Serum total bilirubin and conjugated bilirubin determination assay

Summary

Bound bilirubin in serum can react directly with diazo reagent to produce azo bilirubin, under the same conditions, free bilirubin must have an accelerator, so that bilirubin hydrogen bonding is destroyed in the reaction of diazo reagent, caffeine, sodium malate as an accelerator, sodium nitrate to maintain the PH and also have an accelerating effect, ascorbic acid (or sodium azide) destroys the remaining diazo reagent, aborting the azotization of the junction bilirubin tube, to prevent the slow reaction of free bilirubin, add alkaline sodium tartrate to shift the maximum absorbance from 530nm to 598nm. Bilirubin slow reaction, adding alkaline sodium tartrate so that the maximum absorbance from 530nm to 598nm, non-bilirubin yellow pigments and other red pigments, and brown pigments produced by the absorbance down to a negligible amount, so that the sensitivity and specificity increased, the final green color is a mixture of blue alkaline azo bilirubin and caffeine and p-aminobenzene sulfonate between the yellow pigment formed. This experiment is from the Mudanjiang Medical College undergraduate 5-year laboratory guide for laboratory testing majors

Operation method

Serum total bilirubin and conjugated bilirubin determination assay

Move

I. Experimental reagents;

1. Caffeine a sodium malate reagent, weigh anhydrous sodium acetate 41.0g (CH3COONa-3H2O6 3.0g) sodium benzoate 38.0g, disodium ethylenediaminetetraacetic acid (EDTA Na2) 0.5g, dissolved in 500ml of distilled water, and then add 25.0g of caffeine stirring to make the dissolution (the addition of caffeine does not take place after the heating of the solubilization), make up to 1 liters of distilled water. Mix well, filter with filter paper, place in brown bottle and store at room temperature.

2. alkaline sodium tartrate solution: weigh 75g of sodium hydroxide sodium tartrate (Na2C4H4O6-2H2O) 263.0g with distilled water to dissolve and make up to 1 liter, mix well. Place in a plastic bottle and store at room temperature.

3. 5.0g / L sodium nitrite solution: weighing sodium nitrite (NaNO2) 5.0g, dissolved in distilled water and diluted to 100 ml, mix well, stored in a brown bottle, stored in the refrigerator, stable for not less than 3 months, for 10 times diluted into 50g / L, stored in the refrigerator, stable for not less than 2 weeks.

4. 50g/L p-aminobenzenesulfonic acid solution: weigh p-aminobenzenesulfonic acid (NH2C6H4SO3H-H2O) 5.0g dissolved in 800ml of distilled water, add 15ml of concentrated HCl, and make up to 1 liter with distilled water.

5. diazo reagent: take 5.0g/L sodium nitrite solution 0.5ml and 5.0g/L p-aminobenzenesulfonic acid solution, 20ml mixed before use. 6. 50g/L sodium azide solution: weigh sodium azide 059 dissolved in distilled water and diluted to 100ml. 7. sodium azide solution: take sodium azide 5.0g dissolved in 800ml distilled water, add concentrated HCl 15ml.

7. bilirubin standard solution: 171umol/dL (10ml/dL) accurately weigh 10mg of bilirubin that meets the requirements of the addition of 1ml of dimethyl alum, stirring with a glass, so that the suspension is made, add 0.05 mol / L sodium carbonate solution 2ml to make the bilirubin completely dissolved. Move into 100 volumetric flasks, with diluted with serum washed several times and into the volumetric flask, slowly add 0.1 mol / L hydrochloric acid 2 ml while shaking (do not shake, so as not to produce bubbles), and finally diluted with serum to make up the full 300 ml, the preparation process should be protected from light, storage containers, wrapped in black paper, placed in a refrigerator at 4 ℃ for 3 days, required to do as soon as possible after matching the standard curve.

Second, the experimental operation;

Fully mixed wavelength 600nm, the control tube zero, read the optical density of each tube, or zero with water. Read the absorbance of the measuring tube and the control tube, and use the difference between the absorbance of the measuring tube and the absorbance of the control tube (Au-Ac) to draw the corresponding bilirubin concentration on the standard curve.

For the standard curve, dilute the bilirubin storage solution with different concentrations according to the following table.

Mix the above tubes well (but do not produce air bubbles), according to the serum total bilirubin assay method, each concentration for three parallel tubes, each concentration for the standard control tube, with the respective standard control tube to zero. Read the absorbance of each standard tube, each tube should also be subtracted from the absorbance of the total bilirubin of the serum used for dilution, and then plot the standard curve with the corresponding bilirubin concentration.

Reference value: serum total bilirubin 5.1-19.0 umol/L (0.3-1.1 mg/dL)

Serum bound bilirubin (1 min) 1.7--6.8 umol/L (0.1-0.4mg/dL)

Caveat

l. The method determines the serum total bilirubin at 10-37℃, no change in temperature de influence, the color development is very stable in 2 hours.

2. The method has high sensitivity (molar absorbance coefficient is 74380±866L-mol-1-cm-1) and can avoid the interference of other colored substances.

3. Mild hemolysis has no effect on this method, but severe hemolysis can make the results low.

4， the use of sodium azide as an anti-titration agent of the quality control serum, can cause the azo reaction is incomplete, or even do not show color.

5. bilirubin is sensitive to light, standards and specimens should be protected from light.

6. Lipid blood to enterolysis pigment has interference in the determination, should try to take fasting blood.

7， bilirubin greater than 100mg / L specimen can reduce the amount of specimen, or diluted with saline and redo.

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