Methodology for selection of human antibodies to membrane proteins from a phage-display library.

2.50
Hdl Handle:
http://hdl.handle.net/10541/95487
Title:
Methodology for selection of human antibodies to membrane proteins from a phage-display library.
Authors:
Sawyer, C; Embleton, Jim; Dean, C
Abstract:
We describe a simple antigen capture technique for the selection of a specific human antibody to p185erbB-2, a transmembrane glycoprotein, from a library of human Fab genes expressed on the surface of bacteriophage. Magnetic beads coated with the rat antibody ICR55 have been used to capture erbB-2 antigen from Triton X-100 extracts of SKOV3 cells. The antigen-coated beads have then been used to select bacteriophage displaying human Fab with affinity for p185erbB-2. After 4 rounds of selection, 65 phage clones were isolated which bound specifically to p185erbB-2 in a capture assay. Nine of the clones which gave the strongest reaction in an ELISA were selected for further development and the Fab genes were subcloned into the expression vector pUC119his6mycXba and electroporated into E. coli TG1. Colonies were grown, induced and the supernatants tested for the presence of secreted human Fab. Supernatants from two of the 9 clones contained human Fab and one of these bound specifically to erbB-2 in a capture assay, stained the membranes of the erbB-2 overexpressing cell lines BT474 and SKBR3 and immunoprecipitated a protein of molecular weight 185 000 kDa from SKOV3 cells. We conclude that a membrane antigen captured by specific monoclonal antibody can be used successfully to select phage displaying human antibodies specific for the antigen.
Affiliation:
Section of Immunology, McElwain Laboratories, Institute of Cancer Research, Belmont, Sutton, Surrey, UK.
Citation:
Methodology for selection of human antibodies to membrane proteins from a phage-display library. 1997, 204 (2):193-203 J. Immunol. Methods
Journal:
Journal of Immunological Methods
Issue Date:
26-May-1997
URI:
http://hdl.handle.net/10541/95487
DOI:
10.1016/S0022-1759(97)00048-3
PubMed ID:
9212837
Type:
Article
Language:
en
ISSN:
0022-1759
Appears in Collections:
All Paterson Institute for Cancer Research

Full metadata record

DC FieldValue Language
dc.contributor.authorSawyer, Cen
dc.contributor.authorEmbleton, Jimen
dc.contributor.authorDean, Cen
dc.date.accessioned2010-04-01T14:02:38Z-
dc.date.available2010-04-01T14:02:38Z-
dc.date.issued1997-05-26-
dc.identifier.citationMethodology for selection of human antibodies to membrane proteins from a phage-display library. 1997, 204 (2):193-203 J. Immunol. Methodsen
dc.identifier.issn0022-1759-
dc.identifier.pmid9212837-
dc.identifier.doi10.1016/S0022-1759(97)00048-3-
dc.identifier.urihttp://hdl.handle.net/10541/95487-
dc.description.abstractWe describe a simple antigen capture technique for the selection of a specific human antibody to p185erbB-2, a transmembrane glycoprotein, from a library of human Fab genes expressed on the surface of bacteriophage. Magnetic beads coated with the rat antibody ICR55 have been used to capture erbB-2 antigen from Triton X-100 extracts of SKOV3 cells. The antigen-coated beads have then been used to select bacteriophage displaying human Fab with affinity for p185erbB-2. After 4 rounds of selection, 65 phage clones were isolated which bound specifically to p185erbB-2 in a capture assay. Nine of the clones which gave the strongest reaction in an ELISA were selected for further development and the Fab genes were subcloned into the expression vector pUC119his6mycXba and electroporated into E. coli TG1. Colonies were grown, induced and the supernatants tested for the presence of secreted human Fab. Supernatants from two of the 9 clones contained human Fab and one of these bound specifically to erbB-2 in a capture assay, stained the membranes of the erbB-2 overexpressing cell lines BT474 and SKBR3 and immunoprecipitated a protein of molecular weight 185 000 kDa from SKOV3 cells. We conclude that a membrane antigen captured by specific monoclonal antibody can be used successfully to select phage displaying human antibodies specific for the antigen.en
dc.language.isoenen
dc.subjectCultured Tumour Cellsen
dc.subject.meshAnimals-
dc.subject.meshAntibodies, Monoclonal-
dc.subject.meshBacteriophages-
dc.subject.meshCloning, Molecular-
dc.subject.meshEnzyme-Linked Immunosorbent Assay-
dc.subject.meshFluorescent Antibody Technique, Indirect-
dc.subject.meshGene Library-
dc.subject.meshHumans-
dc.subject.meshImmunoglobulin Fab Fragments-
dc.subject.meshMembrane Proteins-
dc.subject.meshRats-
dc.subject.meshReceptor, erbB-2-
dc.subject.meshRecombinant Proteins-
dc.subject.meshTumor Cells, Cultured-
dc.titleMethodology for selection of human antibodies to membrane proteins from a phage-display library.en
dc.typeArticleen
dc.contributor.departmentSection of Immunology, McElwain Laboratories, Institute of Cancer Research, Belmont, Sutton, Surrey, UK.en
dc.identifier.journalJournal of Immunological Methodsen

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