A MICROTITER ENZYME-LINKED-IMMUNOSORBENT-ASSAY FOR PROTEIN-TYROSINE PHOSPHATASE

We report the development of an enzyme-linked immunosorbent assay (ELI SA) 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 anti body labelling. PTPase activity of agarose conjugated PTP-1B was demon strated 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 mug protein based on the unit of PTPase activity from the convent ional assay system. The PTP-1B activity was shown by the ELISA system to be completely inhibitable by Poly (Glu,Tyr)4:1 at 100 mug/ml. We us ed the ELISA system to detect PTPase activity in lysates of cultured c ells. The PTPase activity of cell lysates of MDA-MB 468 breast carcino ma cells as obtained by the ELISA were compared with those obtained by a standard P-32i release assay using radio-labelled Raytide(TM) as PT Pase substrate. The decrease in P-Tyr concentration was dependent on t he time of incubation with the lysate and on lysate concentration and compared well with the release of P-32i in the radioactive assay syste m. Orthovanadate as well as heat denaturation inhibited the PTPase act ivity of the cell lysates in both the assay systems. The assay present ed here is a simple immunological system capable of measuring activity of purified PTPases as well as PTPase levels in cell and tissue extra cts.