Serum L-T-glutamate transpeptidase (GGT) assay experiment

Summary

This method uses L-T-glutamic acid α-naphthylamine as a substrate, and under GGT catalysis, the T-glutamic acid group is transferred to bis-glycopeptide fractionated on, while releasing free α-naphthylamine, which reacts with diazoxide reagent to produce a red compound.

Operation method

Serum L-T-glutamate transpeptidase (GGT) assay experiment

Principle

This method uses L-T-glutamic acid α-naphthylamine as a substrate, and under GGT catalysis, the T-glutamic acid group is transferred to bis-glycopeptide fractionated on, while releasing free α-naphthylamine, which reacts with diazoxide reagent to produce a red compound.

Move

I. Experimental reagents:

1. 150mmol/L Tris, 60mmol/L bis-glycopeptide solution: weigh Tris 9.1g, bis-glycopeptide 3.96g, dissolve in 400ml distilled water, then add distilled water to 500ml, add less chloroform to prevent preservation, and keep it in the refrigerator, the PH of this solution is about 8.0 (25℃).

2. 1mol/L HCL

3. Substrate buffer wave (8mmol/L L-T-glutamic acid α-naphthylamine, 54mmol/L bis-glycopeptide): weigh L-T-glutamic acid α-naphthylamine (no crystals, MW272.3) 217mg, put in a small burner, add 1mol/L hydrochloric acid about 4ml, so that completely dissolved, and then add the above Tris bis-glycopeptide solution of 90ml at 25 ℃ with lmoL/L hydrochloric acid to pH 8.0 (25 ℃). Hydrochloric acid to PH8.0±0.1, then add water to 10Oml (this solution at 37 ℃, PH is 7.8-7.9), stored in the refrigerator.

4. 2g/L p-aminobenzenesulfonic acid acetic acid solution (containing 20% acetic acid), stored in a refrigerator.

5. 1g/L aqueous sodium nitrite solution, stored in a refrigerator.

6. Color developer: 4ml of lg/L aqueous sodium nitrite solution, add 100ml of 2g/L p-aminobenzenesulfonic acid solution, mix well and use on the same day.

Laboratory operation: Take 16mm×100mm test tube and operate according to the table:

The above tubes are mixed well and placed for 10min, at the wavelength of 530nm, the optical diameter of the colorimetric cup is 1.0cm, the absorbance is measured by zeroing with the control tube, and the standard curve is checked.

Standard curve drawing:

l. 1.5mmol/L α-naphthylamine standard solution: weigh α-naphthylamine 214.8mg accurately, put it into 1000ml beaker and add 5ml anhydrous ethanol to dissolve, then add 6O0ml distilled water, mix well immediately, quantitatively transfer it into 100Oml volumetric flask, add distilled water to the scale, mix well and put it into a brown bottle, keep it in refrigerator.

2. 0.15mmol/L α-naphthylamine standard solution, 1.5mmol/L α-naphthylamine standard solution with substrate buffer for 1:10 dilution.

Operate according to the table:


The above tubes were mixed thoroughly, placed for 10min, spectrophotometer wavelength 530nm, colorimetric cup aperture 1cm, a pair of illuminated tubes zero, read the absorbance, absorbance as the vertical axis, GGT (u/L) as the horizontal axis, make a graph, this is the standard curve.

Reference value: Adult male: 3--17u/L.

Adult female: 2--13u/L

Caveat

1. More than 300 units of serum should be diluted and redone, and the result should be multiplied by the number of dilutions.

2. 1-2 blank tubes may be made for each batch of specimens, but separate blank tubes must be made for jaundice and hemolyzed specimens.

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