Recombinant renewable polyclonal antibodies

Abstract

Abbreviations: CTBP, C-terminal binding protein; ELISA, enzyme linked immunosorbant assay; HCDR3, Heavy chain complementarity determining region 3; HPA, Human Protein Atlas; scFv, single chain Fv; PrESTs, Protein epitope signature tag; rrpAbs, recombinant renewable polyclonal antibodies; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TEV, tobacco etch virusOnly a small fraction of the antibodies in a traditional polyclonal antibody mixture recognize the target of interest, frequently resulting in undesirable polyreactivity. Here, we show that high-quality recombinant polyclonals, in which hundreds of different antibodies are all directed toward a target of interest, can be easily generated in vitro by combining phage and yeast display. We show that, unlike traditional polyclonals, which are limited resources, recombinant polyclonal antibodies can be amplified over one hundred million-fold without losing representation or functionality. Our protocol was tested on 9 different targets to demonstrate how the strategy allows the selective amplification of antibodies directed toward desirable target specific epitopes, such as those found in one protein but not a closely related one, and the elimination of antibodies recognizing common epitopes, without significant loss of diversity. These recombinant renewable polyclonal antibodies are usable in different assays, and can be generated in high throughput. This approach could potentially be used to develop highly specific recombinant renewable antibodies against all human gene products.

Deep sequencing in library selection projects: what insight does it bring?

“…Deep sequencing of selection outputs reveals that this is a combination of libraries not being as diverse as anticipated, and also that the recovery of unique clones poses a sampling problem: when only 96–384 clones are tested in different selection experiments, sometimes not even the ten most abundant clones can be identified [12,75]. Furthermore, when 96/384 clones are randomly picked, most are duplicates of abundant clones, while others represent single copies of far less abundant clones [76]. In our experience, such rare clones may individually comprise less than 0.001% of the selection output, and yet still be positive for the target, indicating that the only way to identify the full spectrum of binding antibodies after selection is to sequence and rank the complete output.…”

“…Although appropriate controls and negative selections usually allow the elimination of such apparently cross-reactive antibodies, NGS can also be used to identify them after selection. In a recent paper [89], polyclonal antibodies selected from a large naïve library [90] created by recombination [91] using phage/yeast display [76,92] against a series of in vitro biotinylated proteins were found to be strongly cross-reactive with other targets. Careful analysis of the cross-reactivity revealed the polyclonal antibody pool recognized proteins biotinylated using a particular kit (Lightening Link), but not if biotinylated with other kits or in vivo .…”

pMINERVA: A donor–acceptor system for the in vivo recombineering of scFv into IgG molecules

“…We are building a platform that takes advantage of fundamental molecular biology principles to enable the rapid identification of specific Abs. Our screening platform is being developed with single-chain variable fragment Abs (scFvs) but the technology is applicable to other scaffolds, including both Fabs (4) and yeast display libraries (12,(25)(26)(27). Here, we describe a vector system that enables high-throughput conversion of Ab fragments to full length immunoglobulin G (IgG) molecules that can be directly validated in standard immunoassays.…”

A Functional Idiotype/Anti-Idiotype Network Is Active in Genetically Gluten-Intolerant Individuals Negative for Both Celiac Disease–Related Intestinal Damage and Serum Autoantibodies

“…The four CGGI subjects whose biopsies served to derive the yeast-displayed Ab libraries were related to the CD patients (three parents and one sibling). To validate the results obtained with pooled sera, we collected individual sera from 10 additional CGGI subjects (F = 7, M = 3; average age 28.8 ), 10 CD patients (F = 6, M = 4; average age 12 [9][10][11][12][13][14]), 10 HLA + healthy subjects (F = 2, M = 8; average age 32 [22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40]), and 10 HLA 2 healthy subjects (F = 5, M = 5; average age 29.8…”