Appl Microbiol Biotechnol (2009) 84:1087–1094 neutralize LT toxicity was determined by preincubating 100 µl 1 µg/L LT in Medium 199 without serum (Invitrogen) with a twofold dilution series of 10 mg/L VHH for 1 h at 37 °C. These mixtures were then transferred to the Vero cell monolayers. After a further 24-h incubation at 37 °C in a 5% CO2 atmosphere, the percentage of cells showing a CPE was microscopically determined. The VHH concentration required to inhibit the CPE on 75% of the cells was then determined. ELISAs GM1, LT, CT, and the CT subunits A (CTA) and B (CTB) were all obtained from Sigma. For all ELISAs, high-binding polystyrene 96-well plates (Greiner, Solingen, Germany) were coated overnight at 4 °C with proteins in 50 mM carbonate/bicarbonate buffer, pH 9.6. LT was biotinylated using aminohexanoyl-biotin-N-hydroxysuccinimide (Zymed, San Francisco, CA) at a protein to biotin ratio of 100 on a weight basis. The ability of VHHs to bind LT or CT holotoxins or their subunits was determined by incubation of plates coated with 1 mg/L of these antigens with twofold dilution series of 10 mg/L VHH in ELISA-buffer (1% skimmed milk; 0.05% Tween-20; 0.5 M NaCl; 2.7 mM KCl; 2.8 mM KH2PO4; 8.1 mM Na2HPO4; pH 7.4). Bound VHH was detected with peroxidase-conjugated polyclonal rabbit immunoglobulins anti-llama-immunoglobulin (Frenken et al. 2000) and staining with 3,3′,5,5′ tetramethylbenzidine (TMB). The VHH concentration resulting in an extinction at 450 nm of 0.3 was then determined by nonlinear regression analysis. The ability of anti-LT VHHs to inhibit binding of LT to GM1 was determined using an inhibition ELISA. Serial twofold dilution series of 10 mg/L VHH were preincubated with 0.1 mg/L biotinylated LT in 100 μl PBS containing 1% ovalbumin (Sigma; grade V) and 0.05% Tween-20 for 1 h at room temperature. These mixtures were then transferred to a plate coated with 0.2 mg/L GM1 and incubated for another hour. Bound biotinylated LT was then detected using peroxidase-conjugated streptavidin (Jackson Immunoresearch, West Grove, PA) and staining with TMB. The 50% inhibitory concentration (IC50) was calculated by interpolating the VHH concentration resulting in 50% of the extinction value obtained by a control incubation without VHH. SDS-PAGE VHHs (1 μg) were subjected to reducing SDS-PAGE, using precast gels (Novex, San Diego, CA), and stained using Sypro Orange and a STORM fosfor imager (Molecular Dynamics, Sunnyvale, CA). 1089 In vitro FMDV neutralization assay VHH concentrations required for neutralization of FMDV O1 Manisa were determined as described previously (Harmsen et al. 2007) using 20 tissue culture infective doses required to infect 50% of the wells (TCID50) in duplicate assays using monolayers of secondary swine kidney cells and serial twofold dilutions of 10 mg/ml VHHs. When using endoglycosidase-H-treated VHH(2)s, we used a starting concentration of 1 mg/ml VHH(2). Nucleotide sequence accession numbers Nucleotide sequences of selected anti-LT VHHs were submitted to the EMBL database under accession numbers AJ849409–AJ849413, AJ849415–AJ849427, AJ849431– AJ849438 and AJ849442 (Fig. 1). Results Isolation of LT-binding VHHs We isolated 180 clones producing VHHs that bound LT in ELISA by phage display selection from an immunized llama. After fingerprinting and sequence analysis, we obtained 27 unique VHHs (Fig. 1). Antibody variable domains, including VHHs, contain three complementarity- determining regions (CDRs) that are often directly involved in antigen binding (Muyldermans et al. 2001). CDR3 generally is most variable (Harmsen et al. 2000). Many of the 27 clones had similar CDR3 sequences, allowing the dissection into 11 CDR3 groups (A-K, Fig. 1). Antigenic specificity of LT-binding VHHs The 27 unique VHHs were expressed in yeast and purified. The VHH concentration required to inhibit 75% (IC75) of the toxic effect of LT in a Vero cell assay ranges from more than 10 mg/L for non-neutralizing clones to 0.0017 for clone LT109 (Table 1). Most neutralizing clones also inhibited binding of LT to its cellular receptor GM1, as determined by ELISA. Clone LT109 again had the lowest IC50 (Table 1). The subunit specificity of LT- binding VHHs was determined using the commercially available subunits of the related CT. Most clones cross- reacted sufficiently to CT holotoxin to enable such determination (Table 1). These VHHs required a similar VHH concentration for binding to the CT B-subunit and holotoxin, suggesting that they bind the B-subunit. The subunit specificity of LT104 and LT139 could not be determined due to insufficient cross reaction with CT (Table 1).