Competitive immunoassay for recombinant hirudin using capillary electrophoresis with laser-induced fluorescence detection.
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.
Ban
E
E
Bioanalysis and Biotransformation Research Center, Korea Institute of Science and Technology, Cheongryang, Seoul, South Korea.
Nam
H S
HS
Yoo
Y S
YS
D000906:Antibodies / Q000276:immunology; D019075:Electrophoresis, Capillary / Q000379:methods*; D006629:Hirudins / Q000032:analysis* / Q000276:immunology; D007118:Immunoassay / Q000379:methods*; D007834:Lasers; D011994:Recombinant Proteins / Q000032:analysis / Q000276:immunology; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*
D000906:Antibodies; D006629:Hirudins; D011994:Recombinant Proteins