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| Ultrasensitive quantification of crustacean tropomyosin by immuno-PCR Radomirovic, M.; Gligorijevic, N.; Stanic-Vucinic, D.; Rajkovic, A.; Cirkovic Velickovic, T.C. (2023). Ultrasensitive quantification of crustacean tropomyosin by immuno-PCR. International Journal of Molecular Sciences 24(20): 15410. https://dx.doi.org/10.3390/ijms242015410
In: International Journal of Molecular Sciences. MDPI AG: Basel. ISSN 1661-6596; e-ISSN 1422-0067, meer
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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. |
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