Enzymic inhibition assay for methotrexate with a discrete analyzer, the ABA-100

We adapted an inhibition assay for methotrexate, involving dihydrofolate reductase from bovine liver, for use with a discrete analyzer (the ABA-100). The analyzer was used both for dilution and a 5-min pre-incubation of the sample with NADPH--enzyme reagent, and for the assay itself. The standard curve was linear between 10 and 120 microgram/L. Without pre-incubation the standard curve was nonlinear. The presence of albumin in the NADPH--enzyme reagent enhanced both enzyme activity and stability. Within-run precision (CV) was 2.0% (n = 24), run-to-run precision 7.1% (n = 49). Results obtained on patients' samples (29 sera, 15 urines, 18 cerebrospinal fluids) by the present method and a radioimmunoassay method did not differ statistically (p greater than 0.05) when the paired data were analyzed by use of the sign test and Wilcoxon's ranked sign test.