Dmitriev D A, Massino Y S, Segal O L, Smirnova M B, Pavlova E V, Kolyaskina G I, Gurevich K G, Gnedenko O V, Ivanov Y D, Archakov A I, Osipov A P, Dmitriev A D, Egorov A M
Department of Chemical Enzymology, School of Chemistry, Lomonosov Moscow State University, Moscow, 119992 Russia.
Biochemistry (Mosc). 2002 Dec;67(12):1356-65. doi: 10.1023/a:1021805909314.
The interaction between two different monoclonal antibodies (Mabs) and their corresponding bispecific antibodies (Babs) with immobilized antigens was investigated using an optical biosensor (IAsys). The analyzed panel of affinity-purified antibodies included two parental Mabs (one of which was specific to human IgG (hIgG), and another one to horseradish peroxidase (HRP)), as well as Babs derived thereof (anti-hIgG/HRP). Babs resulting from the fusion of parental hybridomas bear two antigen-binding sites toward two different antigens and thus may interact with immobilized antigen through only one antigen-binding site (monovalently). Using an IAsys biosensor this study shows that the bivalent binding of Mabs predominates over the monovalent binding with immobilized HRP, whereas anti-hIgG parental Mabs were bound monovalently to the immobilized hIgG. The observed equilibrium association constant (K(ass)) values obtained in our last work [1] by solid-phase radioimmunoassay are consistent with those constants obtained by IAsys. The K(ass) of anti-HRP Mabs was about 50 times higher than that of anti-HRP shoulder of Babs. The dissociation rate constant (k(diss)) for anti-HRP shoulder of Babs was 21 times higher than k(diss) for anti-HRP Mabs. The comparison of the kinetic parameters for bivalent anti-HRP Mabs and Babs derived from anti-Mb/HRP and anti-hIgG/HRP, allowed to calculate that 95% of bound anti-HRP Mabs are bivalently linked with immobilized HRP, whereas only 5% of bound anti-HRP Mabs are monovalently linked. In general, the data obtained indicate that Babs bearing an enzyme-binding site may not be efficiently used instead of traditional antibody-enzyme conjugates in the case of binding of bivalent Mabs.
使用光学生物传感器（IAsys）研究了两种不同的单克隆抗体（Mabs）及其相应的双特异性抗体（Babs）与固定化抗原之间的相互作用。分析的亲和纯化抗体组包括两种亲本单克隆抗体（其中一种对人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）。亲本杂交瘤融合产生的双特异性抗体对两种不同抗原具有两个抗原结合位点，因此可能仅通过一个抗原结合位点（单价）与固定化抗原相互作用。本研究使用IAsys生物传感器表明，单克隆抗体的二价结合比与固定化HRP的单价结合占优势，而抗hIgG亲本单克隆抗体与固定化hIgG单价结合。我们上一项工作[1]通过固相放射免疫测定获得的观察到的平衡缔合常数（K(ass)）值与通过IAsys获得的那些常数一致。抗HRP单克隆抗体的K(ass)比双特异性抗体抗HRP肩部的K(ass)高约50倍。双特异性抗体抗HRP肩部的解离速率常数（k(diss)）比抗HRP单克隆抗体的k(diss)高21倍。对二价抗HRP单克隆抗体以及源自抗Mb/HRP和抗hIgG/HRP的双特异性抗体的动力学参数进行比较，可以计算出95%结合的抗HRP单克隆抗体与固定化HRP二价连接，而只有5%结合的抗HRP单克隆抗体单价连接。一般来说，获得的数据表明，在二价单克隆抗体结合的情况下，带有酶结合位点的双特异性抗体可能无法有效地替代传统的抗体 - 酶缀合物。
