Method of detecting multiple cell factors simultaneously by HTFR (homogeneous time-resolved fluorescence) technique

A technology of cytokines and cells, applied in biological testing, material inspection products, fluorescence/phosphorescence, etc., can solve problems such as expensive, difficult to popularize, and rising detection costs, and achieve low false negative rate, stable signal, and low cost. controllable effect

Inactive Publication Date: 2019-05-24
浠思(上海)生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] To sum up, the existing detection methods require special instruments and are expensive, and some even require specially customized consumables, such as MSD, and also need to be washed, which leads to a sharp increase in detection costs and is difficult to popularize

Method used

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  • Method of detecting multiple cell factors simultaneously by HTFR (homogeneous time-resolved fluorescence) technique
  • Method of detecting multiple cell factors simultaneously by HTFR (homogeneous time-resolved fluorescence) technique
  • Method of detecting multiple cell factors simultaneously by HTFR (homogeneous time-resolved fluorescence) technique

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] like Figure 4 as shown, Figure 4 Shown is the release of cytokines by dexamethasone under the combined action of propylene glycol methyl ether acetate (PMA) and ionomycin.

[0048] Figure 4 Interleukin 2 (IL2), Interleukin 6 (IL6), Tumor Scurvy Factor α (TNFα) and Interferon detected by HTRF in the same PBMC sample stimulated with propylene glycol methyl ether acetate (PMA) and ionomycin γ(IFNγ), from Figure 4 As seen in the left panel, this stimulation significantly increased the release of all cytokines, as expected.

[0049] In this example, propylene glycol methyl ether acetate is a small molecular organic compound that can directly penetrate the cell membrane and activate PKC enzymes without the need for activation of receptors on cells. Ionomycin is a calcium ionophore that activates calcium ion release, thereby activating downstream signaling pathways.

[0050] These two compounds act together to fully activate cytokine release from all cells in PBMCs, wit...

Embodiment 2

[0054] like Figure 5 As shown, under the action of dexamethasone, cytokines in B cells were released.

[0055] In this example, LPS is a specific stimulating factor for B cells in PBMCs. The experimental results obtained by using HTRF are as expected. After it stimulates B cells, the secretion of IL1beta, IL6 and TNFalpha cytokines is significantly increased. After co-stimulation with dexamethasone, more than 60% of the stimulating effect of LPS can be effectively reversed.

Embodiment 3

[0057] like Image 6 As shown, under the action of dexamethasone, cytokines in T cells were released.

[0058] In this example, CD3 and CD28 antibodies are widely used to stimulate T cells in PBMC, and after stimulating T cells, the secretion of IL2 and IFNgamma is significantly increased. The experimental results obtained with HTRF are as expected. After co-stimulation with dexamethasone, more than 90% of the stimulating effect of CD3 / CD28 can be effectively reversed.

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Abstract

The invention discloses a method of detecting multiple cell factors simultaneously by HTFR (homogeneous time-resolved fluorescence) technique and belongs to the field of detection of cell factors. Themethod herein comprises the steps of I, setting cell density with a cell culture board, and culturing cells with a culture system; II, treating the cells with a drug, and acquiring a strongest signalby optimizing the stimulation time; III, collecting cell supernate, and configuring standard curves if the cell supernate requires diluting; IV, adding 16 ul of the cell supernate into a detection board, adding 4 ul of a marked detection antibody mixture, and incubating at room temperature; V, acquiring a reading with a microplate reader, and calculating concentrations of the cell factors in a sample respectively through their respective standard curves. The method herein has the advantage that the novel method is suitable for detecting multiple factors just through reagents.

Description

technical field [0001] The invention belongs to the field of cytokine detection, and in particular relates to a method for simultaneously detecting multiple cytokines using HTRF technology. Background technique [0002] PBMC, peripheral blood mononuclear cells, has become an important model in the study of tumor and tumor immunity. The T cells, B cells, NK cells and other monocytes contained in it are widely used as the immune response model after the body receives treatment. Used in fields ranging from scientific research to drug development, these responses include T cell activation, cytokine storms, and more. Therefore, multiple cytokine detection is used to monitor the response of the immune system after treatment, which is of great significance in drug research. [0003] At present, the most commonly used methods for detecting multiple cytokines are: flow CBA, MSD, and Luminex. Among them, CBA is a micro-sample multi-index flow cytometric protein quantification techno...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N21/64
Inventor 王维娜
Owner 浠思(上海)生物技术有限公司
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