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X-linked lymphoproliferative syndrome diagnostic kit and application thereof

A diagnostic kit and the technology of the kit, which are applied in the field of medical diagnosis, can solve the problems that the test results are easily affected by the subjective factors of the tester, the accuracy is poor, and the precision is low.

Inactive Publication Date: 2017-08-11
BEIJING FRIENDSHIP HOSPITAL CAPITAL MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The object of the present invention is to provide a diagnostic kit for X-linked lymphoproliferative syndrome and a detection method using the kit to detect the abundance of SAP protein. The accuracy is poor, and the test results are easily affected by the subjective factors of the tester.

Method used

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  • X-linked lymphoproliferative syndrome diagnostic kit and application thereof
  • X-linked lymphoproliferative syndrome diagnostic kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] S11. Detection of SAP protein expression in NK and cytotoxic T (CTL) cells by flow cytometry

[0078] ① Ethylenediamine-tetraacetic acid (EDTA) or heparin sodium (Heparin Sodium) anticoagulated venous whole blood 2ml, Ficoll density gradient centrifugation to separate peripheral blood mononuclear cells (PBMC), and count them, adjust the cell concentration to 3.5× 10 6 / ml suspension for later use.

[0079] ②Add 200ul diluted cells to each tube, this tube is generally used as a sample tube. Add 16ul CD3-FITC, 12ul CD56-APC, 12ul CD8a-Percp Cy5.5 to each tube, incubate at room temperature for 15 minutes in the dark, then add 2ml flow buffer (1×PBS solution of 2.0% FBS and 2.0mM EDTA) to each tube ), centrifuged at 1200 rpm for 5 minutes, and discarded the supernatant.

[0080] ③Fixation: Dilute 4× fixative (eBioscience, #00-5521-00) to 1×, add 2ml to each tube, vortex and mix, fix at 4 degrees in the dark for 50 minutes, centrifuge at 1200 rpm for 5 minutes, discard c...

Embodiment 2

[0104] S21. Detection of SAP protein expression in NK and cytotoxic T (CTL) cells by flow cytometry

[0105] ① Ethylenediamine-tetraacetic acid (EDTA) or heparin sodium (Heparin Sodium) anticoagulated venous whole blood 2ml, Ficoll density gradient centrifugation to separate peripheral blood mononuclear cells (PBMC), and count them, adjust the cell concentration to 4.5× 10 6 / ml suspension for later use.

[0106] ②Add 200ul diluted cells to each tube, this tube is generally used as a sample tube. Add 24ul CD3-FITC, 8ul CD56-APC, 8ul CD8a-Percp Cy5.5 to each tube, incubate at room temperature in the dark for 25 minutes, then add 2ml flow buffer (2.0% FBS and 2.0mM EDTA in 1×PBS solution ), centrifuged at 1200 rpm for 5 minutes, and discarded the supernatant.

[0107] ③Fixation: Dilute 4× fixative (eBioscience, #00-5521-00) to 1×, add 2ml to each tube, vortex and mix, fix at 4 degrees in the dark for 50 minutes, centrifuge at 1200 rpm for 5 minutes, discard clear.

[0108] ...

Embodiment 3

[0117] S31. Detection of SAP protein expression in NK and cytotoxic T (CTL) cells by flow cytometry

[0118] ① Ethylenediamine-tetraacetic acid (EDTA) or heparin sodium (Heparin Sodium) anticoagulated venous whole blood 2ml, Ficoll density gradient centrifugation to separate peripheral blood mononuclear cells (PBMC), and count them, adjust the cell concentration to 4.0× 10 6 / ml suspension for later use.

[0119] ②Add 200ul diluted cells to each tube, this tube is generally used as a sample tube. Add 20ul CD3-FITC, 10ul CD56-APC, 10ul CD8a-Percp Cy5.5 to each tube, incubate at room temperature in the dark for 20 minutes, then add 2ml flow buffer (1×PBS solution of 2.0% FBS and 2.0mM EDTA ), centrifuged at 1200 rpm for 5 minutes, and discarded the supernatant.

[0120] ③Fixation: Dilute 4× fixative (eBioscience, #00-5521-00) to 1×, add 2ml to each tube, vortex and mix, fix at 4 degrees in the dark for 50 minutes, centrifuge at 1200 rpm for 5 minutes, discard clear.

[0121...

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Abstract

The invention relates to the technical field of medical diagnosis, and specifically relates to an X-linked lymphoproliferative syndrome diagnostic kit and application thereof. The kit comprises anti-human CD 3, CD56, CD8a and SAP flow fluorescent antibodies, an isotype control fluorescent antibody of the anti-human SAP flow fluorescent antibody, an anti-human SAP Western Blot antibody, and a secondary antibody corresponding to the anti-human SAP Western Blot antibody. A method for detecting SAP protein abundance in CTL and NK cells based on the diagnostic kit is quick to detect, high in flux, high in accuracy, and small in human factor influence.

Description

technical field [0001] The invention relates to the technical field of medical diagnosis, in particular to a diagnostic kit for X-linked lymphoproliferative syndrome and its application. Background technique [0002] SAP (SLAM associated protein) protein is a small-molecule adapter protein with an SH2 domain, which plays an important role in signal transduction in the signaling receptors of the lymphocyte activation molecule (SLAM) family. As a signal adapter and regulatory switch in the cytosol, the SAP protein regulates the response of the body's immune cells to Epstein-Barr virus by interacting with SLAM-related factors. When the SH2D1A gene is mutated and affects the transcription of genetic information and the expression of SAP protein, it will change the function of SAP protein, so that through the relationship between SAP protein and SLAM family factors, it will further affect the activation of immune cells and the activation of antiviral immune signals. Transmission...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N15/14
CPCG01N15/14G01N33/68G01N2015/1006
Inventor 王昭高卓张嘉王旖旎王晶石吴林赖雯苑金志丽孟硕宋悦崔婷婷
Owner BEIJING FRIENDSHIP HOSPITAL CAPITAL MEDICAL UNIV
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