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

众所周知，免疫后血清的平均亲和力一般随着时间的推移而增加。这种现象被称为免疫反应的亲和力成熟。基因分析表明，抗NP抗体重链33位Trp*Leu交换对抗NP抗体亲和力成熟起着至关重要的作用。根据亲和力测定和动力学分析，抗NP抗体重链33位Trp*Leu交换具有较高的缔合速率常数(Kon)。我们用选择性标记的Fab类似物进行了核磁共振分析，以阐明这种“动力学成熟”的结构基础。NP-AmTEMPO结合实验的结果表明，H1、H3、L1和L3环形成了一次反应抗体Fab(N1G9)和二次反应抗体Fab(B2)的抗原结合部位，两种Fab的抗体结合部位的结构非常相似。通过对Fab(N1G9)和Fab(B2)的氢-氚交换率和横向弛豫速率的比较分析，我们得出结论：在没有半抗原的情况下，Fab(B2)的抗原结合部位存在构象柔性，这使得半抗原分子很容易进入抗原结合部位。因此，我们认为，次级反应抗体抗原结合部位存在的构象灵活性使双分子半抗原抗体结合速率常数净增加。这种动力学成熟机制被认为是最有效的亲和成熟过程之一。

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.