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6-glucose-6-phosphate dehydrogenase mutant and application thereof in preparing detection reagent

A technology for glucose phosphate and detection reagents, which is applied in the field of enzyme 6-phosphate glucose dehydrogenase and its application in detection kits, and can solve the problems of cumbersome operation, long time-consuming and short validity period of enzyme-linked immunosorbent assay

Inactive Publication Date: 2019-08-27
BEIJING STRONG BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are many defects in these detection methods, such as radioimmunoassay isotopes have radioactive contamination, short validity period, inconvenient operation and many other disadvantages, and enzyme-linked immunosorbent assay is cumbersome and time-consuming, which is not suitable for clinical application. use on
Although chemiluminescence has good sensitivity, it needs supporting special equipment, and the high cost of putting it into use is not conducive to popularization

Method used

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  • 6-glucose-6-phosphate dehydrogenase mutant and application thereof in preparing detection reagent
  • 6-glucose-6-phosphate dehydrogenase mutant and application thereof in preparing detection reagent
  • 6-glucose-6-phosphate dehydrogenase mutant and application thereof in preparing detection reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Embodiment 1. Synthesis of Glycocholic Acid Derivatives

[0073] Add glycocholic acid (1.0eq), maleimidoethylamine (1.0g, 1.0eq) and triethylamine (3.0eq) into a dry and clean 25mL two-necked bottle;

[0074] Then add dimethylformamide (5mL) and stir until completely dissolved, add dichloroethane (1.25eq), and stir at 25°C for 2h;

[0075] HPLC monitoring, until the completion of the reaction;

[0076] Add the above reaction mixture into water (25mL), add ethyl acetate 20mL×3 for extraction;

[0077] Combined organic phases, anhydrous Na 2 SO 4 After drying and concentrating under reduced pressure, the resulting oil was purified by column chromatography to obtain 1.04 g of milky white powdery solid, yield 45%, M+: 602.72.

[0078] The function of this embodiment is to make CG have a group that can bind to enzymes, and the technical effect of this application does not depend on specific hapten derivatives.

Embodiment 2

[0079] Example 2. Coupling of Glycocholic Acid Derivatives to G6PDH Molecules

[0080] According to the G6PDH-glycocholic acid conjugate of the present application, the coupling is carried out in the following manner: the sulfhydryl reactive group (such as maleimide group) on the glycocholic acid derivative molecule co-operates with the thiol group on the G6PDH molecule. price combination.

[0081] 1. Solution preparation:

[0082] Glycocholic acid derivative solution: 10 mg / ml of the glycocholic acid derivative prepared in Example 1 was dissolved in DMF;

[0083] G6PDH solution: 6.7mg / mL G6PDH (mutant or wild type of this application), PB 100mmol, NaCl100mmol, pH=8.0;

[0084] Coupling solution: 100mM PB / K, 100mM EDTA, 150mM NaCl, pH=7.2;

[0085] Desalting solution: 100 mM PB / K, 100 mM EDTA, 150 mM NaCl, pH=7.2.

[0086] 2. Coupling operation: 1.6ml G6PDH solution, 6ml coupling solution and 0.40ml glycocholic acid derivative solution were reacted at room temperature (20 ...

Embodiment 3

[0088] Embodiment 3. Preparation of kit

[0089] Prepare the following kit for detecting glycocholic acid, which comprises:

[0090] Reagent R1, comprising:

[0091] 100mM PB buffer, pH 7.2

[0092] 15mM Glucose 6-phosphate

[0093] 15mM β-nicotinamide adenine dinucleotide

[0094] 0.1mg / L Glycocholic acid antibody

[0095] 200mM NaCl

[0096] 0.5g / L bovine serum albumin

[0097] 0.1g / L Tween20

[0098] 1g / L sodium azide;

[0099] Reagent R2, including:

[0100] 100mM PB buffer, pH 7.2

[0101] 0.1mg / L G6PDH-CG conjugate

[0102] 0.5g / L bovine serum albumin

[0103] 0.1g / L Tween 20

[0104] 1g / L sodium azide;

[0105] Calibrator: 100mM PB buffer, pH 7.2, and 0, 2.5, 5.0, 10, 20, 40mg / L glycocholic acid (or add as needed);

[0106] Quality control: 100mM PB buffer, pH 7.2, and 1.5, 8.0, 25, 35mg / L glycocholic acid (or add as needed).

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Abstract

The application relates to a 6-glucose-6-phosphate dehydrogenase mutant and an application thereof in preparing detection reagent. Specifically, the 6-glucose-6-phosphate dehydrogenase mutant comprises one of the following mutants or combination thereof: D306C, D375C, and G426C in comparison with the wild type 6-glucose-6-phosphate dehydrogenase. The detection kit prepared from the 6-glucose-6-phosphate dehydrogenase mutant has the advantages of being strong in specificity, high in sensitivity, convenient to operate, short in detection time, accurate in quantification, and suitable for high-flux detection.

Description

technical field [0001] This application relates to the field of biological detection, in particular to an enzyme 6-phosphate glucose dehydrogenase (G6PDH for short) with multi-site mutation and its application in detection kits. Background technique [0002] Haptens, certain small molecular substances (molecular weight less than 4000Da), which alone cannot induce an immune response, that is, they are not immunogenic, but when they are cross-linked or combined with macromolecular proteins or non-antigenic polylysine and other carriers Afterwards, immunogenicity can be obtained and an immune response can be induced. These small molecular substances can be combined with the response effect products and have antigenicity. They are only immunoreactive, not immunogenic, and are also called incomplete antigens. [0003] A hapten can combine with the corresponding antibody to produce an antigen-antibody reaction, but it cannot stimulate the human or animal body to produce antibodie...

Claims

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Application Information

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IPC IPC(8): G01N21/31G01N35/00
CPCG01N21/31G01N35/00Y02A50/30Y02P20/55
Inventor 龚俊祁金祥肖兰萍刘希
Owner BEIJING STRONG BIOTECH INC
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