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antibody icon Antibody Plasmid Collection

Addgene Receives a NIH BRAIN Initiative Grant to Create an Open-Access Recombinant Antibody Resource! Coming in early 2022.

Antibody schematic

Antibodies are produced by the immune system to help defend against invaders such as bacteria, viruses, or even perceived threats such as proteins in peanuts or plant pollen. They are large proteins that bind to an antigen, a particular part of a foreign substance. In addition to being useful for our bodies to fight off intruders, antibodies are also extremely useful to researchers in a lab. When an antibody binds tightly to a specific antigen, scientists can use it to pull out a protein of choice from a mix of other proteins, visualize a protein under a microscope, or detect when the protein is present in a sample--just to name a few.

A standard antibody is made up of 4 protein chains: two large heavy chains and two small light chains. These four domains come together to form a “Y” shape, as shown in the picture to the right. The two arms of the Y structure are responsible for binding the antigen, and are called the antigen binding fragments (Fab). The tips of the Fab fragments contain highly variable regions, shown in light blue and orange, that bind to a specific antigen, triggering an immune response. The other part of the antibody is the constant region, shown in dark blue and orange, which is the same within each class of antibody. Antibodies are divided into five major classes, IgM, IgG, IgA, IgD, and IgE, based on their constant region structure and immune function.

Traditional sources of antibodies for research purposes include animals or hybridoma cells, but are difficult to generate due to expense and time. An alternative approach is to use synthetically produced recombinant antibodies, created by cloning antibody components into plasmids and expressing these in bacteria, mammalian cells, yeast, plants, or insect cells. Benefits include consistency between lots and the ability to optimize the antibody’s antigen binding sequence to improve binding and reproducibility.

Some scientists choose to work with full length recombinant monoclonal antibodies (R-mAbs) expressed in mammalian cells. Monoclonal antibodies are derived from an individual clone targeting a single antigen. Examples of R-mAbs are the monoclonal antibodies (NeuroMabs) extensively validated for neuroscience research applications from the NeuroMab/Trimmer Lab Recombinant mAb Plasmid Collection. Once the plasmids are transiently transfected into mammalian tissue culture cells, the R-mAbs are secreted into the culture media and can be collected for use.

Other scientists may choose to work with a smaller fragment of an antibody, which could offer increased stability and ease of production due to their small size. These recombinant antibody fragments can be used in experiments such as immunoprecipitation and super-resolution microscopy. Two commonly used plasmid-based antibody fragments are described below:

Antibody and scFv schematics

ScFvs (single-chain variable fragment) include parts of the variable regions of heavy (VH) and light chains (VL) fused together to form a single polypeptide. Unlike full length antibodies, which are often produced in mammalian cell cultures, scFvs are often produced in bacterial cell cultures such as E. coli. While full-length antibodies generally do not fold properly in the cytoplasm, soluble scFv antibodies have been successfully expressed. The Vale lab has created several scFv fragments which bind to the GCN4 peptide, from the SunTag system, fused to sfGFP for imaging.

Hcab and nanobody schematics

Nanobodies are small single chain antibodies that are derived from an unusual type of IgG antibody called a heavy chain antibody (HCab), which are unique to camels, llamas, alpacas and other camelids. In terms of structure, HCabs are like a pared down version of a standard IgG antibody. Their small size also allows better tissue penetration and decreases the distance between a fluorescent tag and the target antigen, which can lead to higher resolution for super-resolution microscopy. Examples include secondary antibodies from the Gorlich lab and GFP nanobodies from the Cepko lab.

Cloning and Expressing Antibodies

The following table highlights plasmid collections that can be used to create and express plasmid-based antibodies.

Description Article PI
One-step assembly of antibody heavy- and light-chain DNAs into a single mammalian expression vector A tool kit for rapid cloning and expression of recombinant antibodies. Sci Rep. 2014 Jul 30;4:5885. Andrew Beavil
Cloning and mammalian expression of mouse and human IgG monoclonals with reduced immune effector functions Expression of IgG Monoclonals with Engineered Immune Effector Functions. Methods Mol Biol. 2018;1827:313-334. Daniel Christ
pComb3 phage display vector system for producing human antibodies Synthetic Human Antibodies: Selecting and Evolving Functional Proteins. METHODS: A Companion to Methods in Enzymology 8, 94–103 (1995). Carlos Barbas
Variant of the pComb3 phage display vector system for isolation of scFv, diabody and Fab fragment libraries from chickens Methods for the generation of chicken monoclonal antibody fragments by phage display. J Immunol Methods. 2000 Aug 28;242(1-2):159-81. Carlos Barbas
Vector system for expression, dimerisation and detection of recombinant antibody fragments in the form of scFvs A simple vector system to improve performance and utilisation of recombinant antibodies. BMC Biotechnol. 2006 Dec 7;6:46. John McCafferty
CRISPR system to rapidly engineer the constant immunoglobulin domains to obtain recombinant hybridomas, which secrete antibodies as Fab' fragments, isotype-switched chimeric antibodies, and Fc-silent mutants. Functional diversification of hybridoma-produced antibodies by CRISPR/HDR genomic engineering. Sci Adv. 2019 Aug 28;5(8):eaaw1822. Scheeren and Verdoes
Backbones for mouse recombinant antibodies A panel of recombinant monoclonal antibodies against zebrafish neural receptors and secreted proteins suitable for wholemount immunostaining. Biochem Biophys Res Commun. 2015 Jan 2;456(1):527-33. Gavin Wright
pET-30-based vector dedicated to efficient scFv expression, which circumvents the problem of in-frame amber codons in scFvs A new expression vector facilitating production and functional analysis of scFv antibody fragments selected from Tomlinson I+J phagemid libraries. Immunol Lett. 2015 Oct;167(2):95-102. Joanna Bereta
Sybody Generation Toolbox for making synthetic nanobodies (sybodies) against membrane protein targets. Synthetic single domain antibodies for the conformational trapping of membrane proteins. Elife. 2018 May 24;7. and Generation of synthetic nanobodies against delicate proteins. Nat Protoc. 2020 Apr 8. Markus Seeger

Antibody Plasmids

The table below highlights plasmids that feature expression of antibodies, nanobodies or ScFvs. Use the search bar or sort buttons to find antibody plasmids for:

  • Common antigens such as GFP or mCherry
  • Monocolonals, Nanobodies, Sybodies, or ScFVs
  • Secondary antibodies (search for "anti-mouse" or "anti-rabbit")
ID Plasmid Description Gene/Insert PI

Other Resources

Addgene Blog Posts

External Resources

  • The Human Protein Atlas - Open access project that aims to map all the human proteins in cells, tissues, and organs.
  • The Antibody Society - An international, non-profit association representing individuals and organizations involved in antibody-related research and development.
  • Developmental Studies Hybridoma Bank - Sells monoclonal antibodies at cost to facilitate basic science.
  • Geneva Antibody Facility - Provides the academic community with easy, affordable access to well-characterized rAbs to generate better and more reproducible data, in a collaborative way.
  • The Antibody Registry - Gives researchers a way to universally identify antibodies used in their research.
  • Neuromab - Provides a unique neuroscience-based approach to generating mouse monoclonal antibodies (mAbs) optimized for use in mammalian brain (NeuroMabs).
  • NCI Office of Cancer Clinical Proteomics Research Antibody Portal - Provides access to a large number of standardized renewable affinity reagents (to cancer-associated targets) and accompanying characterization data.
  • SAbDab - Database of antibody structures that updates on a weekly basis.

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