Competitive protein binding assay for methotrexate

Abstract

A competitive protein binding assay has been developed for methotrexate based on the tight binding of this drug to Lactobacillus casei dihydrofolate reductase (= tetrahydrofolate dehydrogenase; 5,6,7,8 tetrahydrofolate: NADP+ oxidoreductase; EC 1.5.1.3). Free drug may be separated from that bound to reductase by adsorption with dextran albumin coated charcoal. Scatchard plot analysis of the enzyme drug interaction confirmed the presence of a single homogeneous class of binding sites with an association constant K(a) of 2.1 x 108 M-1. This high affinity binding permits detection of methotrexate in the range of 0.3-30 pmol with a coefficient of variation of 15% or less. The predominant circulating folate, 5 methyl tetrahydrofolate, and the clinically useful rescue agent leucovorin (5 formyl tetrahydropteroyl glutamic acid) do not interfere with the assay, nor does the methotrexate metabolite 4 amino 4 deoxy 10 methylpteroic acid. Assay of clinical samples, including plasma and cerebrospinal fluid, showed close agreement between the previously described enzyme inhibition assay and the more rapid competitive binding method.