A fully automated, continuous-flow radioimmunoassay for methotrexate.

We describe a fully automated continuous-flow radioimmunoassay for methotrexate. [125I]Histamine-labeled methotrexate was used as tracer. Anti-methotrexate serum was coupled to a magnetizable solid-phase and the bound and free fractions were separated with an electromagnetic field. The assay is precise (CV less than 2.5%) and rapid (30 samples per hour), incubation volume is small (about 160 micro L), and incubation brief (10 min). The accurate timing inherent in the system obviates the need to attain equilibrium, so that assay of each sample takes only 15 min. The assay is sensitive (1--100 microgram/L). There is no significant carryover between samples of high and low concentration. Results by the automated method correlated well with those by both a manual assay in which the same reagents and separation technique are used (r - 0.99) and a competitive protein-binding assay (r = 0.96).