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Application of reagents for detecting kyn content

A content and reagent technology, applied in the field of biological detection, can solve the problem that lymphocytes are not well characterized, and achieve the effect of accurate judgment

Active Publication Date: 2020-09-18
THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, despite the unprecedented clinical success of programmed cell death receptor 1 (PD-1) antibodies targeting many types of tumors, PD-1 has been shown to be active in tumor-specific cytotoxic CD8 cells in the tumor microenvironment. + The underlying mechanisms of how this is regulated in lymphocytes (CTLs) are still not well characterized

Method used

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  • Application of reagents for detecting kyn content
  • Application of reagents for detecting kyn content
  • Application of reagents for detecting kyn content

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1: Tumor-specific CD8 + PD-1 expression in T cells is induced by IFN-γ-stimulated TRC

[0064] The steps and corresponding results of the technical solution adopted in this embodiment are as follows:

[0065] (1) OVA-CTLs activated by anti-CD3 / CD28 beads were cultured alone or together with OVA-B16 cells from conventional culture flasks (differentiated OVA-B16) or OVA-B16 TRCs grown in 3D fibrin matrices for 24 Hour. PD-1 expression was determined by flow cytometry. Measurement results such as figure 1 As shown in A.

[0066] (2) The co-culture supernatant or complete medium (negative control) of CTLs with differentiated OVA-B16 cells or OVA-B16 TRCs was concentrated by freeze-drying and incubated with anti-CD3 / CD28 beads-activated OVA-CTLs for 24 Hour. PD-1 expression was determined by flow cytometry. Measurement results such as figure 1 Shown in B.

[0067] (3) CD3 / CD28 bead-activated OVA-CTL were cultured for 24 hours and the supernatant was anal...

Embodiment 2

[0073] Example 2: Identifying that kynurenine produced by TRC mediates upregulation of PD-1 and that IFN-γ is upregulated in TRC amino acid transporter SLC1A5 to promote Trp uptake and Kyn production

[0074] The steps and corresponding results of the technical solution adopted in this embodiment are as follows:

[0075] (1) OVA-CTLs activated by anti-CD3 / CD28 beads were co-cultured with OVA-B16 cells from conventional culture flasks or OVA-B16 TRCs grown in 3D fibrin matrix for 24 hours. Kyn in the supernatant was determined by ELISA. Measurement results such as figure 2 As shown in A.

[0076] (2) CD3 / CD28 bead-activated OVA-CTL were co-cultured with OVA-B16TRC for 24 hours in the presence of neutralizing anti-IFN-γ antibody or isotype control. Kyn levels in supernatants were determined by ELISA. Measurement results such as figure 2 Shown in B.

[0077] (3) OVA-B16 cells or OVA-B16 TRC grown in culture flasks were treated with IFN-γ (10 ng / ml) for 24 hours. The ...

Embodiment 3

[0088] Example 3: Kyn activates PD-1 upregulated aryl hydrocarbon receptor (AhR) in T cells

[0089] The steps and corresponding results of the technical solution adopted in this embodiment are as follows:

[0090] (1) CD8 isolated from the spleen + T cells were stimulated with 200 μM Kyn or Kyn+anti-CD3 / CD28 beads for 48 h. CD8 + T cells were fixed, stained with anti-AhR antibody and imaged by confocal microscopy. Imaging results such as image 3 As shown in A. Error bars (Bar), 10 [mu]M.

[0091] (2) Same as (1) except that the cytosolic and nucleoprotein fractions were separated prior to immunoblot analysis of AhR. Intensities were calculated relative to the rest / Kyn group. Calculated as image 3 Shown in B.

[0092] (3) Resting CD8 cells were treated with PBS or Kyn (200μM) for 48h + CD8 activated by T cells and anti-CD3 / CD28 beads + Immunoblot analysis of AhR expression in T cells. The AhR ratio of Nuc / Cyt is calculated relative to the Kyn(-) group. Analysi...

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Abstract

The disclosure provides an application of a reagent for detecting Kyn content, and specifically provides an application of a reagent for detecting Kyn content in preparing a reagent or a kit for diagnosing or auxiliarily diagnosing whether a subject has a disease accompanied by an increase in PD-1 expression, or whether the subject has a high risk of suffering from the disease. The technical solution provided by the disclosure can perform accurate in vitro diagnosis on whether or not the subject has a tumor, preferably, whether or not has a tumor having an increase in the expression level of PD-1.

Description

technical field [0001] The disclosure belongs to the field of biological detection and relates to the application of detection reagents for biomarkers in body fluids. Specifically, the present disclosure relates to the application of detection reagents that can detect Kyn in body fluids. Background technique [0002] Tumors remain one of the deadliest threats to human health. Solid tumors are responsible for most of those deaths. Despite significant advances in medical treatment of certain tumors, overall 5-year survival rates for all tumors have only improved by about 10% over the past 20 years. Tumors or malignancies grow and metastasize rapidly in an uncontrolled manner, making timely detection and treatment extremely difficult. [0003] Tumor immunotherapy is another tumor treatment method that has a clear effect on the treatment of tumors after surgery, radiotherapy, and chemotherapy. It is to resist and kill tumor cells by activating the body's own immune system. It...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/574G01N33/68
CPCG01N33/574G01N33/68
Inventor 黄波刘玉英
Owner THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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