Immunofluorescence kit for detecting PD-L1 and CD8 antigens and application method

A technology of PD-L1 and immunofluorescence, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., to achieve the effect of convenient material collection

Pending Publication Date: 2019-12-31
CYTTEL BIOSCI BEIJING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The purpose of the present invention is to solve the problem that there is no simultaneous detection of PD-L1 expression level in CTC and the proportion of PD-L1 and CD8 positive cells in PBMC by immunofluorescence method in the prior art, and propose a method for detecting PD-L1 and CD8 antigen Immunofluorescence kit and application method

Method used

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  • Immunofluorescence kit for detecting PD-L1 and CD8 antigens and application method
  • Immunofluorescence kit for detecting PD-L1 and CD8 antigens and application method
  • Immunofluorescence kit for detecting PD-L1 and CD8 antigens and application method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0130] Materials: negatively enriched blood CTC smears, density gradient centrifuged PBMC smears.

[0131] Experimental steps:

[0132] 1. Extract 3.5ml of peripheral blood into an ACD (sodium citrate) anticoagulant tube; use Cyttel's human peripheral blood leukocyte removal kit to negatively enrich tumor cells, and use the CS3 density gradient centrifugation in the kit to obtain PBMC, and fix them on the carrier Slide;

[0133] 2. Wash slides with CYP1 for 3 minutes x 3 times, 100-150 μL each time, to ensure that the entire sample area is covered;

[0134] 3. Absorb the excess liquid on the slide, add CYPP for 5 minutes, and wash the slide with CYP1 as above for 3 minutes×1 time; absorb the excess liquid, add 100-150 μl of blocking solution to seal at room temperature for 25-30 minutes;

[0135] 4. Absorb excess blocking solution, add 100 μl of diluted PD-L1 antibody + CD45 antibody and CD8 antibody respectively, and incubate in a humid chamber at room temperature in the da...

Embodiment 2

[0143] Materials: 1 tube of appropriate amount of anticoagulated blood, which is enriched by membrane filtration and then detected for protein.

[0144] Experimental steps:

[0145] 1. Take an appropriate amount of peripheral blood and put it into a blood collection tube containing anticoagulant, and shake it slightly to mix;

[0146] 2. Add the suspension to the membrane filtration separation tumor cell technology device, and slowly pass through the filter and membrane;

[0147] 3. After the filtration is completed, continue to add 50ml of 0.01M PBS to the membrane filtration device, wash the cell suspension attached around the tube wall into the membrane filtration device, and let it pass through the filter and membrane;

[0148] 4. Fix the cells on the filter membrane;

[0149] 5. Perform the same operation as in Example 1 to detect the protein.

Embodiment 3

[0151] Materials: 1 tube of appropriate amount of anticoagulated blood, which is enriched by microfluidic method and then detected for protein.

[0152] Experimental steps:

[0153] 1. The appropriate amount of blood drawn is enriched by microfluidic chips of various principles;

[0154] 2. After enrichment, the samples were subjected to protein immunofluorescence detection.

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Abstract

The present invention relates to an immunofluorescence kit for detecting PD-L1 and CD8 antigens and a detection method using the kit. The kit comprises the following reagents: buffer solutions CYP1, CYP2 and CYPP, a blocking solution, a specific antibody for detecting PD-L1, a corresponding fluorescent second antibody, a fluorescently labeled CD45 antibody, a fluorescently labeled CD8 antibody, anantibody dilution solution and a nuclear staining solution. The invention also provides a method for carrying out antigen detection by using the kit. A detection result of the kit can guide actual drug use, and not only can reflect the whole tumor comprising carcinoma in situ and possibly existent invisible micro-metastasis load, but also can reflect the expression condition of the PD-L1 in CTC;a reference can be provided for precise selection of therapeutic drugs according to the proportion of PD-L1 and CD8 positive cells in PBMC; and the kit is more convenient than a detection technology based on tumor tissues in material acquisition.

Description

technical field [0001] The invention relates to a medical detection method, in particular to the detection of cancer cells in body fluids and the detection of immune-related cells in PBMCs, in particular to the programmed death ligand (programmed death-ligand 1, PD-L1) antigen in circulating tumor cells The kit for immunofluorescence detection and the detection of the ratio of PD-L1 and CD8 in PBMC is suitable for the detection of PD-L1 and CD8 in different types of samples, and also relates to the application method of the kit in the detection of PD-L1 and CD8. Background technique [0002] At present, the incidence of cancer is relatively high, which seriously threatens human health. Some cancers are difficult to detect early and are often diagnosed at an advanced stage, such as lung cancer, colorectal cancer, ovarian cancer, and pancreatic cancer. Correspondingly, the 5-year survival rate of advanced cancer is significantly lower than that of early-stage cancer, and earl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/533G01N33/574
CPCG01N33/533G01N33/57484
Inventor 郭志敏樊晓婷郭素杰
Owner CYTTEL BIOSCI BEIJING
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