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[Isolation of single chain antibodies against cell surface molecules by pathfinder selection].
Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 2004 Aug; 26(4):405-9.ZY

Abstract

OBJECTIVE

To isolate single chain antibody fragments (scFv) against cell surface molecules by pathfinder selection from an anti-KG1a cell scFv phage library.

METHODS

The anti-KG1a scFv library was enriched by KGla cell panning for three rounds, or unenriched, then processed for pathfinder selection respectively using anti-CD34 monoclonal antibody as pathfinder molecule. ScFv phage clones were randomly picked and identified by binding KG1a cells using immunofluorescein and flow cytometry. The KG1a+ clones were further identified by KG1a, HL60, U937, and CEM cell lines and ELISA. Their antigenic molecules on cell surface were digested by chymopapain and analyzed by flow cytometry. DNAs from ten positive clones were sequenced. The scFv clones with different primary structure were used to analyze the molecular weight of their antigens by Western blot.

RESULTS

One hundred and two KG1a+ scFv phage clones were isolated from 144 enriched and 96 unenriched scFv phage library respectively, among which 47 bound KG1a, HL60, U937, and CEM cells, 55 bound KG1a cells exclusively. None of 28 KG1a+, HL60-, U937-, and CEM- scFv clones bound to the CD34 antigen, as confirmed by ELISA, although most of their antigens were sensitive to chymopapain digestion. DNA sequences from ten positive clones showed that they were from four different clones. They bound antigens with different molecular weight.

CONCLUSIONS

One hundred and two scFv phage clones specific for hematopoietic stem and progenitor cells have been isolated from an anti-KG1a cell scFv phage library. The pathfinder selection has showed advantages to improve the screening efficacy of scFv phage clones against antigens, which present at very low densities on the cell surface.

Authors+Show Affiliations

National Key Laboratory of Experimental Hematology, Institute of Hematology, CAMS and PUMC, Tianjin 300020, China.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

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

Language

chi

PubMed ID

15379265

Citation

Liu, Jun-xia, et al. "[Isolation of Single Chain Antibodies Against Cell Surface Molecules By Pathfinder Selection]." Zhongguo Yi Xue Ke Xue Yuan Xue Bao. Acta Academiae Medicinae Sinicae, vol. 26, no. 4, 2004, pp. 405-9.
Liu JX, Meng L, Xu J, et al. [Isolation of single chain antibodies against cell surface molecules by pathfinder selection]. Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 2004;26(4):405-9.
Liu, J. X., Meng, L., Xu, J., Jia, H. R., & Song, Z. X. (2004). [Isolation of single chain antibodies against cell surface molecules by pathfinder selection]. Zhongguo Yi Xue Ke Xue Yuan Xue Bao. Acta Academiae Medicinae Sinicae, 26(4), 405-9.
Liu JX, et al. [Isolation of Single Chain Antibodies Against Cell Surface Molecules By Pathfinder Selection]. Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 2004;26(4):405-9. PubMed PMID: 15379265.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - [Isolation of single chain antibodies against cell surface molecules by pathfinder selection]. AU - Liu,Jun-xia, AU - Meng,Lei, AU - Xu,Jing, AU - Jia,Hai-rong, AU - Song,Zeng-xuan, PY - 2004/9/24/pubmed PY - 2005/2/16/medline PY - 2004/9/24/entrez SP - 405 EP - 9 JF - Zhongguo yi xue ke xue yuan xue bao. Acta Academiae Medicinae Sinicae JO - Zhongguo Yi Xue Ke Xue Yuan Xue Bao VL - 26 IS - 4 N2 - OBJECTIVE: To isolate single chain antibody fragments (scFv) against cell surface molecules by pathfinder selection from an anti-KG1a cell scFv phage library. METHODS: The anti-KG1a scFv library was enriched by KGla cell panning for three rounds, or unenriched, then processed for pathfinder selection respectively using anti-CD34 monoclonal antibody as pathfinder molecule. ScFv phage clones were randomly picked and identified by binding KG1a cells using immunofluorescein and flow cytometry. The KG1a+ clones were further identified by KG1a, HL60, U937, and CEM cell lines and ELISA. Their antigenic molecules on cell surface were digested by chymopapain and analyzed by flow cytometry. DNAs from ten positive clones were sequenced. The scFv clones with different primary structure were used to analyze the molecular weight of their antigens by Western blot. RESULTS: One hundred and two KG1a+ scFv phage clones were isolated from 144 enriched and 96 unenriched scFv phage library respectively, among which 47 bound KG1a, HL60, U937, and CEM cells, 55 bound KG1a cells exclusively. None of 28 KG1a+, HL60-, U937-, and CEM- scFv clones bound to the CD34 antigen, as confirmed by ELISA, although most of their antigens were sensitive to chymopapain digestion. DNA sequences from ten positive clones showed that they were from four different clones. They bound antigens with different molecular weight. CONCLUSIONS: One hundred and two scFv phage clones specific for hematopoietic stem and progenitor cells have been isolated from an anti-KG1a cell scFv phage library. The pathfinder selection has showed advantages to improve the screening efficacy of scFv phage clones against antigens, which present at very low densities on the cell surface. SN - 1000-503X UR - https://www.unboundmedicine.com/medline/citation/15379265/[Isolation_of_single_chain_antibodies_against_cell_surface_molecules_by_pathfinder_selection]_ L2 - https://medlineplus.gov/stemcells.html DB - PRIME DP - Unbound Medicine ER -