A quantitative luminescence assay for nonradioactive nucleic acid probes

In histochemical work with digoxigenin- or biotin-labeled nucleic acid probes, reproducibility of in situ hybridization depends on accurate measurement of the amount of nonradioactive 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[3.3.1.1(3,7)]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.