Abstract：
Somatic hypermutation (SHM) is one of the driving forces that increases antibody (Ab) affinity. We studied the effects of SHM on thermostability and affinity using three single-chain Fv fragments (scFvs) of anti-(4-hydroxy-3-nitrophenyl)acetyl Abs, namely 9TG, 9T7, and E11. 9TG has a germline structure that lacks SHM and is an ancestor of 9T7 with 11 mutations. E11, which has 21 mutations, is a mature Ab and has its own ancestor. The thermostabilities and antigen-Ab interactions were analyzed by circular dichroism (CD), differential scanning calorimetry (DSC), and isothermal titration calorimetry (ITC). Far-UV CD spectra showed that all scFvs were folded into a structure referred to as immunoglobulin-fold and were unfolded by heating at different melting temperatures. Comparison of thermodynamic parameters obtained from DSC and ITC revealed that the magnitude of stabilization free energy at 37 °C was in the order, 9TG > 9T7 > E11, while that of the free energy of interaction with antigen was 9TG < 9T7 < E11, suggesting that Abs make a trade-off between stability and affinity during affinity maturation.
摘要：
体细胞超突变（SHM）是增加抗体（Ab）亲和力的驱动力之一。我们使用抗（4-羟基-3-硝基苯基）乙酰基Ab的三个单链Fv片段（scFvs）研究了SHM对热稳定性和亲和力的影响，即9TG，9T7，和E11。9TG具有缺乏SHM的种系结构,并且是具有11个突变的9T7的祖先。E11有21个突变,是成熟的Ab，有自己的祖先。通过圆二色性（CD）分析热稳定性和抗原-Ab相互作用，差示扫描量热法(DSC),和等温滴定量热法(ITC)。远-UVCD光谱显示所有scFv折叠成称为免疫球蛋白折叠的结构,并通过在不同的熔化温度下加热而展开。从DSC和ITC获得的热力学参数的比较表明，在37°C时稳定自由能的大小顺序为，9TG>9T7>E11，而与抗原相互作用的自由能为9TG<9T7
