Valuation method for specific killing function of specific cytotoxicity T lymphocyte

A technology of cytotoxicity and lymphocytes, applied in biochemical equipment and methods, microbial determination/inspection, measuring devices, etc., can solve the problems of inapplicability of long-term repeated and accurate measurement of CTL, inability to detect in real time, high cost, etc. To improve the effect of cellular immunotherapy

Inactive Publication Date: 2019-10-22
BEIJING UNIV OF TECH
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  • Abstract
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  • Claims
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Problems solved by technology

Common CTLs evaluation methods in this field are as follows: 1. Radioactive isotope Cr51 release test: This method has a wide range of applications, but it has radioactive pollution, damage to the body, and requires special instruments and the purchase of Cr salts; and due to the half-life of Cr51 It is relatively short, and the reagent will decay a lot when it is in hand, which will eventually lead to inaccurate results. It is not suitable for long-term repeated and accurate measurement of CTL
2.3H incorporation method: the advantages and disadvantages are the same as the radioisotope Cr51 release experiment
3. MTT colorimetric method: the advantages are cheap and convenient, but the repeatability is very poor and inaccurate; 4. CCK-8: the principle is the same as that of MTT colorimetric method, but there is no need to remove the supernatant, and directly add CCK-8 , does not need DMSO to dissolve formazan, which is simpler and more reproducible than the MTT step, but because WST-1 in CCK-8 is reduced to water-soluble formazan by intracellular dehydrogenase, and the effector cells in the CTL reaction In the same system as the target cells, formazan will be produced, and judging the intensity of the CTL reaction only based on the absorbance may make the result inaccurate; 5. LDH method: the advantage is that it does not require special equipment and is relatively cheap, but the workload is large The disadvantage is that the spontaneous release of the background is too high, leading to inaccurate results
7. ELISPOT, this method has high accuracy and is measured according to the cytokines released after CTL killing, but the cost is very high, and special instruments are required for measurement, or it can be measured under a microscope
8. Soluble peptide tetramer method: the premise is that the antigen to be used must have a clear epitope, and a peptide tetramer targeting the epitope must be synthesized; this method has high precision, but the popularity rate is low and the cost is expensive
Other methods, such as granzyme measurement, ELISA measurement of INF-gamma, etc., all have the disadvantages of labeling cells, inaccurate detection, high cost, and unstable results. At the same time, they all detect killing at a single time point and cannot be used continuously real-time detection

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  • Valuation method for specific killing function of specific cytotoxicity T lymphocyte
  • Valuation method for specific killing function of specific cytotoxicity T lymphocyte
  • Valuation method for specific killing function of specific cytotoxicity T lymphocyte

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Experimental program
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experiment example

[0101] Experimental example, verification data of the evaluation method of the present invention in terms of accuracy, efficiency and economy

[0102] In the above method of the present invention, live cell imaging provides a visualization platform, which can dynamically observe the process of recognition and specific killing of target cells by LMP2-specific CD8+ T cells, so as to determine their specific killing specificity. RTCA provides a real-time label-free long-term monitoring platform, which can accurately evaluate the real-time killing efficiency and maintenance killing time of LMP2-specific CD8+ T cells on target cells. Therefore, an evaluation system consisting of live-cell imaging and RTCA can accurately evaluate the killing function of LMP2-specific CD8+ T cells in vitro.

[0103] In the present invention, the live cell imaging technology is mainly a qualitative analysis of the killing function of LMP2-specific CD8+ T cells; and RTCA is a quantitative analysis of t...

experiment example 1

[0110] Experimental example 1. rAd-LMP2 vaccine immunized mice to induce LMP2-specific CD8+ CTLs

[0111] Female BALB / c and C57BL / 6 mice aged 4-6 weeks were selected and randomly divided into two groups: PBS group and rAd-LMP2 group, 5 mice in each group. Dilute the rAd-LMP2 vaccine to 2x109VP / ml and inject 100ul intramuscularly. PBS intramuscular injection of 100ul each. They were immunized three times at 0, 2, and 4 weeks, and at the 5th week, the spleen was taken to separate lymphocytes. ELISpot results showed that: rAd-LMP2 immunized BALB / c mice induced 405 SFC / 106 spleen lymphocytes; rAd-LMP2 immunized C57BL / 6 mice induced SFC / 106 spleen lymphocytes was 488 ( figure 1 ).

experiment example 2

[0112] Experimental Example 2 Miltenyi Magnetic Bead Separation of CD8+ T Cells

[0113] After the isolated mouse spleen lymphocytes were fully lysed with red blood cell lysate, washed three times with 1xPBS, the cells were resuspended with RPMI1640-10% FBS and counted with trypan blue. Take 1x107 spleen lymphocytes and put them into a 2ml EP tube and centrifuge at 1500rpm for 5 minutes. Resuspend the cell pellet with 40ul buffer. Add 10ul of biotin-antibody mixture, mix thoroughly, and incubate in a refrigerator at 4°C for 5min. Add 30ul of buffer solution, add 20ul of anti-biotin microbeads, mix well, and incubate at 4°C for 10min. Add buffer to a volume of 500 ul, mix well and apply to a column for magnetic separation to collect sorted CD8+ T cells. After resuspending with RPMI1640-10% FBS, count the hope blue.

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Abstract

The invention provides a valuation method for a specific killing function of a specific cytotoxicity T lymphocyte, which belong to the field of immunotherapy. The method comprises the following steps:(1) treating the target cells with specific cytotoxicity T lymphocyte constructed aiming at the target cells to-be-killed; (2) detecting the killing process of specific cytotoxicity T lymphocyte on the target cells by Live-Cell Imaging; and (3) employing real-time unlabeled cell analysis (RTCA) to detect the killing efficiency and killing ability of specific cytotoxicity T lymphocyte to target cells; wherein steps (2) and (3) are not ordered, or can be carried out at the same time.

Description

technical field [0001] The invention relates to the field of immunotherapy, in particular to a method for evaluating the specific killing function of specific cytotoxic T lymphocytes. [0002] Background of the invention [0003] EBV-specific CD8+ T cells have been successfully used to prevent and treat EBV-related tumors, but the effect is poor in nasopharyngeal carcinoma (NPC), CD8+ T cell responses can be detected in NPC patients, and in Hypofrequency and functional unresponsiveness problems are present in NPC patients. [0004] LMP2 has continuous expression and does not cause B lymphocyte transformation, so it is not carcinogenic, and is often selected as an ideal target antigen for EBV-related tumor immunotherapy. LMP2 can induce strong CD8+CTLs, and some CD8+T cell epitopes have been identified in NPC patients. The level of LMP2 cell immunity is related to the occurrence and prevention of NPC. [0005] CTLs-based immunotherapy is only effective in some NPC patients. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/02
CPCG01N33/4833
Inventor 曾毅葛玉洋周玉柏周志祥滕智平
Owner BEIJING UNIV OF TECH
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