Preparation of polyclonal antibodies for nateglinide (NTG) and development of a sensitive chemiluminescent immunoassay to detect NTG in tablets and serum
Preparation of polyclonal antibodies for nateglinide (NTG) and development of a sensitive chemiluminescent immunoassay to detect NTG in tablets and serum
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那格列奈 (NTG) 多克隆抗体的制备以及检测片剂和血清中 NTG 的灵敏化学发光免疫分析方法的开发
DOI：
10.1016/j.talanta.2015.09.008
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发表时间：
2015
期刊：
影响因子：
6.1
通讯作者：
Xi Rimo
中科院分区：
文献类型：
--
作者：
Zheng Lei;Wang Jing;Zhang Jie;Song Zhaorui;Dong Yaqing;Wang Yufen;Tong Zhongsheng;Deng Chuan;Yin Yongmei;Meng Meng;Xi Rimo
本研究制备了抗糖尿病药物那格列奈（NTG）的多克隆抗体，并建立了灵敏的化学发光免疫分析法（CLIA）检测片剂和血清中NTG的含量。以乙基碳二亚胺(EDC)/羟基琥珀酰亚胺（NHS）和羰基二咪唑(CDI)/4-二甲氨基吡啶（DMAP）为偶联剂，分别合成了两种免疫原。当EDC/NHS激活时，免疫原中更多的NTG分子与载体蛋白结合。采用辣根过氧化物酶(HRP)-鲁米诺- h2o2 -对碘酚增强体系进行CLIA分析。与CDI/DMAP组相比，EDC/NHS组抗体滴度、灵敏度更高，检测线性范围更宽，可作为下一步研究对象。所建立的CLIA方法在结构相似的类似物中对NTG具有良好的选择性。该方法在缓冲液中可检出0.35 ng mL - 1NTG，在血清中可检出2.1 ng mL - 1NTG，在片剂中可检出0.84 ng mL - 1NTG。在5.0 ~ 400µg mL−1范围内，CLIA法与HPLC法测定结果一致（r=0.9986）。CLIA方法一次可检测78个样品，样品只需要稀释预处理即可。综上所述，本研究为在处方控制和药代动力学研究中对NTG进行高通量筛选提供了一种灵敏的检测方法。
In this study, we prepared polyclonal antibodies against anti-diabetic drug nateglinide (NTG), and established a sensitive chemiluminescent immunoassay (CLIA) to detect NTG in tablets and serum. Two kinds of immunogens were synthesized using ethylcarbodiimide (EDC)/hydroxysuccinimide (NHS) and carbonyldiimidazole (CDI)/4-dimethylaminopyridine (DMAP) as coupling reagents respectively. When activated by EDC/NHS, more molecules of NTG coupled with carrier protein in immunogens. A horseradish peroxidase (HRP)-luminol-H2O2system withp-iodophenol enhancement was applied in the CLIA analysis. The antibodies in EDC/NHS group showed higher titer, sensitivity and wider detection linear range than those in CDI/DMAP group, and were chosen for next studies. The developed CLIA assay exhibited good selectivity towards NTG among structually similar analogs. The method could detect as low as 0.35 ng mL−1NTG in buffer, 2.1 ng mL−1NTG in serum and 0.84 ng mL−1NTG in tablets. The CLIA method provided consistent results with HPLC method (r=0.9986) in determination of NTG from 5.0 to 400 µg mL−1. The CLIA method could detect 78 samples in one assay, and the samples need only dilution in pretreatment. As a summary, this research offers a sensitive assay for high-throughout screening of NTG in formulation control and pharmacokinetic studies.