After blocking with 5% skim milk in Tris-buffered saline (TBS, pH 7.4), NC strips were incubated with individual lysates containing VH/VHH at 25 C for 1h. of peptide-based pertussis vaccines. Additionally, such toxin-specific nanobodies have a potential for test-driven development of a ready-to-use therapeutic in passive immunization for mitigation of disease severity. . In recent years, there has been an upsurge of whooping cough among elderly people  whose vaccination-induced protective immunity waned-off due to the lack of natural boosters caused by a decrease of circulating pathogens as a result of mass vaccination . This pertussis-causative pathogen Ciwujianoside-B secretes several virulence factors among which is the adenylate cyclase-hemolysin toxin (CyaA) that plays an important role during the early phase of infection [3,4]. CyaA is a 1706-residue long bi-functional protein which consists of an N-terminal adenylate cyclase (AC) catalytic domain (residues 1C400) and a C-terminal pore-forming or hemolysin (Hly) domain (residues 401C1706) . Upon entry into the host cells, catalytic function of the AC domain is activated by endogenous calmodulin, leading to supra-physiological levels of cAMP that would result in cell death and disruption of the host innate immune responses [5,6]. The CyaA-Hly domain which contains a hydrophobic pore-forming subdomain (residues 500C700) has the ability to form cation-selective channels causing lysis of target cells [7,8]. There is also an RTX (Repeat-in-ToXin) subdomain (residues 1006C1613) which harbors ~40 repeats of Gly-Asp-rich nonapeptides  and is organized into five structurally similar blocks (Blocks I-V) connected by linker sequences (Linkers 1C4) of variable lengths [10,11]. CyaA is stabilized by extracellular Ca2+ Ciwujianoside-B ions which serve as a structure-stabilizing bridge in a -roll structure within Ciwujianoside-B each RTX-Block region [10,11,12]. Moreover, CyaA is synthesized as an inactive precursor which requires a palmitoyl group be added at Lys983 by CyaC acyltransferase [7,13,14]. The CyaA-RTX subdomain is involved in toxin binding to target cells through the M2-integrin receptor (also known as CD11b/CD18) expressed on the surface of cells in the myeloid lineage, e.g., neutrophils and macrophages . CyaA also exerts its hemolytic activity against sheep erythrocytes, although they lack the M2-intergrin receptor, suggesting the possibility of an alternative pathway for target cell recognition via the RTX subdomain [8,11]. In addition, we have shown that the 126-kDa truncated CyaA-Hly Ciwujianoside-B fragment still retains high hemolytic activity independent of the phospholipase-A2 and botulinum neurotoxin-type A [17,18,19,20,21]. Here, attempts were made to generate CyaA-Hly-specific nanobodies from a humanized-camel VH/VHH phage-display library. After single-round bio-panning against CyaA-Hly, a total of forty phage-transformed clones were selected and subjected to PCR analysis for initial verification of the presence of VH/VHH-coding sequences. Among these selected clones, thirty-four clones were were therefore verified for their binding capability to CyaA-Hly via indirect ELISA and Western blotting. As shown in Figure 1a, lysates from eleven clones (~40%) containing VH/VHH proteins gave significant OD405 signals to the immobilized CyaA-Hly toxin above the BSA control, reflecting their high-binding activity against the target toxin. Nevertheless, subsequent analysis via Western blotting revealed that only lysates from four of these ELISA-positive clones could give rise to an intense binding signal to SDS-PAGE-separated CyaA-Hly seen as 126-kDa immuno-reactive bands (Figure 1b). The results suggest that these four CyaA-Hly-specific nanobodies were able to recognize a sequential epitope of the denatured target protein whereas the remaining ELISA-positive nanobodies GSS apparently recognized conformation-dependent epitopes that were abolished Ciwujianoside-B by SDS denaturation. Open in a separate window Figure 1 (a) Indirect ELISA results of lysates from selected clones expressing VHs/VHHs that give OD405 signals to the immobilized CyaA-hemolysin.