Antibody Phage Display: Technique and Applications

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Research Techniques Made Simple: Immunofluorescence Techniques

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Research Techniques Made Simple: Immunofluorescence Techniques

Presentation transcript:

Antibody Phage Display: Technique and Applications Christoph M. Hammers, John R. Stanley Journal of Investigative Dermatology Volume 134, Issue 2, Pages 1-5 (February 2014) DOI: 10.1038/jid.2013.521 Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Antibody phage display (APD). (a) Diagram showing how APD is performed. (b) Interrelatedness of Ig (here, IgG), single-chain variable fragment (scFv), and Fab. Importantly, both scFv and Fab reflect the original specificity of the Ig they are derived from because variable heavy (VH) and variable light (VL) chains form the interface with the antigen. (c) Phage display systems are derived from wild-type bacteriophage of Escherichia coli. Monovalency of the displayed protein (turquoise) ensures selection for high-affinity mAbs in phagemid vector-based systems (e.g., pComb3X). Helper phages display a fusion protein as well after propagation from phage-infected cells and are also selected during panning (but they do not possess the sequence of the displayed protein within). A defective helper phage origin of replication ensures preferential production and packaging of the desired phage particles, requiring addition of new helper phage in every panning round. Journal of Investigative Dermatology 2014 134, 1-5DOI: (10.1038/jid.2013.521) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Monovalent mAbs (in the form of scFv) cause blister formation typical of pemphigus foliaceus in human skin. After injection of purified antidesmoglein 1 scFv, human skin specimens were cultured for 24 hours. Histology and direct immunofluorescence (IF) are shown. 3-07/1e and 3-30/3h caused superficial epidermal blisters. All scFv, except 3-094/O1808, bound to the cell surface of epidermal keratinocytes. Immunoprecipitation and indirect IF experiments suggested that 3-094/O1808 binds to the proprotein but not to the mature protein (data not shown). mAb, monoclonal antibody; scFv, single-chain variable fragment. Reprinted with permission from Ishii et al. (2008). Journal of Investigative Dermatology 2014 134, 1-5DOI: (10.1038/jid.2013.521) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Multiple PF and PV sera compete for the same or nearby epitopes targeted by an anti-Dsg-1 monoclonal scFv (3-30/3h) derived from a PF patient. 6 PF sera, 5 mucocutaneous PV sera, and 3 mucosal PV sera (and a normal control, N) were used to block binding of the 3-30/3h to Dsg1. All PF and mucocutaneous PV sera inhibited binding of 3-30/3h, but mucosal PV patients’ sera (featuring anti-Dsg3 antibodies only) did not. Dsg, desmoglein; PF, pemphigus foliaceus; PV, pemphigus vulgaris; scFv, single-chain variable fragment. Reprinted with permission from Ishii et al. (2008). Journal of Investigative Dermatology 2014 134, 1-5DOI: (10.1038/jid.2013.521) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Three anti-Dsg3 scFvs (PNP-A1/B1/C1) isolated from a PNP patient bind to the EC2 and EC3 domains of Dsg3 in immunoprecipitation/immunoblot analysis of the patient’s and a PV patient’s sera. The PNP patient’s sera recognized Dsg3 epitopes EC1–4, but the mAbs derived by phage display recognized only EC2 and EC3. In classic PV patients, the main epitopes are EC1 and EC2. These findings also demonstrate that antibody phage display may miss some pathologic clones (here, such mAbs that target EC1 and EC4) for various reasons, as discussed in the last paragraph of this article. Dsg, desmoglein; PNP, paraneoplastic pemphigus; PV, pemphigus vulgaris; scFv, single-chain variable fragment. Reprinted with permission from Saleh et al. (2012). Journal of Investigative Dermatology 2014 134, 1-5DOI: (10.1038/jid.2013.521) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Px44-hTRAIL binds to human epidermal keratinocytes and causes apoptosis of tumor cells. (a) When injected intradermally into human skin, the fusion protein binds to the cell surface of epidermal keratinocytes as detected by immunofluorescence with anti-hemagglutinin (HA) and anti-hTRAIL antibodies. (b) TUNEL staining of actinic keratosis-like lesions in K14-activated Fyn transgenic mice after intratumoral injection of the fusion protein shows apoptosis of tumor cells (green fluorescence signal). hTRAIL, human tumor necrosis factor–related apoptosis-inducing ligand. Reprinted with permission from Kouno et al. (2013). Journal of Investigative Dermatology 2014 134, 1-5DOI: (10.1038/jid.2013.521) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Journal of Investigative Dermatology 2014 134, 1-5DOI: (10. 1038/jid Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Journal of Investigative Dermatology 2014 134, 1-5DOI: (10. 1038/jid Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions