of resveratrol, measured by a reduction of
hexacyanoferrate (III), while greatly increasing its solubility in an aqueous environment, thus increasing potential bioavailability and activity of resveratrol in circulation. By direct interaction and protection of fibrinogen, resveratrol may serve as an important antioxidant for prevention of
thrombosis. The antioxidative effect of resveratrol may also protect and thus keep the desired characteristics of fibrinogen during the application of this protein as a food additive.
PB  - University of Belgrade - Faculty of Chemistry
C3  - FoodEnTwin Symposium “Novel analytical approaches in food and environmental sciences”, Book of Abstracts, 16th-18th June, 2021, Belgrade, Serbia
T1  - Resveratrol and fibrinogen interactions
SP  - 15
EP  - 15
UR  - https://hdl.handle.net/21.15107/rcub_cer_7284
ER  - 

@conference{
author = "Gligorijević, Nikola and Radomirović, Mirjana and Rajković, Andreja and Nedić, Olgica and Ćirković Veličković, Tanja",
year = "2021",
abstract = "The French paradox describes a lower incidence of cardiovascular problems despite a high intake of
saturated fats. This phenomenon was associated with higher consumption of red wine, only to be later discovered that the presence of several antioxidants, including resveratrol, are responsible for it. We investigated if resveratrol has a more direct role in protection from harmful oxidation and development of thrombosis, presumably through binding to important proteins of the blood coagulation process. Spectrofluorimetric analysis demonstrated binding of resveratrol to fibrinogen, the main protein in the coagulation process, which also has an important application as a food additive in making of fibrin gels. Various spectroscopic methods have demonstrated that binding of
resveratrol does not unfold or destabilize fibrinogen since both near and far-UV CD spectra as well as its melting temperature remained unchanged. A mutuallyprotective effect against the free radical-
induced oxidation of resveratrol and fibrinogen was found. The presence of fibrinogen caused a very
small masking effect of the antioxidative potential of resveratrol, measured by a reduction of
hexacyanoferrate (III), while greatly increasing its solubility in an aqueous environment, thus increasing potential bioavailability and activity of resveratrol in circulation. By direct interaction and protection of fibrinogen, resveratrol may serve as an important antioxidant for prevention of
thrombosis. The antioxidative effect of resveratrol may also protect and thus keep the desired characteristics of fibrinogen during the application of this protein as a food additive.",
publisher = "University of Belgrade - Faculty of Chemistry",
journal = "FoodEnTwin Symposium “Novel analytical approaches in food and environmental sciences”, Book of Abstracts, 16th-18th June, 2021, Belgrade, Serbia",
title = "Resveratrol and fibrinogen interactions",
pages = "15-15",
url = "https://hdl.handle.net/21.15107/rcub_cer_7284"
}

Gligorijević, N., Radomirović, M., Rajković, A., Nedić, O.,& Ćirković Veličković, T.. (2021). Resveratrol and fibrinogen interactions. in FoodEnTwin Symposium “Novel analytical approaches in food and environmental sciences”, Book of Abstracts, 16th-18th June, 2021, Belgrade, Serbia
University of Belgrade - Faculty of Chemistry., 15-15.
https://hdl.handle.net/21.15107/rcub_cer_7284

Gligorijević N, Radomirović M, Rajković A, Nedić O, Ćirković Veličković T. Resveratrol and fibrinogen interactions. in FoodEnTwin Symposium “Novel analytical approaches in food and environmental sciences”, Book of Abstracts, 16th-18th June, 2021, Belgrade, Serbia. 2021;:15-15.
https://hdl.handle.net/21.15107/rcub_cer_7284 .

Gligorijević, Nikola, Radomirović, Mirjana, Rajković, Andreja, Nedić, Olgica, Ćirković Veličković, Tanja, "Resveratrol and fibrinogen interactions" in FoodEnTwin Symposium “Novel analytical approaches in food and environmental sciences”, Book of Abstracts, 16th-18th June, 2021, Belgrade, Serbia (2021):15-15,
https://hdl.handle.net/21.15107/rcub_cer_7284 .