Exploration of Specific Nanobodies As Immunological Reagents to Detect Milk Allergen of β-Lactoglobulin without Interference of Hydrolytic Peptides,Journal of Agricultural and Food Chemistry

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Exploration of Specific Nanobodies As Immunological Reagents to Detect Milk Allergen of β-Lactoglobulin without Interference of Hydrolytic Peptides
Journal of Agricultural and Food Chemistry ( IF 5.7 ) Pub Date : 2022-11-22 , DOI: 10.1021/acs.jafc.2c06175
Yaozhong Hu ₁ , Yi Wang ₁ , Linqing Nie ₁ , Jing Lin ₁ , Sihao Wu ₁ , Shijie Li ₁ , Jing Wu ₁ , Xuemeng Ji ₁ , Huan Lv ₁ , Serge Muyldermans ₂ , Shuo Wang ₁

Affiliation

Milk proteins are widely used for food supplementation, despite the potential risk of food allergy, especially against β-lactoglobulin (BLG), which makes BLG surveillance critical. Possible interaction of detecting antibodies with BLG-derived peptides will result in unprecise inspection. Thus, in this study, it was proposed to generate nanobodies (Nbs) and validate the immunological detection of intact BLG rather than hydrolytic peptides. Nbs were successfully retrieved and characterized with high stability and target specificity. A competitive enzyme-linked immunosorbent assay (cELISA) was developed with a linear range from 39 to 10,000 ng/mL and a detection limit (LOD) of 4.55 ng/mL, with a recovery of 86.30%–95.09% revealed by analysis of spiked samples. Meanwhile, a sandwich ELISA (sELISA) was established with Nb82 and BLG polyclonal antibody (pAb-BLG) providing a linear range from 29.7 to 1250 ng/mL and an LOD of 13.82 ng/mL with a recovery of 87.82%–103.97%. The interaction of selected Nbs with BLG-derived peptides was investigated by Nb structure modeling and BLG docking. No binding on hydrolytic peptides was revealed, confirming the precision of Nb-mediated immunoassays. In summary, this study successfully identified BLG-specific Nbs for immunoassay development and guaranteed the monitoring of intact BLG without interference of hydrolytic peptides, providing experimental evidence that our Nbs recognize intact food allergen.

中文翻译：

探索特异性纳米抗体作为免疫试剂在不受水解肽干扰的情况下检测乳球蛋白β-乳过敏原

尽管存在食物过敏的潜在风险，尤其是对 β-乳球蛋白 (BLG) 过敏，但牛奶蛋白仍广泛用于食品补充剂，这使得 BLG 监测变得至关重要。检测抗体与 BLG 衍生肽的可能相互作用将导致不精确的检查。因此，在这项研究中，建议生成纳米抗体 (Nbs) 并验证完整 BLG 而不是水解肽的免疫学检测。Nbs 被成功回收并表征为具有高稳定性和目标特异性。开发了一种竞争性酶联免疫吸附测定 (cELISA)，其线性范围为 39 至 10,000 ng/mL，检测限 (LOD) 为 4.55 ng/mL，加标分析显示回收率为 86.30%–95.09%样品。同时，使用 Nb82 和 BLG 多克隆抗体 (pAb-BLG) 建立夹心 ELISA (sELISA)，线性范围为 29.7 至 1250 ng/mL，LOD 为 13.82 ng/mL，回收率为 87.82%–103.97%。通过 Nb 结构建模和 BLG 对接研究了所选 Nb 与 BLG 衍生肽的相互作用。未发现与水解肽的结合，证实了 Nb 介导的免疫测定的准确性。总之，这项研究成功地确定了用于免疫测定开发的 BLG 特异性 Nbs，并保证在不受水解肽干扰的情况下监测完整的 BLG，为我们的 Nbs 识别完整的食物过敏原提供了实验证据。通过 Nb 结构建模和 BLG 对接研究了所选 Nb 与 BLG 衍生肽的相互作用。未发现与水解肽的结合，证实了 Nb 介导的免疫测定的准确性。总之，这项研究成功地确定了用于免疫测定开发的 BLG 特异性 Nbs，并保证在不受水解肽干扰的情况下监测完整的 BLG，为我们的 Nbs 识别完整的食物过敏原提供了实验证据。通过 Nb 结构建模和 BLG 对接研究了所选 Nb 与 BLG 衍生肽的相互作用。未发现与水解肽的结合，证实了 Nb 介导的免疫测定的准确性。总之，这项研究成功地确定了用于免疫测定开发的 BLG 特异性 Nbs，并保证在不受水解肽干扰的情况下监测完整的 BLG，为我们的 Nbs 识别完整的食物过敏原提供了实验证据。

更新日期：2022-11-22

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