Competitive immunoassay for recombinant hirudin using capillary electrophoresis with laser-induced fluorescence detection

被引:25
作者
Ban, E [1 ]
Nam, HS [1 ]
Yoo, YS [1 ]
机构
[1] Korea Inst Sci & Technol, Bioanal & Biotransformat Res Ctr, Seoul 130650, South Korea
关键词
immunoassays; hirudin; polypeptides;
D O I
10.1016/S0021-9673(01)00829-9
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
A competitive immunoassay based on capillary electrophoresis (CE) with laser-induced fluorescence (LIF) has been developed for the determination of recombinant hirudin (r-hirudin) in biological mixtures. Hirudin, a thrombin inhibitor, is a polypeptide of 65 amino acids. To check purity levels and perform pharmacokinetic studies of (r-hirudin), specific and reproducible analysis methods are demanded. The work involved the development of separation conditions allowing for routine analysis of plasma samples. In this study, r-hirudin was labeled with fluorescein isothiocyanate (FITC), and FITC-labeled r-hirudin was purified using high-performance liquid chromatography. The purified product was then mixed with the sample followed with the addition of anti-hirudin antibody. Free, antibody-bound, and tagged r-hirudin could be separated within 5 min by CE analysis using uncoated fused-silica capillary with high reproducibility. The developed method can be used to determine r-hirudin with good precision and a detection limit lower than 20 nM. This result demonstrates the feasibility of the CE-LIF immunoassay method for the determination of r-hirudin in plasma samples. (C) 2001 Elsevier Science B.V. All rights reserved.
引用
收藏
页码:337 / 344
页数:8
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