Sayyadi Nima, Care Andrew, Connally Russell E, Try Andrew C, Bergquist Peter L, Sunna Anwar
Macquarie University, Department of Chemistry and Biomolecular Sciences, Sydney, NSW 2109, Australia.
Macquarie University, ARC Centre of Excellence for Nanoscale BioPhotonics, Sydney, NSW 2109, Australia.
Sci Rep. 2016 Jun 10;6:27564. doi: 10.1038/srep27564.
Luminescent lanthanide chelates have been used to label antibodies in time-gated luminescence (TGL) bioimaging. However, it is a challenging task to label directly an antibody with lanthanide-binding ligands and achieve control of the target ligand/protein ratios whilst ensuring that affinity and avidity of the antibody remain uncompromised. We report the development of a new indirect detection reagent to label antibodies with detectable luminescence that circumvents this problem by labelling available lysine residues in the linker portion of the recombinant fusion protein Linker-Protein G (LPG). Succinimide-activated lanthanide chelating ligands were attached to lysine residues in LPG and Protein G (without Linker) and the resulting Luminescence-Activating (LA-) conjugates were compared for total incorporation and conjugation efficiency. A higher and more efficient incorporation of ligands at three different molar ratios was observed for LPG and this effect was attributed to the presence of eight readily available lysine residues in the linker region of LPG. These Luminescence-Activating (LA-) complexes were subsequently shown to impart luminescence (upon formation of europium(III) complexes) to cell-specific antibodies within seconds and without the need for any complicated bioconjugation procedures. The potential of this technology was demonstrated by direct labelling of Giardia cysts and Cryptosporidium oocysts in TGL bioimaging.
发光镧系螯合物已被用于在时间分辨发光(TGL)生物成像中标记抗体。然而,直接用镧系元素结合配体标记抗体,并在确保抗体亲和力和avidity不受影响的同时控制目标配体/蛋白质比例,是一项具有挑战性的任务。我们报告了一种新型间接检测试剂的开发,该试剂可通过标记重组融合蛋白Linker-Protein G(LPG)连接部分中可用的赖氨酸残基,用可检测的发光标记抗体,从而规避了这个问题。将琥珀酰亚胺活化的镧系螯合配体连接到LPG和Protein G(无连接子)中的赖氨酸残基上,并比较所得的发光激活(LA-)缀合物的总掺入率和缀合效率。观察到LPG在三种不同摩尔比下配体的掺入更高且更有效,这种效应归因于LPG连接区域中存在八个容易获得的赖氨酸残基。这些发光激活(LA-)复合物随后被证明能在几秒钟内(在形成铕(III)复合物后)赋予细胞特异性抗体发光,且无需任何复杂的生物缀合程序。通过在TGL生物成像中直接标记贾第虫囊肿和隐孢子虫卵囊,证明了该技术的潜力。