To enable students to master the principle of alkaline phosphoric acid determination, method and preparation of reagents, this experiment is from the Mudanjiang Medical College undergraduate 5-year laboratory guide for laboratory tests.

Operation method

Colorimetric assay for serum alkaline phosphatase (ALI) test

Principle

In a reaction solution at pH 10, alkaline phosphatase catalyzes the hydrolysis of disodium benzene phosphate to produce free phenol and phosphoric acid, and the phenol binds to 4-aminoantipyrine in alkaline solution and is oxidized by potassium ferric chloride to produce a reddish-colored quinone derivative, and the enzyme activity is calculated based on the red color shade.

Move

I. Experimental reagents:

l. 0.1 mol/L carbonate buffer (PH 10.0)

Dissolve anhydrous sodium carbonate 6.39g sodium bicarbonate 3.36, 4-aminoantipyrine combined with 1.5g in 800 ml of distilled water, put this solution into a 1000 ml volumetric flask, add distilled water to the scale, and keep it in a brown bottle.

2. 20mmol/L disodium phosphate solution: first boil 500ml distilled water, eliminate microorganisms, gradually add disodium phosphate 2.18g (such as 2 molecules of water of crystallization should be weighed 2.54g) after cooling, add 2ml of chloroform antiseptic, preserved in the refrigerator, called the substrate solution.

3. Potassium ferricyanide solution: weigh potassium ferricyanide 2.5g, boric acid 17g each dissolved in 400 ml of distilled water until the two liquids are mixed, add distilled water to 1000 ml, placed in a brown bottle to protect from light and save, such as the appearance of blue-green color that is discarded.

4. Phenol standard storage solution (1mg/ml) easy to buy commercial standard solution

5. Phenol standard application liquid 0.05mg/ml: phenol standard storage solution 5m1, add distilled water to 100ml, this night can only be saved for 2-3 days.

Second, the experimental operation: take 16mm × 100mm test tube according to the table for the number and measurement

Steps for serum alkaline phosphatase determination:

Immediately mix, at a wavelength of 51Onm, distilled water to adjust the zero point, read the absorbance of each tube. Measure the absorbance of each tube, subtract the absorbance of the control tube, check the standard curve table, and find out the enzyme activity unit.

Gold's unit definition: 100ml, serum at 37 ℃, and substrate action for 5 minutes, producing 1mg of phenol for a gold's unit

Standard Curve Plotting:

Caveat

1. Adding boric acid to potassium ferricyanide solution has the effect of stabilizing the color development.

2. The substrate should not contain free phenol, if the blank tube shows red color, it means that disodium phosphate starts to decompose and should be discarded.

3. Potassium ferricyanide must be mixed quickly after addition, otherwise the color development is not sufficient.

4. Jaundice serum and blood bath serum are used as control respectively, and the general serum standard can be shared with the control tube.

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