Maltezos Anastasios, Platis Dimitris, Vlachakis Dimitrios, Kossida Sophia, Marinou Marigianna, Labrou Nikolaos E
Laboratory of Enzyme Technology, Department of Biotechnology, School of Food, Biotechnology and Development, Agricultural University of Athens, 75 Iera Odos, GR 118 55, Athens, Greece.
J Mol Recognit. 2014 Jan;27(1):19-31. doi: 10.1002/jmr.2327.
The human anti-human immunodeficiency virus (HIV) antibody 2G12 (mAb 2G12) is one of the most broadly neutralizing antibodies against HIV that recognizes a unique epitope on the surface glycoprotein gp120. In the present work, a limited affinity-ligand library was synthesized and evaluated for its ability to bind and purify recombinant mAb 2G12 expressed in transgenic corn. The affinity ligands were structural fragments of polysulfonate triazine dye Cibacron Blue 3GA (CB3GA) and represent novel lead scaffolds for designing synthetic affinity ligands. Solid phase chemistry was used to synthesize variants of CB3GA lead ligand. One immobilized ligand, bearing 4-aminobenzyl sulfonic acid (4ABS) linked on two chlorine atoms of the triazine ring (4ABS-Trz-4ABS), displayed high affinity for mAb 2G12. Absorption equilibrium, 3D molecular modelling and molecular dynamics simulation studies were carried out to provide a detailed picture of the 4ABS-Trz-4ABS interaction with mAb 2G12. This biomimetic affinity ligand was exploited for the development of a facile two-step purification protocol for mAb 2G12. In the first step of the procedure, mAb 2G12 was purified on an S-Sepharose FF cation exchanger, and in the second step, mAb 2G12 was purified using affinity chromatography on 4ABS-Trz-4ABS affinity adsorbent. Analysis of the antibody preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and enzyme-linked immunosorbent assay showed that the mAb 2G12 was fully active and of sufficient purity suitable for analytical applications.
人抗人免疫缺陷病毒（HIV）抗体2G12（单克隆抗体2G12）是针对HIV的最具广泛中和作用的抗体之一，它识别表面糖蛋白gp120上的一个独特表位。在本研究中，合成了一个有限的亲和配体文库，并评估了其结合和纯化在转基因玉米中表达的重组单克隆抗体2G12的能力。亲和配体是聚磺酸盐三嗪染料汽巴蓝3GA（CB3GA）的结构片段，代表了用于设计合成亲和配体的新型先导支架。采用固相化学方法合成了CB3GA先导配体的变体。一种固定化配体，在三嗪环的两个氯原子上连接有4-氨基苄基磺酸（4ABS）（4ABS-Trz-4ABS），对单克隆抗体2G12表现出高亲和力。进行了吸收平衡、三维分子建模和分子动力学模拟研究，以详细描绘4ABS-Trz-4ABS与单克隆抗体2G12的相互作用。利用这种仿生亲和配体开发了一种简便的单克隆抗体2G12两步纯化方案。在该方法的第一步中，单克隆抗体2G12在S-Sepharose FF阳离子交换剂上进行纯化，第二步中，使用4ABS-Trz-4ABS亲和吸附剂通过亲和色谱法对单克隆抗体2G12进行纯化。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和酶联免疫吸附测定对抗体制剂进行分析，结果表明单克隆抗体2G12具有完全活性且纯度足以用于分析应用。
