A microtiter enzyme-linked immunosorbent assay for protein tyrosine phosphatase.
We report the development of an enzyme-linked immunosorbent assay (ELISA) for protein tyrosine phosphatases (PTPases). PTPase activity, was monitored by quantitating the disappearance of O-phospho-L-tyrosine (P-Tyr) in an ELISA system using antigen capture followed by double antibody labelling. PTPase activity of agarose conjugated PTP-1B was demonstrated using the ELISA system. PTPase activity was sensitive to both PTB-1B concentrations and time of incubation. 1 mU of PTPase activity was defined as that amount of enzyme producing a rate of loss of 0.01 absorbance units/minute with a specific activity of 150 pmol P-Tyr/min per micrograms protein based on the unit of PTPase activity from the conventional assay system. The PTP-1B activity was shown by the ELISA system to be completely inhibitable by Poly (Glu,Tyr)4:1 at 100 micrograms/ml. We used the ELISA system to detect PTPase activity in lysates of cultured cells. The PTPase activity of cell lysates of MDA-MB 468 breast carcinoma cells as obtained by the ELISA were compared with those obtained by a standard 32P(i) release assay using radio-labelled Raytide as PTPase substrate. The decrease in P-Tyr concentration was dependent on the time of incubation with the lysate and on lysate concentration and compared well with the release of 32P(i) in the radioactive assay system. Orthovanadate as well as heat denaturation inhibited the PTPase activity of the cell lysates in both the assay systems. The assay presented here is a simple immunological system capable of measuring activity of purified PTPases as well as PTPase levels in cell and tissue extracts.
Mishra
S
S
University of Maryland Cancer Center, Department of Pathology, Baltimore 21227.
Hamburger
A W
AW
D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D019000:Phosphotyrosine; D017027:Protein Tyrosine Phosphatases / Q000032:analysis*; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000031:analogs & derivatives / Q000737:chemistry
D019000:Phosphotyrosine; D014443:Tyrosine; D017027:Protein Tyrosine Phosphatases