target specificity and less off-target cytotoxicity [2]. Quorum
quenching using antibodies would not only block the signalling
cascade and virulence factors production, but also neutralize
3OC 12 -HSL, a molecule that beyond its action as QS signal
has immunomodulatory effects important forP. aeruginosainfec-
tions [4].
Displaying antibody binding sites (proteins or peptides) on the
surface of bacteriophage by fusing the antibody gene to one of the
phage coat proteins and its selection based on the antigen binding
of individual clones is generalized as phage display. Phage display
antibody libraries are constructed by PCR-based cloning of the
antibody heavy chain variable domain (VH) and light chain variable
domain (VL) repertoires by random pairing into a phage or pha-
gemid vector system and displayed on the surface of bacteriophage
to facilitate the combinatorial selection of the best antibody bind-
ing site. This procedure has been widely used in the antibody
engineering field as a technique to mimic B cells, which in the
body act as self-replicating systems containing antibody genes
encoding a specific antibody displayed on its surface [5]. A key
feature of the phage display approach is the similar linking of
genotype (phagemid vector) and phenotype (phage coat protein-
antibody fragment), which allows immediate access to the
corresponding gene sequences of selected antibodies. The
sequences encoding the epitope binding sites can be easily sub-
cloned into various antibody formats based on downstream appli-
cations. Phage display technologies have been successfully used for
the development of therapeutic antibody candidates over the last
20 years with more and more products now also entering the
diagnostic and research markets [6].
This chapter describes the methods involved in the construc-
tion of an immunized sheep phage display library for the generation
of antibodies with high affinity and sensitivity towards AHL signal
molecules and for the characterization of their binding properties
using ELISA based techniques. The overall procedure used to
generate the anti-AHL antibodies is schematized in Fig.1.In
brief, sheep are immunized with AHLs conjugated to carrier pro-
teins and antibody genes derived from the immunized sheep are
used to generate a phage display antibody library. High sensitivity
single chain antibody fragments (scFv) are isolated from the library
using “smart selection strategies” and reformatted into single chain
antibodies (scAbs).
The neutralization potential and protective effect of anti-
quorum sensing antibodies is validated by monitoring the inhibi-
tion of a main QS-dependent virulence factor ofP. aeruginosa
(i.e., elastase) and ofP. aeruginosavirulence in a simple in vivo
infection model (i.e., slow killing of the nematodeCaenorhabditis
elegans).326 Soumya Palliyil