Multifunctional multivalency: a focused library of polymeric cholera toxin antagonists Huu-Anh Tran, a,b Pavel I. Kitov, a Eugenia Paszkiewicz, a Joanna M. Sadowska, a and David R. Bundle a a Alberta Ingenuity Centre for Carbohydrate Science, Department of Chemistry, University of Alberta, Edmonton, AB T6G 2G2, Canada. Fax: (+1)780-492-7705; Tel: (+1)780-492-8808; E-mail: dave.bundle@ualberta.ca b TheraCarb Inc. 243–3553 31st Street N.W. Calgary, Alberta CANADA T2L 2K7 ELISA Assay for Inhibition of Heat-labile Toxin (LT) To PVC microtiter plates (Gibco BRL Inc.) solution of coating compound 9 (1 μg/mL, 100 μL/well) in PBS buffer was added and incubated at room temperature overnight, then washed (6×) with PBST (0.05 % Tween 20 in phosphate buffer saline, PBS). An inhibitor at decreasing concentrations (starting at 20 mg/mL, dilution factor 3.16; 50 μL/well) was mixed with LT (Sigma, 2 ng/mL) and the mixtures were applied to the plates. After incubation at room temperature for 2 h the plates were washed (6×) with PBST. Rabbit anti-LT polyclonal antibody (Abcam Inc., dilution 1:10000, 100 μL/well) was added, incubated at room temperature for 1 h then the plates were washed (6×) with PBST. Goat anti-rabbit IgG conjugated to horseradish peroxidase (Kirkegaard and Rerry Laboratories, dilution 1:5000, 100 μL/well) was added, incubated at room temperature for 30 min then the plates were washed (6×) with PBST. 3,3:5,5-Tetramethylbenzidine solution (TMB purchased from KPL,100 μL/well) was added. After 15 min incubation the reaction was stopped by addition of H 3 PO 4 (100 μL/well) and absorbance was measured at 450 nm. Table 1S. Inhibitory activities of heterobifunctional polymers against LT. Alkyne derivative 7.n α-Gal polymer IC 50 , μM a β-Gal polymer IC 50 , μM a 5α27 (3%) 0.00099 (h b =0.56) 5β27 (3%) 0.0011 (h b =0.8) 5α27 (5%) 0.00007 (h=0.54) 5β27 (5%) 0.0004 (h=0.95) 5α27 (9%) 0.00015 (h=1.04) 5β27 (9%) 0.00023 (h=1.3) H N O F 7.27 6α27 (6.6%) 0.0006 (h=0.76) 6β27 (6.6%) 0.0017 (h=1.0) 5α28 (5%) 0.0029 (h=1.3) 5β28 (5%) 0.0034 (h=1.1) 5α28 (9%) 0.00007 (h=1.3) 5β28 (9%) 0.00013 (h=1.4) H N O F F 7.28 6α28 (6.6%) 0.00095 (h=0.8) 6β28 (6.6%) 0.001 (h=0.74) 6α28 (15%) 0.00019 (h=0.7) 6β28 (15%) 0.00084 (h=0.7) a activities per pendant ligand. b Hill coefficient S 1 Supplementary Material (ESI) for Organic & Biomolecular Chemistry This journal is (c) The Royal Society of Chemistry 2011