Selection Of Single Domain Antibodies Research Articles

Phage display selection of human single domain antibodies towards karilysin, a metalloproteinase and secreted virulence factor from Tannerella forsythia

Metalloproteases derived from microbial pathogens are important virulence factors contributing to evasion of antimicrobial mechanisms of the innate immune system.Karilysin is a metalloprotease recently discovered in the periodonto-pathogen Tanneralla forsythia and currently no monoclonal antibodies exist against karilysin, which is a gap in the molecular toolbox for structure-function studies of karilysin.In this study we have used phage display for fast selection of single domain antibodies (VHs) towards the karilysin catalytic domain (Kly18) using a human domain library based on a VH framework. Following five panning rounds, phage clones were sequenced, and three unique sequences were identified (termed Kly18-VHI-III). Initial screens identified Kly18-VHII-phage as capable of inhibiting Kly18 proteolytic activity. The free Kly18-VHII was expressed in the periplasmic space of BL21 E. coli using the pET22b (+) vector and purified by IMAC and the inhibition capacity of purified Kly18-VHII was confirmed. The data presented in this study provides input to the molecular toolbox for the study of karilysin and Kly18-VHII could serve as a lead molecule for development of a karilysin-specific inhibitor.

Selection of Single-Domain Antibodies towards Western Equine Encephalitis Virus.

In this work, we describe the selection and characterization of single-domain antibodies (sdAb) towards the E2/E3E2 envelope protein of the Western equine encephalitis virus (WEEV). Our purpose was to identify novel recognition elements which could be used for the detection, diagnosis, and perhaps treatment of western equine encephalitis (WEE). To achieve this goal, we prepared an immune phage display library derived from the peripheral blood lymphocytes of a llama that had been immunized with an equine vaccine that includes killed WEEV (West Nile Innovator + VEWT). This library was panned against recombinant envelope (E2/E3E2) protein from WEEV, and seven representative sdAb from the five identified sequence families were characterized. The specificity, affinity, and melting point of each sdAb was determined, and their ability to detect the recombinant protein in a MagPlex sandwich immunoassay was confirmed. Thus, these new binders represent novel recognition elements for the E2/E3E2 proteins of WEEV that are available to the research community for further investigation into their applicability for use in the diagnosis or treatment of WEE.

Selection of Single Domain Antibodies from Immune Libraries Displayed on the Surface of E. coli Cells with Two β-Domains of Opposite Topologies

Screening of antibody (Ab) libraries by direct display on the surface of E. coli cells is hampered by the presence of the outer membrane (OM). In this work we demonstrate that the native β-domains of EhaA autotransporter and intimin, two proteins from enterohemorrhagic E. coli O157:H7 (EHEC) with opposite topologies in the OM, are effective systems for the display of immune libraries of single domain Abs (sdAbs) from camelids (nanobodies or VHH) on the surface of E. coli K-12 cells and for the selection of high affinity sdAbs using magnetic cell sorting (MACS). We analyzed the capacity of EhaA and intimin β-domains to display individual sdAbs and sdAb libraries obtained after immunization with the extracellular domain of the translocated intimin receptor from EHEC (TirMEHEC). We demonstrated that both systems displayed functional sdAbs on the surface of E. coli cells with little proteolysis and cellular toxicity, although E. coli cells displaying sdAbs with the β-domain of intimin showed higher antigen-binding capacity. Both E. coli display libraries were screened for TirMEHEC binding clones by MACS. High affinity binders were selected by both display systems, although more efficiently with the intimin β-domain. The specificity of the selected clones against TirMEHEC was demonstrated by flow cytometry of E. coli cells, along with ELISA and surface plasmon resonance with purified sdAbs. Finally, we employed the E. coli cell display systems to provide an estimation of the affinity of the selected sdAb by flow cytometry analysis under equilibrium conditions.