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KOMABIOTECH
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Lake Forest, IL 60045
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Exalpha
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SCETI K.K
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EnzyChromTM Acetylcholine Assay Kit (EACL-100)
Quantitative Colorimetric/Fluorimetric Acetylcholine
Determination
DESCRIPTION
ACETYLCHOLINE
is a
neurotransmitter produced in acetylcholinergic
neurons. It plays important roles in skeletal muscle movement,
regulation of smooth and cardiac muscles, as well as in learning, memory
and mood. BioAssay Systems' method provides a simple, direct and
highthroughput assay for measuring acetylcholine in biological samples.
In this assay, acetylcholine is hydrolyzed by acetylcholinesterase to
choline which is oxidized by choline oxidase to betaine and H2O2. The
resulting H2O2 reacts with a specific dye to form a pink colored
product. The color intensity at 570nm or fluorescence intensity (530/585
nm) is directly proportional to the acetylcholine concentration in the
sample.
KEY FEATURES
Use 20
μL samples.
Linear detection range: colorimetric assay 10 to 200 μM, fluorimetric
assay 0.4 to 10 μM
acetylcholine.
APPLICATIONS
Assays:
acetylcholine
in
biological samples such as serum, plasma, urine, saliva, milk, tissue,
and cell culture.
Drug
Discovery/Pharmacology:
effects of
drugs on acetylcholine metabolism.
KIT CONTENTS
Assay
Buffer:
10 mL
ACHE
Enzyme: 120 μL
Enzyme Mix:
120 μL
Dye
Reagent: 120 μL
Standard:
400 μL 2 mM acetylcholine
Storage
conditions.
The kit is shipped on ice. Store all components at -20°C. Shelf life of
three months after receipt.
Precautions:
reagents are for research use only. Normal precautions for
laboratory reagents should be exercised while using the reagents. Please
refer to Material Safety Data Sheet for detailed information.
COLORIMETRIC ASSAY
Sample
treatment:
liquid
samples such as serum and plasma can be assayed directly. Tissue and
cell lysates can be prepared by homogenization in cold 1 x PBS and
centrifugation (5 min at 14,000 rpm). Use clear supernatants for assay.
Milk samples should be cleared by mixing 600 μL milk with 100 μL 6 N
HCl. Centrifuge 5 min at 14,000 rpm. Transfer 300 μL supernatant into a
clean tube and neutralize with 50 μL 6 N NaOH. The neutralized
supernatant is ready for assay (dilution factor n = 1.36).
Note: (1).
SH-containing reagents (e.g.
b–mercaptoethanol,
dithiothreitol, > 5
μM)
are known to interfere in this assay and should be avoided in
sample preparation. (2). This assay is based on an enzyme-catalyzed
kinetic reaction. Addition of Working Reagent should be quick and mixing
should be brief but thorough.
1. Equilibrate all components
to room temperature. Briefly centrifuge the tubes before opening. Keep
thawed tubes on ice during assay.
2. Standards :
mix 24 μL 2 mM Standard with 216 μL dH2O (final 200 μM). Dilute standard
in dH2O as follows.
Transfer 20
μL diluted standards into separate wells of a clear flatbottom 96-well
plate.
Samples :
transfer 20 μL of each sample into separate wells of the plate.
Note: if a sample is known to
contain choline, prepare an extra sample blank well with 20
μL
of the sample.
3. Color
reaction. Prepare enough Working Reagent by mixing, for each
well,
85 μL Assay Buffer, 1 μL ACHE Enzyme, 1 μL Enzyme Mix and 1 μL Dye
Reagent. Add 80 μL Working Reagent to each well.
Note: for samples that contain
choline, prepare a blank control reagent with no ACHE Enzyme (i.e., 85
μL Assay Buffer, 1 μL
Enzyme Mix and 1
μL Dye
Reagent). Add 80 μL
of the control Reagent to each Sample Blank well.
Immediately tap plate to mix.
Incubate 20 min at room temperature.
4. Read optical density at 570nm
(550-585nm).
FLUORIMETRIC ASSAY
The fluorimetric assay procedure
is similar to the colorimetric procedure except that (1) 0, 3, 6 and 10
μM acetylcholine standards and (2) a black 96-well plate are used. Read
fluorescence intensity at lex = 530 nm and l em = 585 nm.
Note:
if the calculated acetylcholine concentration of a sample is higher than
200 μM in the Colorimetric Assay or 10 μM in the Fluorimetric Assay,
dilute sample in water and repeat the assay. Multiply result by the
dilution factor n.
CALCULATION
Subtract blank value (#4) from the
standard values and plot the DOD or DF against standard concentrations.
Determine the slope and calculate the acetylcholine concentration of
Sample,
RSAMPLE and RBLANK
are optical density or fluorescence intensity readings of the Sample and
H2O Blank (or Sample Blank if sample contains choline), respectively.
n is the sample dilution factor.
Conversions:
1 mM acetylcholine equals 14.6 mg/dL, 0.015% or 146 ppm.
MATERIALS REQUIRED, BUT NOT
PROVIDED
Pipetting devices, centrifuge
tubes, clear flat-bottom uncoated 96-well plates, optical density plate
reader; black flat-bottom uncoated 96-well plates, fluorescence plate
reader.
Choline Standard Curves
LITERATURE
1. Vizi, E.S. et al (1985). A
simple and sensitive method of acetylcholine identification and assay.
Bioassay combined with minicolumn gel filtration or high-performance
liquid chromatography. J Pharmacol Methods. 13:201-211.
2. Gilberstadt, M.L. and Russell,
J.A. (1984). Determination of picomole quantities of acetylcholine and
acetylcholine in physiologic salt solutions. Anal Biochem. 138:78-85.
