Circulating nerve cell detection kit and detection method

A technology for detecting kits and nerve cells, which is applied in the field of medical biotechnology research to achieve the effects of low detection cost, simple sampling, and high specificity

Pending Publication Date: 2021-03-09
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, scholars have not yet identified specific biomarkers that can accurately describe the occurrence and development of ischemic s...

Method used

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  • Circulating nerve cell detection kit and detection method
  • Circulating nerve cell detection kit and detection method
  • Circulating nerve cell detection kit and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] density gradient centrifugation

[0052] Such as figure 1 As shown, the method for separating and identifying circulating nerve cells in a blood sample mainly includes the following specific steps:

[0053] a) Density gradient centrifugation: the patient's peripheral blood sample was added to the corresponding volume of antibody for incubation and then density gradient centrifugation was performed.

[0054] b) Adding the cell suspension onto the chip: loading the cell suspension layer containing the target cells after density gradient centrifugation onto the microwell array chip.

[0055] c) Cell fixation, membrane rupture, staining: the cells on the chip are fixed, membrane rupture and then stained to identify circulating nerve cells, wherein the dye is a certain concentration of fluorescently labeled neuron-specific antibody NeuN antibody substance or NeuN The primary antibody and the secondary antibody labeled with fluorescent markers, the antibody CD45 against leu...

Embodiment 2

[0061] immunomagnetic bead method

[0062] Such as figure 2 As shown, the separation and identification method of circulating nerve cells in the peripheral blood of the central nervous system disease mainly includes the following specific steps:

[0063] 1. Lying red blood cells: using red blood cell lysate to lyse the red blood cells in the patient's peripheral blood.

[0064] 2. Negative screening: Immunomagnetic beads modified with CD45 were added to the lysed cell suspension and screened with the aid of a magnetic frame after full incubation.

[0065] 3. Add the cell suspension to the chip: After the negative screening, keep the centrifuge tube on the magnetic frame, extract the cell suspension in the centrifuge tube and load it on the microwell array chip.

[0066] 4. Cell fixation, membrane rupture, and staining: the cells on the chip are fixed, membrane ruptured, and stained to identify circulating nerve cells. The staining agent is a certain concentration of fluores...

Embodiment 3

[0069] Identification and analysis of circulating nerve cells in the peripheral blood of stroke patients is used to monitor the clinical prognosis of stroke patients.

[0070] 1. Incubate 5 ml of patient's peripheral blood (with known patient information) and 75 microliters of CTC-enriched antibody (including CD36 antibody) at room temperature for 20 minutes.

[0071] 2. Add 15 ml of Hank's balanced salt solution (HBSS) containing 2% fetal bovine serum (FBS) to the blood in step 1 and mix thoroughly.

[0072] 3. Add 15ml of density gradient centrifugation solution to the bottom of the centrifuge tube through the small hole in the middle of the centrifuge tube.

[0073] 4. Transfer the well-mixed patient's blood in step 2 to the density gradient centrifuge tube in step 3 along the tube wall. It is best not to disturb the density gradient centrifuge at the bottom of the tube.

[0074] 5. Under the premise of balance, centrifuge at 1200g for 20 minutes.

[0075] 6. After centri...

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Abstract

The invention belongs to the field of medical biotechnology research, and discloses a circulating nerve cell detection kit containing a fluorescently-labeled neuron specific antibody, a fluorescently-labeled white blood cell classic marker antibody and a cell nucleus fluorescent dye on the first hand. Besides, the invention discloses a density gradient centrifugation kit for detecting circulatingnerve cells and a detection method of the kit. Furthermore, the invention discloses an immunomagnetic bead kit for detecting circulating nerve cells and a detection method of the immunomagnetic bead kit. Compared with the conventional detection means, the technical scheme disclosed by the invention has the technical effects of high specificity, simplicity in detection and sampling, lower detectioncost and price and capability of realizing real-time monitoring of a blood sample.

Description

technical field [0001] The invention belongs to the field of medical biotechnology research, and relates to a circulating nerve cell detection kit and a detection method. Background technique [0002] In recent years, the age of onset of stroke in my country has become younger and younger, which will have a serious impact on social and economic development. Studies have shown that stroke is closely related to many diseases, such as diabetes, high blood pressure and heart disease, and even related to genetic factors. Due to the high heterogeneity of the pathogenic factors of stroke, how to make a convenient, fast and accurate diagnosis of stroke is of great significance for the prevention and prognosis monitoring of stroke. The current diagnosis of ischemic stroke is mainly based on clinical diagnosis and neuroimaging diagnosis, which depends largely on local availability and is difficult to detect in the early stages of ischemic stroke development. [0003] However, the cu...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/543G01N21/64
CPCG01N21/6486G01N33/54326G01N33/56966
Inventor 丁显廷张瑜
Owner SHANGHAI JIAO TONG UNIV
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