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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  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, immunomagnetic beads have been widely used in chemiluminescence immunoassay, nucleic ...

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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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