ANTIBODY LIBRARY GENERATION FOR PHAGE DISPLAY

Naïve antibody libraries

Thanks to its expertise, ProteoGenix is able to provide you with diverse high-quality naïve libraries generated through our unique platform of antibody library generation for phage display applications. Our libraries contain a vast repertoire of antibodies in many formats (scFv, Fab, or even VHH) and from many different species.

We have more libraries in scFv format because they are genetically more stable than the Fab format.
But the advantage of the Fab format is that it is possible to select in a more simple way the binders (by avoiding the risk of formation of dimers or trimers).

More libraries of other species may be available upon request.

Screening from a naive library

A screening of the chosen library is carried out by ELISA in order to identify high-affinity binders of the protein of interest. During the interaction between the binders and the protein, binders will remain attached to the well while non-binders will be eliminated by washing.

Binders are eluted and then recovered to infect bacteria (with a helper phage) and increase their quantity. The repetition of this method is called « panning » (or also called « biopanning ») and allows to select the best binders, which are then isolated for validation in ELISA.

At the end, the DNA of the best binders is sequenced thereby allowing to produce them as recombinant proteins. ProteoGenix generally performs 3 to 5 steps of panning to ensure you get the best possible candidates.

Immune antibody libraries

ProteoGenix can generate high-diversity antibody libraries for phage display providing you with the most refined binders possible for your project. The construction of an immune library potentially increases the affinity of your final antibody against the targeted protein.

Construction and screening of an immune library

The library capacity is measured at 10⁹ to 10¹⁶ different binders. We are able to offer you several types of libraries: scFv, Fab and VHH. The library screening is performed to identify binders with the best affinity.
Construction and screening of an immune library can be tailored to your research for high specificity and stability.

The screening process is based on our standard protocol of panning/biopanning and ProteoGenix can adapt this protocol to any request. You can find out more about the stages of biopanning in the screening from a naive library.

After 3 to 5 rounds of biopanning, ProteoGenix provides you with several binders (3 to 10) whilst giving you the option to ask for more if required.

At ProteoGenix, the choice of building and screening an immune library by phage display will allow you to produce monoclonal antibodies with the highest affinity for any species.

For more information or to get a quote for our phage display services with the use of immune libraries, don’t hesitate to contact us.

What is an antibody library?

The creation of the first antibody libraries preceded the development of antibody display technologies. Their creation was made possible by the advance of molecular biology techniques and genomic studies. Today, it is possible to recover the antibody repertoire from any species and from hosts in any immune state (naïve/immune) to build high diversity and highly functional libraries adapted to display technologies. In this article, we explain how these libraries are generated and how they are used in antibody display. Check our frequently asked questions (FAQs) page about phage display for a complete overview of all steps of this robust process for antibody generation.

How were antibody libraries created?

The onset of advanced molecular biology techniques created new possibilities to leverage the natural ingenuity of biological systems. In the late 1980s, this led to the construction of the first antibody libraries. These libraries were created by amplifying antibody encoding genes from lymphocytes, cloning them, and expressing them in bacterial systems.
These early attempts at transferring immune and naïve repertoires to the lab preceded the creation of antibody display techniques. At that time, the libraries would be either expressed as single-chain variable fragments (scFv) or as antibody combinatorial libraries of Fab fragments in bacteria. However, screening these libraries was quite labor-intensive and involved the use of radio-labeled antigens to identify the bacterial clones expressing antibodies with the highest affinity and specificity.
Shortly after these initial breakthroughs, the phage display technology was born. It created not only the possibility to screen libraries efficiently but also the ability to engineer antibodies by random and site-directed mutagenesis.

How are antibody libraries built?

Genomic studies led to the discovery of conserved regions in antibody-encoding genes across different species. This discovery further prompted the development of primers targeting these regions, allowing the amplification of variable light and heavy chains (VL and VH, respectively) for many different host species, and subsequent cloning antibody-encoding genes using standard molecular biology techniques.
These VL and VH repertoires are commonly generated from naïve or immune hosts by harvesting either B lymphocytes (B cells) or peripheral blood mononuclear cells (PBMC) which consist of a mixture of T cells, B cells, and NK cells (natural killer cells), isolated from peripheral blood samples by simple density gradient centrifugation.
This initial harvesting step is followed by mRNA isolation, cDNA synthesis, and antibody gene amplification using a mixture of primers that ensures the highest antibody diversity is captured at this stage. These genes are subsequently cloned into the proper vector.
Phagemid vectors have become the most commonly used approach for antibody library generation. These vectors are typically derived from filamentous phage M13, a single-stranded DNA (ssDNA) phage with a circular genome capable of infecting strains of Escherichia coli harboring the F (fertility) pili. This phage carries all genes necessary for infection, replication, assembly, as well as all structural proteins. In contrast, phagemid vectors encode only a “minimal” version of the M13 phage containing key elements such as:
• a selection marker (i.e. antibiotic resistance gene)
• genes encoding for specific antibody fragments fused to one of the phage’s coat proteins
• the phage’s origin of replication – crucial for phage DNA replication

Antibody-encoding genes in the form of scFv (single-chain fragment variable regions), Fab, or VHH are typically fused to one of M13’s coat proteins – G3P, also known as pIII. About 4-5 copies of G3P are expressed on the tip of fully assembled M13 phages and these copies are responsible for binding to the F pili in E. coli.
However, fused copies of G3P cannot interact with E. coli’s F pili. Because, when all copies of the G3P protein are coupled to antibody fragments, the phage is unable to infect E. coli and, thus, no replication can take place.
The most common approach to circumventing this issue is the use phagemid vectors in conjugation with helper phages. Helpers contain all genes of the M13 phage essential for capsid production, assembly, replication, and budding, including the wild-type unfused GP3.
In E. coli, helper phage vectors compete with the phagemid encoding the G3P-antibody fusion protein. As a result, the majority of phage particles either have no antibody fragment displayed on its surface or display a single G3P-antibody fusion protein alongside wild-type G3P. The mixed phenotype allows the reconciliation of the phage’s role in antibody display with its ability to infect and replicate in E. coli.

The phagemid system also ensures that only a single antibody fragment is displayed per phage particle which, in turn, ensures only the binders with the highest affinity towards a specific antigen are enriched during the multiple rounds of biopanning.

What are the advantages of the phagemid system for antibody library construction?

Antibody libraries based on phagemid vectors have a critical advantage over full phage libraries – a higher transformation efficiency. Due to its “minimal” size, E. coli is more easily transformed by phagemid than by phage vectors. Moreover, since most resulting phage particles derived from phagemid libraries have only a single antibody fragment on its surface (monovalent), the selection of high-affinity antibodies is more efficient than when multivalent formats are used (phage vectors). In other words, the monovalent format ensures that antibodies with weak affinity can be easily washed off during panning campaigns.

However, because phagemid libraries need to be amplified by direct competition with a helper phage with wild-type G3P, the vast majority of the phages will be devoid of antibody fragments (“bald” phages”) but still retain their infectivity.

To circumvent this issue and reduce the abundance of “bald” phages, a trypsin cleavage site is often introduced into the wild-type G3P. This modification ensures that, after panning and enrichment of high-affinity antibody fragments, trypsin treatment can be performed to render “bald” phages non-infective. The same trypsin cleavage site is also added to the linker region between the antibody fragment and G3P, so that antibody fragments can be cleaved from positive phages to allow them to infect E. coli.