Logit-log calibration curves for EMIT assays.

The logit-log and four-parameter logistic procedures when appropriate for calculation of enzyme-multiplied immunoassay (EMIT) data have the advantage that they can be applied regardless of the kinetic analyzer or reaction conditions. To use these procedures correctly one must determine the change in absorbance at an infinite drug concentration (delta A infinity). The marked variation of delta A infinity with equipment and reaction conditions and the difficulty in determining this value have hindered broad use of these otherwise universally applicable procedures. We have evaluated two simple methods for determining delta A infinity, both based on its equivalence to delta A in the absence of specific antibody: (1) cross-kit reaction using antibody/substrate and enzyme-drug reagents from kits for different drugs, and (2) substitution of an antibody-free substrate reagent with composition based on direct analysis. The cross-kit procedure was tested with EMIT assays for phenobarbital, primidone, phenytoin, carbamazepine, ethosuximide, and theophylline. In some cases an unexpected type of cross-reaction occurred, giving an erroneously low value for delta A infinity. The antibody-free substrate reagent always permitted accurate determination of delta A infinity.