Isopropanol was used, and bilirubin was adsorbed with alumina during extraction equal to the interferents (phospholipids and glycerol were also adsorbed, and this extract was released to be used for TG measurement by chemical method). After centrifugation in the supernatant add high iron. Sulfuric acid reagent shows purple-red color, the degree of color development is proportional to the concentration of TG. This experiment is from the Mudanjiang Medical College undergraduate 5-year laboratory guide for testing majors
Operation method
Serum total cholesterol assay
Principle
Isopropanol was used, and bilirubin was adsorbed with alumina during extraction equal to the interferents (phospholipids and glycerol were also adsorbed, and this extract was released to be used for TG measurement by chemical method). After centrifugation in the supernatant add high iron. Sulfuric acid reagent shows purple-red color, the degree of color development is proportional to the concentration of TG. Move I. Experimental reagents: Caveat 1, Fe3＋-Phosphoric acid -sulfuric acid is quite a lot, but the determination of this method is color stable, sensitivity is about 5 times higher than the L-B reaction, but the interference factors of the color reaction is more than the L-B reaction method. 2、Triglycerides and cholesterol can be measured simultaneously by this method. 3， the extraction wave and the colorant mixed with heat, the degree of hair fall and color depth. Therefore, the operation should pay attention to the points in column F.(l) the same batch of specimens need to be measured with the same caliber and thickness of the test tube, in order to facilitate the mixing, the test tube caliber is best larger.(2) Along the wall of the tube to add colorant, must be extracted with isopropyl alcohol serum for two layers, and then quickly mixed, not to be added while shaking, otherwise the color development is incomplete.(3) The colorant must be added to a tube mixed with a tube, can not be compared to add together with the mixing, mixing techniques to be consistent.(4) All test tubes and colorimetric cups must be dry, concentrated H2SO4 must be superior pure airtight storage to prevent water absorption. 5， the accuracy of this method is close to the ALBK method, the recovery rate is close to 100%, repeatability is good, skilled operators within the CV ＜1% day to day ＜2%. For more product details, please visit Aladdin Scientific website.
1, isopropyl alcohol
2, alumina (chromatography with medium-sized alumina, washed several times with distilled water, pour off the fine particles that are not easy to sink, suction filtration after drying in the oven at 110 ℃ activated at least 8h, stored in closed containers or desiccators.
3, ferric chloride storage solution: weighing FeCl3 storage solution and concentrated sulfuric acid (excellent pure) mixed by 1:1 volume, room temperature can be stable for about 2 months.
4, coloring agent; 1g / L (that is, 0.18/dL Fecl storage solution and concentrated sulfuric acid (superior purity) mixed by 1:1 volume, room temperature can be stable for about 2 months.
5, cholesterol standard solution: accurately weigh cholesterol (purity of 99.5% or more) 200mg, with isopropyl alcohol to make 100ml stored in 4 ℃ refrigerator, the pro-use to reach room temperature before you can draw.
Second, the experimental operation:
l, absorb the serum and standard solution (or fixed value serum) 20Oul, respectively, into the plugged centrifuge tube, to the bottom of the tube inhalation of isopropyl acid 5.0ml, dispersed serum, so that the protein precipitation into fine particles, mixed with a stopper and placed in a 6O ℃ water bath for 1min, add alumina (or zeolite composite) 1g with a stopper, will be fixed in the tube lying on the vibration of the vibration of the 10min (can also be used to rotary mixer or hand shaking) Centrifugation.
2, measurement tube and standard tube were added to the corresponding supernatant 1ml, the blank tube with isonitol lml, into the 60 ℃ water bath for 1min, respectively, along the wall of the tube to add 3ml of colorant, mix well and continue to be placed in the 60 ℃ water bath for 15min, and then cooled at room temperature after colorimetry.
3, colorimetric photometer, wavelength 530-550nm, with a blank tube school zero, read the absorbance of each tube, with a 1cm colorimetric cup, the linear range of at least up to 10.3mmol / L (400g / dL).
Calculation:
Serum cholesterol concentration = absorbance of measurement tube / absorbance of standard tube × cholesterol concentration of standard liquid.