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A Rapid and Specific Radioimmunoassay for Methotrexate¹

Division of Tumor Immunology, Sidney Farber Cancer Center [V. R.,R. S.], and Department of Pharmacology [V. R.], Harvard Medical School, Boston, Massachusetts 02115

A sensitive and precise radioimmunoassay for methotrexate has been developed using antibody induced in rabbits, tritium-labeled methotrexate, and a nitrocellulose membrane separation technique. Antibody specificity was characterized by comparing the effectiveness of various related compounds to displace labeled methotrexate from the antibody-binding site. Assay of serum samples from persons receiving the drug was rapid and easy to perform. In a pharmacokinetic study of methotrexate, corresponding results were obtained when measurements were made by either enzymic assay or by radioimmunoassay. Drug concentrations could also be monitored in the cerebrospinal fluid and urine of patients on high-dose methotrexate therapy followed by citrovorum factor rescue. The system measured as little as 0.1 to 1 pmole of methotrexate, depending upon the antiserum used, and naturally occurring folates did not interfere with these determinations.

¹ This work was supported in part by Grants CA-06516 from National Cancer Institute and RR-05526 from the Division of Research Sources, NIH.

Received 11/ 8/74. Accepted 2/14/75.