Immunoassay of digoxin by differential centrifugation

We describe an immunoassay of digoxin that exploits the differential sedimentation rate between two types of latex particles in a microcentrifugal analyzer. Conventional polystyrene latex particles (relative density = 1.05), sensitized with antibody, are first suspended with the sample and then mixed with digoxin-sensitized latex particles (relative density = 1.5). In the absence of digoxin in the sample, the two particle types bind to each other and, when subjected to centrifugal force, are cleared from the solution simultaneously. In the presence of digoxin, the binding between the particles is inhibited in proportion to the concentration of the drugs; the particles therefore can be differentially sedimented in 1 min. We measured the resulting absorbance to quantify the number of less-heavy particles and used the assay to generate a linear standard curve from 0 to 5 micrograms/L. Because the detection limit of the assay depends on the concentrations of both particle types, we optimized the assay by using two-dimensional analysis. Interference from rheumatoid factor was negligible up to 800 kilo-int. units/L. Precision studies indicated CVs between 5% and 13% within-run and between 4% and 9% for run-to-run in this endpoint assay.