Serum amylase (AMS) assay test

Summary

Through this experiment, trainees can master the principle of amylase determination, the preparation of reagents and learn the determination method. This experiment is from the laboratory guide of Mudanjiang Medical College, undergraduate 5-year testing program.

Operation method

Serum amylase (AMS) measurement assay

Principle

Serum or plasma α-amylase catalyzes the hydrolysis of α-1.4 glucoside Ken in the starch molecule to produce glucose, maltose, and dextrins containing branched chains of α-1.6 glycosidic bonds, and under the condition of sufficient substrate (known concentration), the starch not hydrolyzed by the addition of iodine solution after the reaction is incorporated into a blue compound, and the shade of its color is compared with that of blanks, etc., which have not been subjected to an enzymatic reaction, so that the enzyme's unit of activity can be deduced.

Move

I. Experimental reagents:

1. 0.4g/L buffered starch solution: dissolve 9g sodium chloride, 22.6g anhydrous sodium phosphate (or Na2HPO4-12H2O 56.94g) and 12.5g anhydrous potassium dihydrogen phosphate in about 500ml distilled water, heat to boiling, and take a small beaker, accurately weigh 0.4g of soluble starch, add about 10ml of distilled water, so that the solution becomes a paste, and then add to the boiling solution. The solution, wash the beaker and pour, cold to room temperature, add 37% formaldehyde solution 5ml, diluted to 1 liter with distilled water, the solution PH 7.0 ± 0.1, placed in the refrigerator to save.

2. 0.1 mol / L iodine storage solution: with about 400 ml of distilled water to dissolve 1.7835 g of potassium iodate and 22.5 g of potassium iodide, slowly add 4.5 ml of concentrated hydrochloric acid, stirring while adding, diluted with distilled water to 500 ml of fully mixed, stored in a brown bottle tightly corked in the refrigerator for preservation.

3. 0.01mol/L iodine application solution: take iodine storage solution, dilute 10 times with distilled water, store in brown bottle and place in refrigerator for one month.

Experimental operation: serum first with saline for 10 times dilution, according to the operation of the

Mix well, use 660nm wavelength, 1.0nm aperture colorimetric cup, distilled water to adjust the zero, read the optical density of each tube.

Unit definition: 100ml serum amylase, hydrolyze 5mg starch at 37℃ for 15 minutes as a unit.

Calculation: Amylase unit = (AB-AU) × / AB × 800

Reference value: 80--180 U urine 100--1200 U

Caveat

1. oxalate. Citrate, EDTA, Na2 and sodium cyanide inhibit AMS activity, heparin does not.

2. The enzyme activity is linear with the hydrolysis of the substrate at 40 OU or less. If the absorbance of the assay tube is less than half of the absorbance of the blank tube, the serum should be diluted by a greater factor or the amount of diluted serum added should be reduced, and the result of the assay should be multiplied by the factor of dilution.

3. This method is used in the determination of amylase in other body fluids, and urine should be diluted 20 times first.

4. Saliva contains high concentration of amylase and must be prevented from being brought in.

5. If the starch solution appears turbid or flocculent, it means that the starch solution is contaminated or deteriorated and cannot be used again.

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