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Monoclonal antibody of hepatoma-derived growth factor and use thereof

A monoclonal antibody and double-antibody sandwich technology, applied in anti-growth factor immunoglobulin, anti-animal/human immunoglobulin, instruments, etc., can solve the problem of low specificity, high polyclonal antibody background, no anti-HDGF monoclonal Cloning of antibodies and other issues to achieve the effect of simplified experimental steps, clear staining, and accurate positioning

Inactive Publication Date: 2008-11-12
曹伯良 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, polyclonal antibodies often have the disadvantages of high background and low specificity, and the immunohistochemical results are not easy to standardize.
[0007] At present, there is no anti-HDGF monoclonal antibody, and there is no report that HDGF can be detected in the serum of tumor patients

Method used

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  • Monoclonal antibody of hepatoma-derived growth factor and use thereof
  • Monoclonal antibody of hepatoma-derived growth factor and use thereof
  • Monoclonal antibody of hepatoma-derived growth factor and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] The preparation of embodiment 1 HDGF monoclonal antibody

[0042] 1. Preparation of recombinant human HDGF protein

[0043] Take 3 equal amounts of cryopreserved human liver cancer tissues (from the First Affiliated Hospital of Nanjing Medical University), add 1ml Trizol and homogenate, extract total RNA according to the method in the manual, reverse transcribe into cDNA, and amplify by PCR. Increased HDGF fragments. The forward primer is 5'-gcggatccatgtcgcgatccaaccggcagaag-3', the reverse primer is 5'-cggaattcggcaggctctcatgatctctgatgcc-3', and the reaction conditions are: hot start at 94°C for 5min, 94°C for 1min, 60°C for 1min, 72°C for 2min, 40 cycles After that, it was extended for 10 minutes at 72°C. After the product was subjected to agarose electrophoresis, it was found that a fragment with a length of about 700bp was amplified. After sequencing and BLAST comparison, it was confirmed that the sequence was completely correct as a human HDGF fragment.

[0044] A...

Embodiment 2～6

[0056] Embodiment 2~6 is the experiment that HDGF monoclonal antibody is used for various immunoassays

Embodiment 2W

[0057] Embodiment 2Western western blot experiment

[0058] 1. Method

[0059] After performing SDS-PAGE with recombinant His-HDGF (0.1 μg / lane), the protein was transferred to a nitrocellulose membrane. After blocking with 5% skimmed milk powder at room temperature for 1 hour, the membrane was cut into 3 equal parts according to the swimming lanes, and the purified 2F12 monoclonal antibody (1:10000) prepared in Example 1 and the last immunized mouse serum diluted 1:5000 were added respectively. And 1:1000 anti-His antibody (Invitrogen) as Yangshen, overnight at 4°C. After the primary antibody was poured off the next day, the membrane was washed 3 times with PBST, and horseradish peroxidase-labeled goat anti-mouse enzyme-labeled secondary antibody (1:2000) was added to incubate at room temperature for 90 minutes, the secondary antibody was poured off, and the membrane was washed with PBST. Four times, signal detection was performed with an ECL luminescence system (PIERCE). ...

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Abstract

The invention relates to anti-liver cancer derived growth factor monoclonal antibody and an application of the same. The invention makes the specific monoclonal antibody of anti-hepatoma derived growth factor (HDGF), wherein the monoclonal antibody can be used to make HDGF detection reagent suitable for a plurality of detection method.

Description

Technical field: [0001] The invention relates to an anti-hepatoma-derived growth factor monoclonal antibody. Specifically, it relates to a specific monoclonal antibody against human liver cancer-derived growth factor (hereinafter abbreviated as HDGF). The invention also relates to the application of using the monoclonal antibody to prepare HDGF detection reagents for various detection methods. Background technique: [0002] Early detection and correct diagnosis of malignant tumors play a decisive role in the treatment of cancer patients. [0003] HDGF is a liver cancer-derived growth factor (hepatoma-derived growth factor, HDGF). HDGF related protein (HDGF related protein, HRPs) is a newly discovered growth factor family, composed of HDGF, HRP1, HRP2, HRP3, HRP4 and LEDGF / p75 / p52. [0004] HDGF plays an extremely important role in tumor pathology, and it may affect the occurrence and development of tumors. Animal experiments have confirmed that HDGF has a tumorigenic eff...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/22G01N33/577
Inventor 曹伯良张爱霞
Owner 曹伯良
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