Lumi-Phos® 530 is a ready-to-use formulation containing Lumigen PPD (4-methoxy-4-(3-phosphatephenyl)spiro[1,2-dioxetane-3,2′-adamantane], disodium salt) for chemiluminescent detection of alkaline phosphatase. A unique co-surfactant system promotes energy-transfer to the fluorescent head groups causing the generation of luminescence with a broad maximum at 530 nm.
Lumi-Phos 530 is useful for chemiluminescent detection of alkaline phosphatase conjugates in immunoassays and DNA probe assays. It provides ultrasensitive detection with magnetic particles, beads, tubes, microtiter plates and on nylon and PVDF membranes. Lumi-Phos 530 is 10,000 times more sensitive than colorimetric substrates in solution. As little as 0.001 attomoles of enzyme can be detected.
Addition of Lumi-Phos 530 to a tube or microtiter well at 37°C containing alkaline phosphatase results in chemiluminescence which increases with time and after 40-50 minutes reaches a plateau. With low concentrations of enzyme, the light intensity remains constant for several hours. The luminescence intensity at any time point is a direct measure of the concentration of the enzyme.
For experiments on nylon or PVDF membranes with Lumi-Phos 530, the membrane is saturated with the material and incubated at room temperature or 37°C for periods ranging from 30 min to overnight. After incubation an image is recorded with X-ray film or a CCD camera.
Although the dioxetane is quite stable in the formulations, for extended periods we recommend that the solutions be stored at 4°C. Exposure of Lumi-Phos 530 to intense light should be avoided. The bottle should be brought to room temperature before dispensing. Care should be taken to minimize the chance of contamination with enzyme.
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