Dmitriev Dmitriy A, Massino Yulia S, Segal Olga L
Department of Chemistry, Division of Chemical Enzymology, Moscow State University, Moscow, Russian Federation.
J Immunol Methods. 2003 Sep;280(1-2):183-202. doi: 10.1016/s0022-1759(03)00271-0.
A resonant mirror biosensor (IAsys) protocol is described for the comparative kinetic analysis of the ability of monoclonal antibodies (Mabs) and bispecific antibodies (Babs) to bind immobilized antigens. The protocol has been optimized and validated using the panel of affinity-purified antibodies, including two parental Mabs, one specific to human immunoglobulin G (hIgG) and another specific to horseradish peroxidase (HRP), and a Bab derived thereof by cell fusion (anti-hIgG/HRP Bab). The real-time kinetic analysis of antigen-antibody interactions using this protocol allows to demonstrate the differences in the avidity of bivalently binding Mabs and monovalent Babs. As shown in our previous study [J. Immunol. Methods 261 (2002) 103], the observed equilibrium association constants (Kass) determined by IAsys using this protocol yield figures almost overlapping with those obtained by solid-phase radioimmunoassay (RIA). The described protocol is suited for the investigation of the effects of valency on the binding properties of antibodies. It also may be applied for the selection of Mabs and Babs with desired features, for different fields of application.
描述了一种用于比较单克隆抗体（Mabs）和双特异性抗体（Babs）结合固定化抗原能力的共振镜生物传感器（IAsys）实验方案。该方案已使用一组亲和纯化抗体进行了优化和验证，包括两种亲本单克隆抗体，一种特异性针对人免疫球蛋白G（hIgG），另一种特异性针对辣根过氧化物酶（HRP），以及通过细胞融合产生的由其衍生的双特异性抗体（抗hIgG/HRP双特异性抗体）。使用该方案对抗抗原-抗体相互作用进行实时动力学分析，可以证明二价结合单克隆抗体和单价双特异性抗体在亲和力上的差异。正如我们之前的研究[《免疫学方法杂志》261（2002）103]所示，通过IAsys使用该方案测定的观察到的平衡缔合常数（Kass）得出的数值几乎与通过固相放射免疫分析（RIA）获得的数值重叠。所描述的方案适用于研究价态对抗体结合特性的影响。它也可用于选择具有所需特性的单克隆抗体和双特异性抗体，用于不同的应用领域。
