Elucidation of the structural mechanisms of the affinity maturation in the immune response of anti-NP antibodies.

已知血清的平均亲和力通常在免疫后随时间增加。这种现象被称为免疫反应的亲和力成熟。已经证明，初级应答抗NP抗体NIG 9的可变区不携带体细胞突变，并且二次应答抗NP抗体是体细胞突变的并且对NP具有高亲和力。通过基因分析，已经显示在重链的位置33处的Trp*Leu交换在抗NP抗体的亲和力成熟中起关键作用。基于从抗NP抗体的亲和力测量和动力学分析获得的数据，在重链的位置33处具有Trp*Leu交换的二次应答抗体显示出更高的缔合速率常数（kon）。我们通过使用选择性地用N标记的Fab类似物进行NMR分析以阐明这种类型的“动力学成熟”的结构基础<15>。NP-AmTEMPO结合实验的结果表明，H1、H3、L1和L3环形成初级应答抗体Fab（N1 G9）和次级应答抗体Fab（B2）两者的抗原结合位点。两种Fab抗体结合位点的结构相似。通过对Fab（N1 G9）和Fab（B2）的氢氘交换速率和横向弛豫速率的比较分析，我们可以得出结论：在半抗原缺失的情况下，Fab（B2）的抗原结合位点存在构象柔性，使得半抗原分子更容易进入抗原结合位点。因此，我们认为，存在于二级反应抗体的抗原结合位点的构象灵活性，使双分子半抗原-抗体结合速率常数净增加。这种类型的动力学成熟机制被认为是最有效的亲和力成熟过程之一。

It is known that the average affinity of serum generally increases with time after immunization. This phenomenon is called affinity maturation of immune response. It has been demonstrated that the variable region of the primary response anti-NP antibody, NIG9 carry none somatic mutations, and the secondary response anti-NP antibodies are somatically mutated and have a high affinity for NP.By gene analysis it has been shown that a Trp*Leu exchange at position 33 of the heavy chain is plays a crucial role in the affinity maturation of anti-NP antibodies.On the basis of the data obtained from affinity measurements and kinetic analyzes of anti-NP antibodies, the secondary response antibodies, which have a Trp*Leu exchange at position 33 of the heavy chain, show higher rate constant for association (kon). We performed the NMR analyzes for elucidating the structural basis of this type of "kinetic maturation" by using Fab analogues selectively labeled with ^<15>N.The result of the NP-AmTEMPO binding experiments indicate that H1, H3, L1, and L3 loop form the antigen binding site of both the primary response antibody, Fab (N1G9), and the secondary response antibody, Fab (B2), and the construction of the antibody combining sites ofboth Fabs were closely similar. On the basis of the comparative analyzes of the hydrogen-deuterium exchange rates and transverse relaxation rates between Fab (N1G9) and Fab (B2), we are able to conclude that a conformational flexibility exists in the antigen binding site of Fab (B2) in the absence of the hapten, which leads hapten molecule easily accessible into antigen binding site. We therefore suggest that the conformational flexibility existing in the antigen binding site of the secondary response antibodies gives net increase in bimolecular hapten-antibody association rate constant. This type of kinetic maturation mechanism is thought to be one of the most efficient affinity maturation process.