蛋白质酪氨酸磷酸酶活性的荧光法和时间分辨免疫荧光法检测

Fluorometric and time-resolved immunofluorometric assays for protein-tyrosine phosphatase activity.

作者信息

Galvan B, Christopoulos T K

机构信息

Department of Chemistry and Biochemistry, University of Windsor, Ontario, Canada.

出版信息

Clin Biochem. 1996 Apr;29(2):125-31. doi: 10.1016/0009-9120(95)02026-8.

DOI:10.1016/0009-9120(95)02026-8

PMID:8601320

Abstract

OBJECTIVE

To develop sensitive nonisotopic assays for protein-tyrosine phosphatase (PTP) activity.

METHODS

The fluorometric assay is based on the fact that phosphotyrosine but not tyrosine forms highly fluorescent complexes with Tb3+. Thus, PTP activity can be followed by measuring the decrease of fluorescence due to hydrolysis of phosphotyrosine. The time-resolved immunofluorometric assay employs tyrosine-phosphorylated substrates, immobilized on microtitre wells. After incubation with PTP, the remaining phosphotyrosine residues are reacted with an antiphosphotyrosine antibody. The immunocomplexes formed are detected with an alkaline phosphatase (ALP)-labeled second antibody. The phosphate ester of 5' fluorosalicylate (FSAP) is used as substrate. The fluorosalicylate produced forms highly fluorescent complexes with Tb3+ - EDTA in alkaline solution. The fluorescence is measured with a time-resolved fluorometer.

RESULTS

The truncated form of the T-cell protein tyrosine phosphatase (TCdeltaC11 PTP) was determined in the range 1100-36,500 U/L by the fluorometric assay and 36-7100 U/L by the time-resolved immunofluorometric assay.

CONCLUSIONS

The two nonisotopic assays should prove beneficial for the determination and study of various PTP.

摘要

目的

开发用于蛋白质酪氨酸磷酸酶（PTP）活性的灵敏非同位素检测方法。

方法

荧光检测法基于磷酸酪氨酸而非酪氨酸能与Tb3+形成高荧光复合物这一事实。因此，可通过测量由于磷酸酪氨酸水解导致的荧光降低来追踪PTP活性。时间分辨免疫荧光检测法采用固定在微量滴定孔上的酪氨酸磷酸化底物。与PTP孵育后，剩余的磷酸酪氨酸残基与抗磷酸酪氨酸抗体反应。用碱性磷酸酶（ALP）标记的二抗检测形成的免疫复合物。5'氟水杨酸磷酸酯（FSAP）用作底物。产生的氟水杨酸在碱性溶液中与Tb3+-EDTA形成高荧光复合物。用时间分辨荧光计测量荧光。