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Liquid reagent for determining N-acetyl-beta-D-glucosaminidase

A glucosamine, liquid technology, applied in the preparation of sugar derivatives, sugar derivatives, sugar derivatives, etc., can solve the problems of poor substrate solubility and stability, difficult to meet clinical test requirements, and inconvenient clinical use, etc. The method is simple, easy to operate, and the effect of chromogen interference is small

Active Publication Date: 2010-06-16
NINGBO MEDICAL SYSTEM BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The solubility and stability of the former substrate are poor, and when NAG activity is determined with it as the substrate, the color of p-nitrophenol, the decomposition product of the substrate, is very close to the color of urine under alkaline conditions, and must be deducted during detection. The absorbance of urine itself at 405nm, otherwise it will lead to large measurement errors, and it is difficult to meet the requirements of clinical testing
The detection sensitivity of the latter is only 1 / 3 of that of the former, and it is usually provided as a dry powder kit for clinical use. The mixture of substrate and buffer needs to be incubated at 60°C for more than 6 hours to dissolve, and the mixture can only be stable for 7 days at 4°C. It is very inconvenient to use

Method used

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  • Liquid reagent for determining N-acetyl-beta-D-glucosaminidase
  • Liquid reagent for determining N-acetyl-beta-D-glucosaminidase
  • Liquid reagent for determining N-acetyl-beta-D-glucosaminidase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 is the synthesis of 5-[4-(3-methoxy-phenylene)-rhodanine]-3-ammonium acetate-N-acetylamino-β-D-glucoside.

[0035] Example 1

[0036] (1) Dissolve 3.11g of N-acetylglucosamine (purchased from Guangzhou Weber Chemical Co., Ltd.) in 10ml of acetic anhydride, add 0.1ml of perchloric acid, stir at 30°C for 2h, cool to below 8°C, add red phosphorus 0.5 g, slowly drop 2.25g of bromine, react at 10°C for 3h, then add 5ml of ice water, then filter to remove red phosphorus, extract the obtained filtrate twice with 5ml of dichloromethane, combine the organic phases, and wash once with saturated sodium bicarbonate solution , and then washed once with water to obtain a dichloromethane solution containing compound C:

[0037] (2) Add 0.45 g of tetrabutylammonium bromide to the dichloromethane solution containing compound C obtained in step (1), mix 1.07 g of vanillin and 2 g of sodium carbonate with 10 ml of water to form a solution, and mix the above Add the solution co...

Embodiment 2

[0042] Example 2 The surfactant is Tween-20, and the stabilizer is BSA.

[0043] Component A (R1): pH 5.0 citric acid-trisodium citrate buffer solution 200mmol / L

[0044] Tween-20 2g / L

[0045] NaN 3 1g / L,

[0046] Component B (R2):

[0047] 5-[4-(3-Methoxy-Benzene)-Rhodanine]-3-Ammonium Acetate-N-Acetamido-β-D-Glucoside Solution

[0048] 3mmol / L

[0049] BSA 10mmol / L

[0050] Tween-20 2g / L

[0051] NaN 3 1g / L.

[0052] When measuring the sample, use the fixed time two-point method, the temperature is 37°C, R1: sample: R2 = 300: 20: 100 (volume ratio), the measurement wavelength is 505nm, R1 is added to the standard or sample (add the standard when calibrating Or calibrator, add sample when detecting sample), after incubating for 300s, read the absorbance at the first point, then add R2 and continue to incubate for 150-300s, then read the absorbance at the second point.

Embodiment 3

[0053] Example 3 Brij 35 was selected as the surfactant, and β-cyclodextrin was selected as the stabilizer.

[0054] Component A (R1): pH 5.0 glycine buffer solution 50mmol / L

[0055] Brij 35 0.5g / L

[0056] NaN 3 1g / L,

[0057] Component B (R2): 5-[4-(3-Methoxy-Benzene)-Rhodanine]-3-Ammonium Acetate-N-Acetamido-β-D-Glucoside Solution

[0058] 4mmol / L

[0059] β-cyclodextrin 100mmol / L

[0060] Brij 35 0.5g / L

[0061] NaN 3 1g / L.

[0062] The assay method of embodiment 3 is identical with embodiment 2.

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Abstract

The invention discloses a liquid reagent for determining N-acetyl-beta-D-glucosaminidase, which takes 5-(4-(3-methoxyl-phenylmethene-)-rhodanine-3-qmmonium acetate-N-acetylamino-beta-D-glucoside as substrate and is prepared by adding proper buffer solution, surfactant, preservative, stabilizer and the like. The liquid reagent has the advantages of large solubility of the substrate, strong stability, high detection sensitivity, convenient clinical detection, low detection cost and small outside interference for the detection result of N-acetyl-beta-D-glucosaminidase.

Description

technical field [0001] The invention relates to a liquid reagent for measuring N-acetyl-β-D-glucosaminidase. Background technique [0002] N-acetyl-β-D-glucosaminidase (English name: N-acteyl-β-D-glucosaminidase; English abbreviation: NAG; EC 3.2.1.30) is one of the intracellular lysosomal hydrolases with a molecular weight of 140,000. NAG in the blood cannot pass through the glomerular filter, and the content of NAG is higher in the nephron, especially in the epithelial cells of the proximal convoluted tubule. [0003] Studies have shown that NAG has important detection value in acute renal tubular damage caused by diabetes, hypertension, early renal tubular damage, drug nephrotoxicity, infection, shock, renal transplant rejection, etc. Simple screening tool. There are many kinds of urine enzymes, among which NAG determination is currently the most widely used urine enzyme diagnosis item. It responds sensitively to renal tubular injury. In addition, using urine as a spe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/31C07H15/26C07H1/00
Inventor 邹炳德邹继华沃燕波胡治宝
Owner NINGBO MEDICAL SYSTEM BIOTECHNOLOGY CO LTD
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