Abstract: A radiochemical ligand binding assay for methotrexate is provided. A binder factor comprising a partially purified dihydrofolic acid reductase preparation is employed. The binder factor is conveniently prepared by homogenizing a factor containing animal organ such as liver, and extracting with isotonic saline and ammonium sulfate. A binder cofactor, NADPH.sub.2, is also employed in the binding reaction. The procedure contemplates both direct and sequential assay techniques, and it is not interfered with by vast excesses of many natural folate derivatives.

Abstract: There is provided a rapid and less costly radioisotopic assay for measuring the concentration of folate in blood serum. This procedure utilizes .sup.3 H-pteroylmonoglutamate, unlabeled 5-methyl-tetrahydrofolic acid, and a partially purified folate binder, such as for example a folate binder extracted from hog kidney. The procedure involves radioisotopically relating the bound amounts of a labeled folate and a known folate at various concentrations of the known folate in a system containing a predetermined amount of the labeled folate, a predetermined amount of the binder factor for the folates, and a predetermined amount of defolated test serum.