Institute for Biological Sciences, National Research Council of Canada, 100 Sussex Drive, Ottawa, Ontario, Canada, K1A 0R6.
Sensors (Basel). 2009;9(7):5351-67. doi: 10.3390/s90705351. Epub 2009 Jul 7.
Antibody engineering has allowed for the rapid generation of binding agents against virtually any antigen of interest, predominantly for therapeutic applications. Considerably less attention has been given to the development of diagnostic reagents and biosensors using engineered antibodies. Recently, we produced a novel pentavalent bispecific antibody (i.e., decabody) by pentamerizing two single-domain antibodies (sdAbs) through the verotoxin B subunit (VTB) and found both fusion partners to be functional. Using a similar approach, we have engineered a bispecific pentameric fusion protein consisting of five sdAbs and five cellulose-binding modules (CBMs) linked via VTB. To find an optimal design format, we constructed six bispecific pentamers consisting of three different CBMs, fused to the Staphylococcus aureus-specific human sdAb HVHP428, in both orientations. One bispecific pentamer, containing an N-terminal CBM9 and C-terminal HVHP428, was soluble, non-aggregating, and did not degrade upon storage at 4 °C for over six months. This molecule was dually functional as it bound to cellulose-based filters as well as S. aureus cells. When impregnated in cellulose filters, the bispecific pentamer recognized S. aureus cells in a flow-through detection assay. The ability of pentamerized CBMs to bind cellulose may form the basis of an immobilization platform for multivalent display of high-avidity binding reagents on cellulosic filters for sensing of pathogens, biomarkers and environmental pollutants.
抗体工程使得针对几乎任何感兴趣的抗原快速生成结合剂成为可能，主要用于治疗应用。然而，用于诊断试剂和生物传感器的工程抗体的开发却受到了相当少的关注。最近，我们通过五聚化毒素 B 亚基（VTB）将两个单域抗体（sdAb）五聚化，从而产生了一种新型的五价双特异性抗体（即 decabody），并且发现两个融合伙伴都是功能性的。使用类似的方法，我们设计了一种由五个 sdAb 和五个纤维素结合模块（CBM）组成的双特异性五聚体融合蛋白，通过 VTB 连接。为了找到最佳的设计格式，我们构建了六个双特异性五聚体，其中包含三个不同的 CBM，融合到金黄色葡萄球菌特异性人 sdAb HVHP428 上，采用两种方向。一种双特异性五聚体，包含 N 端 CBM9 和 C 端 HVHP428，可溶、不聚集，并且在 4°C 下储存超过六个月也不会降解。这种分子具有双重功能，因为它可以结合纤维素基过滤器和金黄色葡萄球菌细胞。当双特异性五聚体浸渍在纤维素过滤器中时，它可以在通过检测试验中识别金黄色葡萄球菌细胞。五聚化 CBM 结合纤维素的能力可能为基于纤维素的高亲和力结合试剂在纤维素过滤器上的多价展示提供基础，从而用于病原体、生物标志物和环境污染物的检测。
