Gen5 version required: 1.0 or higher
Basis for the Assay:
Enzyme linked immunosorbent assays (ELISA) have been used to quantitate a wide range of compounds and pathogens for almost 40 years. One of the most commonly used enzyme conjugates is Alkaline Phosphatase (AP). (2’-[2-benzothiazoyl]-6’-hydroxybenzothiazole phosphate (BBTP) serves as a substrate for AP. Unreacted BBTP is weakly fluorescent, but in the presence of alkaline phosphatase it is converted to a highly fluorescent product, BBT, which has excitation and emission maxima of 435 nm and 575 nm respectively. ELISA assays that employ an alkaline phosphatase conjugate can be used with AttoPhos® substrate system.
The protocol calls for a fluorescence measurement using a 440/30 excitation filter and a 560/40 emission filter.
The experiment data file contains data from several unknown alkaline phosphatase samples using AttoPhos as the substrate.
Plate configuration assumes that samples will be run in duplicate. The plate includes seven AP standards ranging from 0 to 0.05 DEA Units/well, and two blank wells. Thus, 40 samples can be run on a microplate in duplicate, in addition to the standard curve.
The report is configured to provide the (1) raw fluorescence, (2) blanked fluorescence, (3) calibration curve, (4) curve fitting results, and (5) unknown sample concentrations.