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Preparation method and application of programmed death receptor 1 antibody magnetic bead

A technology of programmed death and magnetic beads, applied in the field of biomedicine, can solve the problems of affecting anti-tumor effect, toxic and side effects, etc.

Inactive Publication Date: 2018-09-07
XUZHOU YES BIOTECH LAB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the systemic application of immune checkpoint inhibitors or antibodies brings more toxic side effects, which may affect any organ system, usually affecting the skin, gastrointestinal tract, liver and endocrine system, etc.
Temporary use of immunosuppressive drugs attenuates these side effects, possibly compromising antitumor effects

Method used

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  • Preparation method and application of programmed death receptor 1 antibody magnetic bead
  • Preparation method and application of programmed death receptor 1 antibody magnetic bead
  • Preparation method and application of programmed death receptor 1 antibody magnetic bead

Examples

Experimental program
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Effect test

preparation example Construction

[0046] The preparation method of carboxyl magnetic beads comprises the following steps:

[0047] (1) After mixing the Mag magnetic beads evenly, take 100 μL Mag magnetic beads into a 1 mL centrifuge tube, remove the supernatant by magnetic separation, perform magnetic separation and wash twice with 200 μL MEST solution, and then remove the supernatant; the MEST solution The concentration is 100mM 2-(N-morpholine)ethanesulfonic acid, the pH of the MEST solution is 5.0, and the volume fraction of the MEST solution is 0.05% Tween 20 (that is, 0.5mL Tween 20 is added to 1L MES solution);

[0048] (2) Quickly add 100 μL of the prepared 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution and 100 μL of N-hydroxysuccinimide solution to the centrifuge tube containing the magnetic beads, Vortex and mix well to suspend the magnetic beads, activate at 25°C for 30 minutes, and keep the magnetic beads in suspension during activation; the 1-(3-dimethylaminopropyl)-3-ethylcar...

Embodiment 1

[0060] Example 1 Preparation of PD-1 Antibody Magnetic Beads

[0061] 1 EDC / NHS activated carboxyl magnetic beads conjugated mouse anti-human PD-1 monoclonal antibody

[0062] 14 strains of anti-human PD-1 monoclonal antibodies successfully developed by the anogen-yes team: 7E5, 1H8, 1C4, 9A5, 9B4, 7C9, 3E5, 7G12, 9E11, 5B2, 5H7, 2H7, B1C4, 2C4. The carboxyl magnetic beads with a diameter of 2um developed by biomedical are coupled to prepare PD-1 antibody magnetic beads.

[0063] A. Activation of carboxyl groups on the surface of magnetic beads

[0064] 1. After mixing the magnetic beads, put 100 μL Mag magnetic beads into a 1 mL centrifuge tube, remove the supernatant by magnetic separation, and wash twice with 200 μL MEST solution (100 mM MES, pH 5.0, 0.05% Tween 20) for magnetic separation, then transfer Remove the supernatant;

[0065] 2. Quickly add 100 μL of freshly prepared EDC solution (10-50 mg / mL, use MEST / ethanol / ultrapure water as dispersant) and 100 μL NHS (10-...

Embodiment 2

[0086] Example 2 Application of immunomagnetic beads to separate and remove PD-1 positive T cells

[0087] Isolation of PBMC from Peripheral Blood Mononuclear Cells

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Abstract

The invention discloses a preparation method of a programmed death receptor 1 (PD-1) antibody magnetic bead. The preparation method comprises the following steps: respectively coupling 14 anti-human PD-1 monoclonal antibodies with a carboxyl magnetic bead to prepare a PD-1 antibody magnetic bead, wherein the 14 anti-human PD-1 monoclonal antibodies are from the Canada YES company biotechnology research limited company, and the clone numbers of the 14 anti-human PD-1 monoclonal antibodies are respectively 7E5, 1H8, 1C4, 9A5, 9B4, 7C9, 3E5, 7G12, 9E11, 5B2, 5H7, 2H7, B1C4 and 2C4. The inventionalso discloses the application of the above PD-1 antibody magnetic bead in the field of CIK (cytokine induced killer), CART (Chimeric Antigen Receptor T-Cell Immunotherapy) and TIL (tumor infiltrationlymphocyte) immunotherapy for rejecting PD-1 positive cells. According to the preparation method disclosed by the invention, a coupling scheme of antibodies and magnetic beds is optimized, so that the coupling amount of the antibody on the surface of the magnetic bead can achieve 1ml of magnetic bead+1mg of antibody, and a fluorescence signal on the surface of the magnetic bead is enhanced afterthe antibody is coupled. The antibody magnetic bead prepared with the preparation method can be used for capturing PD-1 positive cells.

Description

technical field [0001] The invention relates to a preparation method and application of programmed death receptor 1 (PD-1) antibody magnetic beads, belonging to the technical field of biomedicine. Background technique [0002] The immune system can recognize and remove foreign components expressing neoantigens to maintain homeostasis, and cancerous abnormal cells are also targeted for removal. T cell-mediated cellular immune response plays an important regulatory role, forming an anti-tumor immune cycle consisting of multi-step cascade reactions. There are multiple factors in the anti-tumor immune cycle that can enhance or inhibit the immune clearance of tumor cells, including programmed death factor 1 (PD‐1) and its ligands, which constitute an important immunosuppressive checkpoint [3‐6 ], the PD-1 / PD-L1 immune checkpoint. [0003] The programmed death factor PD-1 is a member of the CD28 family. PD-1 is an inducible protein, that is, when T cells are not activated, ther...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K17/14C12N5/0783C12N5/00
CPCC07K17/14C12N5/0087C12N5/0636C12N5/0638
Inventor 叶庆炜
Owner XUZHOU YES BIOTECH LAB
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