Sulfated bile acid enzyme fluorescence capillary analytical method and enzyme fluorescence quantitative reagent kit

A technology of capillary analysis and sulfonation, which is applied in the field of clinical biochemical analysis, can solve the problems of expensive reagents that can only be used once, low sensitivity, and reagent pollution, so as to overcome the weaknesses of easy denaturation and instability, improve sensitivity, and operate short time effect

Inactive Publication Date: 2008-07-16
SICHUAN UNIV
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

[0005] The main problem with existing SBA enzyme colorimetric assays is that there are too many types of enzymes and reagents used (BSS, β-HSD, 3-ketobile acid-Δ 4 -Dehydrogenase, diaphorase, β-NAD, WST-1, ASOD, Tween20, sorbitol, 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), enzyme consumption is large, cost is high, and sensitivity is low. The expensive reagents can only be used once; and the reaction product formazan is not completely soluble in water, and there are different degrees of reagent pollution in the quantitative cuvette after use. If it is reused, it must be washed repeatedly. It is still a problem for the application of automatic analysis equipment

Method used

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  • Sulfated bile acid enzyme fluorescence capillary analytical method and enzyme fluorescence quantitative reagent kit
  • Sulfated bile acid enzyme fluorescence capillary analytical method and enzyme fluorescence quantitative reagent kit
  • Sulfated bile acid enzyme fluorescence capillary analytical method and enzyme fluorescence quantitative reagent kit

Examples

Experimental program
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Effect test

Embodiment 1

[0058] This example is to investigate the effect of the concentration of immobilized BSS (6) in SBA-CBR (2) on the fluorescence intensity. Experimental conditions: excitation wavelength 540nm, emission wavelength 580nm, reaction solution volume 18μL, 500μmol / Lβ-NAD + , 50μmol / L1-MPMS, 50μmol / L resazurin, 5kU / Lβ-HSD(7), pH8.0, reaction temperature 37℃, reaction time 10min; change BSS(6) concentration in the range of 1-10kU / L , using 1.0 μmol / L and 2.0 μmol / L GLCA-S standard solution as the measurement sample respectively, and optimized the concentration of immobilized BSS (6) in SBA-CBR, the results are shown in Figure 2. The fluorescence intensity of the product reached the maximum when the dosage of BSS(6) was 5kU / L.

Embodiment 2

[0060] This example is to investigate the effect of the amount of β-HSD (7) in SBA-CBR (2) on the fluorescence intensity. Based on the above conditions, the dosage of BSS(6) was 5kU / L, and the concentration of β-HSD(7) was changed in the range of 1-10kU / L to investigate its effect on sensitivity. The results are shown in Figure 3. The fluorescence intensity of β-HSD(7) increased gradually in the range of 1-5kU / L; when the dosage of β-HSD(7) was 5kU / L, the fluorescence intensity reached the maximum.

Embodiment 3

[0062] This example is to investigate β-NAD + Effect of concentration on fluorescence intensity. The amount of β-HSD (7) was 5.0 kU / L, and other experimental conditions were the same as in Example 2. Change β-NAD in the range of 50-800μmol / L + concentration, examine β-NAD + The effect of concentration on the sensitivity of the method. The results obtained are shown in Figure 4. β-NAD + In the range of 50-400μmol / L, the fluorescence intensity of the reaction system gradually increases, and the fluorescence intensity reaches the maximum at 400μmol / L; when β-NAD + When the concentration is higher than 400μmol / L, the fluorescence intensity decreases instead.

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Abstract

The invention discloses a sulfated bile acid (SBA) enzyme fluorescence capillary analytical method and an SBA fluorescence fixed amount agent kit (SBA-FCA-Kit) which belongs to the field of a clinic biochemistry inspection field and is suitalbe for the fast screening and diagnosing of liver and gall diseases, more particularly, is suitalbe for the early period discovery of jaundice in neonatal period and congenital biliary atresia. After being mixed with fluorescence reaction liquid, a biology sample is sucked by an SBA capillary biology reactor (SBA-CBR) which contains sulfated bile acid sulfoacid esterase and Beta-hydroxysteroid dehydrogenase to determine the empty fluorescence intensity of the SBA-CBR under a given excitation wavelength and an emission wavelength; then the biology sample is led to continuously react for a certain time and the fluorescence intensity is determined; the amount of the SBA in the sample is fixed by using fluorescence intensity difference value. The SBA-FCA-Kit is composed of an SBA-CBR and fluorescence reaction liquid containing Beta-NAD<+>, an electric transmission body and a diazoresorcinal, etc.

Description

technical field [0001] The invention relates to a sulfonated bile acidase fluorescence capillary analysis method and a SBA enzyme fluorescence quantitative kit for realizing the method, belonging to the field of clinical biochemical analysis. The invention is suitable for the general survey of the hepatobiliary system of healthy people, the rapid screening and diagnosis of the hepatobiliary diseases of clinical patients, and is especially suitable for the early detection of neonatal jaundice, congenital biliary atresia and hepatitis. Background technique [0002] Bile acids are synthesized in the liver. Under normal circumstances, 99% of bile acids exist in the enterohepatic circulation, and very little enters the systemic circulation. However, when the liver and gallbladder are damaged by disease, bile acids flow back into the blood, resulting in a significant increase in bile acids in the blood. As a detoxification mechanism in the body, bile acids in blood are sulfonated...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
Inventor 李永生高秀峰
Owner SICHUAN UNIV
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