Clone LT105 LT106 LT109 LT109 endoHc LT128 LT131 LT132 LT141 LT143 LT101 LT104 LT111 LT116 LT133 LT129 LT139 LT103 LT124 LT130 LT154 LT155 LT156 LT161 LT163 LT165 LT176 LT168 LT170 >10b 3.3 0.0017 0.0084 >10 6.7 >10 3.3 >10 >10 >10 1.1 >10 >10 0.21 >10 0.74 0.12 0.21 0.39 0.02 0.17 0.27 0.07 0.03 1.1 1.4 0.39 ±0 ±0 ±0.0012 ±0.005 ±0 ±4.7 ±0 ±0 ±0 ±0 ±0 ±0 ±0 ±0 ±0.23 ±0 ±0.52 ±0 ±0.23 ±0.03 ±0.009 ±0.06 ±0.21 ±0.05 ±0.01 ±0.00 ±0.39 ±0.03 >10 0.028 0.021 0.06 >10 >10 >10 >10 >10 0.91 7.2 0.67 1.5 8.5 0.81 0.18 0.86 0.27 0.78 0.96 0.21 2.1 2.9 0.18 0.09 1.2 0.03 0.03 ±0 ±0.003 ±0.008 ±0.024 ±0 ±0 ±0 ±0 ±0 ±0.34 ±4.9 ±0.26 ±1.4 ±6.4 ±0.14 ±0.11 ±0.29 ±0.11 ±0.01 ±1.02 ±0.25 ±2.5 ±3.6 ±0.03 ±0.02 ±0.39 ±0.00 ±0.01 LT 0.073 0.032 0.18 NDd 0.002 0.023 0.020 0.038 0.028 0.042 0.010 0.042 0.010 0.059 0.20 0.059 0.16 0.054 0.035 0.048 0.028 0.024 0.20 0.038 0.12 0.040 0.057 0.073 CT 1.3 0.25 0.12 ND 0.098 0.023 0.024 0.019 0.040 4.6 >10 3.1 0.66 4.1 3.6 8.1 0.13 0.037 0.032 0.022 0.014 0.019 0.061 0.026 0.042 0.030 0.042 0.048 CTB 0.26 0.12 0.15 ND 0.027 0.012 0.013 0.016 0.016 1.6 8.6 0.83 0.21 1.6 2.9 >10 0.071 0.033 0.023 0.016 0.010 0.010 0.026 0.024 0.043 0.023 0.038 0.038 1091 CTA >10 >10 >10 ND >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 Appl Microbiol Biotechnol (2009) 84:1087–1094 Table 1 Toxin neutralization and antigenic specificity of LT- binding VHHs Vero cell assay IC75 Mean ± SDa (mg/L) GM1-LT ELISA IC50 Titer in ELISA (mg/L) Mean ± SD (mg/L) a SD, standard deviation b >10, the VHH showed no effect at the highest concentra- tion analyzed (10 mg/L) c endoH, sample that was degly- cosylated by endoglycosidase H treatment d ND, not determined species of approximately 15 kDa which is expected for unglycosylated VHH (Fig. 2a). Deglycosylation of LT109 by endoglycosidase H treatment resulted in only this 15-kDa species, confirming that LT109 is partially glycosylated (Fig. 2q). Densitometric comparison of bands representing glycosylated and deglycosylated LT109 revealed that about 98 % of LT109 is glycosylated. A similar analysis revealed that LT156 is not glycosylated (Fig. 2a). Deglycosylation of LT109 resulted in a fivefold- increased IC75 in a Vero cell assay and a threefold- increased IC50 in a GM1-LT inhibition ELISA (Table 1), indicating that N-glycosylation of LT109 contributes to its LT-neutralizing capacity. Enhancement of virus neutralization by glycosylation of VHHs We next analyzed the effect of VHH glycosylation on virus neutralization. For this purpose, we used two previously isolated FMDV-binding VHHs, M180, and M200, that lack potential N-glycosylation sites and neutralize FMDV in vitro (Harmsen et al. 2007). These VHHs were genetically fused to a porcine IgG binding VHH, VI-12, that contains a high-mannose-type N-glycan at position 28 (Harmsen et al. 2005a). Both yeast-produced genetic fusions (VHH2s) were also efficiently glycosylated as revealed by analysis of endoglycosidase H-treated and untreated samples (Fig. 2b). These glycosylated VHH2s neutralized FMDV at at least fourfold-lower concentrations than observed with the corresponding individual FMDV-binding VHHs and at at least 50-fold-lower concentrations than their deglycosylated counterparts (Table 2). This shows that VHH glycosylation contributes to FMDV neutralization. Discussion After isolating 27 VHHs binding to E. coli LT, a single yeast- expressed clone, LT109, was found to be N-glycosylated at a