FLUOROMETRIC MIMETIC ENZYME-IMMUNOASSAY OF METHOTREXATE

A new fluorometric immunoassay for methotrexate (MTX) was developed us ing the mimetic enzyme Mn(III)-tetrakis(sulfophenyl) porphine (Mn-TPPS 4) as a labelling reagent to catalyze the fluorescence reaction of 4-h ydroxyphenylacetic acid (HPA) and hydrogen peroxide. In the competitiv e assay MTX (antigen) from human serum and Mn-TPPS4 labelled MTX compe titively react with antibody (anti-MTX) coated on a polystyrene 40-wel l plate. The Mn-TPPS4 in the bound fraction, after the separation from the free fractions, was determined by measuring the fluorescence inte nsity between HPA and H2O2 catalyzed by bound Mn-TPPS4 and MTX conjuga te, which was anti-proportional to the concentration of MTX in the ser um. MTX can be determined between 0.5 and 10 mug/well with a detection limit of 100 ng/well.