• eg: metal ions (Ni2+, Cu2+, Co2+ and Zn2+) demonstrated high affinity towards Fc region of the IgG rather than the Fab regions.
Jiangnan University
• 3.Synthetic ligands
• 1. biospecific ligands
• 1.1 Staphylococcal protein A (SpA)
• Crystallographic studies showed that protein A binds to the Fc region of IgG at the junction between its CH2 and CH3 domains. It is also found to bind the heavy chain variable region between CDR2 and CDR3
2、 Pseudobiospecific ligands
2.1、 Hydrophobic charge-induction chromatography (HCIC)
原理：利用高密度的疏水基团实现疏水作用结合；通过 调节溶液 pH 值引入静电排斥作用协助解吸，实现有效 洗脱。 抗体保守区域（Fc区）含有大量疏水氨基酸Trp、Phe等。 常用配基：4-巯基乙基吡啶（MEP）
仿生物特异性配基物
合成配基
Protein A Protein G Protein L
HCIC IMAC
Ligand 8/7
优 选择性强 点
①化学配基价格相对便宜②
配基稳定，再生容易，可多
次重复使用③抗体吸附容量 高较 Protein A 高④洗脱 p H 值在 4 左右⑤亲和力适中， 不易引起抗体结构变异。
可靠，便宜，可以
纯化的抗体范围广， 尤其适合纯化scFv。
缺 配基费用高，再生 选择性差 点 困难，重复使用有
限稳定性差。配基
来源于细菌，有潜 在的污染物如：DNA、 热源、病毒等。抗
体吸附容量不高， 需要低PH洗脱，易 造成抗体聚集。
发展时间短，与抗
体的作用机理不清 楚，不便进行优化。
Jiangnan University
Jiangnan University
• 1.3 Peptostreptococcus magnus protein L
its nanomolar affinity towards kappa (κ) light chains of variable region of antibody. It binds specifically to κ 1, κ 3 and κ 4 subclasses of the antibody light chains while not recognizing κ2 and λ subgroups.
Jiangnan University
抗体的纯化原理与方法
Jiangnan University
Jiangnan University
Jiangnan University
affinity chromatography
1、The use of biospecific ligands in affinity chromatography 2、 Pseudobiospecific ligands 3、 Synthetic ligands
Jiangnan University
• 2.2 Immobilized metal affinity chromatography (IMAC).
• Biomolecules with exposed His, Cys, Ser, Glu, Asp and Trp have affinity towards metal ions.
• Artificial Peptostreptococcus.magnus proteinL (PpL) mimic——ligand 8/7
识别IgG部位 具体类型
Protein L
Artificial protein L
轻链
轻链
Κ1型、κ3型、κ4型 λ 型、Κ1型、κ2型、 κ3型、κ4型
Hale Waihona Puke 生物特异性配基CDR区：决定簇互补区，分别 位于24-34,、50-65、95-102 位氨基酸
1.2 streptococcal protein G(SpG)
• it binds strongly to the Fc region of IgG. It is also reported to possess low affinity towards the CH1 domain of Fab region, thus, allowing purification of Fab fragments through a b-zipper interaction.