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Competitive protein binding assay for methotrexate

C E Myers, M E Lippman, H M Elliot

    Proceedings of the National Academy of Sciences of the United States of America
    |September 1, 1975
    PubMed
    Summary
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    A new competitive protein binding assay accurately detects methotrexate using Lactobacillus casei dihydrofolate reductase. This method is rapid, sensitive, and unaffected by common interfering substances in clinical samples.

    Area of Science:

    • Biochemistry
    • Analytical Chemistry
    • Pharmacology

    Background:

    • Methotrexate is a crucial chemotherapy drug.
    • Accurate quantification of methotrexate is essential for effective treatment and toxicity monitoring.
    • Existing assays may lack speed or specificity.

    Purpose of the Study:

    • To develop a rapid and sensitive competitive protein binding assay for methotrexate.
    • To utilize the high-affinity binding of methotrexate to Lactobacillus casei dihydrofolate reductase.
    • To validate the assay's performance and specificity.

    Main Methods:

    • Developed a competitive protein binding assay using Lactobacillus casei dihydrofolate reductase.
    • Separated bound and free drug using dextran-albumin coated charcoal.

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  • Analyzed enzyme-drug interaction using Scatchard plot analysis.

    • Main Results:

    • The assay demonstrated high-affinity binding with an association constant (Ka) of 2.1 X 10(8) M-1.
    • Achieved detection of methotrexate in the range of 0.3--30 pmol with <15% coefficient of variation.
    • Showed no interference from 5-methyl tetrahydrofolate, leucovorin, or a key methotrexate metabolite.

    Conclusions:

    • The developed assay is a rapid, sensitive, and specific method for methotrexate quantification.
    • The assay shows excellent agreement with existing methods in clinical samples.
    • This assay offers a valuable tool for therapeutic drug monitoring of methotrexate.