Digoxin is widely prescribed for the treatment of cardiac conditions (1). Because of its narrow therapeutic range, digoxin-related toxicity resulting from acute or chronic overdose is common. Metabolites of digoxin as well as related compounds, including digitoxin, tanshinones, bufandienolide, and oleander, can contribute to or independently produce digoxin toxicity (2, 3). Digoxin toxicity can be rapidly and safely reversed by administration of anti-digoxin immune fragments (Fab) such as DIGIBIND®, which has been available in the US since 1986. Therapeutic Fab products act by binding digoxin with high affinity (109–1010 L/mol), favoring movement of digoxin out of tissue and thus promoting elimination. Factors that impact dosing with Fab products include known or suspected digoxin load, patient weight and history, and renal function (4–7). Monitoring the free digoxin concentration after Fab administration may help ensure appropriate dosing, prevent deadly recrudescent toxicity, and determine when digoxin therapy should be resumed (8–10). Monitoring free digoxin in serum is challenged by the positive interference that has been extensively described with DIGIBIND, which interferes with immunoassays by competing with assay capture antibodies. The degree of interference depends on incubation times, washing steps, and the affinity of capture antibody for bound vs free digoxin (11–13). Consequently, monitoring of free digoxin in ultrafiltrates is a popular strategy for managing DIGIBIND-treated patients. Although ultrafiltration eliminates interference produced by large molecules, such as endogenous digoxin-like immunoreactive factors (DLIFs) and DIGIBIND, it does not eliminate interferences produced by small molecules known to interfere with digoxin immunoassays, such as spironolactone (14). In addition, ultrafiltration methods are not standardized, may require matrix-specific calibration, add expense and manual manipulation, and lengthen turnaround time (8, 15, 16). DigiFabTM is a Fab preparation that was approved by the Food and Drug Administration in 2001 for treating potentially life-threatening digoxin toxicity or overdose. Fab is produced by immunization of sheep with digoxin (DIGIBIND) or digoxindicarboxymethylamine (DigiFab), followed by purification of the Fab from blood. The approximate molecular weights of DigiFab (46 000) and DIGIBIND (46 200) are similar, and a single vial of either DIGIBIND or DigiFab will bind 0.5 mg of digoxin in vivo. As such, the clinical claims, dosing recommendations, and administration of DigiFab are identical to those of DIGIBIND. However, clinical studies have monitored DigiFab therapy by measuring digoxin in ultrafiltrates only (17). The present study was designed to determine whether clinically relevant concentrations of DigiFab in serum interfere with 13 digoxin immunoassays and to compare results with DIGIBIND. Single vials of DIGIBIND (38 mg; Glaxo Wellcome Inc.) and DigiFab (40 mg; Protherics, Inc.) were dissolved in 4 mL of type 1 water. DIGIBIND and DigiFab were added to pooled drug-free and DLIF-free serum to obtain final concentrations of 0.1, 0.8, 2, 8, and 16 mg/L. Increasing concentrations of each antidote (1, 2, 4, 8, and 16 mg/L) were also combined with 40 g/L digoxin (Sigma-Aldrich) in pooled serum. Ultrafiltrates were prepared by use of Millipore Centrifree filters (30 kDa) and a fixedangle centrifuge rotor (2000g for 30 min at ambient temperature). Prepared samples were aliquoted and stored frozen (70 C) until analyzed, a practice that should not affect the ability of DIGIBIND to bind digoxin