Recombinant Human Anti-C. difficile TcdB Antibody (PA41) (CAT#: PABC-308)

Figure 1 PA41 inhibits TcdB-induced Rac1 glucosylation in Caco-2 cells but not in vitro.

A, in vitro glucosylation of GST-Rac1 (2 μM) by TcdB-027-GTD (10 nM) in the presence of [¹⁴C]UDP-glucose (24 μM) and increasing concentrations of PA41 mAb. Controls are shown in lane 1 (GTD with Rac1) and lane 2 (Rac1 only). B, Rac1 glucosylation by TcdB(WT) or TcdB(Y323H) in the presence of isotype control or PA41 mAb was assessed in Caco-2 cells. Cells that did not receive toxin or antibody were used as controls. C, experiments in B were quantified by densitometry, and the extent of Rac1 glucosylation was determined by normalizing the unglucosylated and total Rac1 levels. Means ± S.D. (n = 3) are shown. D and E, similar experiment performed with Fab fragments. The antibody concentration was doubled to keep the available number of antigen-binding sites similar between the mAb and Fab experiments. Error bars represent S.D.

Kroh, H. K., Chandrasekaran, R., Zhang, Z., Rosenthal, K., Woods, R., Jin, X., ... & Spiller, B. W. (2018). A neutralizing antibody that blocks delivery of the enzymatic cargo of Clostridium difficile toxin TcdB into host cells. Journal of Biological Chemistry, 293(3), 941-952.

Figure 2 PA41 inhibits pore formation and the subsequent delivery of the GTD into the cytosol.

A, pore formation on biological membranes. TcdB holotoxin alone (1 nM) or preincubated with PA41 mAb or Fab (10 nM) was applied to CHO-K1 cells preloaded with 86Rb+, and rubidium release was compared at pH 7.5 and 4.5. Means ± S.D. (n = 6) are shown. B, Caco-2 monolayers were intoxicated with 3xFLAG-TcdB (25 nM) preincubated with equimolar (25 nM) amounts of control or PA41 mAb. Toxin cleavage assays were performed as described under "Experimental procedures." Blots were probed with antibodies against the toxin (anti-FLAG; detects both internalized holotoxin and free GTD), unglucosylated and total Rac1, and GAPDH. C, the fraction of the internalized toxin that is cleaved and released in each condition was determined by normalizing the free GTD signal to the corresponding internalized holotoxin signal. For each time point, the fractional GTD release measurements were normalized to that of the isotype mAb controls to obtain the relative GTD cleavage. D and E, similar experiments performed with the Fab fragments. Means ± S.D. (n = 3) are shown. Error bars represent S.D.

Kroh, H. K., Chandrasekaran, R., Zhang, Z., Rosenthal, K., Woods, R., Jin, X., ... & Spiller, B. W. (2018). A neutralizing antibody that blocks delivery of the enzymatic cargo of Clostridium difficile toxin TcdB into host cells. Journal of Biological Chemistry, 293(3), 941-952.