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Method for screening estrogen receptor Alpha single chain antibody with phage antibody library

A phage antibody library, estrogen receptor technology, applied in the direction of anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, chemical instruments and methods, anti-animal/human immunoglobulin, etc., can solve the binding Positive clones, non-specific phages, incomplete site blocking, etc., to achieve the effect of enhancing specificity

Inactive Publication Date: 2018-07-06
FUZHOU UNIV
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Problems solved by technology

However, sometimes due to incomplete site blocking, non-specific phage binding occurs and the positive clones screened fail to show good binding ability in immunohistochemical results

Method used

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  • Method for screening estrogen receptor Alpha single chain antibody with phage antibody library
  • Method for screening estrogen receptor Alpha single chain antibody with phage antibody library
  • Method for screening estrogen receptor Alpha single chain antibody with phage antibody library

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Embodiment 1

[0038] A method for screening estrogen receptor alpha single chain antibodies using phage antibody library:

[0039] 1) Immunization of experimental rabbits with ERα protein and extraction of total RNA from spleen: ERα recombinant protein is prepared and purified in the laboratory, and the diluted ERα protein is heat-repaired with EDTA antigen retrieval solution, and labeled as ERα-AR. The repaired protein was used as the immunogen to immunize New Zealand white rabbits, and blood was collected from the ear vein before immunization as a negative control. After the first immunization, immunization is performed every two weeks. After the third immunization, blood is collected from the ear vein. The antibody titer is detected by ELISA, and the titer reaches 10 5 After the last booster immunization, the animals were sacrificed three days later, the spleen cells were extracted, total RNA was obtained, and the purified RNA was used as a template to reverse transcription using Oligo (dT) ...

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Abstract

The invention discloses a method for screening an estrogen receptor Alpha (ER Alpha) single-chain antibody with a phage antibody library. The method comprises the following steps: using an ER Alpha protein as an immunogen for immunizing a New Zealand white rabbit and extracting total RNA of spleen; amplifying VH and VL fragments by RT-PCR, and splicing an scFv gene; constructing a PCANTAB5E-ScFv recombinant plasmid, electrotransforming E. coli TG1 competent cells with the recombinant plasmid to construct a phage single-chain antibody library; using ER Alph-positive breast cancer tissue sections and ER Alpha protein as solid phase antigens for performing affinity enrichment screening on the antibody library; and identifying positive clones with a method of combining a Phage-ElLSA method with breast cancer cell smear immunochemistry. The invention establishes a method for specifically screening ER from the phage antibody library by using the ER Alpha -positive breast cancer tissue sections, and identifying the positive clones with the method of combining the Phage-ElLSA method with the breast cancer cell smear immunochemistry, and provides a new idea for a phage antibody library screening method.

Description

Technical field [0001] The invention belongs to the technical field of phage antibody library screening methods, and specifically relates to a method for screening estrogen receptor alpha single-chain antibodies using a phage antibody library. Background technique [0002] As a biomarker, estrogen receptor (ER) is a kind of nuclear hormone receptor (NR) protein, and it is also a transcription factor with activation function. The currently discovered ER has There are three subtypes: ERα, ERβ, and ERγ. In recent years, researchers have found that estrogen receptor alpha (ERα) has high expression and high activity levels in approximately 70% of breast cancer cases. The expression of ERa is closely related to breast cancer, especially the development of tumors. For example, reducing the expression of ERa in breast cancer treatment tends to have better therapeutic effects. The expression level of ER is closely related to the efficacy of endocrine therapy. ER detection can help pred...

Claims

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Application Information

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IPC IPC(8): C07K16/28C07K16/00
CPCC07K16/005C07K16/2869
Inventor 王航张芸孟春高蕊陈思诗
Owner FUZHOU UNIV
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