Development of Radioimmunoassay for Measurement of Serum Digoxin in Digitalized Patients Using Novel Anti-digoxin Antiserum.

Abstract

Digoxin is the most widely used cardiac glycoside for the treatment of congestive heart failure and atrial fibrillation. Digoxin is known to be converted metabolically to digoxigenin bis-digitoxoside, digoxigenin mono-digitoxoside and digoxigenin by cleavage of the sugar moiety. 1,2) In addition, it is metabolized to dihydrodigoxin by reduction of the double bond in the lactone ring.3)The analysis of digoxin in biological fluids can be performed by immunoassays. The antisera used for these immunoassays are produced by immunization with a haptencarrier protein conjugate, which is coupled to the terminal digitoxose of digoxin using periodate oxidation. 4,5) These antisera show high cross-reactivity with metabolites formed by the successive cleavage of the digitoxose residues. [6][7][8] In the radioimmunoassay (RIA) for determining digoxin concentrations in serum, 3 H-and 125 I-labeled digoxin derivatives have been widely used. [8][9][10] We recently reported the preparation and antigenic properties of digoxin-bovine serum albumin (BSA) conjugates linked at the digitoxose C-3Ј and C-3Љ positions. 11,12) The properties of the antisera were characterized by RIA for [ 3 H]-digoxin. Although the antiserum elicited by digoxin 3Ј-hemisuccinate-BSA conjugate possesses high specificity for digoxin, it still lacks the sensitivity needed for measurement of digoxin concentrations in serum. The sensitivity of RIA increases using a tracer whose specific radioactivity is high. In the present study, we describe development of a type of RIA of digoxin 3Ј-hemisuccinyl-[ 3 H]leucine using the antiserum elicited by digoxin 3Ј-hemisuccinate-BSA conjugate for measuring digoxin in serum from digitalized patients, and compare its performance with commercially available anti-digoxin BSA serum and monoclonal anti-digoxin. , and Dextran T-70 from Pharmacia Fine Chemicals (Uppsala, Sweden). Norit SX and other general reagents were supplied by Wako Pure Chemical Industries (Osaka, Japan). Digoxigenin, and its mono-and bis-digitoxosides were prepared by hydrolysis of digoxin according to the methods of Kaiser and his colleagues. MATERIALS AND METHODS Materials 13)RIA was performed in phosphate saline buffer (pH 7.4) containing K 2 HPO 4 (0.696 g), NaH 2 PO 4 · H 2 O (0.138 g), NaCl (4.39 g), and BSA (1.0 g) in H 2 O (500 ml). A dextrancoated charcoal suspension was prepared by continuously stirring Norit SX (500 mg) and Dextran T-70 (50 mg) in cold phosphate saline buffer (40 ml) for 10 min prior to use.The antiserum (antibody (A)) was prepared by immunizing rabbit with digoxin 3Ј-hemisuccinate-BSA conjugate. 12)The commercial anti-digoxin BSA serum (antibody (B)) and monoclonal anti-digoxin (antibody (C)) were supplied by BioMakor Co. (Rehovot, Israel).Apparatus The melting point was determined with a Yanagimoto micro hot-stage apparatus and is uncorrected. Optical rotation was measured with a JASCO DIP-370 digital polarimeter. FAB-MS measurement was made on a JEOL HX-100 instrument equipped with a FAB ion source using glycerol and NaCl as th...

Rapid and simultaneous measurement of midazolam, 1′-hydroxymidazolam and digoxin by liquid chromatography/tandem mass spectrometry: Application to an in vivo study to simultaneously measure P-glycoprotein and Cytochrome P450 3A activity

“…Several methods for the determination of DG in bio-matrix also have been reported, including immunoassay techniques [17,18], HPLC methods [19,20] and LC-MS/MS methods [21,22]. However, to our knowledge, no analytical method has been reported that is able to simultaneously determine MDZ, 1 -OHMDZ and DG in bio-matrix after co-administration of MDZ and DG.…”

Sensitive and specific LC‐MS assay for quantification of digoxin in human plasma and urine

“…Currently, therapeutic drug monitoring for digoxin incorporates a wide variety of immunoassay techniques including radio immunoassay (Ikeda et al, 2002), microparticle enzyme immunoassay (Azzazy et al, 1997), turbidimetric immunoassay (Datta and Dasgupta, 2004), fluorescence polarization immunoassay (Ijiri et al, 2004) and enzyme-linked chemiluminesent immunosorbent assays (Dasgupta et al, 2006). These techniques depend on the binding specificity of an antibody to digoxin to produce a radioactive, chemiluminescent, turbidimetric or fluorescent signal.…”

Pharmacokinetic interaction with digoxin and glucocorticoids in rats detected by radio-immunoassay using a novel specific antiserum

“…We have reported the preparation and antigenic properties of digitoxin-bovine serum albumin (BSA), Dx-BSA, and h-methyldigoxin-BSA conjugates linked at the digitoxose C-3V and C-3W positions using radio immunoassay (RIA) or enzyme immunoassay (EIA) (Fujii et al, 1991(Fujii et al, , 1994Ikeda and Fujii, 2000;Ikeda et al, 2001Ikeda et al, , 2002Higashi et al, 2002). Our previous results suggested that antisera to these conjugates linked at the digitoxose C-3V position showed higher specificity to metabolites, while a commercially available anti-digitoxin and Dx antisera exhibited remarkable cross-reactivities.…”