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

Authors
  • Widemann, B C
  • Balis, F M
  • Adamson, P C
Type
Published Article
Journal
Clinical chemistry
Publication Date
Feb 01, 1999
Volume
45
Issue
2
Pages
223–228
Identifiers
PMID: 9931044
Source
Medline
License
Unknown

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 micromol/L. The within-run CVs at 0.03 micromol/L and 0.08 micromol/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.

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