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Method for detecting human tumor antigen P185 Her-2 and its application in diagnosing tumor

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

Inactive Publication Date: 2005-12-21
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because the existing E method uses the same standard antigen as A, B, C, and D methods, it usually uses lectin affinity column affinity chromatography, ion exchange chromatography, Superose 12HR chromatography or Sepharose 4B obtained by affinity chromatography, 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

[0013] Embodiment 1, preparation contains soluble p185 HER-2 Antigen cell lysate and cell culture supernatant:

[0014] (1) Preparation of soluble p185-containing HER-2 Antigen cell lysate:

[0015] Collect NIH / 3T3 cells (a kind of mouse fibroblast) and T6-17 cells (NIH / 3T3 cells transfected with human HER-2 gene, which highly express 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.

[0016] (2) Preparation of soluble p185-containing HER-2 Cell culture supernatant for antigen:

[0017] When T6-17 cells were subcultured, they were cultured without serum. When they were congested, the culture supernatant was collected, and the culture su...

Embodiment 2

[0019] Example 2, detection of soluble p185 HER-2 The establishment of double antibody sandwich ELISA method conditions for antigen:

[0020] (1) Cultivate cells obtained by cell surface epitope embedding method that can specifically secrete anti-p185 HER-2 Immortal hybridoma cells of extracellular domain monoclonal antibody, such as A18 and A21, are amplified and implanted into the peritoneal cavity of 6-8 week old Balb / c mice. Antibodies A18 and A21.

[0021] (2) Purify monoclonal antibodies A18 and A21 in ascitic fluid by conventional octanoic acid-ammonium sulfate two-step precipitation method

[0022] (3) Prepare the purified monoclonal antibody A18 labeled with peroxidase by the improved sodium periodate method, i.e. A18-HRP: Dissolve 5 mg of HRP in distilled water, add 0.2ml of newly prepared 0.1M sodium periodate NaIO 4 , stirred at room temperature in the dark for 20 minutes; dialyzed against 1mM, pH4.4 NaAC solution at 4°C overnight; 3 Dialyze the buffer overnigh...

Embodiment 3

[0027] 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

[0028] The concrete steps of this method are:

[0029] (1) Preparation and anti-p185 HER-2 Monoclonal Antibody Crosslinked to Sepharose 4B-Protein G Affinity Beads:

[0030] Mix the purified antibody solution with Sepharose 4B-Protein G gel particles fully swollen by buffer, and incubate overnight at 4°C with rotation; wash twice with 10 times the volume of 0.08M, pH 9.0 sodium tetraborate buffer, Rotate at 3000g each time for 2 minutes; discard the supernatant, resuspend the affinity beads in 10 times the volume of sodium tetraborate buffer, add the solid bifunctional reagent DMP to a final concentration of 20mM, shake gently at room temperature for 30 Minutes; wash once with 10 times volume of 0.2M, pH8.0 ethanolamine buffer; resuspend affinity beads in 10 times volume of the above-mentioned ethanolamine b...

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Abstract

Soluble p185 of the present invention HER-2 The double-antibody sandwich ELISA detection method for antigen is characterized in that the purified p185 prepared by surface epitope embedding method HER-2 The monoclonal antibody is used as the coating antibody to bind to the solid phase support, respectively will contain soluble p185 HER-2 Samples and purified p185 of known concentration HER-2 Standard antigens were incubated with coated antibodies, followed by another purified, horseradish peroxidase-tagged p185 prepared by surface epitope embedding HER-2 Monoclonal antibody is used as a sandwich ELISA detection method for detecting antibodies. The purified p185 HER-2 The standard antigen was anti-p185 prepared by surface epitope embedding method HER-2 Affinity bead pairs covalently crosslinked with Sepharose 4B-ProteinG monoclonal antibodies to the extracellular region containing p185 HER-2 Antigen samples are obtained by affinity chromatography; the detection method of the present invention has the ability to detect soluble p185 HER-2 It has the advantages of strong specificity of antigen and high purity of standard soluble antigen; it can be used for tumor serum diagnosis.

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 Patents(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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