Related Experiment Video
Updated: Aug 23, 2026
Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood
Published on: February 6, 2018
Production of a human single-chain variable fragment antibody against esophageal carcinoma
Ming-Yan Xu1, Xiao-Hu Xu, Geng-Zhen Chen
1Department of Forensic Medicine, Medical College, Shantou University, Shantou 515041, Guangdong Province, China. xmycasey@sina.com.cn
Aim:
To construct a phage display library of human single-chain variable fragment (scFv) antibodies associated with esophageal cancer and to preliminarily screen a scFv antibody against esophageal cancer.
Methods:
Total RNA extracted from metastatic lymph nodes of esophageal cancer patients was used to construct a scFv gene library. Rescued by M13K07 helper phage, the scFv phage display library was constructed. esophageal cancer cell line Eca 109 and normal human esophageal epithelial cell line (NHEEC) were used for panning and subtractive panning of the scFv phage display library to obtain positive phage clones. Soluble scFv was expressed in E.coli HB2151 which was transfected with the positive phage clone, then purified by affinity chromatography. Relative molecular mass of soluble scFv was estimated by Western blotting, its bioactivity was detected by cell ELISA assay. Sequence of scFv was determined using the method of dideoxynucleotide sequencing.
Results:
The size of scFv gene library was approximately 9X10(6) clones. After four rounds of panning with Eca109 and three rounds of subtractive panning with NHEEC cells, 25 positive phage clones were obtained. Soluble scFv was found to have a molecular mass of 31 ku and was able to bind to Eca109 cells, but not to HeLa and NHEEC cells. Variable heavy (VH) gene from one of the positive clones was shown to be derived from the gamma chain subgroup IV of immunoglobulin, and variable light (VL) gene from the kappa chain subgroup I of immunoglobulin.
Conclusion:
A human scFv phage display library can be constructed from the metastatic lymph nodes of esophageal cancer patients. A whole human scFv against esophageal cancer shows some bioactivity.
Insights
Researchers constructed a human single-chain variable fragment (scFv) phage display library from esophageal cancer patient lymph nodes. They identified a bioactive scFv antibody targeting esophageal cancer cells, paving the way for new diagnostics and therapeutics.
Area of Science:
- Immunotechnology
- Oncology
- Molecular Biology
Background:
- Esophageal cancer remains a significant global health challenge.
- Development of targeted therapies and diagnostic tools is crucial.
- Phage display technology offers a powerful platform for antibody discovery.
Purpose of the Study:
- To construct a human single-chain variable fragment (scFv) phage display library from esophageal cancer patient samples.
- To identify and characterize scFv antibodies with specificity for esophageal cancer cells.
- To lay the groundwork for developing novel diagnostic and therapeutic strategies for esophageal cancer.
Main Methods:
- Construction of a scFv phage display library using RNA from metastatic lymph nodes of esophageal cancer patients.
- Panning and subtractive panning using esophageal cancer cell line Eca109 and normal human esophageal epithelial cells (NHEEC).
- Expression, purification, and characterization of soluble scFv, including Western blotting, ELISA, and DNA sequencing.
Main Results:
- A scFv gene library of approximately 9x10^6 clones was successfully constructed.
- 25 positive phage clones were isolated after rigorous panning against cancer cells.
- A specific scFv antibody demonstrated binding to Eca109 cells but not to normal cells, with a molecular mass of 31 ku.
Conclusions:
- A human scFv phage display library can be effectively generated from esophageal cancer patient metastatic lymph nodes.
- A novel, whole human scFv antibody targeting esophageal cancer exhibits preliminary bioactivity.
- This study provides a foundation for further development of targeted esophageal cancer therapies.
