Effect of fab fragment of antidigoxin antibody on digoxin immunoassays

The Fab fragment of antidigoxin antibody is commercially available as Digibind and DigiFab. Digibind has been available in the USA since 1986 (Glaxo Wellcome Inc.), and more recently in 2001, the Food and Drug Administration of the USA approved DigiFab for treating potentially life-threatening digoxin toxicity or overdose. Digibind is produced by immunizing sheep with digoxin followed by the purification of the Fab fragment from blood, whereas DigiFab is prepared by injecting sheep with digoxindicarboxymethylamine followed by purification of fab fragment. The molecular weight of DigiFab (46,000 Da) is similar to that of Digibind (46,200 Da). The approximate dose of Fab fragment is 80 times the digoxin body burden (in mg), or if neither the dose ingested nor the plasma digoxin concentration is known, then 380 mg of Fab fragment should be given. The half-life of the Fab fragment in humans is 12-20 h, but this may be prolonged in patients with renal failure (63). The Fab fragment is also effective in treating digitoxin overdose.

The concentration of digoxin in myocardium is substantially higher than the corresponding digoxin concentration in serum. The Fab binds free digoxin in serum because of its high affinity for digoxin and effectively removes pharmacologically active free digoxin from serum. Therefore, the equilibrium between free and bound digoxin in serum is disturbed and digoxin bound to myocardium is released back in serum and subsequently binds with the Fab fragments. In this process, toxicity because of digoxin can be reversed.

The Fab fragment is known to interfere with serum digoxin measurements using immunoassays, and the magnitude of interference depends on the assay design and the specificity of the antibody used. The MEIA of digoxin (on the AxSYM analyzer) as well as the Stratus digoxin assay show digoxin values that are higher than measured free digoxin concentration in the presence of Fab fragment (64). McMillin et al. studied the effect of Digibind and DigiFab on 13 different digoxin immunoassays. Positive interference in the presence and absence of digoxin was observed with Digibind and DigiFab, although the magnitude of interference was somewhat less with DigiFab. The magnitude of interference varied significantly with each method whereas IMMULITE, Vitros, Dimension, and Access digoxin methods showed highest interference. The magnitudes of interference were in the order of Elecsys, TinaQuant, Integra, EMIT, and Centaur methods whereas minimal interferences were observed with FPIA, MEIA, Synchron, and CEDIA methods (65).

5.1. Elimination of Fab Interference by Ultrafiltration

The molecular weight of the Fab fragment (46,000 or 46,200 Da) is much higher than the cutoff of the Amicon Centrifree filters (30,000) used in the preparation of proteinfree ultrafiltrate for measuring free digoxin concentrations. Therefore, Fab fragment is absent in the protein-free ultrafiltrate, and monitoring free digoxin concentration is not subjected to the interference by the Fab fragment. Jortani et al. reported that analysis of serum ultrafiltrate for digoxin concentration remains the most accurate approach in monitoring unbound digoxin in the presence of the Fab fragment. Moreover, no matrix bias was observed in measuring digoxin concentrations in protein-free ultrafil-trates using immunoassays (66). McMillin et al. commented that patients treated with Digibind can be monitored reasonably by using either MEIA (on AxSYM analyzer) or the Stratus. Another alternative is to measure free digoxin concentration in the proteinfree ultrafiltrate. The immunoassays for direct measurement of digoxin in serum in the presence of Digibind however will overestimate free digoxin concentration (65).

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