Dmitriev Dmitriy A, Massino Yulia S, Segal Olga L, Smirnova Maria B, Pavlova Elena V, Gurevich Konstantin G, Gnedenko Oksana V, Ivanov Yuriy D, Kolyaskina Galina I, Archakov Alexander I, Osipov Alexander P, Dmitriev Alexander D, Egorov Alexey M
Division of Chemical Enzymology, Department of Chemistry, Moscow State University, Moscow, Russian Federation.
J Immunol Methods. 2002 Mar 1;261(1-2):103-18. doi: 10.1016/s0022-1759(01)00558-0.
The interaction between two monoclonal antibodies (mAbs) and their corresponding bispecific antibody (bAb) with immobilized antigens has been examined using a resonant mirror biosensor (IAsys). BAbs were produced by cell fusion. The analysed panel of affinity-purified antibodies included two parental mAbs, one specific to human IgG (hIgG), and another specific to horseradish peroxidase (HRP), and a bAb derived thereof (anti-hIgG/HRP). The real-time analysis showed the drastic differences in the avidity of bivalent anti-HRP mAbs and anti-HRP shoulder of bAbs. Thus, the observed equilibrium association constant (K(ass)) of anti-HRP mAbs was about 50 times higher that of anti-HRP shoulder of bAbs. The ratio of association rate constants (k(ass)) of mAbs and bAbs was about two, due to the statistical factor of two binding sites per bivalent antibody molecule. However, the dissociation rate constant (k(diss)) of anti-HRP shoulder of bAbs was 21 times higher k(diss) of anti-HRP mAbs. The comparison with the theoretical model shows that these observations are consistent only with a situation in which bivalent binding of mAbs with immobilized HRP predominates over monovalent binding. On the contrary, the second parental mAb (anti-hIgG) did not show the increase in avidity due to bivalent binding, compared to the anti-hIgG shoulder of bAbs, suggesting that this mAb was bound monovalently to immobilized hIgG. The K(ass) values determined by solid-phase radioimmunoassay (RIA) yielded figures almost overlapping with those obtained by IAsys. The results of the comparison of bAbs and mAbs are discussed from the viewpoint of the use of bAbs in heterogeneous systems. On the other hand, these data demonstrate that real-time analysis of antibody binding parameters in IAsys biosensor is valuable for the selection of mAbs and bAbs with desired features, for different fields of application.
利用共振镜生物传感器（IAsys）检测了两种单克隆抗体（mAb）及其相应的双特异性抗体（bAb）与固定化抗原之间的相互作用。双特异性抗体通过细胞融合产生。经分析的亲和纯化抗体组包括两种亲本单克隆抗体，一种对人IgG（hIgG）具有特异性，另一种对辣根过氧化物酶（HRP）具有特异性，以及由此衍生的双特异性抗体（抗hIgG/HRP）。实时分析显示，二价抗HRP单克隆抗体和双特异性抗体的抗HRP肩部在亲和力上存在显著差异。因此，观察到的抗HRP单克隆抗体的平衡缔合常数（K(ass)）约为双特异性抗体抗HRP肩部的50倍。由于每个二价抗体分子有两个结合位点的统计因素，单克隆抗体和双特异性抗体的缔合速率常数（k(ass)）之比约为2。然而，双特异性抗体抗HRP肩部的解离速率常数（k(diss)）是抗HRP单克隆抗体的k(diss)的21倍。与理论模型的比较表明，这些观察结果仅与单克隆抗体与固定化HRP的二价结合占主导地位而非单价结合的情况一致。相反，与双特异性抗体的抗hIgG肩部相比，第二种亲本单克隆抗体（抗hIgG）未显示出由于二价结合而导致的亲和力增加，这表明该单克隆抗体以单价形式结合到固定化的hIgG上。通过固相放射免疫测定（RIA）测定的K(ass)值与通过IAsys获得的数值几乎重叠。从双特异性抗体在异质系统中的应用角度讨论了双特异性抗体和单克隆抗体比较的结果。另一方面，这些数据表明，IAsys生物传感器中抗体结合参数的实时分析对于为不同应用领域选择具有所需特性的单克隆抗体和双特异性抗体具有重要价值。
