REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, , 2829-516 Caparica, Portugal.
J R Soc Interface. 2013 Nov 20;11(91):20130875. doi: 10.1098/rsif.2013.0875. Print 2014 Feb 6.
Aminophenyl boronic acids can form reversible covalent ester interactions with cis-diol-containing molecules, serving as a selective tool for binding glycoproteins as antibody molecules that possess oligosaccharides in both the Fv and Fc regions. In this study, amino phenyl boronic acid (APBA) magnetic particles (MPs) were applied for the magnetic separation of antibody molecules. Iron oxide MPs were firstly coated with dextran to avoid non-specific binding and then with 3-glycidyloxypropyl trimethoxysilane to allow further covalent coupling of APBA (APBA_MP). When contacted with pure protein solutions of human IgG (hIgG) and bovine serum albumin (BSA), APBA_MP bound 170 ± 10 mg hIgG g(-1) MP and eluted 160 ± 5 mg hIgG g(-1) MP, while binding only 15 ± 5 mg BSA g(-1) MP. The affinity constant for the interaction between hIgG and APBA_MP was estimated as 4.9 × 10(5) M(-1) (Ka) with a theoretical maximum capacity of 492 mg hIgG adsorbed g(-1) MP (Qmax), whereas control particles bound a negligible amount of hIgG and presented an estimated theoretical maximum capacity of 3.1 mg hIgG adsorbed g(-1) MP (Qmax). APBA_MPs were also tested for antibody purification directly from CHO cell supernatants. The particles were able to bind 98% of IgG loaded and to recover 95% of pure IgG (purity greater than 98%) at extremely mild conditions.
氨苯基硼酸可以与含有顺式二醇的分子形成可逆的共价酯相互作用，作为一种选择性工具，用于结合糖蛋白，如抗体分子，这些抗体分子在 Fv 和 Fc 区域都含有寡糖。在这项研究中，氨苯基硼酸（APBA）磁性颗粒（MPs）被应用于抗体分子的磁性分离。首先，将氧化铁粉 MP 用葡聚糖包覆以避免非特异性结合，然后用 3-缩水甘油氧基丙基三甲氧基硅烷进一步进行共价偶联 APBA（APBA_MP）。当与纯蛋白溶液的人 IgG（hIgG）和牛血清白蛋白（BSA）接触时，APBA_MP 结合 170 ± 10 mg hIgG g(-1) MP，并洗脱 160 ± 5 mg hIgG g(-1) MP，而仅结合 15 ± 5 mg BSA g(-1) MP。hIgG 与 APBA_MP 之间相互作用的亲和常数估计为 4.9×10(5) M(-1)（Ka），理论最大吸附容量为 492 mg hIgG 吸附 g(-1) MP（Qmax），而对照颗粒结合了可忽略不计的 hIgG，估计理论最大吸附容量为 3.1 mg hIgG 吸附 g(-1) MP（Qmax）。APBA_MPs 也直接从 CHO 细胞上清液中用于抗体纯化。在极其温和的条件下，颗粒能够结合 98%负载的 IgG，并回收 95%的纯 IgG（纯度大于 98%）。
