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Method for using a kit for detecting sialidase in in vitro sperm capacitation fluid

A technology of sialidase and sperm capacitation, which is applied in the field of medical testing, can solve problems such as complex technical process, unscientific enzyme activity value, high requirements for experimental equipment and operator skills and experience, and achieve simple operation methods and objective test results Effect

Active Publication Date: 2019-09-24
成都思瑞迪医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the method includes the expression detection of sperm sialidase NEU1 / 3, which requires a fluorescence microscope or flow cytometer, and has the following disadvantages: 1) The technical process is complicated; 2) It requires high skills and experience in experimental equipment and operators; 3 ) Not suitable for use in clinical laboratories and routine technicians; 4) Use abcam's kit, which is expensive
In addition, because the number of sperm in different samples is different, the value of expressed protein is different, and the corresponding enzyme activity value is also different. Therefore, it is not scientific to simply compare the enzyme activity values ​​​​of different samples.

Method used

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  • Method for using a kit for detecting sialidase in in vitro sperm capacitation fluid

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Embodiment 1

[0039] The use method of the kit for detecting sialidase in in vitro sperm capacitation fluid, the kit consists of phosphate buffer or BWW culture fluid, BWW culture fluid, in vitro capacitation fluid, fluorescent reagent, sialidase standard stock solution and detection buffer Composition; the in vitro capacitation fluid is HTF containing HSA 5mg / ml; the detection buffer is a phosphate buffer containing 0.05 mmol / l sodium acetate and 0.25 μg / μl bovine serum albumin; the saliva Acidase standard stock solution is Clostridium perfringens sialidase, concentration is 5U / ml; Described fluorescent reagent is 2'-4-methylumbelliferyl-α-D-N-acetylneuraminic acid sodium; Specifically Proceed as follows:

[0040] A. Sperm washing

[0041] Collect fresh liquefied semen samples, wash the sperm with phosphate buffer solution or BWW culture medium, fully remove the remaining seminal plasma components, and then centrifuge to separate the sperm;

[0042] B. In vitro capacitation of sperm

[...

Embodiment 2

[0048] The use method of the test kit for detecting sialidase in in vitro sperm capacitation fluid, the use method of the test kit for detecting sialidase in in vitro sperm capacitation fluid, the test kit consists of phosphate buffer solution or BWW culture medium, BWW culture medium , in vitro capacitation solution, fluorescent reagent, sialidase standard stock solution and detection buffer; the in vitro capacitation solution is HTF containing HSA 5mg / ml; the detection buffer is containing 0.05 mmol / l sodium acetate and 0.25 μg / μl bovine serum albumin in phosphate buffer; the sialidase standard stock solution is Clostridium perfringens sialidase, and the concentration is 5U / ml; the fluorescent reagent is 2′-4- Methylumbelliferyl-α-D-N-acetylneuraminic acid sodium; the method of using the kit, the specific steps are as follows:

[0049] A. Sperm washing

[0050] Collect fresh liquefied semen samples, wash the sperm with phosphate buffer solution or BWW culture medium, fully ...

Embodiment 3

[0057] The use method of the test kit for detecting sialidase in in vitro sperm capacitation fluid, the use method of the test kit for detecting sialidase in in vitro sperm capacitation fluid, the test kit consists of phosphate buffer solution or BWW culture medium, BWW culture medium , in vitro capacitation solution, fluorescent reagent, sialidase standard stock solution and detection buffer; the in vitro capacitation solution is HTF containing HSA 5mg / ml; the detection buffer is containing 0.05 mmol / l sodium acetate and 0.25 μg / μl bovine serum albumin in phosphate buffer; the sialidase standard stock solution is Clostridium perfringens sialidase, and the concentration is 5U / ml; the fluorescent reagent is 2′-4- Methylumbelliferyl-α-D-N-acetylneuraminic acid sodium; the method of using the kit, the specific steps are as follows:

[0058] A. Sperm washing

[0059] Collect fresh liquefied semen samples, wash the sperm with phosphate buffer solution or BWW culture medium, fully ...

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Abstract

The invention provides a method for evaluating the quality of sperm obtaining energy in vitro. The method specifically includes the following steps of A, collecting a fresh liquefied sperm specimen, washing sperm with a phosphate buffer or a BWW culture solution, sufficiently removing residual seminal plasma components, and separating out sperm in a centrifugal mode; B, adding the BWW culture solution to sperm separated out in the step A, obtaining upstream sperm through an upstream method, counting the number of sperm, incubating sperm for 3-4 hours in a CO2 cell culture box with 5% of in-vitro energy obtaining liquid, conducting centrifugal separation on the obtained supernate, and collecting supernate as a to-be-tested sample; C, measuring the activity of neuraminidase in sperm energy obtaining liquid, and finally obtaining the value of neuraminidase activity / sperm number to evaluate the quality of sperm. By detecting the activity of neuraminidase in sperm in-vitro energy obtaining liquid, diagnosis basis and clinical queries are provided for male infertility patients of unknown clinical reasons.

Description

technical field [0001] The invention belongs to the technical field of medical detection, and in particular relates to a method for using a kit for detecting sialidase in in vitro sperm capacitation fluid. Background technique [0002] With the continuous development of social industry, the total incidence of infertile couples increased significantly. In 2004, the statistics of the European Society of Reproduction: 25% of couples of childbearing age were unable to conceive within one year, 15% of them sought medical treatment, and 50% were infertility caused by male factors. The causes of male infertility are sexual dysfunction (1.7%), varicocele (12.3%), reproductive tract infection (6.6%), congenital dysplasia (2.1%) acquired disease (2.6%), Endocrine disorders (0.6%), immune factors (3.1%), and other abnormalities (3%), but up to 60% to 75% of patients cannot find the cause, which is called idiopathic male infertility, and they only show a few Abnormal sperm quality suc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/34
Inventor 马芳马黔红潘倩
Owner 成都思瑞迪医疗科技有限公司
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