Bioassay for detection of methotrexate in serum.

Objective: A bioassay is developed for the measurement of methotrexate (MTX) in serum. Methods: The assay is based on MTX inhibition of the proliferation of hypoxanthine-guanosine phosphoribosyl transferase (HGPRT) negative mouse B-cells (B9.H). HGPRT negative cells cannot use the salvage pathway of nucleotide synthesis to overcome inhibition by MTX. Results: When B9.H cells are cultured with serial dilutions of serum, inhibition of proliferation is a measure of the amount of MTX in the serum. Circulating folates do not interfere with the assay. Conclusion: This simple assay can detect low concentrations of MTX in serum: it is therefore useful for following the pharmacodynamics of functional MTX after low-dose MTX treatment.