FLUOROMETRIC AND TIME-RESOLVED IMMUNOFLUOROMETRIC ASSAYS FOR PROTEIN-TYROSINE-PHOSPHATASE ACTIVITY

Objective: To develop sensitive nonisotopic assays for protein-tyrosin e phosphatase (PTP) activity. Methods: The fluorometric assay is based on the fact that phosphotyrosine but not tyrosine forms highly fluore scent complexes with Tb3+. Thus, PTP activity can be followed by measu ring the decrease of fluorescence due to hydrolysis of phosphotyrosine . The time-resolved immunofluorometric assay employs tyrosine-phosphor ylated substrates, immobilized on microtitre wells. After incubation w ith PTP, the remaining phosphotyrosine residues are reacted with an an tiphosphotyrosine antibody. The immunocomplexes formed are detected wi th an alkaline phosphatase (ALP)-labeled second antibody. The phosphat e ester of 5' fluorosalicylate (FSAP) is used as substrate. The fluoro salicylate produced forms highly fluorescent complexes with Tb3+-EDTA in alkaline solution. The fluorescence is measured with a time-resolve d fluorometer. Results: The truncated form of the T-cell protein tyros ine phosphatase (TC Delta C11 PTP) was determined in the range of 1100 -36,500 U/L by the fluorometric assay and 36-7100 U/L by the time-reso lved immunofluorometric assay. Conclusions: The two nonisotopic assays should prove beneficial for the determination and study of various PT P.