Human bladder cancer marker ag-cd71 and its antibody abc71 and application

An AG-CD71, bladder cancer technology, applied in the field of tumor immunology, can solve the problems of bleeding, infection, and high cost of urethral cystoscopy

Active Publication Date: 2020-08-07
北京中科健兰集团有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the diagnosis and follow-up of bladder cancer mainly rely on urethral cystoscopy, but invasive urethral cystoscopy is expensive and inconvenient to operate, which not only brings great pain to patients, but also has risks such as infection and bleeding

Method used

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  • Human bladder cancer marker ag-cd71 and its antibody abc71 and application
  • Human bladder cancer marker ag-cd71 and its antibody abc71 and application
  • Human bladder cancer marker ag-cd71 and its antibody abc71 and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Preparation and purification of ABC71 monoclonal antibody:

[0031] (1) Preparation of hybridoma cells

[0032] 1) Animal immunization and cell culture: Homogenize human bladder cancer tissue and extract total protein, immunize Balb / C mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.), and use 50ug of total protein per mouse. Dose for intraperitoneal immunization. Mice were re-immunized every two weeks. After the serum titer of the mouse reaches the requirement, the immunization is boosted again. After 3 days, the spleen of the mouse is taken to prepare a spleen cell suspension for cell fusion. Resuscitated mouse myeloma cells Sp2 / 0 (ATCC CRL-1772) were selected with 8-AG (8 azaguanine) to maintain the sensitivity of cells to HAT.

[0033] 2) Cell fusion: Fusion the splenocyte suspension prepared in step 1) with myeloma cells. For specific methods, refer to "Guidelines for Experimental Immunology" ((USA) J.E. Science Root (USA) D.H. ...

Embodiment 2

[0041] Identification of ABC71 monoclonal antibodies:

[0042] Using the ABC71 monoclonal antibody prepared in Example 1 to perform immunohistochemistry on human bladder cancer tissue and human normal bladder tissue sections, the results are as follows figure 1 , 2 shown. The results showed that human bladder cancer tissue showed positive reaction after ABC71 monoclonal antibody immunohistochemical staining (such as figure 1 shown), while normal human bladder tissue showed negative reaction after immunohistochemical staining with ABC71 monoclonal antibody (such as figure 2 shown).

[0043] Using the ABC71 monoclonal antibody prepared in Example 1, human bladder cancer tissue and human normal bladder tissue were detected by immunohistochemistry, and the results are shown in Table 1. The results showed that the ABC71 antibody had a strong positive reaction with human bladder cancer tissue, but had no cross-reaction with normal human bladder tissue.

[0044] Table 1. Immuno...

Embodiment 3

[0047] Preparation of AG-CD71 antigen:

[0048] 1) Total protein extraction: Grind 100 mg of human bladder cancer tissue into a homogenate, add 2 ml of three decontamination lysates, lyse at 4°C for 10 minutes, centrifuge at 12,000 rpm for 20 minutes, and take the supernatant, which is the total protein of human bladder cancer tissue.

[0049] 2) Immunoprecipitation: Add 50ug of ABC71 monoclonal antibody and incubate at 4°C for 2h.

[0050] 3) Antigen identification: add 50ul Protein G beads and incubate at 4°C for 2h. The beads were washed with PBS, and then the beads were eluted with 0.2 mol / L glycine-HCl solution (PH2.8) for mass spectrometry analysis. The mass spectrometry results are shown in Table 2. The results of mass spectrometry showed that the antigen of ABC71 was the transferrin receptor TFRC. After analyzing the sugar chip data of ABC71 monoclonal antibody (such as image 3 (shown) found that the antigenic epitope recognized by the ABC71 monoclonal antibody is...

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Abstract

The invention mainly belongs to the technical field of tumor immunology, and specifically relates to a novel bladder cancer marker AG‑CD71 and an anti-AG‑CD71 monoclonal antibody ABC71. The human bladder cancer marker AG-CD71 of the present invention is an abnormally glycosylated transferrin receptor TFRC, and the abnormally glycosylated transferrin receptor TFRC means that TFRC carries an antigenic epitope The carbohydrate structure Fuca1‑4(GlcNAcb1‑3)[6OSO3]GlcNAc; and provide an antibody against the human bladder cancer marker AG‑CD71, the antibody is a specific anti-human bladder cancer AG‑CD71 monoclonal antibody Cloning antibody ABC71, the monoclonal antibody is secreted by the hybridoma cell line with the preservation number of CGMCC No.14312. Cellular and histological levels prove that: ABC71 specifically recognizes human bladder cancer cells and human bladder cancer tissues. Anti-human bladder cancer monoclonal antibody ABC71, the antibody has a strong positive reaction with human bladder cancer tissue, but a negative reaction with human normal bladder tissue.

Description

technical field [0001] The invention mainly belongs to the technical field of tumor immunology, and specifically relates to a novel bladder cancer marker AG-CD71 and the anti-AG-CD71 monoclonal antibody ABC71. Cellular and histological levels prove that: ABC71 specifically recognizes human bladder cancer cells and human bladder cancer tissues. The invention also relates to a method for detecting human bladder cancer by immunocytochemistry. The antigen detected by the detection method is the novel marker of human bladder cancer AG-CD71 in the present invention, and the detection antibody is the monoclonal antibody ABC71 against human bladder cancer AG-CD71 in the present invention. Background technique [0002] Bladder cancer refers to malignant tumors that occur on the bladder mucosa. It is the most common malignant tumor of the urinary system and one of the top ten common tumors in the whole body. It accounts for the first place in the incidence of genitourinary system tum...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/705C12N5/20C07K16/30C07K16/28G01N33/68G01N33/574A61K39/395A61K47/68A61P35/00C12R1/91
CPCA61P35/00A61K38/00A61K47/6861C07K16/3038G01N33/57407G01N33/57484G01N2333/70582C07K14/70582A61K47/68A61K2039/505C07K16/2881C07K16/30G01N33/574
Inventor 李翀
Owner 北京中科健兰集团有限公司
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