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.
Kobos
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Corporate New Business Development Division, DuPont Company, Incorporated, Wilmington, Delaware 19880.
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Robertson
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Kielhorn
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D006571:Heterocyclic Compounds / Q000737:chemistry*; D006573:Heterocyclic Compounds, 1-Ring; D008163:Luminescent Measurements; D012815:Signal Processing, Computer-Assisted; D013696:Temperature; D014962:X-Ray Film
D006571:Heterocyclic Compounds; D006573:Heterocyclic Compounds, 1-Ring; C079142:1,2-dioxetane