Neutralization of<i>Burkholderia pseudomallei</i>Protease by Fabs Generated through Phage Display

Nathan, Sheila; Rader, Christoph; Barbas, Carlos F.

doi:10.1271/bbb.69.2302

Cited by 4 publications

(1 citation statement)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It has been shown that when the diversity of an antibody phage display library reaches 1×10 7 individual clones, it will contain 99% of all antibody molecules. The size of antibody libraries that have been constructed so far generally ranges between 10 6 and 10 8 clones [24,25]. The size of our library for B cell lymphoma is ~1.94×10 7 , which is of medium size and should satisfy the needs for covering the diversity of antibodies [26].…”

Section: Discussionmentioning

confidence: 99%

Generation and selection of immunized Fab phage display library against human B cell lymphoma

et al. 2007

View full text Add to dashboard Cite

npgThe approval of using monoclonal antibodies as a targeted therapy in the management of patients with B cell lymphoma has led to new treatment options for this group of patients. Production of monoclonal antibodies by the traditional hybridoma technology is costly, and the resulting murine antibodies often have the disadvantage of triggering human anti-mouse antibody (HAMA) response. Therefore recombinant Fab antibodies generated by the phage display technology can be a suitable alternative in managing B cell lymphoma. In this study, we extracted total RNA from spleen cells of BALB/c mice immunized with human B lymphoma cells, and used RT-PCR to amplify cDNAs coding for the κ light chains and Fd fragments of heavy chains. After appropriate restriction digests, these cDNA fragments were successively inserted into the phagemid vector pComb3H-SS to construct an immunized Fab phage display library. The diversity of the constructed library was approximately 1.94×10 7. Following five rounds of biopanning, soluble Fab antibodies were produced from positive clones identified by ELISA. From eight positive clones, FabC06, FabC21, FabC43 and FabC59 were selected for sequence analysis. At the level of amino acid sequences, the variable heavy domains (V H ) and variable light domains (V L ) were found to share 88-92% and 89-94% homology with sequences coded by the corresponding murine germline genes respectively. Furthermore, reactivity with membrane proteins of the B cell lymphoma was demonstrated by immunohistochemistry and western blotting. These immunized Fab antibodies may provide a valuable tool for further study of B cell lymphoma and could also contribute to the improvement of disease therapy.

show abstract

Section: Discussionmentioning

confidence: 99%

Generation and selection of immunized Fab phage display library against human B cell lymphoma

et al. 2007

View full text Add to dashboard Cite

show abstract

Selection of a buried salt bridge by phage display

Vagt

Jäckel

Samsonov

et al. 2009

Bioorganic & Medicinal Chemistry Letters

View full text Add to dashboard Cite

The pComb3 Phagemid Family of Phage Display Vectors

Rader

2023

Cold Spring Harb Protoc

View full text Add to dashboard Cite

A phagemid is a plasmid that contains the origin of replication and packaging signal of a filamentous phage. Following bacterial transformation, a phagemid can be replicated and amplified as a plasmid, using a double-stranded DNA origin of replication, or it can be replicated as single-stranded DNA for packaging into filamentous phage particles. The use of phagemids enables phage display of large proteins, such as antibody fragments. Phagemid pComb3 was among the first phage display vectors used for the generation and selection of antibody libraries in the 50-kDa Fab format, a monovalent proxy of natural antibodies. Affording a robust and versatile tool for more than three decades, phage display vectors of the pComb3 phagemid family have been widely used for the discovery, affinity maturation, and humanization of antibodies in Fab, scFv, and single-domain formats from naive, immune, and synthetic antibody repertoires. In addition, they have been used for broadening phage display to the mining of nonimmunoglobulin repertoires. This review examines conceptual, functional, and molecular features of the first-generation phage display vector pComb3 and its successors, pComb3H, pComb3X, and pC3C.

show abstract

Neutralization ofBurkholderia pseudomalleiProtease by Fabs Generated through Phage Display

Cited by 4 publications

References 29 publications

Generation and selection of immunized Fab phage display library against human B cell lymphoma

Generation and selection of immunized Fab phage display library against human B cell lymphoma

Selection of a buried salt bridge by phage display

The pComb3 Phagemid Family of Phage Display Vectors

Contact Info

Product

Resources

About