Enzymatic Assays:
Acetylcholinesterase – AChE
Stock solutions:
1. Phosphate buffer 0.2M – titrate 0.2 M Na2HPO4 (basic) with 0.2 M NaH2PO4 (acidic)
to get pH 7.4, Store at 4oC.
2. DTNB 15 mM (X30)– for 100ml : 0.6 gm dithio-bis-nitrobenzoic acid, 150 mg
sodium bicarbonate (Na2CO3) in 0.1M Phosphate buffer (pH 7.4). Store refrigerated
in dark bottle.
3. BuChE Inhibitor (IO) 10mM - ready, commercial
Working solutions:
1. Serum – 1:20 (with PBS) - preliminary calibration is needed
The dilution is done in a round bottom 96 well microtiter plates. Take 10 l serum
into 190 l PBS at 40C .
2. Ellman’s :
0.1 M Phosphate buffer pH 7.4 (1:2 from 0.2M stock)
0.5 mM DTNB ( 1:30 from 15 mM stock)
Inhibitor 50 M (1:200 from 10mM stock) - preliminary calibration is needed
Keep covered with aluminum foil.
Per 10 samples : 3 ml of Ellman’s reagent : 1.5 ml Phosphate buffer 0.2M, 100 l DTNB
15 mM, 15 l inhibitor 10mM
3. Substrate : 20 mM AcSCh (Acetylthiocholine) - 10 mg/1.7ml. Keep on ice until use.
Possible to keep frozen overnight
Assay:
Add to each well In flat bottom 96 well microtiter plates:
1. 10 l serum
2. 180 l Ellman’s reagent
Incubate for 20 min in dark
3. 10 l substrate
Spectrophotometric reading (at 405 nm) every 2 min for 20 min.
Butyrylcholinesterase – BuChE
Stock solutions:
1. Phosphate buffer 0.2M – titrate 0.2 M Na2HPO4 (basic) with 0.2 M NaH2PO4 (acidic)
to get pH 7.4, Store at 4oC.
2. DTNB 15 mM (X30)– for 100ml : 0.6 gm dithio-bis-nitrobenzoic acid, 150 mg
sodium bicarbonate (Na2CO3) in 0.1M Phosphate buffer (pH 7.4). Store refrigerated
in dark bottle.
3. AChE Inhibitor (BW) 10mM - ready, diluted in Ethanol
Working solutions:
1. Serum – 1:20 (with PBS) - preliminary calibration is needed
The dilution is done in a round bottom 96 well microtiter plates. Take 10 l serum
into 190 l PBS at 40C .
2. Ellman’s :
0.1 M Phosphate buffer pH 7.4 (1:2 from 0.2M stock)
0.5 mM DTNB ( 1:30 from 15 mM stock)
Inhibitor 10 M (1:2000 from 20mM stock) - preliminary calibration is needed
Keep covered with aluminum foil.
Per 10 samples : 3 ml of Ellman’s reagent : 1.5 ml Phosphate buffer 0.2M, 100 l DTNB
15 mM, 1.5 l inhibitor 10mM
3. Substrate : 200 mM BuTCh (Butyrylthiocholine) - 63.44 mg/ml DDW. Keep on
ice until use.
Assay:
Add to each well In flat bottom 96 well microtiter plates:
1. 10 l serum
2. 180 l Ellman’s reagent
Incubate for 20 min in dark
3. 10 l substrate
Spectrophotometric reading (at 405 nm) every 2 min for 20 min.
PARAOXONASE – PON1
Stock solutions:
1 Tris-HCl buffer 1M – titrate TRIZMA-base (prepare higher concentration than 1M
about 1.6M) with concentrated HCl to get pH 8.5, dilute to 1M. Store at 4oC.
2 CaCl2 1M
3 NaCl 5M
4 Paraoxon (P-nitriphenol) 327mM - ready liquid, commercial
Working solutions:
4. Serum – 1:10 (with PBS) - preliminary calibration is needed
The dilution is done in a round bottom 96 well microtiter plates. Take 10 l serum
into 190 l PBS at 40C .
5. Substrate mix :
0.26 mM Tris-HCl buffer pH 8.5 (1:2 from 0.2M stock)
25 mM CaCl2 (1:40 from stock 1M)
0.5M NaCl (1:10 from stock 5M)
1.2mM Paraoxon (0.7:200 from stock 327mM)
Assay:
Add to each well In flat bottom 96 well microtiter plates:
1. 10 l serum
2. 190 l substrate mix
Spectrophotometric reading (at 405 nm) every 2 min for 10 min.
ARYLESTERASE
Stock solutions:
1. 0.9 mM CaCl2
9 mM TRIS-HCl Ph8
2. Phenylacetate - ready liquid, commercial
Working solutions:
1. Serum – 1:40 (with PBS) - preliminary calibration is needed
The dilution is done in a round bottom 96 well microtiter plates – UV adequate! Take
10 l serum
2. Buffer mix
20 ml buffer
9.6l Phenylacetate
Assay:
Add to each well in flat bottom 96 well microtiter plates (UV adequate):
1. 10 l serum
2. 200 l substrate mix
Spectrophotometric reading (at 280 nm) every minimal interval for 10 min. – don’t forget
to replace the filter to the correct one!
Calibrations and Controls
1. AChE
Calibrations : IsoOMPA try 10-4 and10-5
Controls: 10-5 BW , 10-5 BW and IO , without serum, without substrate
2. BuChE
Calibrations : BW try 10-4 and10-6
Controls: 10-5 IO , 10-5 IO and BW, without serum, without substrate
3. PON1
Calibrations : Serum try 1:10 and 1:20 and 1:50
Controls: without serum, without substrate
Calculations
Activity nmols/min/ml serum = Slope(A/min)* dilution*1000 (ml) / serum volume
(ml)*Ex()
AChE and BuChE
Ex(405)13.600 per mol per cm – the path length is 0.5 cm => 6,800.
3. PON1