2010-09 ache assay Protocol

1. Compound and equipment:

1) Phosphate: 0.1 M, ph 8.0.
①Begin by making the following volumes of the specified solutions.

Volume (A)
0.2M sodium phosphate monobasic
②Weigh 0.552g nah2po4 and add to 20ml of distilled H2O

Volume (B)
0.2M sodium phosphate dibasic
③Weigh 2.83g Na2HPO4 and add to 100ml of distilled H2O
Combine the two solutions according to the following:
Vol (A) = 5.3ml
Vol (B) = 94.7ml
④The resulting solution will have a final concentration of 0.2M, [ph=8] and will yield 100ml.
⑤Dilute buffer by adding an equal volume of distilled H2O ~ 100ml. The working concentration of the
new solution will be 0.1M phosphate buffer, ph=8.
⑥Filter sterilize with 1L filter system and autoclave for 25min.

2) Substrate: Acetylthiocholine iodide, 0.075 M (21.67 mg/ml). This solution was used successfully for
l0-15 days if kept refrigerated.
3) Reagent.: Dithiobisnitrobenzoic acid (DTNB): 0.01 M of the 5’ 5-dithiobis-2- nitrobenzoic acid ,
39.6 mg were dissolved in 10 ml ph 7.0 phosphate buffer (0.1 M) and 15 mg of sodium
bicarbonate were added. The reagent was made up in buffer of ph 7 in which it was more stable than
in that of ph 8.
4) ???Enzyme: Bovine erythrocyte cholinesterase (Nutritional Biochem. Corp., 20,000 units) was
dissolved in 20 ml of 1% gelatin. This solution was diluted 1 ：200 with water for use, yielding a
solution of 5 units/ml.⇒ maybe for blood samples not for brain tissues
5) Ethopropazine hydrochloride: to inhibit nonspecific cholinesterase activity, added to the incubation
mixture or the assay mixture. For brain ache, butyryl cholinesterase was inhibited by the addition of
10μm ethopropazine to the assay mixture and the change in absorbance was measured at 412 nm for
2min at 30 s intervals.

6) Versa Max Microplate Reader: (Molecular Devices Corp., Sunnyvale, CA).

7) Pipeting (multi-channel) devices. Clear-bottom 96-well plates (e.g. Corning Costar) and
plate reader.
2. Assay method
1) The tissue was homogenized (approximately 20 mg of tissue per ml of phosphate buffer (ph 8.0, 0.1
M) by a pestle (Scienceware, Pequannock, USA) (For muscular tissue, considerable mincing was
necessary before homogenizing).
2) A 0.4-ml aliquot of this homogenate was added to a cuvette containing 2.6 ml of phosphate buffer (ph
8.0, 0.1 M).
3) Of the DTNB reagent, 100 μ1 were added to the photocell. The absorbance was measured at 412 nm;
when this had stopped increasing, the photometer slit was opened so that the absorbance was set to
zero.
4) Of the substrate, 20 μ1 were added. Changes in absorbance were recorded and the change in
absorbance per min. Was calculated.
5) The rates were calculated as follows:

3. Price of compound
1) Ethopropazine hydrochloride ≥98% (HPLC), powder

E5406 Sigma

E5406-50MG 185,000 KRW

E5406-250MG 832,000 KRW

2) 5,5′-Dithiobis(2-nitrobenzoic acid) ≥97.5% (HPLC)

43760 Fluka

43760-1G 39,000 KRW

43760-25G 356,000 KRW

D218200 Aldrich

D218200-1G 44,000 KRW

D218200-5G 113,000 KRW
D218200-10G 223,000 KRW

D8130 Sigma
D8130-500MG 23,000 KRW
D8130-1G 58,000 KRW
D8130-5G 118,000 KRW
D8130-10G 218,000 KRW
D8130-25G 516,000 KRW

3) Acetylthiocholine iodide ≥98% (TLC), powder

A5751 Sigma

A5751-250MG 43,000
A5751-1G 49,000
A5751-5G 111,000
A5751-25G 343,000

01480 Fluka

01480-1G 34,000
01480-5G 88,000
01480-25G 345,000

4) Quantichrom™ Acetylcholinesterase Assay Kit( DACE-100)

$299.00 USD

Quantitative determination of acetylcholinesterase activity by colorimetric (412nm) method.

Procedure: 10 min. Kit size: 100 tests. Detection limit: 10 U/L. Shelf life: 6 months.
Shipping: ambient temp; storage: room temp.

5) Amplite™ Colorimetric Acetylcholinesterase Assay Kit

$195 USD

Product Number: #11400 (200 assays)

Keep in freezer and avoid light
Absorbance microplate readers
Quantichrom™ Acetylcholinesterase Assay Kit( DACE-100) protocol

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. 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. 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.

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

Samplepreparation. 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.
3. Calculation: acetylcholinesterase activity is calculated as follows,

Ache Activity = (OD10 – OD2 / OD CAL – ODH2o ) × n × 200 (U/L)

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).