Dihydrofolate reductase enzyme inhibition assay for plasma methotrexate determination using a 96-well microplate reader

Brigitte C. Widemann, Frank M. Balis, Peter C. Adamson

Research output: Contribution to journalArticle

Abstract

Microplate reader assays offer several advantages over conventional spectrophotometric assays. We adapted the dihydrofolate reductase (DHFR) enzyme inhibition assay for use in a 96-well microplate reader to measure plasma methotrexate (MTX) concentrations. The assay is linear from 0.01 to 0.1 μmol/L. The within-run CVs at 0.03 μmol/L and 0.08 μmol/L MTX were 4.0% and 2.7%, respectively, and the interday (total) CVs were 7.6% and 1.8%. Cross-reactivity with the inactive MTX metabolite 2,4-diamino-N10- methylpteroic acid (DAMPA) was 3.9%, significantly less than that described with commercial immunoassays; with 7-hydroxymethotrexate cross-reactivity was 1.7%. In addition to sensitivity and specificity, the advantages of this assay are small sample volumes, simultaneous analysis of multiple samples, and rapid turnaround. Because of its greater specificity, the DHFR enzyme inhibition assay may be useful when DAMPA is present in plasma samples and HPLC is not available.

Original languageEnglish (US)
Pages (from-to)223-228
Number of pages6
JournalClinical chemistry
Volume45
Issue number2
StatePublished - Feb 19 1999

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Biochemistry, medical

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