Fluorometric mimetic enzyme immunoassay of methotrexate

A new fluorometric immunoassay for methotrexate (MTX) was developed using the mimetic enzyme Mn(III)-tetrakis(sulfophenyl) porphine (Mn-TPPS₄) as a labelling reagent to catalyze the fluorescence reaction of 4-hydroxyphenylacetic acid (HPA) and hydrogen peroxide. In the competitive assay MTX (antigen) from human serum and Mn-TPPS₄ labelled MTX competitively react with antibody (anti-MTX) coated on a polystyrene 40-well plate. The Mn-TPPS₄ in the bound fraction, after the separation from the free fractions, was determined by measuring the fluorescence intensity between HPA and H₂O₂ catalyzed by bound Mn-TPPS₄ and MTX conjugate, which was anti-proportional to the concentration of MTX in the serum. MTX can be determined between 0.5 and 10 µg/well with a detection limit of 100 ng/well.