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Enzymatic Method Measurement of Serum Amylase and Lipase
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Enzymatic Method Measurement of Serum Amylase and Lipase
Shengchuan Chen, Feng Ma*
Institute of Systems Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100005, China
1.Sample preparation
Whole blood from control and experimental acute pancreatitis mice were centrifuged at 1,500 g for 10 min at 4℃ to separate the serum. If you cannot perform the assay immediately, the serum samples should be aliquoted and stored at -80°C.
2.Measurement of serum amylase activity
(1) We recommend performing several dilutions of the samples to ensure the OD660 values are within the linear range (mild acute pancreatitis: 1:50-1:100 dilution; severe acute pancreatitis: 1:500-1:1000 dilution).
(2) Assay procedure:
| Sample tubes (mL) | Blank tubes (mL) | |
Amylase Substrate Mix (Preheat at 37℃ for 5 min) | 0.5 | 0.5 |
Serum samples | 0.1 | |
Mix thoroughly and incubate the plate at 37 °C for 7.5 min | ||
Amylase Assay Buffer | 0.5 | 0.5 |
ddH2O | 3.0 | 3.1 |
| Mix thoroughly, and measure absorbance at OD660 in a 96-well clear plate. | ||
(3) Activity of amylase is calculated as:
Amylase activity (U/dL) = (ODBlank-ODSample)/ ODBlank * 80 * sample dilution fold
(4) Solution:
| Amylase Substrate Mix | 0.4 mg/mL amylon solution |
| Amylase Assay Buffer | 0.01 mol/mLiodine solution (keep away from light) |
(5) Results example:
3. Measurement of serum lipase activity
(1) We recommend performing several dilutions of the sample to ensure the OD580 values are within the linear range (mild acute pancreatitis: no dilution; severe acute pancreatitis: 1:5-1:10 dilution).
(2) Assay procedure:
| Blank wells | Standard wells | Sample wells |
ddH2O (μL) | 4 |
|
|
Standard Sample (μL) |
| 4 |
|
Serum samples (μL) |
|
| 4 |
Reagent 1 (μL) | 200 | 200 | 200 |
Mix thoroughly and incubate the plate at 37 °C for 3~5 min | |||
Reagent 2 (μL) | 50 | 50 | 50 |
Mix thoroughly, incubate the plate at 37 °C for 2 min, and measure absorbance immediately at OD580 in a kinetic mode, every 2 minutes. ΔA580nm = A2 – A1 | |||
(3) Activity of lipase is calculated as:
Lipase activity (U/L) = (ΔASample-ΔABlank)/ (ΔAStandard-ΔABlank) * The activity of Standard sample * sample dilution fold
(4) Solution:
| Reagent 1 | Containing Sodium taurocholate and calcium chloride |
| Reagent 2 (Lipase Substrate) | 1,2-Di-O-lauryl-rac-glycero-3-(glutaric acid 6-methylresorufin ester) |
| Standard Sample | 45.8 U/L |
(5) Results example:
Related files
Enzymatic Method Measurement of Serum Amylase and Lipase.docx 
Enzymatic Method Measurement of Serum Amylase and Lipase.pdf - Chen, S and Ma, F(2021). Enzymatic Method Measurement of Serum Amylase and Lipase. Bio-protocol Preprint. bio-protocol.org/prep1240.
- Huang, C., Chen, S., Zhang, T., Li, D., Huang, Z., Huang, J., Qin, Y., Chen, B., Cheng, G., Ma, F. and Zhou, M.(2019). TLR3 Ligand PolyI:C Prevents Acute Pancreatitis Through the Interferon-β/Interferon-α/β Receptor Signaling Pathway in a Caerulein-Induced Pancreatitis Mouse Model. Frontiers in Immunology 10. DOI: 10.3389/fimmu.2019.00980
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