Rapid radioimmunoassay for methotrexate in biological fluids

We report a radioimmunoassay for the cytostatic folic acid antagonist methotrexate. Antiserum was raised in rabbits immunized with a conjugate of methotrexate and methylated bovine serum albumin. The calibration curve of the assay is linear from 1 to 100 ng methotrexate per 100-mul sample, 0.1 ng methotrexate still inhibiting the binding of the tracer by 20%. The coefficient of variation ranged from 4.04% to 11.58%. The reaction of the antiserum proved to be specific for methotrexate, as only insignificant interference could be demonstrated with folic acid, folinic acid (leucovorin), or tetrahydrofolic acid. With the method, results are available about 20 h after blood sampling at a daily capacity of at least 48 samples (i.e., about 15 determinations), depending on the equipment. Concentrations of methotrexate in plasma and erythrocytes were determined in five patients who were on continuous treatment with the drug. In one patient the concentration/time curves were followed in plasma and erythrocytes by frequent determinations for 24 h after a parenteral dose. The results indicate that the procedure is well suited for clinical investigations.