3. Israel, M. and Lesbats, B.
(1982). Application to mammalian tissues of the chemiluminescent method
for detecting acetylcholine. J Neurochem. 39:248-250. |
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QuantiChromTM Acetylcholinesterase Assay Kit
(DACE-100)
Rapid Colorimetric Determination of
Acetylcholinesterase Activity
DESCRIPTION
ACETYLCHOLINESTERASE
(EC 3.1.1.7, AChE), also known as RBC
cholinesterase, is found primarily in the blood and neural synapses. Low
serum cholinesterase activity may relate to exposure to insecticides or
to one of a number of variant genotypes. AChE catalyzes the hydrolysis
of the neurotransmitter acetylcholine into choline and acetic acid, a
reaction necessary to allow a cholinergic neuron to return to its
resting state after activation. Cholinesterase levels of cells and
plasma are used as a guide in establishing safety precautions relative
to exposure and contact, as well as a guide in determining the need for
workers to be removed from areas of contact with the organic phosphate
insecticides. Simple, direct and automation-ready procedures for
measuring AChE activity are very desirable. BioAssay Systems'
QuantiChromTM Acetylcholinesterase Assay is based on an improved Ellman
method, in which thiocholine produced by the action of
acetylcholinesterase forms a yellow color with
5,5’-dithiobis(2-nitrobenzoic acid). The intensity of the product color,
measured at 412 nm, is proportionate to the enzyme activity in the
sample.
APPLICATIONS
Direct assays of
acetylcholinesterase activity in blood, serum, plasma, and other
biological samples. Evaluation of acetylcholinesterase inhibitors.
KEY FEATURES
Sensitive and accurate .
Detection range 10 to 600 U/L AChE activity in 96-well plate assay.
Convenient.
The procedure involves adding a single working reagent, and reading the
optical density at 2 min and 10 min at room temperature.
High-throughput.
Can be readily automated as a high-throughput 96-well plate assay for
thousands of samples per day.
KIT CONTENTS (100 tests in
96-well plates)
Assay Buffer (pH 7.5): 30 mL
Reagent: 240 mg
Calibrator: 4 mL (equivalent to
200 U/L)
Storage conditions .
Store all reagents at room temperature. Shelf life of at least 6 months
(see expiry dates on labels).
Precautions:
reagents are for research use only. Normal precautions for laboratory
reagents should be exercised while using the reagents. Please refer to
Material Safety Data Sheet for detailed information.
PROCEDURES
Sample preparation.
Blood samples should be diluted 40-fold in the Assay Buffer, e.g.
accurately pipet 5 μL blood and mix thoroughly with 195 μL Assay Buffer.
Tissue or cell lysates are prepared by brief sonication or
homogenization in 0.1M phosphate buffer (pH 7.5), followed by
centrifugation at 14,000 rpm for 5 min. Use supernatant for assay.
Ideally samples should be assayed fresh. If this is not possible,
refrigerate samples and assay them within 24 hours.
Reagent preparation:
the Working Reagent should be prepared freshly and used within 30 min.
Each reaction well requires 2 mg reagent. Calculate the amount of
reagent needed and weigh this amount (mg) in a centrifuge tube. Add 200
μL Assay Buffer per 2 mg reagent.
Vortex to dissolve.
1. Calibrator: transfer
200 μL water and 200 μL calibrator separately into wells of a clear
bottom 96-well plate.
Samples:
add 10 μL sample per well in separate wells.
2. Reaction: transfer
190 μL freshly prepared Working Reagent to all sample wells and tap
plate briefly to mix. Read OD412nm at 2 min and at 10 min in a plate
reader.
3. Calculation:
acetylcholinesterase activity is calculated as follows,
Where OD10 and OD2 are the
OD412nm values of the sample at 10 min and 2 min, respectively. ODCAL
and ODH2O are the OD412nm values of the Calibrator and water at 10 min.
n is the dilution factor (n = 40 for whole blood). The
number “200” is the equivalent activity of the calibrator under the
assay conditions.
Note:
if the calculated AChE activity is higher than 600 U/L, dilute sample in
Assay Buffer and repeat this assay. Multiply the results by the dilution
factor.
Unit definition:
one unit of enzyme catalyzes the production of 1 μmole of thiocholine
per minute under the assay conditions (pH 7.5 and room temperature).
MATERIALS REQUIRED, BUT NOT
PROVIDED
Pipeting (multi-channel)
devices. Clear-bottom 96-well plates (e.g. Corning Costar) and plate
reader.
GENERAL CONSIDERATIONS
1. This assay is based on an
enzyme-catalyzed kinetic reaction. Addition of Working Reagent should be
quick and mixing should be brief but thorough. Use of a multi-channel
pipettor is recommended.
2. For assays in standard 1 mL
cuvet, use 1 mL water and 1 mL Calibrator, 50 μL sample + 950 μL Working
Reagent.
EXAMPLES
Two human blood samples were
assayed in duplicate using the 96- well plate protocol. The AChE
activities were 3,402 ± 163 and 3,660 ± 151 U/L.
Kinetics of 0-1000 U/L
Acetylcholinesterase Reaction in 96-well plate
LITERATURE
1. Magnottl, RA. et al. (1987).
Measurement of Acetylcholinesterase in Erythrocytes in the Field. Clin.
Chem. 33/10, 1731-1 735.
2. Kovarik, Z et al. (2003).
Acetylcholinesterase active centre and gorge conformations analysed by
combinatorial mutations and enantiomeric phosphonates. Biochem. J.
(2003) 373, 33–40.
3. Ordentlich, A. et al.
(1996). The Architecture of Human Acetylcholinesterase Active Center
Probed by Interactions with Selected Organophosphate Inhibitors. J.
Biol. Chem. 271 (20): 11953–11962. |
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