Detection of recombinant hirudin using capillary electrophoresis with laser-induced fluorescence detection

Title
Detection of recombinant hirudin using capillary electrophoresis with laser-induced fluorescence detection
Authors
반은미류재천유영숙
Keywords
recombinant (r-)hirudin; Immunoassay; Capillary electrophoresis; Laser-induced fluorescence
Issue Date
2001-12
Publisher
Microchemical journal
Citation
VOL 70, NO 3, 211-217
Abstract
Hirudin, a thrombin inhibitor, is a polypeptide of 65 amino acids. To check purity levels and perform pharmacokinetic studies of recombinant hirudin Žr-hirudin., a specific and reproducible analysis method is required. Capillary electrophoresis ŽCE. is rapidly becoming an important procedure for the analysis of biological molecules. Recently, CE combined with immunoassay has emerged as a new analytical technique. CE-based immunoassay ŽCEIA. is a sensitive and specific method combining laser-induced fluorescence ŽLIF. and immunoassay. Therefore, in this study, we specifically investigated fluorescence labeling and determination of r-hirudin by CEIA with a LIF detector using labeled r-hirudin and polyclonal antibody. r-Hirudin was labeled with fluorescein isothiocyanate ŽFITC.. FITC-labeled r-hirudin was purified using high-performance liquid chromatography ŽHPLC.. The method is based on preincubation of r-hirudin and antibody for 20 min, followed by CE analysis using an uncoated capillary. Free and bound r-hirudin were separated within 5 min using CE with high reproducibility. This study demonstrated that the CEIA method could be applied to quantitative analysis of r-hirudin in biological fluids. 2001 Elsevier Science B.V. All rights reserved.
URI
http://pubs.kist.re.kr/handle/201004/16726
ISSN
0026-265X
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KIST Publication > Article
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