Oriented Immobilization of Single-Domain Antibodies Using SpyTag/SpyCatcher Yields Improved Limits of Detection
Oriented Immobilization of Single-Domain Antibodies Using SpyTag/SpyCatcher Yields Improved Limits of Detection
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DOI：
10.1021/acs.analchem.9b02096
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发表时间：
2019-08-06
影响因子：
7.4
通讯作者：
Goldman, Ellen R.
中科院分区：
文献类型：
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作者：
Anderson, George P.;Liu, Jinny L.;Goldman, Ellen R.
单域抗体是从骆驼体内发现的非常规重链抗体中重组产生的可变重域抗体，它提供了稳定、表达良好的结合元件，具有良好的亲和力，可以通过蛋白质工程为特定的应用量身定做。复杂的基质，如血浆和血清，会显著降低检测的灵敏度。因此，为了在复杂的基质中实现高灵敏度的检测，一种高效的分析是必不可少的。我们生产了基因连锁的sdAb二聚体和三聚体，每个三聚体的C末端都包括SpyTag。将构建物固定在SpyCatcher偶联的染色磁性微球上，并在三明治分析中表征其作为捕获剂的性能。在对添加到缓冲液中的葡萄球菌肠毒素B的检测中，对定向单体、二聚体和三聚体捕获的能力进行的初步测试表明，定向二聚体形式提供了最佳的灵敏度，同时提供了强大的蛋白质生产。因此，这种形式被用来改进基于sdAb的检测血清中登革病毒(DENV)非结构蛋白1(NS1)的方法。当使用定向二聚体捕获时，从添加到50%正常人血清中的四种DENV血清型中的每一种中检测到NS1至少增加了5倍。然后，我们展示了使用定向二聚体捕获来改进临床样本中NS1的检测的潜力。这一通用方法应该会增强sdAb在任何诊断分析中的实用性，包括那些对高后果病原体的诊断分析。
Single-domain antibodies (sdAb), recombinantly produced variable heavy domains derived from the unconventional heavy chain antibodies found in camelids, provide stable, well-expressed binding elements with excellent affinity that can be tailored for specific applications through protein engineering. Complex matrices, such as plasma and serum, can dramatically reduce assay sensitivity. Thus, to achieve highly sensitive detection in complex matrices a highly efficient assay is essential. We produced sdAb as genetically linked dimers, and trimers, each including SpyTag at their C-terminus. The constructs were immobilized onto dyed magnetic microspheres to which SpyCatcher had been coupled and characterized in terms of their performance as capture reagents in sandwich assays. Initial tests on the ability of oriented monomer, dimer, and trimer captures to improve detection versus unoriented constructs in an assay for staphylococcal enterotoxin B spiked into buffer showed the oriented dimer format provided the best sensitivity while offering robust protein production. Thus, this format was utilized to improve a sdAb-based assay for the detection of dengue virus (DENV) nonstructural protein 1 (NS1) in serum. Detection of NS1 from each of the four DENV serotypes spiked into 50% normal human serum was increased by at least a factor of 5 when using the oriented dimer capture. We then demonstrated the potential of using the oriented dimer capture to improve detection of NS1 in clinical samples. This general method should enhance the utility of sdAb incorporated into any diagnostic assay, including those for high consequence pathogens.