Sensitivity enhancement and the role of orientation in enzyme immunosorbent assay based on half fragment antibodies

The free sulfhydryl groups of the hinge region of monovalent antibody fragments (rIgG) allow the orientation of rIgG on functionalized surfaces in immunosensors. To evaluate the contribution of reduction and orientation on signal enhancement we compared the performance of whole antibodies and their rIgG in ELISA performed on polystyrene or maleimide-functionalized microplates. Monoclonal anti-horseradish peroxidase (anti-HRP) and monoclonal anti-fPSA antibodies (1mg/ml) were reduced with 2-mercaptoethylamine (53mM). Using anti-HRP we confirmed the retention of the antigen binding capacity of rIgG. Moreover, we observed a signal enhancement for rIgG even if randomly absorbed on polystyrene [linear regression slope (95%CI): rIgG 0.524 (0.434-0.614), IgG 0.370 (0.430-0.399); P=0.0016] suggesting that chemical reduction might affect the antigen binding capacity of antibodies. ELISA with anti-fPSA rIgG coated on polystyrene confirmed these observations. Oriented anti-fPSA rIgG on a maleimide surface showed comparable signals to the assay performed on polystyrene for each analyzed concentration of antigen, anyway, with a significant improvement of the repeatability.

单价抗体片段（rIgG）铰链区的游离巯基，可实现免疫传感器功能化表面上rIgG的定向固定。为评估还原反应与定向固定对信号增强的贡献，本研究在聚苯乙烯或马来酰亚胺（maleimide）功能化微孔板上开展酶联免疫吸附实验（ELISA），对比完整抗体及其rIgG片段的检测性能。将浓度为1mg/ml的抗辣根过氧化物酶单克隆抗体（anti-HRP）与抗游离前列腺特异性抗原单克隆抗体（anti-fPSA），以53mM的2-巯基乙胺（2-mercaptoethylamine）进行还原处理。借助anti-HRP，我们验证了rIgG片段保留了抗原结合能力。此外，即便rIgG随机吸附于聚苯乙烯表面，仍可观察到其信号增强效应[线性回归斜率（95%置信区间CI）：rIgG为0.524（0.434~0.614），完整IgG为0.370（0.430~0.399）；P=0.0016]，这提示化学还原反应可能会影响抗体的抗原结合能力。针对聚苯乙烯表面包被anti-fPSA rIgG的ELISA实验验证了上述观察结果。在马来酰亚胺表面定向固定的anti-fPSA rIgG，在各检测抗原浓度下的信号与聚苯乙烯表面的实验结果相当，但其重复性得到了显著提升。