Abstract
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.
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