Synthetic antibody libraries are constructed from scratch using designed synthetic DNA. Precise control over design enables the use of highly optimized human frameworks and the introduction of defined chemical diversity at positions that are most likely to contribute to antigen recognition. We describe complete methods for the design, construction, and application of simplified synthetic antibody libraries built on a single human framework with diversity restricted to four complementarity-determining regions and two amino acids (tyrosine and serine). Despite the extreme simplicity of design, these libraries are capable of generating specific antibodies against diverse protein antigens. Moreover, the same methods can be used to build more complex libraries that can produce synthetic antibodies with affinities and specificities beyond the scope of natural antibodies. Most importantly, these simplified methods rely on standard supplies, equipment, and methods that are accessible to any molecular biology laboratory.
"For a typical library construction, an oligonucleotide library is designed complementary to the ssDNA with flanking regions corresponding to the phagemid vector. The oligonucleotides are then annealed to the vector and the complementary strand is synthesized and ligated together to form a circular, double stranded DNA vector, which is then electroporated into E. coli . "
[Show abstract][Hide abstract] ABSTRACT: Phage display is a powerful technique for profiling specificities of peptide binding domains. The method is suited for the identification of high-affinity ligands with inhibitor potential when using highly diverse combinatorial peptide phage libraries. Such experiments further provide consensus motifs for genome-wide scanning of ligands of potential biological relevance. A complementary but considerably less explored approach is to display expression products of genomic DNA, cDNA, open reading frames (ORFs), or oligonucleotide libraries designed to encode defined regions of a target proteome on phage particles. One of the main applications of such proteomic libraries has been the elucidation of antibody epitopes. This review is focused on the use of proteomic phage display to uncover protein-protein interactions of potential relevance for cellular function. The method is particularly suited for the discovery of interactions between peptide binding domains and their targets. We discuss the largely unexplored potential of this method in the discovery of domain-motif interactions of potential biological relevance.
BioMed Research International 09/2014; 2014:176172. DOI:10.1155/2014/176172 · 2.71 Impact Factor
"Antibodies have been extensively studied and many experimental methods are available for their construction, including hybridoma technology , phage display , yeast surface display , and synthetic libraries  (see  for a review). Immunoinformatics tools have been developed to identify the genes used to create antibodies from nucleotide sequences [12-17], amino acid sequences [17-22], and three-dimensional structures [19,23]. "
[Show abstract][Hide abstract] ABSTRACT: Background
The de novo design of a novel protein with a particular function remains a formidable challenge with only isolated and hard-to-repeat successes to date. Due to their many structurally conserved features, antibodies are a family of proteins amenable to predictable rational design. Design algorithms must consider the structural diversity of possible naturally occurring antibodies. The human immune system samples this design space (2 1012) by randomly combining variable, diversity, and joining genes in a process known as V-(D)-J recombination.
By analyzing structural features found in affinity matured antibodies, a database of Modular Antibody Parts (MAPs) analogous to the variable, diversity, and joining genes has been constructed for the prediction of antibody tertiary structures. The database contains 929 parts constructed from an analysis of 1168 human, humanized, chimeric, and mouse antibody structures and encompasses all currently observed structural diversity of antibodies.
The generation of 260 antibody structures shows that the MAPs database can be used to reliably predict antibody tertiary structures with an average all-atom RMSD of 1.9 Å. Using the broadly neutralizing anti-influenza antibody CH65 and anti-HIV antibody 4E10 as examples, promising starting antibodies for affinity maturation are identified and amino acid changes are traced as antibody affinity maturation occurs.
[Show abstract][Hide abstract] ABSTRACT: In order to comprehensively manipulate the human proteome we require a vast repertoire of pharmacological reagents. To address these needs we have developed repertoires of synthetic antibodies by phage display, where diversified oligonucleotides are used to modify the complementarity-determining regions (CDRs) of a human antigen-binding fragment (Fab) scaffold. As diversity is produced outside the confines of the mammalian immune system, synthetic antibody libraries allow us to bypass several limitations of hybridoma technology while improving the experimental parameters under which pharmacological reagents are produced. Here we describe the methodologies used to produce synthetic antibody libraries from a single human framework with diversity restricted to four CDRs. These synthetic repertoires can be extremely functional as they produce highly selective, high affinity Fabs to the majority of soluble human antigens. Finally we describe selection methodologies that allow us to overcome immuno-dominance in our selections to target a variety of epitopes per antigen. Together these methodologies allow us to produce human monoclonal antibodies to manipulate the human proteome.
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.