A Phadebas® tablet consists of homogeneously interlinked starch polymers, taking the form of globular microspheres of defined size. These Bio-Degradable Starch Microspheres (DSM)are insoluble in water. To the DSMs, a blue dye is covalently bound. When the dye is bound to the microsphere, it remains water insoluble. However, in the presence of alpha Amylase, the DSM is degraded at a speed proportional to the alpha Amylase activity, and the blue dye is liberated. Due to the cross-linkages and the large blue dye, no other enzyme can act upon the substrate. Hence, Phadebas is specific for alpha amylase. The free dye molecule is water soluble and its concentration measurable at 620 nm. Using a fixed assay time, the absorbance reading gives the solution’s Amylase activity, through the standard curve supplied with each kit.
Part of a starch chain with arrows indicating points of action for the alpha amylase enzyme.
In the qualitative Phadebas Forensic Press Test, the diffusion of the liberated dye molecules is used to indicate the likely presence of salivary Amylase. In Phadebas Honey Diastase Test, the reactivity is controlled in a manner that ensures exact reproducibility between batches.
In semi-quantitative assays, such as Analis’s Isoamyl assay, the blue dye is used for visual detection. After electrophoretic separation of the various subclasses of salivary and pancreatic Amylases, Phadebas® tablets are dissolved on top of the gel. Where Amylase activity degrades the DMSs, the free dye diffuses into the gel, while non-degraded DSMs (and dye) is washed away. The remaining colored spots reveal the Amylase pattern, for diagnostic purposes.
3D model of salivary alpha-amylase enzyme.
For questions regarding the Phadebas assay, amylase testing and starch; please contact us at info@phadebas.com.