A quantitative luminescence assay for nonradioactive nucleic acid probes

J Histochem Cytochem. 1996 Jun;44(6):657-60. doi: 10.1177/44.6.8666750.


In histochemical work with digoxigenin- or biotin-labeled nucleic acid probes, reproducibility of in situ hybridization depends on accurate measurement of the amount of non-radioactive label being used. We describe a rapid and sensitive assay for nonradioactive label incorporated into nucleic acids employing a luminogenic substrate for alkaline phosphatase, CSPD (disodium 3-(4-methoxyspirol¿1,2-dioxetane-3,2'-(5'-chloro)tricyclo [,7)]decan¿-4-yl)phenyl phosphate). An alkaline phosphatase-antibody conjugate was bound to digoxigenin-labeled nucleic acids spotted on nylon membranes. Light emission from the reaction of the bound alkaline phosphatase with CSPD was measured with a luminometer. This method allows an accurate determination of digoxigenin incorporated into nucleic acid probes in the range of 0.5-500 fmol of nonradioactive label.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adamantane / analogs & derivatives*
  • Adamantane / metabolism
  • Alkaline Phosphatase / metabolism
  • Animals
  • Cattle
  • DNA Probes / analysis*
  • Digoxigenin / analysis*
  • Luminescent Measurements
  • RNA, Complementary / analysis
  • Steroid 17-alpha-Hydroxylase / genetics
  • Time Factors


  • DNA Probes
  • RNA, Complementary
  • 3-(2'-(spiro-5-chloroadamantane))-4-methoxy-4-(3''-phosphoryloxy)phenyl-1,2-dioxetane
  • Steroid 17-alpha-Hydroxylase
  • Alkaline Phosphatase
  • Digoxigenin
  • Adamantane