Cloning immunoglobulin variable domains for expression by the polymerase chain reaction.

Orlandi, Rosaria; Güssow, Detlef; Jones, Peter; Winter, Greg

doi:10.1073/pnas.86.10.3833

Cited by 604 publications

(303 citation statements)

References 43 publications

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“…cDNA synthesis and PCR were performed sequentially, and the primers used (Orlandi et al, 1989) were slightly modified and specific for amplification of VH and V, domains of IgGl. Briefly, for first-strand synthesis a 25-mL reaction containing 500 ng of total RNA, 10 pmol of MOCG12FOR primer or VL amplification, respectively, 250 mM of each dNTP, 67 mM Tris-HC1 (pH 8 4 , 16.6mM (NH4)2S04, 1.5 mM MgClz, 0.01 Vo Tween-20, and Taq polymerase (2 units).…”

Section: Cloning and Sequencing Of The Mab Tw420 V And Vh Regionsmentioning

confidence: 99%

Cloning, characterization, and modeling of a monoclonal anti‐human transferrin antibody that competes with the transferrin receptor

Orlandini¹,

Santucci²,

Tramontano³

et al. 1994

Protein Science

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In this report we describe the isolation and characterization of a monoclonal antibody against human serum transferrin (Tf) and the cloning and sequencing of its cDNA. The antibody competes with the transferrin receptor (TR) for binding to human Tf and is therefore expected to bind at or very close to a region of interaction between Tf and its receptor. From the deduced amino acid sequence, we constructed a 3-dimensional model of the variable domains of the antibody based on the canonical structure model for the hypervariable loops. The proposed structure of the antibody is a first step toward a more detailed characterization of the antibody-Tf complex and possibly toward a better understanding of the Tf interaction with its receptor. The model might prove useful in guiding site-directed mutagenesis studies, simplifying the experimental elucidation of the antibody structure, and in the use of automatic procedures to dock the interacting molecules as soon as structural information about the structure of the human Tf molecule will be available.

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Section: Cloning and Sequencing Of The Mab Tw420 V And Vh Regionsmentioning

confidence: 99%

Cloning, characterization, and modeling of a monoclonal anti‐human transferrin antibody that competes with the transferrin receptor

Orlandini¹,

Santucci²,

Tramontano³

et al. 1994

Protein Science

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“…Leader and 3 0 -region DNA segments, containing sequences for the mouse Ig heavy chain promoter and a signal peptide as well as the appropriate splicing sites were amplified by PCR from M13VHPCR1 [34]. Overlap-extension PCR was used to join these to the 5 0 and 3 0 ends of the V H and Vj cDNA of anti-VAP-1 antibodies 2D10, TK8-14 and 1G6.…”

Section: Production Of Chimeric Antibodiesmentioning

confidence: 99%

“…Overlap-extension PCR was used to join these to the 5 0 and 3 0 ends of the V H and Vj cDNA of anti-VAP-1 antibodies 2D10, TK8-14 and 1G6. HindIII-BamHI fragments comprising leader-V H -3 0 region or leader-Vj-3 0 region DNA were cloned into a pSVgpt expression vector containing a modified human IgG2 constant region gene (G2Da; [20]) or a pSVhyg expression vector containing a human j constant region gene [34], as appropriate. DNA sequences were confirmed by sequencing.…”

Section: Production Of Chimeric Antibodiesmentioning

confidence: 99%

“…Vectors for the control 2D10 IgG1 and 2D10 IgG2 antibodies were constructed in the same way using wild-type constant region genes [20]. For the control G2Da antibody, ChNPLys, the heavy and light chain variable region DNA were provided from M13VHPCR1 (anti-NIP) and the original pSVhyg j expression vector (anti-lysozyme; [34]). …”

Section: Production Of Chimeric Antibodiesmentioning

confidence: 99%

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Function-blocking antibodies to human vascular adhesion protein-1: A potential anti-inflammatory therapy

et al. 2005

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Human vascular adhesion protein-1 (VAP-1) is a homodimeric 170-kDa sialoglycoprotein that is expressed on the surface of endothelial cells and functions as a semicarbazidesensitiveamineoxidase and as an adhesion molecule. Blockade of VAP-1has been shown to reduce leukocyte adhesion and transmigration in in vivo and in vitro models, suggesting that VAP-1 is a potential target for anti-inflammatory therapy. In this study we have constructed mouse-human chimeric antibodies by genetic engineering in order to circumvent the potential problems involved in using murine antibodies in man. Our chimeric anti-VAP-1 antibodies, which were designed to lack Fc-dependent effector functions, bound specifically to cell surface-expressed recombinant human VAP-1 and recognized VAP-1 in different cell types in tonsil. Furthermore, the chimeric antibodies prevented leukocyte adhesion and transmigration in vitro and in vivo. Hence, these chimeric antibodies have the potential to be used as a new anti-inflammatory therapy.

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“…Ā exible (Gly 4 -Ser) 3 linker was inserted between V H and V L regions (Orlandi et al, 1989). The resulting scFv cDNAs were cloned into the pSW1 plasmid, allowing the secretion of scFvs containing the c-mycderived epitope at the C-terminus end into the periplasm of E. coli (Cochet et al, 1998).…”

mentioning

confidence: 99%

Restoration of transcriptional activity of p53 mutants in human tumour cells by intracellular expression of anti-p53 single chain Fv fragments

Fromentel¹,

Gruel

Venot³

et al. 1999

Oncogene

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We report here the production and the properties of single chain Fv fragments (scFvs) derived from the antip53 monoclonal antibodies PAb421 and 11D3. 11D3 is a newly generated monoclonal antibody which exhibits properties very comparable to those of PAb421. The scFvs PAb421 and 11D3 are able to stably associate with p53 and to restore the DNA binding activity of some p53 mutants in vitro. When expressed in p53 7/7 human tumour cells, the scFv421 is essentially localized in the cytoplasm in the absence of p53, and in the nucleus when exogenous p53 is present. Thus, p53 is also able to stably associate with an anti-p53 scFv in cells. Cotransfection of p53 7/7 human tumour cells with expression vectors encoding the His273 p53 mutant and either scFv leads to restoration of the p53 mutant de®cient transcriptional activity. These data demonstrate that, in human tumour cells, these scFvs are able to restore a function essential for the tumour suppressor activity of p53 and may represent a novel class of molecules for p53-based cancer therapy.

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Cloning immunoglobulin variable domains for expression by the polymerase chain reaction.

Cited by 604 publications

References 43 publications

Cloning, characterization, and modeling of a monoclonal anti‐human transferrin antibody that competes with the transferrin receptor

Cloning, characterization, and modeling of a monoclonal anti‐human transferrin antibody that competes with the transferrin receptor

Function-blocking antibodies to human vascular adhesion protein-1: A potential anti-inflammatory therapy

Restoration of transcriptional activity of p53 mutants in human tumour cells by intracellular expression of anti-p53 single chain Fv fragments

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