Bovine milk is a recognized allergenic food source with β-lactoglobulin (BLG) as its major allergen. Reliable detection of BLG epitopes can, therefore, be a useful marker for the presence of milk in processed food products, and for potential allergenicity. At the present, enzyme-linked immunosorbent assays (ELISA) for the detection of BLG are time-consuming and generally not specific to BLG IgE epitopes. In this study, the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-activated anti-BLG IgE epitope monoclonal antibody (mAb 1G9) was covalently bound onto the KOH-treated microtiter plate surface. Using this mAb-bound plate in sandwich combination with biotinylated anti-BLG polyclonal antibody-labeled gold nanoparticles, a linear dynamic range between 31.25 and 64 × 10³ ng mL⁻¹ with a limit of detection for BLG of 0.49 ng mL⁻¹ was obtained, which is 32 times wider and 16 times more sensitive than conventional sandwich ELISA (sELISA). Total recovery of BLG in spiked food samples was found, without matrix effects. Also in partially hydrolyzed infant formulas, the allergenic BLG residues were detected quantitatively. Compared with conventional and commercial BLG detection sELISAs, our sELISA is reliable, highly BLG epitope-specific, user-friendly, and time-saving and allows accurate detection of potentially allergenic residues in different types of processed foods. This improved sELISA protocol can be easily extended to detect other well-identified and characterized food allergens.

Open image in new window

牛乳是公认的过敏性食物来源，其主要过敏原为β-乳球蛋白（BLG）。因此，可靠地检测BLG表位可以成为加工食品中牛奶的存在以及潜在的致敏性的有用标记。目前，用于检测BLG的酶联免疫吸附测定（ELISA）是耗时的，并且通常不特异于BLG IgE表位。在这项研究中，将1-乙基-3-（3-二甲基氨基丙基）碳二亚胺激活的抗BLG IgE表位单克隆抗体（mAb 1G9）共价结合到KOH处理的微量滴定板表面。将该mAb结合板与生物素化抗BLG多克隆抗体标记的金纳米颗粒夹心组合使用时，线性动态范围介于31.25和64×10 ³  ng mL ^-1之间获得的BLG检测限为0.49 ng mL ^-1，比传统的夹心ELISA（sELISA）宽32倍，灵敏度高16倍。发现加标食品样品中BLG的总回收率没有基质效应。同样在部分水解的婴儿配方食品中，定量检测到了致敏性BLG残留。与常规和商业BLG检测sELISA相比，我们的sELISA是可靠的，高度BLG表位特异性，用户友好且省时的方法，可准确检测不同类型的加工食品中潜在的致敏残留物。这种改进的sELISA方案可以轻松扩展，以检测其他良好识别和表征的食物过敏原。

在新窗口中打开图像