ECAM

BioAssay Systems Amylase ECAM003.
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EnzyChromTM α-Amylase Assay Kit (ECAM-100)

Quantitative Colorimetric Amylase Determination at 585nm
DESCRIPTION
AMYLASE belongs to the family of glycoside hydrolase enzymes that 3. Add 150 µL Detection Reagent to each well. Mix and incubate for 20
break down starch into glucose molecules by acting on α-1,4- min at room temperature (25°C). Read OD585nm (540-610nm) on
glycosidic bonds. The α-amylases (EC 3.2.1.1) cleave at random a plate reader.
locations on the starch chain, ultimately yielding maltotriose and
maltose, glucose and "limit dextrin" from amylose and amylopectin. In
CALCULATION
mammals, α-amylase is a major digestive enzyme. Increased enzyme The Amylase activity is calculated as
levels in humans are associated with salivary trauma, mumps due to ODSAMPLE – ODBUFFER 400 × n
inflammation of the salivary glands, pancreatitis and renal failure. Activity = × (U/L)
ODSTD – ODBUFFER t (min)
Simple, direct and automation-ready procedures for measuring
amylase activity are very desirable. BioAssay Systems’ EnzyChrom
TM ODSAMPLE, ODSTD and ODBUFFER are optical density values of the
α-amylase assay method involves two steps: (1). α-amylase in the sample, the 400 µM glucose standard and Assay Buffer. t is the
sample hydrolyzes starch and the product is rapidly converted to incubation time. t = 15 min in the standard protocol. n is the dilution
glucose by α-glucosidase and hydrogen peroxide by glucose oxidase; factor (n = 50 for serum, 2000 for saliva). One unit of enzyme
(2). hydrogen peroxide concentration is determined with a colorimetric catalyzes the production of 1 µmole of glucose per min under the
reagent. assay conditions.
Note: if the calculated activity is higher than 50 U/L, dilute sample in
APPLICATIONS Assay Buffer and repeat assay. Multiply the results by the dilution
Direct assays of α-amylase activity in blood, saliva, urine, grains and factor.
other agricultural samples.
MATERIALS REQUIRED, BUT NOT PROVIDED
KEY FEATURES Pipeting devices, centrifuge tubes, clear flat-bottom 96-well plates,
Sensitive and accurate. Linear detection range 0.3 to 50 U/L α- plate reader, and optionally membrane filters (e.g. Microcon YM-10
amylase in 96-well plate assay. from Millipore).
Convenient. The procedure involves adding a single working reagent,
incubation for 15 min, followed by the detection reagent and a 20-min GENERAL CONSIDERARIONS
incubation and reading the optical density at 585 nm. For samples known to contain glucose, use a membrane filter (e.g.
Microcon YM-10 from Millipore) to remove glucose: load 50 µL sample in
KIT CONTENTS a Microcon YM-10 (10 kDa cutoff) and add 500 µL Assay Buffer.
Assay Buffer (pH 7.0): 20 mL Substrate: 120 µ L Centrifuge at 14000 rpm for 30 min, check level of sample, ideally the
Detection Reagent: 20 mL Enzyme A: 120 µ L sample level will be less than 50 µL. Add 500 µL Assay Buffer and
Glucose Standard: 1 mL Enzyme B: 120 µ L repeat the centrifugation. Measure final sample volume with a pipetman
and calculate dilution factor n = final sample volume/50 µL.
Storage conditions. Kit is shipped on ice. Store Detection Reagent at
4°C and others at -20°C. Shelf life of at least 6 months (see expiry
dates on labels).
Precautions: reagents are for research use only. Normal precautions 0.8
for laboratory reagents should be exercised while using the reagents. α -Amylase
Please refer to Material Safety Data Sheet for detailed information.
∆ OD585nm
0.6
PROCEDURES
Reagents. Equilibrate all components to room temperature. Keep
thawed Enzyme Mix in a refrigerator or on ice. The substrate may 0.4
have precipitates. Prior to use, vortex tube to dissolve precipitates; R2 = 1.00
gentle swirl the Detection Reagent bottle.
0.2
Sample preparation. Ideally samples are assayed fresh. When stored
frozen, α-amylase is stable for one month. Ascorbic acid, heparin,
EDTA, EGTA, citrate, SDS, Tris (> 8mM) and ethanol (>0.4%)
0.0
interfere and should be avoided in sample preparation. If glucose is 0 10 20 30 40 50
present in the sample, treat the samples as described in GENERAL
CONSIDERATIONS. It is prudent to perform a pilot test with samples α -Amylase (U/L)
at various dilutions. Recommended dilution: serum 50-fold, saliva
2,000-fold in Assay Buffer prior to assay. Standard Curve in 96-well plate assay
1. Prepare 400 µM Glucose Standard by mixing 10 µL of the provided
(300 mg/dL) standard with 406 µL Assay Buffer. Transfer 10 µL
Assay Buffer, 10 µL 400 µM glucose, and 10 µL of each sample LITERATURE
into separate wells of a clear flat-bottom 96-well plate. 1. Gullbault, GG and Rietz, E.B. (1976). Enzymatic, Fluorometric
Assay of a-Amylase in Serum. Clin. Chem. 22(10): 1702:1704.
2. Prepare an appropriate Working Reagent for each well. For
Standards and samples: mix 40 µL Assay Buffer, 0.5 µL Substrate, 2. Mashige F, Ohkubo A, Kamei S, Yamanaka M. (1982). An enzymic
method for urine and serum alpha-amylase assay. Clin. Chem.
1 µL Enzyme A, 1 µL Enzyme B.
28(7):1715-6.
Transfer 40 µL working reagent and blank control reagent to the 3. Marshall JJ, Iodice AP, Whelan WJ. (1977). A new serum alpha-
appropriate sample and blank wells. Tap plate to mix. Incubate for amylase assay of high sensitivity. Clin Chim Acta. 76(2):277-83.
15 min at room temperature (25°C).
2008  by BioAssay Systems · 3423 Investment Boulevard, Suite 11, Hayward, CA 94545, USA · Website: www.bioassaysys.com
Tel: 510-782-9988, Fax: 510-782-1588 · Email: order@bioassaysys.com, info@bioassaysys.com
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BioAssay Systems Amylase ECAM003.

EnzyChromTM α-Amylase Assay Kit (ECAM-100)

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