Programme and Exhibitors

Symposium 1997: Abstracts

Chemiluminescent Detection of Proteins and DNA Probes

I. Bronstein, C. Olesen, J. Mosier, and J.C. Voyta
Tropix, Inc., Bedford, MA 01730, USA

Chemiluminescent 1,2-dioxetane substrates are widely used in ultrasensitive detection of proteins and nucleic acids. Examples of applications include immunoblotting, Southern blotting, ELISA, reporter gene assays and quantitative PCR.

Recently developed systems utilize chemiluminescent dioxetane substrates in the detection of enzymes involved in cellular signal transduction pathways. Specifically, the phosphorylated substrate CDP-Star^TM is utilized in the detection of cellular phosphatases.

Procedures for sensitive assays for calcineurin, PTP-1c, PTP-1b, YOP bacterial phosphatase and LAR phosphatase have now been established. Tyrosine, serine and threonine kinase activities are also detected using chemiluminescent immunoassay methods. These assays are performed with a biotinylated peptide substrate for a specific kinase and an antibody recognizing the phosphorylated epitope. Sensitive assays for src kinase and abl kinase have now been demonstrated. Detection of approximately 1 unit of enzyme activity per well is achieved with peptide substrate concentration lower than 10^-7 M.

These systems exhibit greater sensitivity compared to traditional radioactive or colorimetric assays. Both the phosphatase and kinase assays are performed in microwells and are ideal as high throughput pharmaceutical screening systems.

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