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Method for using the kit for detecting sialidase in sperm

A technology of sialidase and kit, applied in the field of medical testing, which can solve the problems of high price, complex technical process, high requirements for experimental equipment and operators' skills and experience, and achieve the reduction of negative impact, simple operation method, and few types of consumables 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

Since the sialidase activity of the sperm itself is not necessarily related to the enzyme activity of the capacitation fluid, this method cannot detect the sialidase contained in the sperm itself, and cannot evaluate the quality of the sperm itself
At the same time, there are still the following disadvantages in the method: 1) The technical process is complicated; 2) It requires high skills and experience for experimental equipment and operators; 3) It is not suitable for clinical laboratories and routine technicians; 4) Use abcam company kits are expensive

Method used

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  • Method for using the kit for detecting sialidase in sperm

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Effect test

Embodiment 1

[0050] The method of using the kit for detecting sialidase in sperm, the kit includes cell lysate, phosphate buffer or BWW culture fluid, protease inhibitor cocktail, reagent A solution, reagent B solution, bovine serum albumin, fluorescent reagent, saliva Acidase standard stock solution and detection buffer; the cell lysate does not contain enzyme activity inhibitors; the reagent A solution is: 1% BCA disodium salt, 2% anhydrous sodium carbonate, 0.16% sodium tartrate, 0.4% Sodium hydroxide, 0.95% sodium bicarbonate, mixed to adjust the pH value to 11.2-11.3; reagent B solution: 4% copper sulfate; the detection buffer contains 0.05 mmol / l sodium acetate and 0.25 μg / μl bovine serum Albumin phosphate buffer; the fluorescent reagent is 2′-4-methylumbelliferyl-α-D-N-acetylneuraminic acid sodium; the sialidase standard stock solution is Clostridium perfringens Sialidase, the concentration is 5U / ml; the use method of the kit, the specific steps are as follows:

[0051] A. Sperm wa...

Embodiment 2

[0063] The method of using the kit for detecting sialidase in sperm, the kit includes cell lysate, phosphate buffer or BWW culture fluid, protease inhibitor cocktail, reagent A solution, reagent B solution, bovine serum albumin, fluorescent reagent, saliva Acidase standard stock solution and detection buffer; the cell lysate does not contain enzyme activity inhibitors; the reagent A solution is: 1% BCA disodium salt, 2% anhydrous sodium carbonate, 0.16% sodium tartrate, 0.4% Sodium hydroxide, 0.95% sodium bicarbonate, mixed to adjust the pH value to 11.2-11.3; reagent B solution: 4% copper sulfate; the detection buffer contains 0.05 mmol / l sodium acetate and 0.25 μg / μl bovine serum Albumin phosphate buffer; the fluorescent reagent is 2′-4-methylumbelliferyl-α-D-N-acetylneuraminic acid sodium; the sialidase standard stock solution is Clostridium perfringens Sialidase, the concentration is 5U / ml; the use method of the kit, the specific steps are as follows:

[0064] A. Sperm wa...

Embodiment 3

[0076]The method for using the kit for detecting sialidase in sperm is characterized in that: the kit includes cell lysate, phosphate buffer or BWW culture fluid, protease inhibitor cocktail, reagent A solution, reagent B solution, bovine serum albumin, Fluorescence reagent, sialidase standard stock solution and detection buffer; the cell lysate does not contain enzyme activity inhibitors; the reagent A solution is: 1% BCA disodium salt, 2% anhydrous sodium carbonate, 0.16% tartaric acid Sodium, 0.4% sodium hydroxide, 0.95% sodium bicarbonate, mixed to adjust the pH value to 11.2-11.3; reagent B solution: 4% copper sulfate; the detection buffer contains 0.05 mmol / l sodium acetate and 0.25 μg / μl bovine serum albumin phosphate buffer; the fluorescent reagent is 2′-4-methylumbelliferyl-α-D-N-acetylneuraminic acid sodium; the sialidase standard stock solution is gas Clostridium perfringens sialidase, the concentration is 5U / ml; the method of using the kit, the specific steps are ...

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Abstract

The invention provides a method for evaluating semen quality. The method comprises the following specific steps: 1, collecting a fresh liquefied seminal fluid sample, washing semen with a phosphate buffer solution or a BWW culture solution, removing residual seminal plasma constituents completely, and then conducting centrifugal separation on the semen; 2, adding cell lysis buffer and a protease inhibiting agent cocktail to the semen obtained through separation in step 1, mixing the mixture to be uniform, and after no obvious cell sedimentation is found, in other words, complete splitting decomposition is achieved, conducting high speed centrifugation at the temperature of 4 DEG C, wherein supernatant is protein extract; 3, measuring the protein concentration of the protein extract; 4, measuring sialidase activity of the semen, and finally obtaining the sialidase activity value / protein concentration value, so that the process is ended. According to the method, through detection of activity of sialidase in the semen, the diagnosis basis and clinical reference are provided for a patient suffering from male infertility with the clinical reasons unclear.

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 sperm. 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 such as sperm, weak sperm or te...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/34
Inventor 马芳马学李福平
Owner 成都思瑞迪医疗科技有限公司
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