Glycosylated serum protein assay

Summary

Serum glucose undergoes a non-enzymatic glycation reaction with the amino group at the N-terminus of albumin and other serum protein molecules to form a macromolecular ketamine structure. This ketamine structure can be reduced with nitrotetrazolium blue (NBT) in alkaline solution to form methylamine, which can be colorimetrically determined using 1-deoxy-1-morpholino fructose (OMF) as a standard reference. This experiment is from Mudanjiang Medical College, undergraduate 5-year laboratory guide for testing majors.

Operation method

Glycosylated serum protein assay

Principle

Serum glucose undergoes a non-enzymatic glycation reaction with the amino group at the N-terminus of albumin and other serum protein molecules to form a macromolecular ketamine structure. This ketamine structure can be reduced with nitrotetrazolium blue (NBT) in alkaline solution to produce methylamine, and colorimetrically determined with 1-deoxy-1-morpholino fructose (OMF) as the standard reference. This experiment was obtained from the Laboratory Instruction of the 5-Year Professional Laboratory of Mudanjiang Medical College.

Move

I. Experimental reagents:

1, 0.1mmol / L carbonate buffer, PH10.8: anhydrous sodium carbonate 9.54g, sodium bicarbonate 0.84g, dissolved in distilled water and diluted to 1000ml.

2、0.11mol/L NBT reagent: weigh 100mg of nitrozolium chloride blue, used in the above buffer solution and diluted to 1000mL, stored in the refrigerator, can be stable for at least 3 months;

3, 4mmol / L DMF standard solution: weigh DMF99.6g, melted in 40g / L bovine serum albumin solution 100ml.

Second, the experimental operation:

Determination of the tube plus the serum to be examined (plasma) 0.1ml, the blank tube with distilled water 0.1ml, each add 37 ℃ pre-warmed NBT reagent 4ml, mix, placed in a 37 ℃ water bath for exactly 15min, immediately removed, running water cooling (less than 25 ℃), 15 minutes after the cooling of the spectrophotometer wavelength 550nm, 10nm light diameter colorimetric cup with a blank tube to adjust the zero, read the determination of the tube. Find the results from the standard curve, reported in fructosamine mmol/L.

Standard curve preparation:

Take 4mmol / L DMF standard solution with bovine serum protein solution (40g / L) diluted to 1, 2, 3, 4mmol / L, and serum protein, 40g / L with the determination of the same operation of the tube, read the corresponding absorbance of each concentration of DMF to DMF concentration as the horizontal coordinate, absorbance as the vertical coordinate, made of standard curve. The concentration of DMF within 4 mmol/L was linearly related to the absorbance.

Reference value: 1.9-10-0.25mmol/L.

Caveat

1, PH value, reaction temperature and reaction time have a great influence on this experiment and must be strictly controlled.

2、Serum protein can form ketoamine structure through non-enzymatic glycation reaction, and DMF can form 1-deoxy-amino-2-keto ketoamine structure through structural rearrangement from the oxirane structure under the appropriate PH value and temperature, so it is reasonable to use it as the standard reference material. Therefore, it is reasonable to use it as a standard reference. It is more desirable to report the results in mmol/L.

3、Fixed value frozen in refined hemoglobin as a standard, the results are more stable, because different standards when the results are not completely consistent, it is best to establish a laboratory reference value.

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