Method for detecting cell capable of expressing carcinoembryonic antigen, and application thereof

A carcinoembryonic antigen, cell technology, applied in the field of immune detection, to achieve the effect of low-cost detection and sensitive detection

Inactive Publication Date: 2019-09-13
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • Method for detecting cell capable of expressing carcinoembryonic antigen, and application thereof
  • Method for detecting cell capable of expressing carcinoembryonic antigen, and application thereof
  • Method for detecting cell capable of expressing carcinoembryonic antigen, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1 Preparation of anti-carcinoembryonic antigen single-chain antibody fusion virus probe

[0060] (1) Construction of anti-carcinoembryonic antigen single-chain antibody phage library as a probe

[0061] 1. The heavy chain variable region (GenBank: CS681102.1) and the light chain variable region (GenBank: CS681104.1) of the anti-carcinoembryonic antigen single-chain antibody sequence are linked by (Gly4Ser)3, provided by Nanjing Detai Biotechnology Co., Ltd. It was synthesized and cloned between the Sfi I and Not I cloning sites of the pCANTAB 5E vector, and the recombinant plasmid was named pCANTAB 5E-ACEAscFv.

[0062] 2. Transform the recombinant plasmid pCANTAB 5E-ACEAscFv into Escherichia coli TG1 competent cells, spread on an ampicillin-resistant plate, and culture overnight at 37°C.

[0063] 3. Select the positive clones to inoculate into 4mL 2xYT (Tryptone 16g, Yeast Extract 10g, Nacl5g, deionized water to 1L, pH=7.0) on the next day, and culture it on t...

Embodiment 2

[0073] Example 2 Identification of single-chain antibody fusion virus probes against carcinoembryonic antigen

[0074] (1) Indirect ELISA identification of anti-carcinoembryonic antigen single-chain antibody library specificity

[0075] 1. Use antigen coating solution (Na 2 CO 3 0.159g,NaHCO 3 0.293g, dilute to 100mL, pH=9.6) Dilute carcinoembryonic antigen protein to 2μg / mL, add 100μL per well into a 96-well plate, and coat overnight at 4°C.

[0076] 2. Discard excess CEA protein dilution in the 96-well plate the next day, wash 3 times with 1x PBST (1xPBS containing 0.05% Tween 80, v / v), and dry.

[0077] 3. Add 200 μL of 1% BSA (v / v), block at 37°C for 2 hours, wash with 1xPBST three times, and dry.

[0078] 4. Add 100 μL of 100-fold diluted M13KO7-ACEA scFv, and incubate at 37°C for 1 hour.

[0079] 5. After washing 6 times with 1xPBST and drying, add 100 μL of anti-M13 phage capsid protein g8p antibody diluted 100 times in 1xPBS, and incubate at 37°C for 1 hour.

[...

Embodiment 3

[0098] Example 3 Application of anti-carcinoembryonic antigen single-chain antibody fusion virus probe

[0099] (1) M13KO7-ACEAscFv infected Caco-2 cells with different MOI

[0100] 1. Inoculate Caco-2 cells into a 6-well plate at 37°C, 5% CO 2 Cells were cultured in an incubator, and when the cells grew to 70-80%, M13KO7-ACEA scFv was inoculated at MOI of 0.01, 0.05, 0.1, 0.5, 1, 2 and 5, M13KO7 was inoculated at MOI of 1, and infected for 2 hours.

[0101] 2. Discard the supernatant, wash the 6-well plate three times with pre-cooled 1xPBS, extract the whole genome of the cells by TRIzol method, and reverse transcribe.

[0102] 3. Set up the M13KO7-ACEAscFv virus group and the β-Actin internal reference group for real-time fluorescent quantitative PCR.

[0103] The reaction system and conditions of real-time fluorescence quantitative PCR are as follows:

[0104] 20μL reaction system:

[0105]

[0106] The reaction conditions are:

[0107]

[0108] (2) M13KO7-ACEAsc...

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Abstract

The invention relates to a method for detecting a cell capable of expressing a carcinoembryonic antigen, and application thereof. A bacteriophage display technology is used for displaying a single chain antibody capable of resisting the carcinoembryonic antigen on the surface of auxiliary bacteriophage M13KO7 to construct a library of the single chain antibody capable of resisting the carcinoembryonic antigen. The library is used as a probe, the cell capable of expressing the carcinoembryonic antigen is subjected to specific targeted combination, and then, a magnetic nanoparticle modified by an antibody capable of resisting the carcinoembryonic antigen is used for capturing the category of cells. Through PCR (Polymerase Chain Reaction), the g3p gene of M13KO7 is amplified so as to achievea purpose of quickly and sensitively detecting the single cell capable of expressing the carcinoembryonic antigen or low-content circulating tumor cells.

Description

technical field [0001] The invention belongs to the technical field of immune detection, and in particular relates to a method for detecting cells expressing carcinoembryonic antigen and its application. Background technique [0002] Tumor cell markers can be divided into tumor cell markers (miRNA, transcription factors, etc.) and tumor cell membrane markers (epithelial cell adhesion molecules, alpha-fetoprotein, carcinoembryonic antigen, etc.) according to their location distribution. [0003] Carcinoembryonic antigen (CEA) is a GPI-linked cell surface cell adhesion glycoprotein with 28 potential N-glycosylation sites and a molecular weight of 180-200kDa, which is an important tumor marker thing. Carcinoembryonic antigen is produced in the cytoplasm, can be secreted out of the cell through the cell membrane, and is free in body fluids such as tissue fluid or blood. Carcinoembryonic antigen exists in the pancreas, liver, gastrointestinal tissue of 2-6 month embryos, and in...

Claims

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Application Information

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IPC IPC(8): G01N33/574G01N33/543
CPCG01N33/574G01N33/54346
Inventor 周昕侯金秀
Owner YANGZHOU UNIV
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