Improved double-antibody enzyme immunoassay for methotrexate.

We report an enzyme immunoassay procedure for methotrexate measurement that takes less than 3 h to perform. beta-D-Galactosidase (EC 3.2.1.23) from Escherichia coli was conjugated to methotrexate by means of the mixed anhydride reaction. Bound and free labeled drug were separated by a preincubated cubic complex of first and second antibody. The enzyme activity of the bound fraction was measured with o-nitrophenyl-beta-D-galactopyranoside as substrate. The standard curve covered the range 1 to 10 micrograms of methotrexate per liter. One microgram of methotrexate per liter inhibited binding of the tracer by 17%. The assay is specific for methotrexate in the presence of folinic acid (citrovorum factor), folic acid, tetrahydrofolic acid, and other methotrexate metabolities. Intra- and inter-assay CVs were less than 5 and 10%, respectively. Results obtained with this enzyme immunoassay method agreed well with those obtained with an established radioimmunoassay method.