Enhancement of Enzyme-Activated 1,2-Dioxetane Chemiluminescence in Membrane-Based Assays

✍ Scribed by R.K. Kobos; B.A. Blue; C.W. Robertson; L.A. Kielhorn

• Publisher: Elsevier Science
• Year: 1995
• Tongue: English
• Weight: 536 KB
• Volume: 224
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1995.1017

✦ Synopsis

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 (\mathrm{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^{\circ} \mathrm{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. 1995 Academic Press, Inc.