Preparation of polyclonal antibodies for nateglinide (NTG) and development of a sensitive chemiluminescent immunoassay to detect NTG in tablets and serum

• Published in: Talanta (Oxford) 2016-01, Vol.146, p.483-489
• Main Authors: Zheng, Lei, Wang, Jing, Zhang, Jie, Song, Zhaorui, Dong, Yaqing, Wang, Yufen, Tong, Zhongsheng, Deng, Chuan, Yin, Yongmei, Meng, Meng, Xi, Rimo
• Format: Article
• Language: English
• Subjects: Antibodies - immunology; Blood Chemical Analysis; Chemiluminescence immunoassay; Chemistry, Pharmaceutical; Cyclohexanes - analysis; Cyclohexanes - blood; Cyclohexanes - immunology; Immunoassay - methods; Kinetics; Limit of Detection; Luminescent Measurements - methods; Nateglinide; Phenylalanine - analogs & derivatives; Phenylalanine - analysis; Phenylalanine - blood; Phenylalanine - immunology; Polyclonal antibodies; Serum; Tablets
• ISSN: 0039-9140; 1873-3573
• DOI: 10.1016/j.talanta.2015.09.008

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-H2O2 system with p-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 0.35ngmL−1 NTG in buffer, 2.1ngmL−1 NTG in serum and 0.84ngmL−1 NTG in tablets. The CLIA method provided consistent results with HPLC method (r=0.9986) in determination of NTG from 5.0 to 400µgmL−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. [Display omitted] •Two kinds of immunogens were synthesized to produce antibodies of nateglinide.•Effect of coating antigen, antibody dilution and reaction time was studied.•A highly sensitive and specific immunoassay for nateglinide was developed.•The method could be used in rapid detection of nateglinide in tablets and serum.