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Modified N-acetylneuraminic acid aldolase and preparation method and application thereof

A neuraminic acid aldolase and acetyl technology, which is applied in the field of modification methods and kits containing the modified enzyme, can solve the problems of poor reagent stability and the like, and achieves high sensitivity, high enzyme activity and better stability. Effect

Active Publication Date: 2017-01-11
上海睿康生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Because the side chain of N-acetylneuraminic acid aldolase has a carboxyl group, and the carboxyl group has certain oxidative properties, the reagent 2 contains a reduced coenzyme, and long-term storage will cause the reduced coenzyme to be absorbed by the N-acetylneuraminic acid aldolase Oxidation, resulting in poor stability of the reagent

Method used

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  • Modified N-acetylneuraminic acid aldolase and preparation method and application thereof
  • Modified N-acetylneuraminic acid aldolase and preparation method and application thereof
  • Modified N-acetylneuraminic acid aldolase and preparation method and application thereof

Examples

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Embodiment 1

[0037] This embodiment relates to a modified N-acetylneuraminic acid aldolase and a preparation method thereof, wherein the amino acid side chain of the N-acetylneuraminic acid aldolase is modified by alkylation.

[0038] Described preparation method comprises the steps:

[0039] Using triethylamine as a solvent, N-acetylneuraminic acid aldolase and carbodiimide are mixed in a mass ratio of 1:3, stirred overnight at room temperature, and the carboxyl group of the amino acid side chain is removed;

[0040] Add guanidine hydrochloride to the above solution containing N-acetylneuraminic acid aldolase from carboxyl removal, adjust the pH to 5.0-6.0 with glacial acetic acid, and stir overnight at room temperature. The mass ratio of N-acetylneuraminic acid aldolase after carboxyl removal to guanidine hydrochloride is 1:5.

Embodiment 2

[0042] This embodiment provides a detection kit containing the modified N-acetylneuraminic acid aldolase prepared in Example 1, which is composed as follows:

[0043] The components and concentrations of reagent R1 are:

[0044]

[0045]

[0046] The components and concentrations of reagent R2 are:

[0047]

[0048] The SA detection kit described in this example is applicable to various types of automatic biochemical analyzers. Taking Hitachi 7170 automatic biochemical analyzer as an example, its operation is shown in Table 1. Analysis method: rate method, that is, reagent R1; the amount of R2 is 240 μl and 60 μl respectively, and the sample volume is 8 μl; add 8 μl sample to 240 μl reagent R1, add 60 μl R2 after 5 minutes at 37 ° C, delay 120 seconds to start reading, and the reading time is about 180 seconds; The detection wavelengths are respectively main wavelength 340nm and subwavelength 405nm.

[0049] Adopt this reagent and above-mentioned assay method, adopt...

Embodiment 3

[0052] Embodiment 3: Correlation test of detection reagent

[0053] The purpose of this example is to detect the correlation between the reagent prepared in Example 2 of the present invention and the existing reagent.

[0054] Use the inventive reagent of this method (specific formula is the same as embodiment 2) and contrast reagent Japan Wako Pure Pharmaceutical Company's SA reagent, measure 100 parts of human serum (comprising normal and abnormal sample) by respective parameter, carry out correlation analysis to measured value . See the test results figure 2 , X, Y axes are measured values ​​(SA content mg / dl).

[0055] Depend on figure 2 The results show that the correlation coefficient R of the two reagents 2 =0.9928, the regression equation is y=1.0265x-0.926. The results show that this reagent has a good correlation with imported reagents in the determination of patient serum, and has good specificity and accuracy.

[0056] In addition, the reagents in the above...

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Abstract

The invention provides modified N-acetylneuraminic acid aldolase and a preparation method and application thereof. The amino acid side chain of the modified N-acetylneuraminic acid aldolase is modified through alkylation. The preparation method of the modified N-acetylneuraminic acid aldolase comprises the following steps that N-acetylneuraminic acid aldolase is reacted with an alkylation reagent to remove carboxyl in the amino acid side chain; the N-acetylneuraminic acid aldolase without carboxyl is modified with guanidine hydrochloride, and the modified N-acetylneuraminic acid aldolase is obtained. The modified N-acetylneuraminic acid aldolase can be used for preparing a stable SA kit and is higher in enzymatic activity and better in stability in the reagent; meanwhile, the modified N-acetylneuraminic acid aldolase and a reduced coenzyme do not disturb each other, so that the sensitivity of the reagent is better, a smaller amount of sialic acid can be detected, and accuracy is better in sample detection with a relatively low value.

Description

technical field [0001] The present invention relates to the field of in vitro diagnosis, in particular to a modified N-acetylneuraminic acid aldolase and its preparation method and application, in particular to a modification method of N-acetylneuraminic acid aldolase and the modified Enzyme Kit. Background technique [0002] N-acetylneuraminic acid is catalyzed by N-acetylneuraminic acid aldolase to generate N-acetylmannosamine and pyruvate, which is one of the important enzymes in the detection of sialic acid. [0003] Sialic acid is an important part of cell membrane glycoprotein, which is related to many biological functions of organisms, and is closely related to cell malignant transformation, cancer metastasis, invasion, loss of contact inhibition, decreased cell adhesion and tumor antigenicity. Determination of serum sialic acid (SA) concentration can be used as an auxiliary index for tumor diagnosis and an index for curative effect observation. [0004] The disease...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/50G01N35/00
CPCG01N33/50G01N35/00
Inventor 李伟奇李杰房君江张秀文林清玉
Owner 上海睿康生物科技有限公司
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