Phage-displayed synthetic library and screening platform for nanobody discovery
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Curated by eLife
eLife Assessment
This valuable study presents an alternative platform for nanobody discovery using phage-displayed synthetic libraries. The evidence supporting the platform is compelling, which is used to isolate and validate nanobodies targeting Drosophila secreted proteins. By making this library openly accessible, this provides an excellent resource to the wider scientific community. The detailed protocol used in this manuscript, associated with various methods for nanobody screening, provides an alternative and reliable platform for nanobody discovery.
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Abstract
Nanobodies, single-domain antibodies derived from camelid heavy-chain antibodies, are known for their high affinity, stability, and small size, which make them useful in biological research and therapeutic applications. However, traditional nanobody generation methods rely on camelid immunization, which can be costly and time- consuming, restricting their practical feasibility. In this study, we present a phage- displayed synthetic library for nanobody discovery. To validate this approach, we screened nanobodies targeting various Drosophila secreted proteins. The nanobodies identified were suitable for applications such as immunostaining and immunoblotting, supporting the phage-displayed synthetic library as a versatile platform for nanobody development. To address the challenge of limited accessibility to high-quality synthetic libraries, this library will be openly available for non-profit use.
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eLife Assessment
This valuable study presents an alternative platform for nanobody discovery using phage-displayed synthetic libraries. The evidence supporting the platform is compelling, which is used to isolate and validate nanobodies targeting Drosophila secreted proteins. By making this library openly accessible, this provides an excellent resource to the wider scientific community. The detailed protocol used in this manuscript, associated with various methods for nanobody screening, provides an alternative and reliable platform for nanobody discovery.
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Reviewer #1 (Public review):
Summary:
Using highly specific antibody reagents for biological research is of prime importance. In the past few years, novel approaches have been proposed to gain easier access to such reagents. This manuscript describes an important step forward toward the rapid and widespread isolation of antibody reagents. Via the refinement and improvement of previous approaches, the Perrimon lab describes a novel phage-displayed synthetic library for nanobody isolation. They used the library to isolate nanobodies targeting Drosophila secreted proteins. They used these nanobodies in immunostainings and immunoblottings, as well as in tissue immunostainings and live cell assays (by tethering the antigens on the cell surface).
Since the library is made freely available, it will contribute to gaining access to better …
Reviewer #1 (Public review):
Summary:
Using highly specific antibody reagents for biological research is of prime importance. In the past few years, novel approaches have been proposed to gain easier access to such reagents. This manuscript describes an important step forward toward the rapid and widespread isolation of antibody reagents. Via the refinement and improvement of previous approaches, the Perrimon lab describes a novel phage-displayed synthetic library for nanobody isolation. They used the library to isolate nanobodies targeting Drosophila secreted proteins. They used these nanobodies in immunostainings and immunoblottings, as well as in tissue immunostainings and live cell assays (by tethering the antigens on the cell surface).
Since the library is made freely available, it will contribute to gaining access to better research reagents for non-profit use, an important step towards the democratisation of science.
Strengths:
(1) New design for a phage-displayed library of high content.
(2) Isolation of valuble novel tools.
(3) Detailed description of the methods such that they can be used by many other labs.
Weaknesses:
My comments largely concentrate on the representation of the data in the different Figures.
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Reviewer #2 (Public review):
Summary:
In this study, the authors propose an alternative platform for nanobody discovery using a phage-displayed synthetic library. The authors relied on DNA templates originally created by McMahon et al. (2018) to build the yeast-displayed synthetic library. To validate their platform, the authors screened for nanobodies against 8 Drosophila secreted proteins. Nanobody screening has been performed with phage-displayed nanobody libraries followed by an enzyme-linked immunosorbent assay (ELISA) to validate positive hits. Nanobodies with higher affinity have been tested for immunostaining and immunoblotting applications using Drosophila adult guts and hemolymph, respectively.
Strengths:
The authors presented a detailed protocol with various and complementary approaches to select nanobodies and test their …
Reviewer #2 (Public review):
Summary:
In this study, the authors propose an alternative platform for nanobody discovery using a phage-displayed synthetic library. The authors relied on DNA templates originally created by McMahon et al. (2018) to build the yeast-displayed synthetic library. To validate their platform, the authors screened for nanobodies against 8 Drosophila secreted proteins. Nanobody screening has been performed with phage-displayed nanobody libraries followed by an enzyme-linked immunosorbent assay (ELISA) to validate positive hits. Nanobodies with higher affinity have been tested for immunostaining and immunoblotting applications using Drosophila adult guts and hemolymph, respectively.
Strengths:
The authors presented a detailed protocol with various and complementary approaches to select nanobodies and test their application for immunostaining and immunoblotting experiments. Data are convincing and the manuscript is well-written, clear, and easy to read.
Weaknesses:
On the eight Drosophila secreted proteins selected to screen for nanobodies, the authors failed to identify nanobodies for three of them. While the authors mentioned potential improvements of the protocol in the discussion, none of them have been tested in this manuscript.
The same comment applies to the experiments using membrane-tethered forms of the antigens to test the affinity of nanobodies identified by ELISA. Many nanobodies fail to recognize the antigens. While authors suggested a low affinity of these nanobodies for their antigens, this hypothesis has not been tested in the manuscript.
Improving the protocol at each step for nanobody selection would greatly increase the success rate for the discovery of nanobodies with high affinity.
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