开发用于检测生物样品中具有 6'-OH 基团的一系列氨基糖苷类药物的通用免疫分析方法，用于治疗遗传疾病。
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.
机构信息
The Edith and Joseph Fischer Enzyme Inhibitors Laboratory, Schulich Faculty of Chemistry, Technion - Israel Institute of Technology, Haifa 32000, Israel.
出版信息
J Pharm Biomed Anal. 2013 Mar 5;75:33-40. doi: 10.1016/j.jpba.2012.11.014. Epub 2012 Nov 20.
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 to 107% in whole blood samples.
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抗生素（包括庆大霉素和 G418）结构在治疗各种由无义突变引起的遗传疾病方面的潜在治疗价值。这些发现导致了氨基糖苷类抗生素的合成衍生物的快速发展，这些衍生物比临床使用的抗生素更具靶向性和更低毒性。药物设计和开发方面的新兴进展需要迫切需要开发一种快速、简便和准确的方法，以确定各种生物衍生基质中的这些潜在治疗剂。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分析，以检测广泛的天然和合成氨基糖苷衍生物，这些衍生物目前被认为是治疗各种遗传疾病的潜在治疗剂。在大多数具有有效生物活性的化合物中都存在一个通用的双环支架 NB82，它被用作免疫原，用于免疫两只兔子。通过一系列化学步骤，NB82 通过戊二酸接头选择性地在 N-1 位置与载体蛋白偶联。三种代表性化合物 NB82、NB84 和 NB124 的识别灵敏度（I₅₀）值分别确定为 10±3ng mL⁻¹、0.5±0.04μg mL⁻¹和 1±0.12μg mL⁻¹。NB82 的检测限为 1±0.3ng mL⁻¹，NB84 的检测限为 20±7ng mL⁻¹，NB124 的检测限为 15±8ng mL⁻¹。开发的测定法进一步用于监测小鼠血清中的治疗化合物。血清实验表现出与 PBS 校准实验观察到的相似检测限，表明测定法的灵敏度没有受到干扰，全血样本的回收率相当高，范围为 92%至 107%。
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