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A kind of tumor-specific antigen and its application

A tumor-specific, antigen-based technology for biomedical applications

Active Publication Date: 2019-08-20
叶尚勉
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, no studies have reported detection of topoisomerase expression as a tumor marker for cancer diagnosis or screening

Method used

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  • A kind of tumor-specific antigen and its application
  • A kind of tumor-specific antigen and its application
  • A kind of tumor-specific antigen and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Separation, purification and screening of tumor-specific antigen TOP-1-40

[0040] 1. Materials and Methods

[0041] (1) Tissue homogenate:

[0042] The lung cancer tissue was homogenized in lysate (0.5% NP40, 0.15M NaCl, 5mM EDTA, 50mM Tris, 1mMPMSF) with a tissue homogenizer. Then centrifuge at 12000 rpm at 4°C, and combine the supernatants.

[0043] (2) Separation of tumor antigens by two-dimensional electrophoresis:

[0044] The experiment included isoelectric focusing electrophoresis in the first dimension and SDS-PAGE electrophoresis in the second dimension. The first-dimension isoelectric focusing electrophoresis steps are as follows:

[0045] ·Take the hydration loading buffer (1ml / tube) frozen at -20°C and dissolve at room temperature. Add 0.01g DTT and Bio-Lyte 4-72.5ml to the small tube, and mix well.

[0046] ·Remove 400ml from the mixed hydration loading buffer, add 100ml sample, and mix well.

[0047] · Take the IPG prefabricated gel strip...

Embodiment 2

[0099] Example 2 Preparation of rabbit anti-human TOP-1-40 polyclonal antibody

[0100] 1. Materials and Methods

[0101] Use the target protein separated and purified by two-dimensional electrophoresis as an immunogen.

[0102] • Dilute the target protein to 500 μg / ml with physiological saline. For the first time, it was mixed with complete Freund's adjuvant at a ratio of 1:1, and then immunized by subcutaneous injection in New Zealand white rabbits. Afterwards, mix the purified target protein with incomplete Freund's adjuvant at a ratio of 1:1, and continue to immunize animals, with an interval of 1-2 weeks, for 4 consecutive times. The antibody titer against the target protein was detected by antibody capture enzyme-linked immunosorbent method, and after the titer reached a certain height, a booster immunization was carried out at the end.

[0103] · Three days later, blood was collected from the inferior vena cava of the animal, and the serum was separated by centrifuga...

Embodiment 3

[0109] Example 3 Immunohistochemical detection of the content of TOP-1-40 in cancer

[0110] 1. Materials and Methods

[0111] Cancer tissue samples for this study included colorectal cancer (40 cases), non-small cell lung cancer (20 cases), gastric cancer (20 cases), breast cancer (35 cases), endometrial cancer (20 cases), esophageal cancer ( 15 cases), ovarian cancer (10 cases), liver cancer (10 cases) and bladder cancer (10 cases), and corresponding normal tissues. These samples were all confirmed by pathological examination of Department of Pathology, Sichuan Provincial People's Hospital.

[0112] Using anti-TOP-1-40 polyclonal antibody, the expression of TOP-1-40 in various cancer tissues was detected by conventional immunohistochemical method and compared with corresponding normal tissues. The specificity of the anti-TOP-1-40 polyclonal antibody has been demonstrated by Western blotting and immunofluorescence (see Example 2).

[0113] The operation of the immunohistoc...

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Abstract

The invention discloses a tumor specific antigen and application thereof. The amino acid sequence of the tumor specific antigen TOP-1-40 is shown as SEQ ID NO:1. A protein dimensional electrophoresis method is used for being combined with a cancer serological immunoblotting and antibody capture enzyme-linked immunoassay method for separating and obtaining the tumor specific antigen from cancer cell strain cells and cancer tissues. Through the specific polyclonal antibody for resisting the antigen, the antigen is determined to be a protein fragment, with the molecular weight being about 40kd, of DNA topoisomerase I by using the methods of a recombinant protein method, an immunoblotting method, a fluorescent-immunohistochemistry method, an antibody capture enzyme-linked immunoassay method and the like; the antigen is named as TOP-1-40. The antigen content is generally increased in common cancer tissues; the antigen cannot be detected in corresponding normal tissues, or the content is very low. The self antibody concentration of the antigen in the serum is detected by the antibody capture enzyme-linked immunoassay method; the antigen can be used for the early stage screening of common cancer. The detection method has 95 to 100 percent of specificity and 61 to 66 percent of sensibility. The tumor specific antigen has good prospects in clinic application.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a tumor-specific antigen and its application. Background technique [0002] Cancer is one of the major diseases that endanger human health. According to domestic and foreign investigation reports, the death cases caused by cancer account for the first place in the number of deaths due to diseases. The main reason is that cancer is not easy to be detected early, and most of the clinically diagnosed cases have reached the middle and late stages of the disease. The existing clinical methods for treating cancer have limited curative effect on advanced cancer patients, but have remarkable effect on early-stage cancer patients. Therefore, one of the best ways to treat cancer is to be able to detect it early. [0003] Medical imaging such as CT, B-ultrasound, X-ray and pathological biopsy cannot be discovered until the cancer cells have developed to a certain extent. Detection...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/90G01N33/68G01N33/574G01N33/543
Inventor 叶尚勉
Owner 叶尚勉
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