Human anti-self antibodies with high specificity from phage display libraries.

Griffiths, Andrew D.; Malmqvist, Magnus; Marks, James D.; Bye, Jacqueline M.; Embleton, M. J.; McCafferty, John; Baier, Michael; Holliger, K P; Gorick, B.D.; Hughes‐Jones, N. C.

doi:10.1002/j.1460-2075.1993.tb05706.x

Cited by 451 publications

(212 citation statements)

References 100 publications

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“…These tissues are frequently obtained from immunologically naive, healthy donors [5,6]. The resultant non-immune libraries have proved useful sources of functional antibodies to a variety of antigens.…”

Section: Introductionmentioning

confidence: 99%

Isolation of human anti-c-erbB-2 Fabs from a lymph node-derived phage display library

Clark

Hawkins²,

Papaioannou

et al. 1997

Clinical and Experimental Immunology

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SUMMARYAn immunoglobulin phage display library constructed from a tumour-associated pericolic lymph node was panned against the extracellular domain of the oncoprotein c-erbB-2. Sixteen independent clones were confirmed as positive binders based on ELISA analysis of soluble Fabs. Nucleotide sequencing demonstrated that the V H region of 12 clones belonged to four different V gene families, and the clones demonstrated varying degrees of somatic mutation compared with germ-line sequences. Fab fragments were examined for cross-reactivity by ELISA and shown to be negative against a panel of irrelevant self and non-self antigens, including bovine serum albumin (BSA), mouse immunoglobulin, tetanus toxoid, heregulin-PE40-FLAG and insulin. Reactivity of Fabs in vitro was verified by immunocytochemistry, which showed binding to the c-erbB-2 over-expressing breast cancer cell line SKBR3 but not to the lowexpressing cell line MDA-MB-231. We conclude that a single lymph node library of moderate diversity (2 × 10 7 k light chain and g heavy chain clones), when derived from an individual whose colorectal tumour over-expressed c-erbB-2, can be successfully panned to isolate a number of unique Fabs specific for this antigen. The nature of the anti-c-erbB-2 Fabs recovered from this library suggests that they may have resulted from a humoral immune response in the individual, and that in vivo antibody responses to tumour-associated antigens may be exploited in vitro for the production of tumour-specific recombinant antibodies.

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Section: Introductionmentioning

confidence: 99%

Isolation of human anti-c-erbB-2 Fabs from a lymph node-derived phage display library

Clark

Hawkins²,

Papaioannou

et al. 1997

Clinical and Experimental Immunology

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“…The use of large repertoires of antibody fragments displayed on phage has allowed the isolation of antibodies of different binding specificities without the need for immunization (for a review see [1]), including those that are difficult to raise by immunization, for example, human self-antigens [2,3] or highly conserved intracellular proteins [4]. Here we have explored the use of this technology for isolation of antibodies binding to compounds that are unstable under physiological condtions.…”

Section: Introductionmentioning

confidence: 99%

Phage antibodies against an unstable hapten: Oxygen sensitive reduced flavin

et al. 1996

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It is difficult to raise antibodies against haptens and antigens that are unstable under the physiological conditions of the serum. Here we have used a phage antibody library to isolate antibody fragments against oxygen sensitive reduced flavin, by selection of the phage under anaerobic and reducing conditions at pH 5 and a pre-elution step with the oxidized flavin. The binding of the reduced hapten to one of the antibody fragments was characterised by time-resolved polarised fluorescence, and shown to be highly specific for the reduced flavin.

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“…However, for a streamlined parallel application, addition of steps is always critical. A convenient way to confirm the binding of selected antibody fragments independent from the phage background is the use of the supE system, which allows to switch from pIIIfusion to free antibody fragment (Griffiths et al, 1993). Parallelised ELISA has been demonstrated for 10.000 clones/day (Hallborn and Carlsson, 2002).…”

Section: Characterisation Of In Vitro Selected Antibodiesmentioning

confidence: 99%

Perspectives for systematic in vitro antibody generation

Konthur

Hust

Dübel

2005

Gene

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After the completion and refinement of the human genome, the characterization of individual gene products in respect of their functions, their modifications, their cellular localization and regulation in both space and time has generated an increased demand for antibodies for their analysis. Taking into account that the human genome contains¨25,000 genes, and that their products are found in different splice variants and produce proteins with post-translational modifications, it can be estimated that at least 100,000 different protein products have to be investigated to gain a complete picture of what's going on in the proteome of a cell.Antibodies are preferred tools helping with the characterization and detection of proteins as well as with elucidating their individual functions. The generation of antibodies to all available human protein products by immunization and/or the hybridoma technology is not only logistically and financially enduring, but may prove to be a difficult task, as quite a number of interesting targets may evade the immune response of experimental animals, for example, allosteric variants dependent on fragile interactions to cofactors, highly conserved antigens etc. For this reason, alternative methods for the generation of antibodies have to supplement these approaches. In vitro methods for antibody generation are seen to offer this capability. In addition, they may provide a cost effective and large scale production alternative for detection reagents for the research community in their own right. Among in vitro techniques, phage display has been evolved as the most efficient option for tackling this problem and approaches optimised for automation are emerging.Maximum benefit for proteomic research could be generated by judicious and preferably international coordination of the ongoing efforts to combine the strengths of the well established animal based approaches and the novel opportunities offered by in vitro methods. D

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Human anti-self antibodies with high specificity from phage display libraries.

Cited by 451 publications

References 100 publications

Isolation of human anti-c-erbB-2 Fabs from a lymph node-derived phage display library

Isolation of human anti-c-erbB-2 Fabs from a lymph node-derived phage display library

Phage antibodies against an unstable hapten: Oxygen sensitive reduced flavin

Perspectives for systematic in vitro antibody generation

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