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Quantitative determination method for seminal plasma glutathione peroxidase activity and kit

A technology for quantitative determination of glutathione peroxide, which is applied in the field of in vitro detection of sperm biochemical indicators, can solve the problems of inability to meet the needs of clinical diagnosis and treatment, false reduction of test results, and great potential safety hazards.

Pending Publication Date: 2022-02-11
珠海高瑞特医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But H 2 o 2 Or tert-butylated hydrogen peroxide is volatile in aqueous solution and has poor stability, and tert-butylated hydrogen peroxide is flammable and explosive, which poses a great safety hazard
In addition, although the detection results of this method are more accurate than the endpoint method, the endogenous peroxidase in the semen samples of some patients with reproductive tract infection can be compared with H 2 o 2 Or tert-butyl hydroperoxide reaction, which reduces the false detection results and cannot meet the needs of clinical diagnosis and treatment

Method used

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  • Quantitative determination method for seminal plasma glutathione peroxidase activity and kit
  • Quantitative determination method for seminal plasma glutathione peroxidase activity and kit
  • Quantitative determination method for seminal plasma glutathione peroxidase activity and kit

Examples

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Embodiment 1

[0024] 1. Preparation of reagents

[0025] Reagent composition: Reagent 1, Reagent 2 and Reagent 3.

[0026] Reagent preparation:

[0027] The preparation steps of reagent 1 are: 1mmolGSH, 1mmolEDTA-Na 2 1. Dissolve 1 mmol of sodium azide in 100 ml of phosphate buffer solution with a concentration of 0.05 mol / L and pH=7.0, and store at 5°C.

[0028] The preparation steps of reagent 2 are: 700U GRD and 0.08mmol NADPH are dissolved in 100ml of phosphate buffer solution with a concentration of 0.05mol / L and pH=7.0, and stored at 5°C.

[0029] The preparation steps of reagent 3 are as follows: dissolve 1.7 mmol cumene hydroperoxide in 100 ml phosphate buffer solution with a concentration of 0.05 mol / L and pH=9.0, and store at 5°C.

[0030] 2. Quantitative assay method for glutathione peroxidase activity in seminal plasma, using fully liquefied seminal fluid specimens as raw material, the steps are:

[0031] The fully liquefied semen samples were centrifuged at 1000 g for 15 mi...

Embodiment 2

[0083] A quantitative detection kit for glutathione peroxidase activity in seminal plasma, comprising reagent 1, reagent 2 and reagent 3; wherein, the preparation steps of reagent 1 are: 1mmolGSH, 1mmolEDTA-Na 2 1. Dissolve 1 mmol of sodium azide in 100 ml of phosphate buffer solution with a concentration of 0.05 mol / L and pH=7.0, and store at 5°C. The preparation steps of reagent 2 are: 700U GRD and 0.8mmol NADPH are dissolved in 100ml of phosphate buffer solution with a concentration of 0.05mol / L and pH=7.0, and stored at 5°C. The preparation steps of reagent 3 are as follows: dissolve 1.7 mmol cumene hydroperoxide in 100 ml phosphate buffer solution with a concentration of 0.05 mol / L and pH=9.0, and store at 5°C. The three reagents are separately packed in bottles.

[0084] The method of using the kit is carried out according to the assay method in the above-mentioned Example 1.

[0085] As an alternative, the preparation steps of the individual reagents can be adjusted, ...

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Abstract

The invention discloses a quantitative determination method for seminal plasma glutathione peroxidase activity and a kit, and the method comprises the following steps: taking a seminal fluid specimen, centrifuging, taking supernate into a centrifuge tube to obtain a seminal plasma specimen, adding a reagent 1 and a reagent 2 into the seminal plasma specimen, uniformly mixing, and determining an absorbance value A (determination 1), wherein the reagent 1 is a phosphate buffer solution containing reduced glutathione, EDTA-Na2 and sodium azide, and the reagent 2 is a phosphate buffer solution containing glutathione reductase and NADPH; adding a reagent 3 into the seminal plasma specimen, carrying out uniform mixing and reaction, and measuring the absorbance value A and the absorbance value 2, wherein the reagent 3 is a phosphate buffer solution containing cumene hydroperoxide; obtaining a blank control group in the same step, measuring an absorbance value A blank 1, adding a reagent 3, uniformly mixing, reacting, and measuring an absorbance value A blank 2; and calculating the measured value according to the following formula: GSH-P* activity (n mol / min / mL) = [(deltaA measurement-deltaA blank) / epsilon / d * V total * 106] / V sample / T.

Description

technical field [0001] The invention belongs to the technical field of in vitro detection of sperm biochemical indicators, and relates to a quantitative determination method and kit for seminal plasma glutathione peroxidase activity. Background technique [0002] A large amount of published evidence shows that oxidative stress plays a role in the impairment of sperm function, and the increase of reactive oxygen species often leads to decreased sperm motility, impaired sperm DNA integrity, acrosome reaction, and decreased sperm-egg fusion ability. Glutathione Peroxidase (GSH-Px) present in seminal plasma and sperm is an antioxidant substance that can specifically catalyze the reaction between reduced glutathione and active oxygen to generate oxidized glutathione Glycerin, thereby protecting sperm from active oxygen damage and maintaining their normal function. GSH-Px in sperm is mainly located in the cytoplasm, but because sperm contains less cytoplasm, the body mainly relie...

Claims

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Application Information

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IPC IPC(8): G01N21/31
CPCG01N21/31
Inventor 张道兵
Owner 珠海高瑞特医疗科技有限公司
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