Development and comparison of Western blot, dot blot and ELISA for mussel tropomyosin quantification
Апстракт
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 bet...ween 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.
Кључне речи:
Tropomyosin / allergens / food / ELISA methodИзвор:
Proceedings - XI Conference of Serbian Biochemical Society "Amazing Biochemistry", 22.09.2022. Novi Sad, Serbia, 2022, 2022, 141- Publisher:, 2022, 125-125Издавач:
- Serbian Biochemical Society
- University of Belgrade - Faculty of Chemistry
Финансирање / пројекти:
- Министарство науке, технолошког развоја и иновација Републике Србије, институционално финансирање - 200168 (Универзитет у Београду, Хемијски факултет) (RS-MESTD-inst-2020-200168)
- European Union’s Horizon 2020 research and innovation programme under grant agreement No 96517 (IMPTOX)
- Science Fund of the Republic of Serbia, Program DIASPORA, #6504499, ShellPCR
Институција/група
IHTMTY - 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 .