Method for detecting neuraminidase influencing sperm functions

A technology of sialidase and detection method, which is applied in the field of medical detection, can solve the problems of inapplicability to clinical application, detection and reporting of sialidase activity, and failure to detect sialidase smoothly, so as to ensure accuracy and avoid dead sperm Effect

Active Publication Date: 2013-12-18
成都思瑞多医疗科技有限公司
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  • Application Information

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Problems solved by technology

However, the literature only discloses the expression detection of sialidase in sperm, and does not further report the detection of sialidase activi

Method used

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  • Method for detecting neuraminidase influencing sperm functions

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Embodiment 1

[0044] The sialidase detection method affecting sperm function is characterized in that: the first step is to visually quantify the expression of sialidase in sperm by analyzing the fluorescence intensity of single sperm, or to visually observe the expression of sialidase in sperm by using fluorescence imaging under a microscope. If the sialidase is highly expressed in the sperm, proceed to the second step, using the in vitro capacitation fluid to capacious the sperm, and detect the sialidase activity with a fluorescent method.

Embodiment 2

[0046] The sialidase detection method affecting sperm function is characterized in that: the first step is to visually quantify the expression of sialidase in sperm by analyzing the fluorescence intensity of single sperm, or to visually observe the expression of sialidase in sperm by using fluorescence imaging under a microscope. If the sialidase is highly expressed in the sperm, proceed to the second step, using the in vitro capacitation fluid to capacious the sperm, and detect the sialidase activity with a fluorescent method.

[0047] The in vitro capacitation solution is HSA 5mg / ml HTF.

Embodiment 3

[0049] The implementation mode of this embodiment is basically the same as embodiment 1, on this basis:

[0050] Follow the steps below to detect the expression of sperm sialidase NEU1:

[0051] A Collect fresh liquefied semen samples, centrifuge to separate the sperm, and wash the sperm twice with PBS;

[0052] B Count the number of sperm and place on ice for 20 min;

[0053] C adopts 0.1% Triton X-100 pretreated the sperm, washed the sperm twice with PBS, and pretreated the sperm again with 3% PFA;

[0054] D uses 1% BSA-PBS solution to block;

[0055] E Incubate with 1ug / ul anti-human NEU1 antibody for 1 h, wash the sperm twice with PBS;

[0056] F At room temperature, the sperm were incubated with conventional fluorescent secondary antibodies at a ratio of 1:1000 for 1 h;

[0057] G Use flow cytometry or fluorescence microscope to make sperm cell smears, and observe the expression of sialidase in sperm.

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Abstract

The invention provides a method for detecting neuraminidase influencing sperm functions. The method is a novel laboratory detecting method for assessing quality and functions of a sperm and can provide detection and clinical consultations for male infertility patients without clear clinical reasons for infertility. The method is characterized in that expressions of neuraminidase in the sperm are visually quantized through single sperm fluorescence strength analysis or are visually observed by means of fluorescence imaging under a microscope, and if the neuraminidase is highly expressed in the sperm, capacitation of the sperm is achieved through external capacitation liquid, and neuraminidase activity is detected in a fluorescence method.

Description

technical field [0001] The invention belongs to the technical field of medical detection, and in particular relates to a method for detecting sialidase that affects sperm function. 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...

Claims

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Application Information

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IPC IPC(8): G01N33/573G01N33/531
Inventor 马芳许文明徐克惠岳焕勋蒋敏欧阳运薇林丽
Owner 成都思瑞多医疗科技有限公司
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