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Method for detecting human tumor antigen P 185 Her-2 and its application in diagnosing tumor

A p185her-2, antigen technology, applied in the preparation methods of peptides, chemical instruments and methods, biological testing and other directions, can solve the problems of low antigen purity and insufficient specificity, and achieve high antigen purification, strong specificity, The effect of reducing steric hindrance

Inactive Publication Date: 2003-02-19
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing E method usually adopts lectin affinity column affinity chromatography, ion exchange chromatography, Superose 12 HR chromatography or Obtained by Sepharose 4B affinity chromatography method, the specificity is not strong enough, and the purity of the antigen is low

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0010] Embodiment 1, preparation contains soluble p185 HER-2 Antigen cell lysate and cell culture supernatant: (1) prepare soluble p185 HER-2 Antigen cell lysate: Collect NIH / 3T3 cells (a kind of mouse fibroblasts) and T6-17 cells (NIH / 3T3 cells transfected with human HER-2 gene) that grow to more than 80% full , the cell surface highly expresses p185 HER-2 ), wash once with cold 0.01M, pH7.2 PBS, add cold lysis buffer consisting of 50mM Tris-HCl pH7.5, 1% Trion X-100, 150mM NaCl and 8mMPMSF, and shake in ice bath for 30 minutes , collect the lysate, centrifuge at 4° C., 12000 rpm for 10 minutes, and the supernatant of T6-17 cells is the cell lysate containing p185 crude antigen. NIH / 3T3 cell lysate was used as negative control. (2) Preparation of soluble p185-containing HER-2 Cell culture supernatant for antigen:

[0011] When T6-17 cells were subcultured, they were cultured without serum. When they were congested, the culture supernatant was collected, and the culture s...

Embodiment 2

[0013] Example 2, detection of soluble p185 HER-2 The establishment of the double antibody sandwich ELISA method conditions for the antigen: (1) The cultured cell surface epitope embedding method can specifically secrete anti-p185 HER-2 Immortalization of ectodomain mAbs

[0014] Hybridoma cells, such as A18 and A21, were expanded and implanted into the peritoneal cavity of 6-8 week old Balb / c mice.

[0015] After water, take out the ascites, which is the crude monoclonal antibodies A18 and A21. (2) The monoclonal antibodies A18 and A21 in ascites were purified by conventional octanoic acid-ammonium sulfate two-step precipitation method (3) The purified monoclonal antibody A18 labeled with peroxidase was prepared by the improved sodium periodate method.

[0016] A18-HRP: Dissolve 5mg of HRP in distilled water, add 0.2ml of freshly prepared 0.1M sodium periodate NaIO 4 ,

[0017] Stir at room temperature in the dark for 20 minutes; dialyze overnight at 4°C against 1mM, pH 4...

Embodiment 3

[0026] Embodiment 3, using anti-p185 HER-2 Purification of p185 from monoclonal antibody cross-linked with Sepharose 4B-Protein G affinity beads HER-2 Antigen method

[0027] The specific steps of the method are: (1) preparation and anti-p185 HER-2 Sepharose 4B-Protein G affinity beads cross-linked with monoclonal antibodies: mix the purified antibody solution with Sepharose 4B-Protein G gel particles fully swelled by buffer, and incubate overnight at 4°C with rotation; use 10 times the volume of 0.08 M, sodium tetraborate buffer of pH 9.0 was washed twice by centrifugation at 3000g for 2 minutes each time; the supernatant was discarded, and the affinity beads were resuspended in 10 times the volume of sodium tetraborate buffer, and added Solid bifunctional reagent DMP, to a final concentration of 20mM, shake slightly at room temperature for 30 minutes; centrifuge and wash once with 10 times the volume of 0.2M, pH8.0 ethanolamine buffer; resuspend the affinity beads in 10 ti...

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Abstract

A dual-antibody sandwith ELISA detecting method for human soluble p185HER-2 antigen features that the purified and coated P185HER-2 monoclonal antibody is bound to solid supporter, the specimen containing soluble p185HER-2, the purified standard p185HER-2 antigen and the coated antibody are incubated, and another purified and coated p185HER-2 monoclonal antibody marked by horseradish perioxidase is used as detecting antibody. It can be used for serum diagnosis of tumor.

Description

Technical field: [0001] The invention relates to the preparation of tumor antigen, the immunological detection technology of soluble tumor marker and its application in tumor diagnosis. Background technique: [0002] p185 HER-2 It is an important tumor cell surface transmembrane glycoprotein encoded by the oncogene neu / erbB-2 / HER-2, and p185 is overexpressed in more than ten kinds of cancers such as breast, ovary, lung, stomach, and liver HER-2 protein. According to the report of American "Science" magazine (Science, 1989, 244:707-712), more than 30% of the tumor tissue cells of breast and ovarian cancer patients have p185 HER-2 High expression, such patients have high tumor malignancy and poor prognosis. "British Journal of Cancer" (Br J Cancer, 1994, 70, 739-742) reported that the detection of p185 HER-2 It is of great significance to the differential diagnosis, prognosis and treatment of various tumors. Especially for breast cancer, p185 HER-2 Has been international...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/14C12N7/02G01N33/531G01N33/574G01N33/96
Inventor 刘兢李平吴强姚阳官伟宁杨峰
Owner UNIV OF SCI & TECH OF CHINA
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