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Method and kit for stably detecting sialic acid by enzyme method

A technology of sialic acid and kit, which is applied in the field of enzymatic detection of sialic acid, which can solve the problems of few applicable models, inability to meet clinical detection needs, and short stability period

Active Publication Date: 2011-05-25
BEIJING STRONG BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The stability period of this method is short, and most of them are dry powder reagents. Due to the lack of popularization of automatic biochemical analyzers in the past, the amount of reagents is large, the stability time after dry powder reconstitution is short, and there are few applicable models, resulting in high application costs for users and unable to meet the needs of clinical testing.

Method used

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  • Method and kit for stably detecting sialic acid by enzyme method
  • Method and kit for stably detecting sialic acid by enzyme method
  • Method and kit for stably detecting sialic acid by enzyme method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Embodiment 1 adopts the SA detection kit of single reagent

[0085] The reagent of the kit adopts a single reagent, and the components and concentrations of the reagents are as follows:

[0086] Neuraminidase 50KU / L

[0087] Neuraminic acid aldolase 50KU / L

[0088] Oxidized coenzyme NAD 0.1mmol / L

[0089] Mannosamine dehydrogenase 50KU / L

[0090] MES buffer 100mmol / L

[0091] Polyethylene glycol 0.05%

[0092] Sodium Azide 0.05%

[0093] The pH of the reagent is 7.0, where the stated percentages are weight to volume.

[0094] The method of using a single reagent is as follows:

[0095] Table 1: How to use a single reagent

[0096]

sample

standard blank

standard

serum

60ul

standard

60ul

60ul

Reagent I

1800ul

1800ul

1800ul

[0097] Mix well and react at 37°C for 10 minutes, adjust to zero with pure water, and measure the absorbance change of each tube.

[0098] Calculati...

Embodiment 2

[0101] Embodiment 2 adopts the SA detection kit of double reagent

[0102] The kit’s reagents use dual reagents, and the components and concentrations of reagent 1 are as follows:

[0103] Neuraminidase 80KU / L

[0104] Oxidized coenzyme NAD 4mmol / L

[0105] HEPES buffer 100mmol / L

[0106] Polyethylene glycol 0.1%

[0107] Sodium Benzoate 0.1%

[0108] Reagent 1 has a pH of 6.5, where the percentages are weight to volume.

[0109] The composition and concentration of reagent 2 are as follows:

[0110] Neuraminic acid aldolase 10KU / L

[0111] Mannosamine dehydrogenase 60KU / L

[0112] HEPES buffer 400mmol / L

[0113] Glycerol 5%

[0114]Sodium Azide 0.1%

[0115] Reagent 2 has a pH of 7.2, where the percentages are weight to volume.

[0116] The dual reagents are used as follows:

[0117] Table 2: How to use the dual reagents

[0118]

[0119]

[0120] Mix well and react at 37°C for 10 minutes, adjust to zero with pure water, and measure the absorbance change of...

Embodiment 3

[0124] Embodiment 3 adopts the SA detection kit of double reagent

[0125] The kit’s reagents use dual reagents, and the components and concentrations of reagent 1 are as follows:

[0126] Neuraminidase 100KU / L

[0127] Oxidized coenzyme NAD 4mmol / L

[0128] HEPES buffer 100mmol / L

[0129] Polyethylene glycol 0.1%

[0130] Sodium Benzoate 0.1%

[0131] Reagent 1 has a pH of 6.5, where the percentages are weight to volume.

[0132] The composition and concentration of reagent 2 are as follows:

[0133] Neuraminic acid aldolase 10KU / L

[0134] Mannosamine dehydrogenase 100KU / L

[0135] HEPES buffer 400mmol / L

[0136] Glycerol 5%

[0137] Sodium Azide 0.1%

[0138] Reagent 2 has a pH of 7.2, where the percentages are weight to volume.

[0139] Its usage method is as the kit in Example 2.

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Abstract

The invention relates to a method and a kit for stably detecting sialic acid by an enzyme method. The method comprises the following steps of: generating N-acetylneuraminic acid from bound sialic acid under the catalysis of neuraminic acid aldolase; transforming the N-acetylneuraminic acid into N-acetylmannosamine under the catalysis of neuraminidase; reacting the product with mannosamine dehydrogenase and nicotinamide adenine dinucleotide (NAD) to generate reduced nicotinamide adenine dinucleotide (NADH); and measuring the sialic acid content by detecting the amount of the generated NADH. The invention also relates to the kit prepared according to the method. The method and the kit are conveniently and quickly used, the sialic acid is not required to be redissolved by dissolving liquid, is stable for a long time and has small inter-bottle variation, the method and the kit are suitable for various biochemical analyzers, secondary pollution is avoided, and a production process method is simplified.

Description

technical field [0001] The invention relates to a stable enzymatic method for detecting sialic acid and a kit. Specifically, the present invention relates to a method for detecting sialic acid content by detecting NADH production, and a single-reagent or double-reagent liquid kit prepared according to the method. Background technique [0002] Sialic acid (SA) is one of many natural neuraminic acid derivatives. Its parent structure is an endocyclic amino acid composed of nine carbon atoms. It is a general term for a family of compounds, and its chemical name is N-acetylneuraminic acid. It gets its name from the fact that it was originally isolated from the submandibular gland mucin. [0003] Sialic acid is distributed in mammals, especially in vascular endothelial cells. Serum total sialic acid (total sialic acid, TSA) content is about 1.5-2.5mmol / L, including free sialic acid and bound sialic acid. The content of free sialic acid is low, about 1-3umol / L; the sialic acid b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/52C12Q1/25C12Q1/32
Inventor 杜爱铭刘希刘瑶
Owner BEIJING STRONG BIOTECH INC
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