Center of Excellence for Molecular Food Sciences and Department of Biochemistry, University of Belgrade-Faculty of Chemistry, 11000 Belgrade, Serbia.
Center for Chemistry, University of Belgrade-Institute of Chemistry, Technology and Metallurgy, National Institute of the Republic of Serbia, 11000 Belgrade, Serbia.
Int J Mol Sci. 2023 Oct 21;24(20):15410. doi: 10.3390/ijms242015410.
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.
原肌球蛋白是贝类中的主要过敏原。本研究开发了一种超灵敏的免疫聚合酶链反应（immuno-PCR）方法，用于定量食物中的甲壳类动物原肌球蛋白。该方法将夹心酶联免疫吸附试验（ELISA）与标记 DNA 的实时聚合酶链反应（rtPCR）扩增相结合。单克隆抗 TPM 抗体作为捕获抗体，多克隆兔抗虾原肌球蛋白抗体作为检测抗体，天然虾原肌球蛋白作为标准。双股氨基 DNA 与二级抗兔抗体共价结合，随后通过 rtPCR 进行扩增和定量。免疫-PCR 的定量灵敏度比类似的 ELISA 高 20 倍，LOQ 为 19.8pg/mL。所开发的免疫-PCR 方法对甲壳类动物原肌球蛋白的检测具有高度特异性，在广泛的浓度范围内具有高度精确性。在添加蔬菜汤中的回收率为 87.7-115.6%。甲壳类动物原肌球蛋白也在商业食品中进行了定量。该报道的免疫-PCR 测定法是用于定量甲壳类动物原肌球蛋白的最敏感方法，也是用于一般食品过敏原和食品蛋白定量的首个免疫-PCR 测定法。该方法可以轻松适应任何目前用 ELISA 定量的食品过敏原的特异性和超灵敏免疫-PCR 检测，这对于食物过敏的人来说至关重要。
