Development of generic immunoassay for the detection of a series of aminoglycosides with 6'-OH group for the treatment of genetic diseases in biological samples.
Development of generic immunoassay for the detection of a series of aminoglycosides with 6'-OH group for the treatment of genetic diseases in biological samples.
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DOI：
10.1016/j.jpba.2012.11.014
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发表时间：
2013-03-05
影响因子：
3.4
通讯作者：
Baasov, Timor
中科院分区：
文献类型：
--
作者：
Shalev, Moran;Kandasamy, Jeyakumar;Skalka, Nir;Belakhov, Valery;Rosin-Arbesfeld, Rina;Baasov, Timor
关键词：
在过去的二十年中，越来越多的科学证据强调了几种氨基糖苷类抗生素（包括庆大霉素和G418）结构在治疗无义突变引起的各种遗传疾病方面的潜在治疗价值。这些发现导致氨基糖苷类合成衍生物的快速发展，这些衍生物被证明比临床使用的抗生素更具靶向性和毒性更小。药物设计和开发的新进展迫切需要开发一种快速、简单和准确的程序来确定各种生物衍生基质中这些潜在的治疗剂。在这里，我们描述了一种通用多克隆抗体的制备，该抗体用于开发同源和异源免疫测定，用于检测各种天然和合成氨基糖苷衍生物，这些衍生物今天被强调为治疗各种遗传疾病的潜在治疗剂。一种常见的双环支架NB82，存在于大多数具有强生物活性的化合物中，被用作两只兔子免疫的通用免疫半抗原。通过一系列的化学步骤，NB82通过戊二酸连接体的N-1位点选择性地偶联到载体蛋白上。对3种代表性化合物NB82、NB84和NB124的识别灵敏度（I50）分别为10±3 ng mL−1、0.5±0.04 μ mL−1和1±0.12 μ mL−1。NB82的检出限为1±0.3 ng mL - 1， NB84的检出限为20±7 ng mL - 1， NB124的检出限为15±8 ng mL - 1。该方法可进一步用于小鼠血清中治疗性化合物的体内监测。血清实验显示出与PBS校准实验相似的检出限，表明不干扰检测灵敏度，在全血样本中具有相当高的回收率，范围为92-107%。
Over the last two decades, a growing number of scientific evidences highlighted the potential therapeutic value of several structures of aminoglycoside antibiotics (including gentamicin and G418) for the treatment of various genetic diseases caused by nonsense mutations. These findings resulted in a fast evolvement of synthetic derivatives of aminoglycosides which were shown to be more target specific and less toxic than the clinically used antibiotics. The emerging progress in drug design and development has necessitated the urge to develop a fast, easy and accurate procedure for the determination of these potential therapeutic agents in various biologically derived matrices. Here we describe the preparation of a generic polyclonal antibody that was used for the development of homologous and heterologous immunoassays for the detection of a wide range of natural and synthetic aminoglycoside derivatives, highlighted today as potential therapeutic agents for the treatment of various genetic diseases. A common two-ring scaffold, NB82, present in the majority of compounds exhibiting potent biological activity, was used as a generic immunization hapten for the immunization of two rabbits. By using a series of chemical steps, NB82 was selectively conjugated via the N-1 position through glutaric acid linker to a carrier protein. Sensitivity (I50) values for the recognition of three representative compounds NB82, NB84 and NB124 were determined to be 10±3 ng mL−1, 0.5±0.04 μg mL−1 and 1±0.12 μg mL−1, respectively. Limits of detection were determined to be 1±0.3 ng mL−1 for NB82, 20±7 ng mL−1 for NB84 and 15±8 ng mL−1 for NB124. The developed assays were further exploited for the in-vivo monitoring of the therapeutic compounds in mice serum. Serum experimentations exhibited similar detection limits as observed for the PBS calibration experiments, demonstrating no interference with assays sensitivity, with rather high recovery ratios ranging from 92–107% in whole blood samples.
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