Anti-human glycosphingolipid Globo-H monoclonal antibody and preparation method and application thereof

A technology of monoclonal antibody and glycosphingolipid, which is applied in the field of monoclonal antibody to achieve the effect of strong specific binding ability

Inactive Publication Date: 2015-06-10
SUZHOU ZHONG YING MEDICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no monoclonal antibody against human glycosphingolipid Globo-H, so the prepa

Method used

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  • Anti-human glycosphingolipid Globo-H monoclonal antibody and preparation method and application thereof
  • Anti-human glycosphingolipid Globo-H monoclonal antibody and preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Example 1 Separation, purification and enrichment of glycosphingolipid Globo-H

[0030] (1) Extraction of glycosphingolipid Globo-H from tumor tissue of patients with liver cancer:

[0031] Take the liver cancer tissue sample and grind it into pieces, add the solvent chloroform:methanol (1:1, v / v) and ultrasonically shake for one hour, then centrifuge to get the supernatant, repeat four times, then add isopropanol:hexane:first-grade water ( 55:25:20, v / v / v) After ultrasonic vibration for one hour, centrifuge to get the supernatant, repeat four times, and dry in a centrifugal desiccator.

[0032] (2) Separation of neutral lipids in tumor tissues of patients with liver cancer:

[0033] First put the SephadexA25 gel solution into the column, and then equilibrate it with chloroform:methanol:water (30:60:8, v / v / v) solution, then add the dried lipid sample in step (1) to the chromatography column, and use Chloroform:methanol:water (30:60:8, v / v / v) eluted to obtain neutral g...

Embodiment 2

[0038] Example 2 Glycosphingolipid Globo-H immunized mice

[0039] Inject mice with 50 μg glycosphingolipid Globo-H combined with 0.5 mL Freund’s complete adjuvant in the intraperitoneal cavity, immunize mice again with 50 μg glycosphingolipid Globo-H combined with 0.5 ml Freund’s incomplete adjuvant after 2 weeks, and 400 μg glycosphingolipid after 4 weeks Mice were immunized intraperitoneally with lipid Globo-H. Blood was collected from the retroorbital canthus vein, and the serum was frozen at -20°C. After 4 days, the mice were sacrificed to obtain the spleen for cell fusion.

Embodiment 3

[0040] Example 3 Glycosphingolipid Globo-H immunization induces specific serum antibodies:

[0041] After immunizing mice with glycosphingolipid Globo-H three times, the anti-glycosphingolipid Globo-H antibody induced in its serum was detected by conventional ELISA method: 15 μg / mL glycosphingolipid Globo-H was dissolved in the coating solution (0.1 NaCO3, PH9.6, 0.1% BSA) coated in 96-well ELISA plate at 4°C, blocked with 5% milk-PBS; add 1:100 diluted serum in turn, HRP-labeled goat anti-mouse IgG, OPD, color development stop , measured OD490nm, showing the successful induction of specific serum antibodies.

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Abstract

The invention relates to an anti-human glycosphingolipid Globo-H monoclonal antibody and a preparation method and application thereof. The anti-human glycosphingolipid Globo-H monoclonal antibody can specifically bind glycosphingolipid Globo-H in tumor tissues of liver cancer patients. The anti-human glycosphingolipid Globo-H monoclonal antibody has strong specific binding ability with human glycosphingolipid Globo-H, and can be well used in preparation of diagnostic reagents for diagnosis of liver cancer and the like.

Description

technical field [0001] The invention relates to the technical field of monoclonal antibodies, in particular to an anti-human glycosphingolipid Globo-H monoclonal antibody, its preparation method and application. Background technique [0002] Tumor-specific sugar chains are currently one of the research hotspots in the field of tumors. 80% of tumor-related proteins are glycoproteins, and various glycolipids are also closely related to tumors. At present, there are few antibodies against tumor-specific glycosphingolipids at home and abroad, and major relevant research institutions are working hard to develop tumor-specific glycosphingolipid antibodies, which has become a potential hotspot. The specificity of antigen-antibody reactions determines that monoclonal antibodies against tumor-specific target antigens will preferentially bind to corresponding tumor tissues, but will not recognize normal tissues. The determination of fucosidase (AFU) in the human body is greatly affec...

Claims

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Application Information

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IPC IPC(8): C07K16/18C12N5/20C12N15/06G01N33/577G01N33/574
Inventor 吴忠福李云森
Owner SUZHOU ZHONG YING MEDICAL TECH
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