Abstract
A highly sensitive homogeneous electrogenerated chemiluminescence (ECL) immunoassay for the determination of anti‐digoxin antibody and digoxin hapten was developed employing Ru(bpy)~2~(dcbpy)NHS (bpy = 2,2′‐bipyridyl; dcbpy = 2,2′‐bipyridine‐4,4′‐dicarboxylic acid; NHS = N‐hydroxysuccinimide ester) as an electrochemiluminescent label and bovine serum albumin (BSA) as a carrier protein. A digoxin hapten was indirectly heavily labelled with Ru(bpy)~2~(dcbpy)NHS through BSA to form Ru(bpy)~2~(dcbpy)NHS–BSA–digoxin conjugate. The ECL intensity of the immunocomplex of the conjugate with anti‐digoxin antibody markedly decreased when the immunoreaction between Ru(bpy)~2~(dcbpy)NHS–BSA–digoxin conjugate and anti‐digoxin antibody took place. Two formats, direct homogeneous immunoassay for anti‐digoxin antibody and competitive immunoassay for digoxin, were developed to determine anti‐digoxin antibody and digoxin, respectively. The anti‐digoxin antibody concentration in the range 7.6 × 10^−8^–7.6 × 10^−6^ g/mL was determined by direct homogeneous format. Digoxin hapten was determined throughout the range 4.0 × 10^−10^–1.0 × 10^−7^ g/mL with a detection limit of 1.0 × 10^−10^ g/mL by competitive format. The relative standard derivation for 6.0 × 10^−9^ g/mL was 4.3%. The method has been applied to assaying digoxin in control human serum. Copyright © 2006 John Wiley & Sons, Ltd.