Methotrexate is a drug used in the treatment of cancer and autoimmune disease. It is designed as an anti-folate to inhibit the metabolism of folic acid. Two distinct mechanisms of action have been described for methotrexate. In cancer treatments, methotrexate competitively inhibits the dihydrofolate reductase (DHFR) by blocking folate binding. DHFR converts dihydrofolate to active tetrahydrofolate. Inhibition of DHFR results in inhibition of the synthesis of purine and pyrimidine bases effectively limiting DNA and RNA synthesis and cancer cell growth. In autoimmune disease and specifically in the treatment of rheumatoid arthritis, methotrexate appears to impact several pathways resulting in inhibition of T cell activation. The effects include suppression of T cell expression of intercellular adhesion molecules, inhibition of methyl transferase activity and increased CD95 sensitivity leading to apoptosis in active T cells.

Monitoring methotrexate levels is important to assure appropriate levels are maintained during therapy or treatment. High levels of methotrexate can lead to toxicity and potential renal failure as well as immunosuppression. Additionally, methotrexate is known to interact with a wide variety of drugs leading to additional complications. Determining the presence of methotrexate in samples from subjects in blinded research studies can assist in the interpretation of study results.

Methotrexate is established as one of the most effective and safe therapeutics for rheumatoid arthritis. The safety profile assures that methotrexate will continue to be used in new studies in combination with other new or established drugs. The same is true in its use as a cancer therapeutic. The Methotrexate ELISA enables monitoring levels of methotrexate in both preclinical and clinical research. The methotrexate assay is also appropriate for the detection of methotrexate contamination after its use as a selective agent for recombinant protein production in mammalian cell lines.