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Cytotoxicity detection reagent composition

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

Active Publication Date: 2018-12-07
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 detection results, provides an improved cytotoxicity detection reagent composition

Method used

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  • Cytotoxicity detection reagent composition
  • Cytotoxicity detection reagent composition
  • Cytotoxicity detection reagent composition

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- V CTS 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 Raji cell density to 1×10 with RPMI medium containing 10% FBS according to the counting results. 6 cells / ml; take 2ml of Raji cells, add 5μl of fluorescence-enhancing ligand (BATDA), ...

Embodiment 3

[0079] Example 3. The influence 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...

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Abstract

The invention provides a cytotoxicity detection reagent composition. The composition is prepared from an europium compound, bis(acetoxy methyl)2,2':6',2''- terpyridyl-6,6''-dicarboxylic acid (BATDA) and albumin. The invention further provides a method for using the cytotoxicity detection reagent composition. The method comprises the following steps: contacting a to-be-detected cell with BATDA, washing, and suspending in a system containing the albumin; treating the to-be-detected cell with the cytotoxic reagent, contacting the europium compound, and detecting the fluorescence value. The invention further provides a kit for detecting cytotoxicity, which comprises the cytotoxic detection reagent composition. The cytotoxicity detection reagent composition can be used for remarkably reducing interference of spontaneous fluorescence background in existing cytotoxic detection reagent based on DELFIA technology and BATDA on a cytotoxic detection result, and effectively improving the accuracyand repeatability of the cytotoxic detection result.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a cytotoxicity detection reagent composition. 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 cytotoxicity ...

Claims

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Application Information

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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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