Phage surface presentation and secretion of antibody fragments using an adaptable phagemid vector

Lah, M; Goldstraw, Alanna; White, Jacinta F.; Dolezal, Olan; Malby, Robyn L.; Hudson, Peter J.

doi:10.3233/hab-1994-51-207

Cited by 18 publications

(4 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…G. Coia and P.J. Hudson, Victoria Park, NSW, Australia), 42 previously linearized with either NotI/NcoI or SfiI/SalI. Ligated products were electroporated into competent TG1 cells (Stratagene, La Jolla, CA) and large-scale plasmid preparations of both libraries (heavy and light chains) prepared.…”

Section: Generation Of Human Ig Combinatorial Phage-fab Libraries Andmentioning

confidence: 99%

Receptor for the globular heads of C1q (gC1q‐R, p33, hyaluronan‐binding protein) is preferentially expressed by adenocarcinoma cells

Rubinstein

Stortchevoi

Boosalis

et al. 2004

Intl Journal of Cancer

View full text Add to dashboard Cite

Combinatorial Ig libraries with phage display allow in vitro generation of human Ig fragments without the need to maintain hybridomas in ongoing cell culture or to select circulating Ig from human serum. Identifying tumor-associated antigens on the surface of intact tumor cells, as opposed to purified proteins, presents a challenge due to the difficulty of preserving complex 3-D epitopic sites on the cell surface, the variable expression of antigens on different malignant cell types and the stereotactic interference of closely associated proteins on the intact membrane surface limiting accessibility to antigenic sites. A combinatorial Ig library of 10 10 clones was generated from the cDNA of PBMCs derived from patients with breast adenocarcinoma. Following subtractive panning, the library was enriched for Ig (Fab fragment) binding to intact adenocarcinoma cells and the resultant Fabs were screened against a cDNA expression library, itself generated from breast cancer cells. Using this approach, we isolated clones from the cDNA library expressing gC1q-R, a glycoprotein comprising the major structure of C1, the first component of the complement system. gC1q-R is a 33 kDa glycoprotein expressed not only on the cell surface but also intracellularly, with motifs that target it to mitochondria and complete homology with HABP and human HeLa cell protein p32, which is copurified with pre-mRNA SF2. Sequencing of the gene encoding tumor-associated gC1q-R did not reveal any consistent tumor-specific mutations. However, histochemical staining with anti-gC1q-R MAb demonstrated marked differential expression of gC1q-R in thyroid, colon, pancreatic, gastric, esophageal and lung adenocarcinomas compared to their nonmalignant histologic counterparts. In contrast, differential expression was not seen in endometrial, renal and prostate carcinomas. Despite high expression in breast carcinoma, gC1q-R was also expressed in nonmalignant breast tissue. Although the precise relation of gC1q-R to carcinogenesis remains unclear, our finding of tumor overexpression and the known multivalent binding of gC1q-R to not only C1q itself but also a variety of circulating plasma proteins as well as its involvement in cell-to-cell interactions suggest that gC1q-R may have a role in tumor metastases and potentially serve in molecule-specific targeting of malignant cells.

show abstract

Section: Generation Of Human Ig Combinatorial Phage-fab Libraries Andmentioning

confidence: 99%

Receptor for the globular heads of C1q (gC1q‐R, p33, hyaluronan‐binding protein) is preferentially expressed by adenocarcinoma cells

Rubinstein

Stortchevoi

Boosalis

et al. 2004

Intl Journal of Cancer

View full text Add to dashboard Cite

show abstract

“…This gene III product directs assembly of the scFvs on the surface of the phage. The scFvs are conveniently cloned between SfiI and NotI restriction sites in suitable phagemid vectors, such as pHFA or pHFA 2 (Lah et al, 1994;Dolezal et al, 1995;Fig. 17.1.4), in frame with and between the 3′ end of the pelB signal-sequence coding region and the 5′ end of the gene III coding sequence.…”

Section: Cloning Scfv Genes Into Phage Display Vectorsmentioning

confidence: 99%

“…Fab fragments are displayed on phage following sequential cloning (see Fig. 17.1.5) of the heavy-and light-chain genes into a phagemid expression vector carrying a bicistronic expression cassette such as pHFA 2 or pHEN (Lah et al, 1994;Dolezal et al, 1995;see Figs. 17.1.4 and 17.1.5). These vectors allow concomitant expression of both heavy-and light-chain gene fragments.…”

Section: Cloning Fab Genes Into Phage Display Vectors Using Sequentiamentioning

confidence: 99%

Bacteriophage Library Construction and Selection of Recombinant Antibodies

1999

Self Cite

View full text Add to dashboard Cite

This unit describes the use of E. coli and bacteriophages to display a diverse library of antibody fragments equivalent in complexity to the mammalian immune repertoire, and subsequent screening of the library for antibody fragments with specific binding affinities. The methods are also used for affinity enhancement (maturation), through the display and selection of improved affinity mutants derived from a single parent antibody. This unit discusses the following key components needed in library construction technology: a repertoire of antibody genes, typically amplified by polymerase chain reaction (PCR) technology; construction of scFv genes by PCR assembly; a method for producing a stable library, using bacteriophage that can both display individual antibodies on the viral surface and carry the gene encoding the antibody; a method of growing phage for selection; a method of selecting the highest-affinity antibody from the phage library; a method for monitoring progress of phage selection; an affinity-enhancement strategy for improving and manipulating the selected antibody; and expression of affinity-enhanced antibodies.

show abstract

“…ro// strains, this allows expression of the fusion product, for incorporation in the phage coat; in non-suppressor strains, this amber serves as a stop codon, yielding soluble antibody fragments. A variety of tags has been described that are appended to the antibody fragment for detection, including the myc-derived tag recognised by antibody 9E10 [20], and the Flag sequence [113]. This set-up will allow the use of unpurified phage antibodies or antibody fragments, present in crude supernatant or periplasmic extracts, for screening assays.…”

Section: 5mentioning

confidence: 99%

Exploring antibody engineering technology for diagnostic applications

Haard¹

View full text Add to dashboard Cite

People interested in the research are advised to contact the author for the final version of the publication, or visit the DOI to the publisher's website.• The final author version and the galley proof are versions of the publication after peer review.• The final published version features the final layout of the paper including the volume, issue and page numbers. Link to publication General rightsCopyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.• Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal.If the publication is distributed under the terms of Article 25fa of the Dutch Copyright Act, indicated by the "Taverne" license above, please follow below link for the End User Agreement:

show abstract

Phage surface presentation and secretion of antibody fragments using an adaptable phagemid vector

Cited by 18 publications

References 23 publications

Receptor for the globular heads of C1q (gC1q‐R, p33, hyaluronan‐binding protein) is preferentially expressed by adenocarcinoma cells

Receptor for the globular heads of C1q (gC1q‐R, p33, hyaluronan‐binding protein) is preferentially expressed by adenocarcinoma cells

Bacteriophage Library Construction and Selection of Recombinant Antibodies

Exploring antibody engineering technology for diagnostic applications

Contact Info

Product

Resources

About