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Subtractive single-chain antibody (scFv) phage-display: tailoring phage-display for high specificity against function-specific conformations of cell membrane molecules

Nature Protocols volume 2, pages 3063–3073 (2007)Cite this article

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Abstract

Phage-display of antibody libraries is a powerful tool to select antibodies for specific epitopes. We describe a strategy for selecting highly specific scFv-clones that discriminate between various conformational states of cell surface receptors. This approach adapts the M13 pIII phage-display technology toward a cell suspension-based strategy, which allows panning against complex, multimeric, fully functional cell membrane epitopes without alteration of structure due to purification or immobilization. As the functional properties are preserved, phage can be specifically depleted or selected for neo-epitopes exposed after physiological alterations of the targeted molecules. This subtractive strategy allows highly specific selection for single-chain antibodies directed against functionally regulated epitopes on the cell surface molecules that can be tailored for diagnostic and therapeutic applications. Using this protocol, activation-specific single-chain antibodies can be obtained within 4–6 weeks.

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Figure 1: Single-chain antibody display on pIII of the M13 phage.
Figure 2: Flow chart of the subtractive panning (depletion/selection) strategy for the generation of conformation-specific single-chain antibodies.
Figure 3: Monitoring for success of the panning process.
Figure 4: Production and purification of selected single-chain antibodies in TG-1 Escherichia coli.

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Change history

  • 05 June 2008

    In the version of this article initially published, some reagents were referred to incorrectly or unclearly. In Step 11, “1 ml buffer” should have been “1 ml HEPES buffer”. In Step 48, “saccharose” should have been “sucrose”. In Box 2, Step 1, “LBG media” should have been “LB media”. Some units were listed incorrectly. In Box 1, Step 3, “1,000 g ml-1” should have been “100 μg ml-1”. In the table in Step 42, the four instances of “ml” should have been “μl”. In Box 2, Step 2, “Inoculate 5 ml LB media with 100 μl” should have been “Inoculate 500 ml LB media with 5 ml”. The relationship between Procedure steps and Figure 4 was unclear, leaving the figure open to misinterpretation. The following sentence has been added to the end of Step 53, before the Critical Step: “Figure 4 shows the successful production and purification of an scFv antibody as assessed by SDS–polyacrylamide gel electrophoresis and western blotting of an scFv cloned into the pHOG-21 vector expressed in TG-1 E. coli (Steps 58–70).” The two labels on Figure 4 that read “36,000 Da” have been changed to “scFv”. In the description of panel b in the legend, “anti-HIS-tag” has been changed to “anti-His-tag”. In Step 15, “XL-1 blue on 5 ml” should have been “XL-1 blue in 5 ml”. In the legend to Figure 3, the citation to ref. 18 should have been to ref. 5. At the end of the legend to Figure 4 and in “Anticipated Results,” the citation “18,20” should have been “4–6”. These errors have been corrected in the HTML and PDF versions of the article.

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Authors and Affiliations

  1. Baker Heart Research Institute, Melbourne, 8008, Victoria, Australia

    Steffen U Eisenhardt, Nicole Bassler & Karlheinz Peter

  2. Department of Cardiology, University of Freiburg, Freiburg, 79106, Germany

    Meike Schwarz

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  1. Steffen U Eisenhardt
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  2. Meike Schwarz
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  4. Karlheinz Peter
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Correspondence to Steffen U Eisenhardt.

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Eisenhardt, S., Schwarz, M., Bassler, N. et al. Subtractive single-chain antibody (scFv) phage-display: tailoring phage-display for high specificity against function-specific conformations of cell membrane molecules. Nat Protoc 2, 3063–3073 (2007). https://doi.org/10.1038/nprot.2007.455

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