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 activitym was monitored by quantitating disappearance of O-phospho-l-tyrosine (P-Tyr) in an ELISA system using antigen captured followed by double antibody labelling. PTPase activity of agarose conjugated PTB-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 μg 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 μg/ml. We used the ELISA system to detect PTPase activity in lysates of cultured cells. The PTPase activity cell lysates of MDA-MB 468 breast carcinoma cells as obtained by the ELISA were compared with those obtained by a standard 32Pi release assay using radio-labelled Raytidetm 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 32Pi 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 PTPase as well as PTPase levels in cell and tissue extracts.