Establishment of Enzyme Immunoassay for Measuring β-Methyldigoxin Levels in Human Serum by Specific Antiserum

• Published in: Biological & pharmaceutical bulletin 2002, Vol.25(10), pp.1251-1257
• Main Authors: Higashi, Yasuhiko, Watanabe, Naomi, Sasaki, Toshiyuki, Fujii, Youichi
• Format: Article
• Language: English
• Subjects: Analysis; Animals; anti-digoxin antiserum; anti-β-methyldigoxin 3′-hemisuccinate–BSA antiserum; Biological and medical sciences; cross-reactivity; enzyme immunoassay; General pharmacology; Humans; Immune Sera - analysis; Immunoenzyme Techniques - methods; Male; Medical sciences; Medigoxin - analysis; Medigoxin - blood; Pharmacology. Drug treatments; phenyl boric acid column; Rabbits; Serum Albumin, Bovine - analysis; β-methyldigoxin
• ISSN: 0918-6158; 1347-5215
• DOI: 10.1248/bpb.25.1251

We investigated the specificity of obtained antisera to β-methyldigoxin by the enzyme immunoassay. Three types of hapten-bovine serum albumin (BSA) conjugates were synthesized to obtain high specific antisera to β-methyldigoxin. The haptens were linked to the carrier protein through hemisuccinate at C-3′ and C-3″ positions in the digitoxose chain and at C-12 position in the aglycone. Anti-β-methyldigoxin 3′-hemisuccinate–BSA antiserum showed a low detection limit (0.2 ng/ml) and possessed high specificity for β-methyldigoxin, exhibiting low cross-reactions with digoxigenin bisdigitoxoside (8.3%), dihydrodigoxin (4.8%), digitoxin (1.5%), and digoxigenin monodigitoxoside (0.95%), except for cross-reaction with digoxin (43%). Compared with commercial anti-digoxin antiserum, clinically used to monitor β-methyldigoxin concentration in human serum, cross-reaction data of anti-β-methyldigoxin 3′-hemisuccinate–BSA antiserum showed higher specificity for β-methyldigoxin. The intra-assay and inter-assay variations using this antiserum were less than 6.9% and 8.1%, respectively. The recovery tests were good, within the range of 96.2—104.3%. Phenyl boric acid (PBA) column treatment was effective to rapidly and selectively separate β-methyldigoxin from the mixture of β-methyldigoxin and its metabolites in human serum. The recovery tests of β-methyldigoxin with PBA column in human serum were about 110% and interference of metabolites of β-methyldigoxin was negligible. These results suggest that anti-β-methyldigoxin 3′-hemisuccinate–BSA antiserum and PBA column treatment are useful to more precisely monitor the unchanged type of β-methyldigoxin concentration in human serum.