TITLE:
Development of Highly Specific Enzyme Immunoassay for Monitoring Serum Digitoxin Level in Patients
AUTHORS:
Yasuhiko Higashi, Yukari Ikeda, Mayumi Douno, Youichi Fujii
KEYWORDS:
Digitoxin, Digitoxin 3’-Hemisuccinate-β-D-Galactosidase, Digitoxin 3’-Hemisuccinate-Bovine Serum Albumin, Antiserum, Enzyme Immunoassay, Cross-Reactivity
JOURNAL NAME:
Journal of Analytical Sciences, Methods and Instrumentation,
Vol.6 No.2,
June
8,
2016
ABSTRACT: We previously developed radioimmunoassays (RIAs) for digitoxin and digoxin using antisera raised against digitoxin 3’-hemisuccinate-bovine serum albumin and digoxin 3’-hemisuccinate-bovine serum albumin conjugates, respectively. Very recently, we converted the RIA for digoxin to an enzyme immunoassay (EIA) system. Here, we aimed to convert the RIA for digitoxin to an EIA suitable for measuring serum digitoxin level in patients, using digitoxin 3’-hemisuccinate-β-D-galactosidase as an enzyme-labeled antigen. The developed EIA showed a quantification range of 1 to 70 ng/mL and exhibited high specificity for digitoxin, with low cross-reactivity to digitoxin metabolites. Compared with a commercial anti-digitoxin antiserum clinically used to monitor serum digitoxin level in patients, our antiserum showed much higher specificity for intact digitoxin. Intra- and inter-assay variations were less than 10.0% and 8.5%, respectively. Recovery was within the range of 93.7% - 107.5%. Mean digitoxin concentrations measured in serum samples (n = 26) from digitoxin-treated patients by EIA using our new antiserum and the commercial anti-digitoxin antiserum were 11.0 and 13.8 ng/mL, respectively. The present EIA, which is superior to RIA in terms of convenience and disposal of waste materials, is expected to be practically useful for clinical monitoring of intact digitoxin in serum.