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A kind of cytotoxicity detection reagent combination

A cytotoxicity and detection kit technology, applied in the biological field, can solve the problems affecting the accuracy and reliability of cytotoxicity detection results, poor repeatability, etc., and achieve the effect of improving repeatability and accuracy and reducing interference

Active Publication Date: 2021-07-27
SHANGHAI CELL THERAPY GRP CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is to overcome the current existing fluorescence enhancing ligand bis(acetoxymethyl) 2,2':6',2"-terpyridine-6,6"-dicarboxylic acid (bis( acetoxymethyl)2,2':6',2″-terpyridine-6,6″-dicarboxylate, BATDA) is used to enhance the autofluorescence of lanthanide europium in the application of DELFIA technology to detect cytotoxicity, the background level is too high, Poor repeatability, which affects the accuracy and reliability of cytotoxicity test results, provides an improved combination of cytotoxicity test reagents

Method used

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  • A kind of cytotoxicity detection reagent combination
  • A kind of cytotoxicity detection reagent combination
  • A kind of cytotoxicity detection reagent combination

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1 Preparation of CD19CAR-T cells

[0063] The CD19CAR-T cells used in the present invention are prepared according to the method for preparing CAR1920-2-T disclosed in CN105330750A, which is incorporated herein by reference in its entirety.

Embodiment 2

[0064] Example 2 The influence of human serum albumin on the detection effect of CD19CAR-T cells killing target cells

[0065] 1. Preparation of culture medium and detection reagents: The DELFIA lysis buffer in the EuTDA cytotoxicity detection kit was incubated in a 37°C water bath, and the BATDA reagent and DELFIA europium solution were placed at room temperature; the RPMI medium containing 10% FBS was mixed with AIM- VCTS medium was incubated in a 37°C water bath for later use;

[0066] 2. Load the target cells with the fluorescence-enhancing ligand BATDA: place the Raji cell culture medium in a centrifuge at 1500 rpm for 4 minutes, remove the supernatant, and add 1 ml of RPMI serum-free cell culture medium (RPMI+10% FBS) containing 10% FBS to resuspend After counting the cells, adjust the density of Raji cells to 1×10 cells / ml with RPMI medium containing 10% FBS according to the counting results; take 2 ml of Raji cells and add 5 μl of fluorescence-enhancing ligand (BATDA...

Embodiment 3

[0079] Example 3 Effect of human serum albumin on the detection effect of CD19CAR-T cells killing target cells

[0080] With reference to Example 2, remove the two groups of samples that added DMSO in the system, only set RPMI+10%FBS and RPMI+10%FBS+50%HSA in step 2, and adjust the effect-target ratio to E:T= 8:1, the rest are the same as in Example 2, and the killing effect and autofluorescence level of CD19CAR-T cells are calculated.

[0081] The result is as image 3 and Figure 4 shown. image 3 It shows the fluorescence level value of the effector cells killing the target cells Raji cells corresponding to the detection results of the killing effect of the CD19CAR-T cells prepared in Example 1 under the two medium conditions of RPMI+10%FBS and RPMI+10%FBS+50%HSA . From image 3 It can be seen that after adding 50% HSA in the culture medium (that is, the final concentration is 25%), the killing effect of the effector cells is significantly increased. And under the con...

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Abstract

The invention provides a cytotoxicity detection reagent composition, the components of which include europium compounds, bis(acetoxymethyl) 2,2':6',2"-terpyridine-6,6"-dicarboxylate acid (BATDA) and albumin. The present invention also provides a method for using the combination of cytotoxic detection reagents, comprising: contacting the cells to be detected with BATDA, washing, and suspending in a system containing albumin; treating the cells to be detected with cytotoxic reagents, contacting the europium compound, Check the fluorescence value. The present invention also provides a kit for detecting cytotoxicity, which includes the aforementioned cytotoxicity detection reagent composition. The cytotoxicity detection reagent composition of the present invention obviously reduces the interference of the autofluorescent background in the existing cytotoxicity detection reagents based on DELFIA technology and BATDA on the cytotoxicity detection results, and effectively improves the accuracy and accuracy of the cytotoxicity detection results. repeatability.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a combination of cytotoxicity detection reagents. Background technique [0002] Cytotoxicity (cytotoxicity) detection is widely used in biological, clinical and pharmaceutical research. Cytotoxicity detects any cell death induced by biologically active substances through biologically relevant mechanisms, so as to screen candidate molecule libraries, identify reaction mechanisms, and characterize the biological activity of drug products, etc. In immunotherapy, immune cells such as NK cells and T cells are isolated from plasma, expanded and / or activated in vitro and reinfused back to the patient. However, even if the operation method is the same at present, the effect after reinfusion is often inconsistent. Therefore, before performing immunotherapy, it is an essential step to determine the cell viability and activity of immune effector cells (immune effector cells) through cytotoxic...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/539G01N33/58
CPCG01N33/539G01N33/582G01N2458/40A61K2039/5156A61K39/0011A61K2039/5158C07K14/7051C07K2319/03
Inventor 尹锋金华君黄晨郑美美刘迪程静波钱其军
Owner SHANGHAI CELL THERAPY GRP CO LTD
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