SpringerProtocols: Abstract: Human Recombinant Fab Antibodies with T-Cell Receptor-Like Specificities Generated from Phage Display Libraries

Human Recombinant Fab Antibodies with T-Cell Receptor-Like Specificities Generated from Phage Display Libraries

By: Jan Engberg³, Ali F. Yenidunya³, Rikke Clausen³, Liselotte B. Jensen³, Peter Sørensen³, Pernille Kops³, Erik Riise³

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In this chapter we describe efficient procedures for the construction, expression and screening of comprehensive libraries of human Fab antibody fragments displayed on the surface of filamentous phage. Phagemid vectors are used for placing randomly paired light (L) and heavy (H) chain coding regions under transcriptional control of Plac. The H chain coding region is fused in-frame with the phage gene, ΔgIII, coding for a truncated version of the phage surface protein pIII (ΔpIII). After superinfection with helper phage and induction of Plac, Fd (composed of V_H and C_H1 domains) and κ-or α-L chains assemble into Fab fragments in the periplasm of the Escherichia coli host strain, and the Fab-ΔpIII protein complex is displayed at one end of the phage by displacing one (or more) of the wild-type pIII proteins. Enrichment of Fab phages with affinity for a selected “antigen” is then carried out by successive rounds of affinity purification using “antigen”-coated immunotubes or plastic beads followed by reinfection of E. coli cells with the eluted bound phages (see refs. 1–10). An outline of the method is illustrated in Fig. 1.

Affiliation(s): (3) Department of Pharmacology, The Royal Danish School of Pharmacy, Copenhagen, Denmark

Book Title: Recombinant Antibodies for Cancer Therapy: Methods and Protocols

Series: Methods in Molecular Biology | Volume: 207 | Pub. Date: Sep-16-2002 | Page Range: 161-177 | DOI: 10.1385/1-59259-334-8:161

Subject: Cancer Research

References

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