Enhancement of enzyme-activated 1,2-dioxetane chemiluminescence in membrane-based assays

Processes for enhancing the chemiluminescent signal generated by enzyme-activated 1,2-dioxetanes have been developed for membrane-based assays in which detection is done using a charge-coupled device (CCD) camera or X-ray film. The enhancement is demonstrated using slot-blots of biotinylated lambda DNA in conjunction with an avidin-alkaline phosphatase conjugate. For detection with a CCD camera, the nylon membrane is dried after processing and incubation in dioxetane substrate solution and heated to temperatures of 50 to 80 degrees C during detection. Up to a 100-fold signal increase is obtained using this enhancement process compared to the conventional detection procedure, in which the blot is kept saturated with substrate solution in a sealed plastic bag during detection. For detection with X-ray film, a fivefold increase in signal intensity is realized by drying the membrane before exposure to the film. These enhancement processes greatly reduce the time required for detection in membrane-based assays.