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Anti-Golgi apparatus protein monoclonal antibody and use

A technology of monoclonal antibody and polyclonal antibody, applied in application, anti-animal/human immunoglobulin, genetic engineering, etc. , time-consuming operation and other problems, to achieve the effect of simple operation, clear dyeing and accurate quantification

Inactive Publication Date: 2009-04-15
曹伯良 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the two detection methods of Western and mass spectrometry have the following disadvantages: first, it is not easy to carry out accurate quantification; Due to large samples, high-throughput screening and applied research, it is difficult to use GP73 for clinical diagnosis of liver cancer patients
[0007] At present, there is no preparation of anti-GP73 polyclonal antibody and monoclonal antibody using recombinant human GP73 protein as an antigen, and there is no report on the detection of GP73 in serum of liver cancer patients by double antibody sandwich ELISA

Method used

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  • Anti-Golgi apparatus protein monoclonal antibody and use
  • Anti-Golgi apparatus protein monoclonal antibody and use
  • Anti-Golgi apparatus protein monoclonal antibody and use

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Embodiment 1G

[0047] The preparation of embodiment 1GP73 monoclonal antibody

[0048] 1. Preparation of recombinant human GP73 protein

[0049]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. Increase the GP73 fragment. The forward primer is 5'-GC GGATCC CTC CAG ACA CGG ATC ATG GAG CTG GAA GGC-3', the reverse primer is 5'-GCA AGC TTG GAG TGT ATG ATT CCG CTT TTC ACG CTG-3', and the reaction conditions are: After hot start at 94°C for 5 minutes, 1 minute at 94°C, 1 minute at 60°C, 2 minutes at 72°C, and 10 minutes at 72°C after 40 cycles. After the product was subjected to agarose electrophoresis, it was found that a fragment with a length of about 1100 bp was amplified. After sequencing and comparing with BLAST, it was confirmed that the sequence was completely correct as...

Embodiment 2

[0062] Example 2 Preparation of Anti-GP73 Polyclonal Antibody

[0063] The immunized animals were 2 adult female New Zealand white rabbits weighing 3kg. For the first immunization, 100 μg of His-GP73 protein was mixed with 0.5 ml of complete Freund's adjuvant (Sigma) and PBS to a total volume of 1 ml, and thoroughly mixed by ultrasound. Take 5 points from each white rabbit, 0.2ml per point, and inject multiple points under the skin. Thereafter, every three weeks, the Freund's complete adjuvant was changed to incomplete adjuvant (Sigma), and the animals were immunized for the second and third times in the same way. On the 10th day after the third immunization, blood was collected from the ear vein and the serum was separated, and the titer was measured by indirect ELISA (the method was the same as that of the mouse serum titer), and the result showed that the serum titer reached more than 1:128000. Animals were sacrificed and serum was collected. After pooling the sera of th...

Embodiment 3～6

[0064] Embodiment 3~6 is the experiment that GP73 monoclonal antibody is used for various immunoassays

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Abstract

The invention relates to an anti-Golgi protein antibody and an application thereof. The invention recombines human GP73 protein immunity animal to obtain an anti-GP73 polyclonal antibody and a monoclonal antibody which specifically aims at GP73 and builds a plurality of methods for detecting GP73 in clinical tissue sections and serum samples, such as immunohistochemical stain and double antibody sandwiched ELISA method and the like. Tests prove that the polyclonal and monoclonal antibodies can be used for preparing a plurality of GP73 detecting agents of different detecting methods.

Description

Technical field: [0001] The present invention relates to anti-Golgi protein antibody and application thereof. It specifically relates to specific monoclonal and polyclonal antibodies against human Golgi protein (hereinafter referred to as GP73), and the application of using the monoclonal and polyclonal antibodies to prepare various GP73 detection reagents with different detection methods. Background technique: [0002] At present, the diagnosis of liver cancer mainly relies on the detection of serum tumor markers, imaging and histological examination, among which alpha-fetoprotein (AFP) is the most important laboratory index for clinical diagnosis of primary hepatocellular carcinoma. Although AFP is the first choice index for diagnosing liver cancer, its sensitivity ranges from 33% to 85% (56.3% on average), and its specificity ranges from 30.9% to 80% (55.4% on average). In the early stage of liver cancer, about 30% to 50% of the patients had negative or low levels of AFP...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N15/13G01N33/577
Inventor 曹伯良张爱霞
Owner 曹伯良
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