Extraction and quantification of tropomyosin in selected samples of shellfish
Аутори
Radomirović, Mirjana Ž.Gligorijević, Nikola
Stanić-Vučinić, Dragana
Rajković, Andreja
Ćirković-Veličković, Tanja
Конференцијски прилог (Објављена верзија)
Метаподаци
Приказ свих података о документуАпстракт
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 gallop...rovincialis) 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.
Извор:
XXI EuroFoodChem conference, Virtual Congress, 22nd-24th November, 2021. In: Book of Abstracts, 2021, 118-118Издавач:
- Sociedade Portuguesa de Química, Av. Da República, 45 – 3º Esq, 1050-187 Lisboa – Portugal
Финансирање / пројекти:
- Министарство науке, технолошког развоја и иновација Републике Србије, институционално финансирање - 200168 (Универзитет у Београду, Хемијски факултет) (RS-MESTD-inst-2020-200168)
- ShellPCR - Development of Elisa and Immuno-PCR for Sensitive and Specific Detection of Shellfish Tropomyosin (RS-ScienceFundRS-Dijaspora-6504499)
- FoodEnTwin-Twinning of research activities for the frontier research in the fields of food, nutrition and environmental omics (EU-H2020-810752)
Колекције
Институција/група
Hemijski fakultet / Faculty of ChemistryTY - 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 .