Time-gated luminescence assay using nonmetal probes for determination of protein kinase activity-based disease markers

Anal Biochem. 2012 Mar 15;422(2):79-88. doi: 10.1016/j.ab.2011.12.048. Epub 2012 Jan 8.

Abstract

A novel nonmetal optical probe ARC-1063 whose long-lifetime luminescence is induced by association with the target protein kinase is used for the measurement of the concentration of catalytic subunit of protein kinase A (PKAc) in complicated biological solutions. High affinity (K(D) = 10 pM toward PKAc) and unique optical properties of the probe enable its application for the measurement of picomolar concentrations of PKAc in the presence of high concentrations of other proteins. The described assay is applicable in the high-throughput format with the instrument setups designed for lanthanide-based time-gated (time-resolved) luminescence methods. The assay is used for demonstration that extracellular PKAc (ECPKA) is present in plasma samples of all healthy persons and cancer patients but great care must be taken for procedures of treatment of blood samples to avoid disruption, damage, or activation of platelets in the course of plasma (or serum) preparation and conservation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biomarkers / blood*
  • Blood Platelets / cytology
  • Breast Neoplasms / blood*
  • Case-Control Studies
  • Catalytic Domain
  • Colorectal Neoplasms / blood*
  • Cyclic AMP-Dependent Protein Kinases / blood*
  • Estonia
  • Female
  • High-Throughput Screening Assays
  • Humans
  • Kinetics
  • Lanthanoid Series Elements
  • Limit of Detection
  • Luminescence
  • Luminescent Measurements / methods*
  • Platelet Activation
  • Specimen Handling

Substances

  • Biomarkers
  • Lanthanoid Series Elements
  • Cyclic AMP-Dependent Protein Kinases