药学学报, 2021, 56(9): 2378-2382
引用本文:
朱小雨, 原梅, 罗素兰, 车津晶. 基于夹心酶联免疫吸附方法的大鼠及比格犬血浆中海洋药物GeXIVA[1,2]定量分析研究[J]. 药学学报, 2021, 56(9): 2378-2382.
ZHU Xiao-yu, YUAN Mei, LUO Su-lan, CHE Jin-jing. Development and validation of antibody sandwich enzyme-linked immunosorbent assay method for quantitation of GeXIVA[1,2] in plasma of rats and Beagle dogs[J]. Acta Pharmaceutica Sinica, 2021, 56(9): 2378-2382.

基于夹心酶联免疫吸附方法的大鼠及比格犬血浆中海洋药物GeXIVA[1,2]定量分析研究
朱小雨1,2,3, 原梅1, 罗素兰2,3, 车津晶1*
1. 军事医学研究院毒物药物研究所, 北京 100850;
2. 广西大学医学院, 广西 南宁 530004;
3. 海南大学生命科学院, 海南 海口 570228
摘要:
GeXIVA[1,2]是利用cDNA克隆技术从我国南海海域的将军芋螺中鉴定的一种新型芋螺毒素,已开发成为新型镇痛药物。本研究建立并验证了大鼠及比格犬血浆中海洋药物芋螺毒素GeXIVA[1,2]的抗体夹心酶联免疫吸附(ELISA)检测方法。制备鼠单克隆抗体4B2及生物素标记兔多克隆抗体2#,使用棋盘法优化抗体配对浓度、最小稀释比例、温育温度、温育时间,建立抗体夹心ELISA检测方法,并进行验证。动物实验经军事科学院军事医学研究院动物伦理与使用委员会批准(编号:IACUC-DWZX-2020-698)。结果表明建立的ELISA方法在大鼠及比格犬血浆中的定量范围均在1.25~80 ng·mL-1,精密度、准确度、选择性、特异性、稳定性、稀释线性、钩状效应均满足生物样本分析要求。本方法可用于新型海洋药物GeXIVA的临床前药代动力学研究。
关键词:    GeXIVA[1,2]      多肽      夹心ELISA      生物分析      方法学验证     
Development and validation of antibody sandwich enzyme-linked immunosorbent assay method for quantitation of GeXIVA[1,2] in plasma of rats and Beagle dogs
ZHU Xiao-yu1,2,3, YUAN Mei1, LUO Su-lan2,3, CHE Jin-jing1*
1. Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China;
2. Medical College of Guangxi University, Nanning 530004, China;
3. School of Life Science, Hainan University, Haikou 570228, China
Abstract:
GeXIVA[1,2] is a new type of conotoxin recently discovered in the transcriptome of Conus generalis and it is expected to be used clinically as a new type of analgesic. This study established and verified a sandwich enzyme-linked immunosorbent assay method for the marine drug GeXIVA[1,2] in the plasma of rats and Beagle dogs. The mouse monoclonal antibody 4B2 and biotin-labeled rabbit polyclonal antibody 2# were developed. The checkerboard method was used to optimize the antibody pairing concentration, minimum dilution ratio, incubation temperature, and incubation time to establish an antibody sandwich ELISA detection method. Verify the established testing methods. The established ELISA method has a quantitative range of 1.25-80 ng�mL-1 in rat and Beagle plasma. The precision, accuracy, selectivity, specificity, stability, dilution linearity, and hook effect all meet the requirements for biological sample analysis. All the procedures for the animal experiments were approved by the Animal Ethics Committee of the Institute (Permit Number:IACUC-DWZX-2020-698). This method can support the preclinical pharmacokinetic study of the marine drug GeXIVA.
Key words:    GeXIVA[1,2]    peptide    sandwich ELISA    bioanalysis    methodology validation   
收稿日期: 2021-05-10
DOI: 10.16438/j.0513-4870.2021-0696
基金项目: 海南省重大科技计划项目(ZDKJ2016002).
通讯作者: 车津晶,Tel:86-10-66930633,E-mail:chejinjing80@126.com
Email: chejinjing80@126.com
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