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Method and kit for detecting PD-L1 expression level

A PD-L1 and expression level technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of not being able to truly reflect the expression level of PD-L1, difficulties in evaluating PD-L1, and differences in expression levels

Pending Publication Date: 2019-09-20
JIAXING YUNYING MEDICAL INSPECTION CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for some patients who cannot or are difficult to obtain tumor tissue, the assessment of PD-L1 becomes very difficult
In addition, the current immunohistochemical method also faces some problems. For example, in terms of detection technology, different detection antibodies, platforms and different threshold settings will have different effects on the detection results; in terms of biology, due to intra-tumor and tumor The test results may not truly reflect the expression level of PD-L1; in terms of tissue sources, for cytology specimens, archived specimens and fresh specimens, the expression level of PD-L1 in the primary site and the metastases will be different

Method used

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  • Method and kit for detecting PD-L1 expression level
  • Method and kit for detecting PD-L1 expression level
  • Method and kit for detecting PD-L1 expression level

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1, extracting the target RNA and internal reference RNA.

[0076] 1.1 Place the blood sample in a centrifuge tube (self-prepared), and centrifuge at 1200 rpm for 20 minutes at room temperature.

[0077] 1.2 Take all the plasma in the upper layer into a centrifuge tube (prepared by yourself), extract 1 mL of the plasma into a T1 centrifuge tube, and centrifuge the remaining plasma at 2100 rpm for 20 minutes at room temperature.

[0078] 1.3 Take the upper layer of plasma and store it at -80°C or directly extract ctDNA and precipitate it into platelets.

[0079] 1.4 Sequentially suspend platelets with 1 mL of plasma from step 1.2.

[0080] Incubate in 1.5 boiling water for 5 minutes, and ice-bath for 5 minutes.

[0081] 1.6 Briefly centrifuge, keep the centrifuge tube containing RNA on ice, mash, add 600ul Buffer A, vortex and mix, add 200ul Buffer B, vortex and mix, centrifuge at 15000rpm 4°C for 20 minutes.

[0082] 1.7 Keep the RNA-containing centrifuge tube...

Embodiment 2

[0088] Example 2. The target RNA and internal reference RNA were reverse transcribed into target cDNA and internal reference cDNA respectively.

[0089] The reverse transcription system is as follows:

[0090] 5×RT Buffer 4μL

[0091] Primer final concentration 200nM

[0092] dNTP 1mM

[0093] super RT 200U

[0094] RNA 6μL

[0095] Make up DEPC water to 20 μL.

[0096] 2.1 Take 11.5 μL sample RNA, 1 μL super RT reverse transcriptase, 1 μL reverse transcription primer, 2 μL dNTP mixture, 4 μL buffer, 0.5 μL RNase inhibitor, add to a sterile centrifuge tube, and mix well.

[0097] 2.2 Start the PCR program: keep warm at 42°C for 1 hour, incubate at 70°C for 10 minutes; after the reaction, briefly centrifuge and cool at 10°C for 10 minutes.

[0098] 2.3 The reverse transcription product can be directly used in the subsequent PCR amplification reaction.

Embodiment 3

[0099] Example 3. The target cDNA and internal reference cDNA were subjected to PCR amplification reaction.

[0100] The PCR amplification system is as follows:

[0101] Dilute 10×PCR Buffer to 1×

[0102] dNTP 0.2mM

[0103] Template 2uL

[0104] Each primer 200-400nM

[0105] 100-400nM for each probe

[0106] Hot start Taq enzyme 1U

[0107] MgCl2 2-5mM

[0108] Total volume 20uL.

[0109] Take 18 μL of buffer solution, a mixture of 0.2 μM PCR-specific primers and 0.2 μM probes, 1 μL of reverse-transcribed sample cDNA, and 1 μL of Tac enzyme, add them to a sterile centrifuge tube, and mix well.

[0110] The conditions of the PCR amplification reaction are as follows:

[0111]

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Abstract

The present application discloses a method and kit for detecting a PD-L1 expression level. The method comprises obtaining a body fluid sample of a mammal, wherein the body fluid sample contains a target RNA and an internal reference RNA derived from a gene of PD-L1; extracting the target RNA and the internal reference RNA from the body fluid sample; reversely transcribing the target RNA and the internal reference RNA into the target cDNA and the internal reference cDNA, separately; carrying out PCR amplification reaction of the target cDNA and the internal reference cDNA; and according to the number of target cDNA and the number of internal reference cDNA after amplification, determining the expression level of PD-L1 in the mammal. The detection method of the present application can detect the PD-L1 expression level by collecting body fluid samples of mammals, and perform PD-L1 immunotherapy prediction, thereby guiding PD-L1 immunotherapy.

Description

technical field [0001] This application relates to the field of biotechnology, in particular to a method and kit for detecting the expression level of PD-L1. Background technique [0002] At present, drugs based on PD-L1 (Programming Death-Ligand 1) immunotherapy have been approved by the FDA and recommended by NCCN guidelines, becoming the first-line treatment option for advanced non-small cell lung cancer (NSCLC). In PD-L1-based immunotherapy, the efficacy of PD-1 / PD-L1 inhibitors is closely related to the expression level of PD-L1. [0003] Currently, immunohistochemical methods are mainly used to monitor the expression level of PD-L1. Specifically, specific antibodies (such as 28-8, 22C3, SP263, and SP142) are used to detect the expression level of PD-L1 in non-small cell lung cancer tissue samples by means of hybridization and color development. However, for some patients whose tumor tissue cannot or is difficult to obtain, the assessment of PD-L1 becomes very difficu...

Claims

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Application Information

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IPC IPC(8): C12Q1/6851
CPCC12Q1/6851C12Q1/6886C12Q2600/158C12Q2600/166C12Q2531/113C12Q2545/101C12Q2561/113
Inventor 张道允巩子英孙永华叶建伟
Owner JIAXING YUNYING MEDICAL INSPECTION CO LTD
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