Automated Panning and Screening Procedure on Microplates for Antibody Generation from Phage Display Libraries

Turunen, Laura; Takkinen, Kristiina; Söderlund, Hans; Pulli, Timo

doi:10.1177/1087057108330113

Cited by 54 publications

(36 citation statements)

References 29 publications

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“…The selection assay should be able to differentiate between targeted and non-targeted binding phages, while a screening assay should accurately identify individual clones in an automated fashion with high specificity [2, 23]. In this study, small peptide- and glycopeptide-based targets were chosen to select binders for a specific epitope.…”

Section: Discussionmentioning

confidence: 99%

A Combinatory Antibody–Antigen Microarray Assay for High-Content Screening of Single-Chain Fragment Variable Clones from Recombinant Libraries

et al. 2016

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We have developed a combinatory antibody–antigen microarray for direct screening of multiple single-chain fragment variable (scFv) clones with no need for pre-purification or enrichment before screening. The straightforward workflow allows for early selection of binders to predefined peptide and glycopeptide targets. A capture antibody is contact printed on microarray slides, side by side with the antigens of interest. A large number of scFv clones, in supernatants, are printed on top of the capture antibody and the antigen in a “spot-on-spot” print. The printed scFv clones, which bind to the capture antibody, are detected using biotinylated antigen, while the binding of scFv clones to the printed antigen is detected through a mouse anti-tag antibody. Two different analyses are thus performed on the same slide, generating two kinds of information: one on the ability of an individual scFv clone to bind to the soluble form of the antigen, which may favour selection for higher affinity rather than avidity, while the other allows the identification of large numbers of clones, simultaneously, due to the binding of scFv clones to densely presented antigens, thus providing an overall increased hit rate. The functionality of the new screening approach was illustrated through the generation of antibodies against peptides from the chaperone complex Ku70/Ku80 and the GalNAcα-serine/threonine epitope on the IgA1 alpha chain hinge region. In total, 659 scFv clones were screened with a hit rate of approximately 20%. This approach allowed the identification of functional antibodies in both cases, illustrating the usefulness and capacity of this combinatory microarray screening technique for efficient analysis and validation of antibodies at an early stage of antibody generation.

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Section: Discussionmentioning

confidence: 99%

A Combinatory Antibody–Antigen Microarray Assay for High-Content Screening of Single-Chain Fragment Variable Clones from Recombinant Libraries

et al. 2016

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“…The VTT naïve human scFv phage display libraries V H V k and V H V l (both containing approximately 10 8 clones) have been cloned from a pool of human lymphocytes of 50 healthy donors (Reinman et al, 2003;Pulli et al, 2005;Weber et al, 2005;Turunen et al, 2009). scFv libraries were enriched against a testosterone-3-CMO-BSA conjugate (TES-BSA, Sigma) in two panning rounds.…”

Section: Isolation Of the 5f2 Anti-testosterone Clonementioning

confidence: 99%

The testosterone binding mechanism of an antibody derived from a naïve human scFv library

Niemi

Takkinen

Amundsen

et al. 2011

J of Molecular Recognition

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A testosterone binding scFv antibody was isolated from a naïve human library with a modest size of 10(8) clones. The crystal structure of the Fab fragment form of the 5F2 antibody clone complexed with testosterone determined at 1.5 Å resolution shows that the hapten is bound deeply in the antibody binding pocket. In addition to the interactions with framework residues only CDR-L3 and CDR-H3 loops interact with testosterone and the heavy chain forms the majority of the contacts with the hapten. The testosterone binding site of the 5F2 antibody with a high abundance of aromatic amino acid residues shows similarity with an in vitro affinity matured antibody having around 300 times higher affinity. The moderate affinity of the 5F2 antibody originates from the different orientation of the hapten and few light chain contacts. This is the first three-dimensional structure of a human steroid hormone binding antibody that has been isolated from a naïve human repertoire.

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“…Phage display is well suited for this application due to its rapid and in vitro nature. In particular, robust antibody phage display methods, which employ highly diverse (>10 9 ) single-chain fragment variable (scFv) or fragment antigen binding (Fab) libraries, have recently been developed 5–8 and these methods have proven amenable to high-throughput automation 9, 10 . Advances in high-throughput phage display have created the pressing need for novel strategies such as new helper phages 11–13 , library diversification strategies 8, 14 , and reformatting methods for downstream expression 15, 16 to upgrade and improve the antibody generation pipeline.…”

Section: Introductionmentioning

confidence: 99%

An Improved Single-Chain Fab Platform for Efficient Display and Recombinant Expression

Koerber

Hornsby

Wells

2015

Journal of Molecular Biology

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Antibody phage display libraries combined with high-throughput selections have recently demonstrated tremendous promise to create the next generation of renewable, recombinant antibodies to study proteins and their many post-translational modification states; however many challenges still remain, such as optimized antibody scaffolds. Recently, a single-chain Fab (scFab) format, in which the carboxy-terminus of the light chain is linked to the amino-terminus of the heavy chain, was described to potentially combine the high display levels of a single-chain Fv with the high stability of purified Fabs. However, this format required removal of the interchain disulfide bond to achieve modest display levels and subsequent bacterial expression resulted in high levels of aggregated scFab, hindering further use of scFabs. Here, we developed an improved scFab format that retains the interchain disulfide bond by increasing the linker length between the light and heavy chains to improve display and bacterial expression levels to 1–3 mg per liter. Furthermore, rerouting of the scFab to the co-translational signal recognition particle (SRP) pathway combined with reengineering of the signal peptide sequence results in display levels 24-fold above the original scFab format and 3-fold above parent Fab levels. This optimized scFab scaffold can be easily reformatted in a single step for expression in a bacterial or mammalian host to produce stable (81°C Tm), predominantly monomeric (>90%) antibodies at a high yield. Ultimately, this new scFab format will advance high-throughput antibody generation platforms to discover the next generation of research and therapeutic antibodies.

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Automated Panning and Screening Procedure on Microplates for Antibody Generation from Phage Display Libraries

Cited by 54 publications

References 29 publications

A Combinatory Antibody–Antigen Microarray Assay for High-Content Screening of Single-Chain Fragment Variable Clones from Recombinant Libraries

A Combinatory Antibody–Antigen Microarray Assay for High-Content Screening of Single-Chain Fragment Variable Clones from Recombinant Libraries

The testosterone binding mechanism of an antibody derived from a naïve human scFv library

An Improved Single-Chain Fab Platform for Efficient Display and Recombinant Expression

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