Mukhametova Lilia I, Eremin Sergei A, Arutyunyan Dmitrii A, Goryainova Oksana S, Ivanova Tatiana I, Tillib Sergei V
Lomonosov Moscow State University, Faculty of Chemistry, Moscow, 119234, Russia.
Institute of Gene Biology, Russian Academy of Sciences, Moscow, 119334, Russia.
Biochemistry (Mosc). 2022 Dec;87(12):1679-1688. doi: 10.1134/S0006297922120227.
Due to its unique structure and properties, human breast milk lactoferrin (hLF) has many nutritional and health-promoting functions in infants, including protection against inflammation and bacterial infections. The lack of LF in breastmilk or formula can result in the weakening of the infant's immune system. Noncompetitive polarization fluorescence immunoassay (FPIA) is a promising method for hLF quantification in milk and dairy products, which does not require the separation of the bound and free protein and allows to avoid time-consuming sample preparation. The use of fluorescently labeled single-domain camelid antibodies (nanobodies) for protein recognition in FPIA makes it possible to quantify relatively large antigens, in particular, hLF. In this work, we used previously obtained fluorescein isothiocyanate (FITC)-conjugated anti-hLF5 and anti-hLF16 nanobodies, which selectively recognized two different human lactoferrin epitopes, but did not bind to goat lactoferrin. The kinetics of hLF interaction with the FITC-labeled nanobodies was studied. The dissociation constant (K) for the anti-LF5 and antiLF16 nanobodies was 3.2 ± 0.3 and 4.9 ± 0.4 nM, respectively, indicating the high-affinity binding of these nanobodies to hLF. We developed the FPIA protocol and determined the concentration of FITC-labeled anti-hLF5 and anti-hLF16 nanobodies that provided the optimal fluorescence signal and stable fluorescence polarization value. We also studied the dependence of fluorescence polarization on the hLF concentration in the noncompetitive FPIA with FITC-anti-hLF5 nanobody. The detection limit for hLF was 2.1 ± 0.2 µg/ml and the linear range for determining the hLF concentration was 3-10 µg/ml. FPIA is commonly used to assay low-molecular-weight substances; however, the use of fluorescently labeled nanobodies allows quantification of high-molecular-weight proteins. Here, we demonstrated that FPIA with fluorescently labeled nanobodies can be used for hLF quantification in milk.
由于其独特的结构和性质，人乳乳铁蛋白（hLF）在婴儿中具有许多营养和促进健康的功能，包括预防炎症和细菌感染。母乳或配方奶中缺乏乳铁蛋白会导致婴儿免疫系统减弱。非竞争性偏振荧光免疫分析（FPIA）是一种用于定量牛奶和乳制品中hLF的有前途的方法，它不需要分离结合蛋白和游离蛋白，并且可以避免耗时的样品制备。在FPIA中使用荧光标记的单域骆驼抗体（纳米抗体）进行蛋白质识别，使得定量相对较大的抗原，特别是hLF成为可能。在这项工作中，我们使用了先前获得的异硫氰酸荧光素（FITC）偶联的抗hLF5和抗hLF16纳米抗体，它们选择性地识别两种不同的人乳铁蛋白表位，但不与山羊乳铁蛋白结合。研究了hLF与FITC标记的纳米抗体相互作用的动力学。抗LF5和抗LF16纳米抗体的解离常数（K）分别为3.2±0.3和4.9±0.4 nM，表明这些纳米抗体与hLF具有高亲和力结合。我们开发了FPIA方案，并确定了提供最佳荧光信号和稳定荧光偏振值的FITC标记的抗hLF5和抗hLF16纳米抗体的浓度。我们还研究了在使用FITC-抗hLF5纳米抗体的非竞争性FPIA中荧光偏振对hLF浓度的依赖性。hLF的检测限为2.1±0.2μg/ml，测定hLF浓度的线性范围为3-10μg/ml。FPIA通常用于分析低分子量物质；然而，使用荧光标记的纳米抗体可以定量高分子量蛋白质。在这里，我们证明了使用荧光标记纳米抗体的FPIA可用于定量牛奶中的hLF。
