Two-step enzymatic determination method for cholesterol ester in serum
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A technology of cholesteryl ester and determination method, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, color/spectral characteristic measurement, etc., which can solve the problems of inability to realize automation, increase the cost of reagents, reduce errors, and increase the burden Effect
Inactive Publication Date: 2011-02-16
李立和
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Embodiment 1
[0039] Composition of reagents:
[0040] a. Reagent I:
[0041] Each liter of Tris-HCl buffer (pH7.6) contains 150mmol of Tris, 3.0mmol of 2,4-dichlorophenol, 4.2mmol of sodium cholate, 0.12g of TritonX-100, 1100U of ascorbate oxidase, 1200U of cholesterol oxidase, POD 1200U, 4-aminoantipyrine 1.6mmol, Proclin-300 200μl.
[0042] b. Reagent II:
[0043] Each liter of Tris-HCl buffer (pH7.7) contains 150 mmol of Tris, 1600 U of cholesterol esterase, and 200 μl of Proclin-300.
[0044] c. Standard solution: 3.2mmol / L cholesteryl ester standard serum.
[0045] Among them, Triton X-100 is a surfactant, Proclin-300 is a liquid high-efficiency preservative, and sodium cholate is a bacteriostatic agent.
Embodiment 2
[0047] In reagent I, 2,4-dichlorophenol was changed to 4-chlorophenol. Content unchanged. All other ingredients remain unchanged, and reagent II remains unchanged.
Embodiment 3
[0049] Measurement procedure
[0050] Two-reagent method: On the Japanese OLYMPUS AU2700 fully automated biochemical analyzer, the instrument automatically adds 2 μl of sample to 150 μl of reagent I and mixes, incubates at 37°C for 3 minutes, adds 50 μl of reagent II and mixes, and incubates at 37°C for 5.1 minutes, fully automatic The analyzer detects at a wavelength of 500nm. The instrument automatically calculates the cholesterol ester results. See Table 1 for details
[0051] Table 1. Automatic biochemical analyzer test conditions of the present invention
[0052]
standard solution
serum sample
[0053] Standard volume SV(μl)
2
2
Add reagent I volume R 1 V 1 (μl)
150
150
Warm time
The instrument automatically warms up for 3 minutes
Determination (OD 1 value)
Add reagent II volume R 2 V 2 (μl)
50
50
Warm time
The instrument automatically warms up for 5.1 minutes ...
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Abstract
The invention discloses a two-step enzymatic determination method for cholesterol ester in serum, which belongs to the field of methods for testing materials through color change of test reaction results by utilizing visible light. The technical scheme of the invention is as follows: a reagent II only contains effective components of hydrolase of the cholesterol ester, and bi-color original substances and other effective components are contained in a reagent I; and the determination method is as follows: firstly carrying out warm bath on the serum and the reagent I for 3-5min at 37 DEG C, leading free cholesterol in the serum to be reacted with the reagent I to generate quinonimine, adding the reagent II, then carrying out the warm bath for 4-7min at 37 DEG C, generating the cholesterol after hydrolysis of the cholesterol ester, and carrying out reaction for generating the red quinonimine. An instrument is used for detecting at the wavelength of 500nm-520nm, the quinonimine generated by the reaction of the reagent I is taken as blank, and the quinonimine generated by the reaction of the reagent II can be used for determining the content of the cholesterol ester. The method can not be affected by the endogenous free cholesterol during the detection, and the using method and the range are the same with the original two-step enzymatic method, thereby being the cholesterol ester detection method with quickness and higher accuracy.
Description
technical field [0001] The invention belongs to a method for measuring enzymes; or a method for testing materials by using visible light to produce color changes as a result of a test reaction, in particular to a two-step method for quickly and accurately detecting cholesterol esters in serum with a biochemical analyzer Enzyme assay method. Background technique [0002] Cholesterol exists in two forms in serum, one is free cholesterol (FC) and the other is cholesteryl ester (CHE) of cholesterol and fatty acid. The normal value of cholesteryl ester is 2.34-3.38mmol / L (accounting for 60-80% of total cholesterol). Measuring cholesteryl ester (CHE) has clinical significance, and its increase is common in diseases such as biliary obstruction, hypothyroidism, hyperlipidemia and atherosclerosis. Decreases are common in conditions such as severe liver disease, hyperthyroidism, severe infections, and malnutrition. Elevated cholesteryl esters are more likely to suffer from cardiova...
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