Development of solid-phase enzyme-linked immunosorbent assays for the determination of epidermal growth factor receptor and pp60c-src tyrosine protein kinase activity

Enzyme-Linked Immunosorbent Assay for trkA Tyrosine Kinase Activity

“…pare closely to those previously reported for the two enzymes (17,18). In order to establish a broad screen for identifying inhibitors of tyrosine kinases, a nonsaturating nucleotide condition was used in the kinase strate ATP is 7.5 { 2.0 mM for trkA (Fig.…”

An SH2 Domain-Based Tyrosine Kinase Assay Using Biotin Ligase Modified with a Terbium(III) Complex

“…Alternatively, non-radioactive ELISA-type assays have been used with the phosphotyrosine antibody and second-antibody carrying appropriate enzymes. 23,24 However, ELISA-type assays entail laborious procedures with multiple incubation and washing steps, and the enzymatic signal amplification reduces the reproducibility of data. To circumvent these problems, a kinase assay has been developed with phosphotyrosine antibody labeled with europium chelates; 27 the sensitivity of this assay is comparable to, or better than, those of radioactive assays, and the procedure is quite simple.…”

“…[7][8][9][10][11][12][22][23][24][25][26][27][28][29] The commonly used methods for the high-sensitivity detection of kinase activity are solid-phase assays. [22][23][24][27][28][29] For example, a scintillation proximity assay with the radioactive substrate has proved to be a reliable and accurate method suited for highthroughput applications. 22 However, it is desirable to use nonradioactive techniques due to environmental concerns.…”

“…The results demonstrate that the activity of the kinase at concentrations lower than 10 pg/μL can be reliably determined; the detection limit was estimated to be 2 and 1 pg/μL for 2 and 6 h reactions, respectively, where the detection limit was defined as the concentration of the kinase at which an increase of luminescence intensity corresponding to 3 × the SD (standard deviation) of the background signal was observed. Enzyme concentrations routinely used in Src kinase assays based on the radioactive techniques and enzyme-linked immunosorbent assays (ELISA) are from a few pg/μL to tens pg/μL; [22][23][24] therefore, our method can be fully utilized as an alternative to those conventional assays. The high sensitivity of our assay is considered to be derived from the luminescence property of the Tb 3+ complex and the labeling method based on S. tokodaii biotinylation, where multiple copies of a fluorophore can be specifically introduced to the target protein under physiological conditions.…”

A Solid-Phase Assay for the Determination of Protein Tyrosine Kinase Activity of c-src Using Scintillating Microtitration Plates

“…Alternatively, scintillating microtiter plates served as the basis to nonisotopic, ELISA-type assays have been used where evaluate enzyme activity. The procedure eliminates substrates are coated to the wells of a microtiter plate detection with phosphotyrosine antibodies, tedious (7,8). The phosphorylated peptide is detected by an separation of phosphorylated peptides with phospho-anti-phosphotyrosine antibody and a secondary anticellulose membranes, and extensive washing steps.…”