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After the inhibition of salivary amylase with a combination of 2 monoclonal antibodies, pancreatic alpha-amylase activity is assayed by a coupled kinetic colorimetric procedure which gives rise to 415 nm absorption proportional to the paranitrophenol released by the action of alpha-amylase and alpha-glucosidase.
In this procedure, the first incubation step results in the inhibition of human salivary alpha-amylase by 2 monoclonal antibodies which do not affect pancreatic alpha-amylase. After a second incubation with the substrate (G7)-p-nitrophenyl (G1)-alpha-D-maltohepatoside, the activity of pancreatic alpha-amylase is measured. The alpha-amylase cleaves the substrate into fragments G2, G3, and G4. Further fragment hydrolysis by alpha-glucosidase yields p-nitrophenol and glucose. Enzyme activity is determined by the rate of increase of absorbance at 415 nm. Chromophore production is equimolar.
The simplified reaction is as follows:
5 ET-G7PNP + 5 H20 -------> 2 ET-G5 + 2 G2PNP + 2 ET-G4
+ 2-G3PNP + ET-G3 + G4 PNP
2 G2PNP + 2 G3PNP + 14 H20 --------->5 PNP + 14G
Abbreviations: ET = ethylidene
G = glucose
PNP = p-nitrophenol
(Tietz NW, Burlina A, Gerhardt W, et al: Multicenter evaluation of a specific pancreatic isoamylase assay based on a double monoclonal-antibody technique. Clin Chem 1988;34:2096-2102)
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