Deckers Susanne, Braren Ingke, Greunke Kerstin, Meyer Nadine, Rühl Dana, Bredehorst Reinhard, Spillner Edzard
Institute of Biochemistry and Molecular Biology, University of Hamburg, Martin-Luther-King Platz 6, Hamburg, Germany.
Biotechnol Appl Biochem. 2009 Jan;52(Pt 1):79-87. doi: 10.1042/BA20080032.
Nowadays, recombinant antibody and phage display technology enable the efficient generation of immunotools and a subsequent manipulation for optimized affinity, specificity or overall performance. Such advantages are of particular interest for haptenic target structures, such as TNT (2,4,6-trinitrotoluene). The toxicity of TNT and its breakdown products makes a reliable and fast detection of low levels in aqueous samples highly important. In the present study, we aimed for the generation of scFvs (single-chain antibody fragments) specific for the TNT-surrogate TNP (2,4,6-trinitrophenyl) and their subsequent production as monoclonal avian IgY immunoglobulins providing improved assay performance. Therefore we subjected a human synthetic scFv library to selection following different strategies. TNP-specific human antibody fragments could be identified, characterized for their primary structure and evaluated for production as soluble scFv in Escherichia coli. Additionally, a murine TNP-specific antibody fragment was obtained from the hybridoma 11B3; however, the prokaryotic expression level was found to be limited. To generate and evaluate immunoglobulin formats with superior characteristics, all recombinant antibody fragments then were converted into two different chimaeric bivalent IgY antibody formats. After expression in mammalian cells, the IgY antibodies were assessed for their reactivity towards TNT. The IgY antibodies generated on the basis of the combinatorial library proved to be useful for detection of TNT, thereby emphasizing the high potential of this approach for the development of detection devices for immunoassay-based techniques.
如今，重组抗体和噬菌体展示技术能够高效生成免疫工具，并对其进行后续操作以优化亲和力、特异性或整体性能。这些优势对于半抗原靶标结构（如TNT（2,4,6-三硝基甲苯））尤为重要。TNT及其分解产物的毒性使得可靠且快速地检测水性样品中的低含量TNT极为重要。在本研究中，我们旨在生成对TNT替代物TNP（2,4,6-三硝基苯基）具有特异性的单链抗体片段（scFvs），并随后将其制备为单克隆禽IgY免疫球蛋白，以提高检测性能。因此，我们采用不同策略对人源合成scFv文库进行筛选。可以鉴定出TNP特异性人源抗体片段，对其一级结构进行表征，并评估其在大肠杆菌中作为可溶性scFv的生产情况。此外，从杂交瘤11B3中获得了鼠源TNP特异性抗体片段；然而，发现其原核表达水平有限。为了生成并评估具有卓越特性的免疫球蛋白形式，随后将所有重组抗体片段转化为两种不同的嵌合二价IgY抗体形式。在哺乳动物细胞中表达后，评估IgY抗体对TNT的反应性。基于组合文库生成的IgY抗体被证明可用于检测TNT，从而强调了这种方法在开发基于免疫测定技术的检测设备方面的巨大潜力。
