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Production of a human single-chain variable fragment antibody against esophageal carcinoma.
World J Gastroenterol. 2004 Sep 15; 10(18):2619-23.WJ

Abstract

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.

Authors+Show Affiliations

Department of Forensic Medicine, Medical College, Shantou University, Shantou 515041, Guangdong Province, China. xmycasey@sina.com.cnNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

15309706

Citation

Xu, Ming-Yan, et al. "Production of a Human Single-chain Variable Fragment Antibody Against Esophageal Carcinoma." World Journal of Gastroenterology, vol. 10, no. 18, 2004, pp. 2619-23.
Xu MY, Xu XH, Chen GZ, et al. Production of a human single-chain variable fragment antibody against esophageal carcinoma. World J Gastroenterol. 2004;10(18):2619-23.
Xu, M. Y., Xu, X. H., Chen, G. Z., Deng, X. L., Li, J., Yu, X. J., & Chen, M. Z. (2004). Production of a human single-chain variable fragment antibody against esophageal carcinoma. World Journal of Gastroenterology, 10(18), 2619-23.
Xu MY, et al. Production of a Human Single-chain Variable Fragment Antibody Against Esophageal Carcinoma. World J Gastroenterol. 2004 Sep 15;10(18):2619-23. PubMed PMID: 15309706.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Production of a human single-chain variable fragment antibody against esophageal carcinoma. AU - Xu,Ming-Yan, AU - Xu,Xiao-Hu, AU - Chen,Geng-Zhen, AU - Deng,Xiao-Ling, AU - Li,Jonathan, AU - Yu,Xiao-Jun, AU - Chen,Mei-Zhen, PY - 2004/8/17/pubmed PY - 2004/12/17/medline PY - 2004/8/17/entrez SP - 2619 EP - 23 JF - World journal of gastroenterology JO - World J Gastroenterol VL - 10 IS - 18 N2 - 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. SN - 1007-9327 UR - https://www.unboundmedicine.com/medline/citation/15309706/Production_of_a_human_single_chain_variable_fragment_antibody_against_esophageal_carcinoma_ L2 - https://www.wjgnet.com/1007-9327/full/v10/i18/2619.htm DB - PRIME DP - Unbound Medicine ER -