Lumi-Phos® Plus is a novel, ready-to-use formulation containing Lumigen PPD (4-methoxy-4-(3-phosphatephenyl)spiro[1,2-dioxetane-3,2′-adamantane], disodium salt) and the patented enhancer shown below in 2-amino-2-methyl-1-propanol buffer (pH 9.6). This formulation provides highly sensitive chemiluminescent detection of alkaline phosphatase with DNA probes or antibody conjugates in solution and on solid supports such as membranes. Light emission is maximal at 470 nm with Lumi-Phos Plus.
Lumi-Phos Plus provides enhanced chemiluminescence characterized by:
- low background
- fast signal ramp-up
- high intensity light production
- consistent, reproducible reaction in homogeneous aqueous solution and on nylon membranes
- high stability and long shelf life
Add a sufficient volume of Lumi-Phos Plus to a microtiter well (ca. 100 µL) or tube containing alkaline phosphatase or conjugate already equilibrated at 37°C. The ratio of the volume of any sample solution in the tube or well to Lumi-Phos Plus should not exceed 1:10. The resulting chemiluminescence increases with time and reaches a plateau after approximately 30 minutes. With low concentrations of enzyme, the light intensity remains constant for more than an hour. The luminescence intensity at any time point on the rising portion of the chemiluminescence curve and at the plateau is an accurate measure of the concentration of enzyme over at least 5 orders of magnitude of alkaline phosphatase concentration in solution.
In the absence of an alkaline phosphatase conjugate at 37°C, a small background chemiluminescent signal may be observed which should reach a constant value as soon as the temperature has stabilized. If it does not, some source of alkaline phosphatase contamination has occurred. Non-enzymatic chemiluminescence background is linearly related to the amount of Lumi-Phos Plus solution and is also dependent on temperature. Precise temperature control assures uniform chemiluminescence measurements.
The dioxetane, Lumigen PPD, is quite stable in the formulation. Lumi-Phos Plus can be stored at 25°C for one week with no detectable change in performance. For extended periods, it should be stored at 4°C and protected from exposure to intense light. Under these conditions Lumi-Phos Plus is indefinitely stable. Care should be taken to minimize the chance of contamination with enzyme through handling. Bottles of Lumi-Phos must be completely equilibrated to room temperature bef
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