Unexpected suppression of immunoassay results by cross-reactivity: now a demonstrated cause for concern.

More than other analytical procedures, immunoassays have afforded a wealth of knowledge about biochemical physiology over the last 40 years. These assays have posed major analytical challenges, which most likely stem from the delicate balance between chemical equilibrium and our ever-increasing quest for speed of analysis, simplicity of procedure, and improved reliability. In attempts to enhance these criteria, we often face limitations imposed by the fundamental principles of thermodynamics: balancing chemical equilibrium, the underlying kinetics, and that ever-present variable, time. Cross-reactivity in competitive immunoassays can be measured by various methods, including 50% displacement, equal displacement, or the gradient approach (1 ). Typically, a cross-reactant is conceptually regarded as an interferent causing positive bias in the assay results. When an immunoassay is “rushed” (i.e., the signal is measured well before reaching equilibrium), the effect of the cross-reactant is enhanced, in essence increasing the positive bias (2 ). That is the expected phenomenon. The unexpected is the observation that cross-reactivity can also lead to suppression in recovery (3 ), i.e., a negative bias. In other words, the result obtained for a constant amount of the analyte in a sample is lower when the cross-reactant is present than when it is absent. This indicates that rigorous characterization of cross-reactivity requires studies in the presence of the analyte (1 ). Suppression of assay results induced by cross-reactivity was first described in Clinical Chemistry in 1996 for digoxin immunoassays (4 ). In retrospect, however, previous observations hinted at this problem. Kanan et al. (5 ) reported that in plasma obtained from patients suspected of having increased concentrations of digoxin-like immunoreactive factor, recovery of digoxin was lower in one assay than in others. Additional immunoassays have also been shown to be subject to suppression of results caused by cross-reactivity (6, 7). In addition to digoxin-like immunoreactive factor, other cross-reactants, such as progesterone (7 ), digitoxin (8 ), or oleandrin (3 ), have been reported to suppress recovery of digoxin in various immunoassays. A proposed mechanism for the observed suppression of results lies in the physical design (i.e., architecture) of the assay and is fundamentally based on the fact that for binding of small ligands to antibodies, the rates of association are comparable for primary ligand and crossreactant. It is the rate of dissociation that is different and accounts for the lower binding affinity of the cross-reactant molecule (2 ). The microparticle enzyme immunoassay (MEIA) for digoxin (Abbott Diagnostics) demonstrates a thermodynamic justification for this phenomenon. In this assay, digoxin in the sample (shown as gray boxes in Fig. 1A) binds to anti-digoxin polyclonal antibodies complexed to microparticles (MP in Fig. 1A). An aliquot of this reaction mixture is transferred to the matrix cell where the microparticle binds to glass fiber. The matrix cell is then washed to remove the unbound material. The enzyme (alkaline phosphatase) complexed to digoxin serves as the tracer binding to unoccupied antibody sites. The instrument measures the conversion of substrate (4-methylumbelliferyl phosphate) to a fluorescent product by the enzyme. The amount of product formed is inversely proportional to the concentration of digoxin present in the sample. A cross-reactant (shown as hatched boxes in Fig. 1B) such as progesterone competes with digoxin for binding to the antibody sites with similar association rate constants. However, during the wash step, the dissociation rate for the cross-reactant is greater