Establishment of enzyme immunoassay for measuring beta-methyldigoxin levels in human serum by specific antiserum.
We investigated the specificity of obtained antisera to beta-methyldigoxin by the enzyme immunoassay. Three types of hapten-bovine serum albumin (BSA) conjugates were synthesized to obtain high specific antisera to beta-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-beta-methyldigoxin 3'-hemisuccinate-BSA antiserum showed a low detection limit (0.2 ng/ml) and possessed high specificity for beta-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 antidigoxin antiserum, clinically used to monitor beta-methyldigoxin concentration in human serum, cross-reaction data of anti-beta-methyldigoxin 3'-hemisuccinate-BSA antiserum showed higher specificity for beta-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 beta-methyldigoxin from the mixture of beta-methyldigoxin and its metabolites in human serum. The recovery tests of beta-methyldigoxin with PBA column in human serum were about 110% and interference of metabolites of beta-methyldigoxin was negligible. These results suggest that anti-beta-methyldigoxin 3'-hemisuccinate-BSA antiserum and PBA column treatment are useful to more precisely monitor the unchanged type of beta-methyldigoxin concentration in human serum.
Higashi
Yasuhiko
Y
Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Hokuriku University, Kanazawa, Japan. y-higashi@hokuriku-u.ac.jp
Watanabe
Naomi
N
Sasaki
Toshiyuki
T
Fujii
Youichi
Y
D000818:Animals; D006801:Humans; D007106:Immune Sera / Q000032:analysis; D007124:Immunoenzyme Techniques / Q000379:methods*; D008297:Male; D008520:Medigoxin / Q000032:analysis / Q000097:blood*; D011817:Rabbits; D012710:Serum Albumin, Bovine / Q000032:analysis
D007106:Immune Sera; D012710:Serum Albumin, Bovine; D008520:Medigoxin