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.