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Kit for rapidly and quantitatively assaying PD-L1 (programmed death ligand-1) by chemiluminescent magnetic bead enzyme-linked immunosorbent assay

A technology of PD-L1 and enzyme-linked immunoassay, which is applied in the field of rapid quantitative detection kit of chemiluminescent magnetic bead enzyme-linked immunoassay, can solve the problems of no literature reports, etc., and achieve the effect of low cost, enhanced stability and high sensitivity

Inactive Publication Date: 2018-06-12
珠海霍普金斯医药研究院股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, immunomagnetic beads have been widely used in chemiluminescence immunoassay, nucleic acid extraction and other fields, but there is no literature report on the magnetic bead ELISA of PD-L1

Method used

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  • Kit for rapidly and quantitatively assaying PD-L1 (programmed death ligand-1) by chemiluminescent magnetic bead enzyme-linked immunosorbent assay
  • Kit for rapidly and quantitatively assaying PD-L1 (programmed death ligand-1) by chemiluminescent magnetic bead enzyme-linked immunosorbent assay
  • Kit for rapidly and quantitatively assaying PD-L1 (programmed death ligand-1) by chemiluminescent magnetic bead enzyme-linked immunosorbent assay

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preparation example Construction

[0023] The preparation method of the standard product is as follows: take the PD-L1 antibody standard product, dilute it with 0.05mol / L PBS buffer solution of pH7.6, and prepare 100ng / mL, 50ng / mL, 25ng / mL, 12.5ng / mL respectively , 6.25ng / mL, 3.125ng / mL, 0ng / mL gradient standard solution.

[0024] The preparation method of the immunomagnetic beads coated with the mouse anti-human PD-L1 antibody I:

[0025] The molar reaction ratio of divalent iron salt and ferric salt is 1:(1-2) (reaction formula is: Fe 2+ +2Fe 3+ +8OH - =Fe 3 o 4 +4H 2 (0), the molar reaction ratio of total iron salt and lye is 1:(7-9), the reaction time is 1-2h, and the reaction temperature is 170-180° C. to obtain magnetic beads;

[0026] Wash the magnetic beads with 0.02-0.07mol / L MES buffer, then add 1-2% glutaraldehyde / 0.02-0.07mol / L PBS at pH5.0-7.0 for 0.5-2h, then 0.02-0.07 mol / L MES buffer for washing to obtain activated magnetic beads;

[0027] Incubate activated magnetic beads with mouse ant...

Embodiment 1

[0042] 1) Preparation of immunomagnetic beads: the magnetic beads are prepared by chemical co-precipitation method, the molar reaction ratio of ferrous salt and ferric salt is 1:1.5, and the molar reaction ratio of all iron salts and lye is 1: 8. The reaction time is 1h, and the reaction temperature is 175°C; wash the above magnetic beads with 0.05mol / L MES buffer, then add 1.25% glutaraldehyde / 0.05mol / L PH6.0PBS for 1h, then 0.05 mol / L MES buffer solution to obtain activated magnetic beads; take activated magnetic beads and mouse anti-human PD-L1 antibody I to incubate at 25°C for 1 hour at the ratio of 250ug antibody / mg magnetic beads, after magnetic separation, add 2% BSA blocks free radicals, and MES washes again to obtain immunomagnetic beads;

[0043] 2) Preparation of enzyme-labeled antibody: Dissolve 10 mg of horseradish peroxidase in 0.2 mL of 1.25% glutaraldehyde / PH6.8 PBS, react at room temperature for 18 hours; use 0.01mol / L PBS of pH7.2, dialyze overnight at 4°C ...

Embodiment 2

[0048] 1) Preparation of immunomagnetic beads: the magnetic beads are prepared by chemical co-precipitation method, the molar reaction ratio of ferrous salt and ferric salt is 1:1, and the molar reaction ratio of all iron salts and lye is 1: 7. The reaction time is 1h, and the reaction temperature is 170°C; wash the above-mentioned magnetic beads with 0.02mol / L MES buffer solution, and then add 1% glutaraldehyde / 0.02mol / L PBS with pH5.0 and shake for 0.5h , and then washed with 0.02mol / L MES buffer to obtain activated magnetic beads; take activated magnetic beads and mouse anti-human PD-L1 antibody I at a ratio of 250ug antibody / mg magnetic beads and incubate for 0.5h at 25°C. After magnetic separation, Add 2% BSA to block free radicals, and wash again with MES to obtain immunomagnetic beads;

[0049] 2) Preparation of enzyme-labeled antibody: Dissolve 8 mg of horseradish peroxidase in 0.1 mL of 1% glutaraldehyde / PH 6.0 PBS for 15 hours at room temperature; use 0.01 mol / L of P...

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Abstract

The invention belongs to the technical field of assay, and particularly relates to a kit for rapidly and quantitatively assaying PD-L1 (programmed death ligand-1) by chemiluminescent magnetic bead enzyme-linked immunosorbent assay. The kit for rapidly and quantitatively assaying PD-L1 by chemiluminescent magnetic bead enzyme-linked immunosorbent assay disclosed by the invention consists of standards, mouse anti-human PD-L1 antibody I-coated immunomagnetic beads, an enzyme-labeled mouse anti-human PD-L1 antibody II, chemiluminescent substrates, and auxiliary reagents. The kit for rapidly and quantitatively assaying PD-L1 by chemiluminescent magnetic bead enzyme-linked immunosorbent assay disclosed by the invention has the advantages of high specificity, good stability, high sensitivity andlower cost, and is suitable for large-scale clinic popularization.

Description

technical field [0001] The invention belongs to the technical field of testing, and in particular relates to a rapid quantitative detection kit for PD-L1 by chemiluminescence magnetic bead ELISA. Background technique [0002] Programmed death molecule 1 (PD-1) is mainly expressed in activated T lymphocytes and B lymphocytes. Programmed death molecule 1 ligand-1 (PD-L1), also known as B7 homolog molecule (B7-H1), is the ligand of PD-1. The combination of PD-L1 and PD-1 can inhibit the proliferation and activation of lymphocytes. However, the tumor microenvironment will induce infiltrating T cells to highly express PD-1 molecules, and tumor cells will highly express PD-1 ligands PD-L1 and PD-L2, resulting in the continuous activation of the PD-1 pathway in the tumor microenvironment. Cell function is inhibited, unable to kill tumor cells, resulting in tumor immune escape. Antibodies to PD-1 can block this pathway and partially restore T cell function. Studies have shown th...

Claims

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Application Information

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IPC IPC(8): G01N33/53G01N33/543G01N21/76
CPCG01N33/53G01N21/76G01N33/54326
Inventor 刘天赫曹文强赵柏松
Owner 珠海霍普金斯医药研究院股份有限公司
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