Human urothelial carcinoma specific antibody and application thereof

A urothelial cancer, antibody technology, applied in the field of tumor immunology, can solve the problems of nuclear matrix protein, telomerase and microsatellite instability, unsatisfactory sensitivity or specificity, etc.

Active Publication Date: 2014-02-19
中科健兰(北京)医学研究院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Molecular markers used clinically in recent years, such as nuclear matrix protein, telomerase, and microsatellite instability, have unsatisfactory sensitivity or specificity

Method used

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  • Human urothelial carcinoma specific antibody and application thereof
  • Human urothelial carcinoma specific antibody and application thereof
  • Human urothelial carcinoma specific antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1: Preparation and purification of DTUC1 monoclonal antibody

[0019] (1) Preparation of hybridoma

[0020] Balb / C mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were immunized with human urothelial carcinoma tissue homogenate, and injected intraperitoneally at a dose of 100 μg / 500 μl / mouse. Mice were re-immunized 2 weeks later with the same injection volume and method. After the mouse serum titer reaches the requirement, the cell fusion is prepared, and the mice are boosted three days before the fusion. Mouse myeloma cells SP2 / 0 (ATCC: CRL-1772) were prepared at the same time as the mice were immunized. The hybridoma technology (Kohler and Milstein1975) was used to fuse the sensitized B lymphocytes with myeloma cells, and they were selectively cultured with HAT medium (purchased from Invitrogen).

[0021] Next, detect hybridoma cell culture supernatant by ELISA method: Coat 96-well plate with human urothelial carcinoma t...

Embodiment 2

[0026] Example 2: Identification of DTUC1 monoclonal antibody

[0027] The DTUC1 monoclonal antibody prepared in Example 1 was immunohistochemically stained on human urothelial carcinoma tissue sections (obtained from Cancer Hospital, Chinese Academy of Medical Sciences) according to conventional methods, and the results were as follows: figure 1 , 2 shown. The results showed that human urothelial carcinoma tissues were positively stained with DTUC1 monoclonal antibody, secondary antibody (anti-mouse IgG‐HRP) and DAB substrate (Wuhan Boster Bioengineering Co., Ltd.), while normal human urothelial tissues After staining with DTUC1 monoclonal antibody, secondary antibody and substrate, the reaction was negative.

[0028] Urothelial cancer cell lines 5637, UMUC3, SW780, J82, T24, RT4 and other cells were detected by flow cytometry according to conventional methods with DTUC1 monoclonal antibody. The results are shown in Table 1. The results showed that DTUC1 monoclonal antib...

Embodiment 3

[0034] Example 3: Preparation of immunodiagnostic reagents for human urothelial carcinoma

[0035] Using the DTUC1 antibody described in the present invention, combined with anti-human TNFRSF25 antibody B‐8 (purchased from Santa Cruz Biotechnology, Inc.), a highly sensitive double-antibody sandwich ELISA method was developed to detect human urine Urothelial carcinoma tumor antigen. The main method is to use B‐8 antibody (purchased from Santa Cruz Biotechnology, Inc.) as the stationary phase, and horseradish peroxidase (HRP)-labeled DTUC1 antibody as the detection antibody to detect urothelial carcinoma tumors in human urine Antigen TNFRSF25. Experiments have proved that the immunodiagnostic reagent for urothelial carcinoma of the present invention has the following positive effects compared with the prior art: (1) direct detection of urine without damage. (2) High sensitivity and strong specificity. The double-antibody sandwich enzyme-linked immunosorbent assay is used to de...

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Abstract

The invention provides a new human urothelial carcinoma tumor marker-human tumor necrosis factor receptor superfamily TNFRSF25 and a hybridoma cell generating a monoclonal antibody resisting the tumor marker and a monoclonal antibody DTUC1 secreted thereby, wherein the monoclonal antibody DTUC1 is secreted by the hybridoma with collection number CGMCC No.8118. The monoclonal antibody DTUC1 is in positive reaction with the human urothelial carcinoma tissue and urothelial carcinoma cell lines such as 5637, UMUC3, SW780, J82, T24 and RT4, and is not in cross reaction with the human normal urothelium tissue and other non-urothelial carcinoma cells; according to mass spectrometric detection, the identified antigen is TNFRSF25. The invention also provides an in-vitro diagnosis kit comprising the monoclonal antibody DTUC1 and a method for detecting the content of tumor markers in urine by use of the monoclonal antibody DTUC1.

Description

Technical field [0001] The invention is the field of tumor immunology.Specifically, the present invention involves a monoclonal antibody DTUC1 of TNFRSF25, an anti -human urinary tract.The antibody can identify TNFRSF25 at the level of biochemical and nuclear tissue levels.The present invention also involves a method of dual -anti -sandwiches, an immune adsorption detection to detect human urinary tract cancer.The fixing phase in this detection method is the B‐8 antibody (purchased from Santa Cruz Biotechnology, Inc.) And detecting DTUC1 in the invention to detect TNFRSF25 content in urine. Background technique [0002] Urinary epithelial cancer refers to tumors that occur with epithelial cells covering the urinary tract, including bladder urinary tract epithelial cancer, ureter urinary epithelial cancer, and pelvic urinary epithelial cancer. They account for most of the urinary system tumorCancer accounts for more than 90%of the incidence of bladder cancer [1].The diagnosis of u...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/574C07K16/24C12N5/20C12R1/91
CPCC07K16/2878C12N5/163G01N33/57407G01N33/577G01N2800/34
Inventor 李翀栗刚
Owner 中科健兰(北京)医学研究院
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