Journal: MicrobiologyOpen
Article Title: Recombinant Limosilactobacillus ( Lactobacillus ) delivering nanobodies against Clostridium perfringens NetB and alpha toxin confers potential protection from necrotic enteritis
doi: 10.1002/mbo3.1270
Figure Lengend Snippet: Neutralization of NetB activity by L. reuteri secreted Nbs. (a) A twofold dilution series of precipitated VHH antibodies (5 µM) was preincubated with recombinant NetB, after which 1% chicken erythrocytes was added. The optical density of 100% hemolysis was obtained by diluting the chicken erythrocytes in distilled water. As a control, chicken erythrocytes were incubated with NetB, but without Nbs was used. This resulted in 100% hemolysis (OD 570 = 0.54). A NetB positive control (NetB in PBS) resulted in a mean OD 570 of 0.52 and a PBS negative control (PBS with no NetB and Nbs) yielded a mean OD 570 of 0.05. As the initial amounts of L. reuteri and E. coli purified Nbs used for the assay were different, normalized OD 570 values are shown. (b) Western blot analysis binding of anti‐NetB Nb to NetB in the culture supernatant from different C. perfringens clinical isolates. 1, Ladder; 2, NetB positive control (5 µg); 3, C. perfringens JP1011 overnight culture supernatant (10 µl); 4, C. perfringens JP1011 overnight culture supernatant, 10× concentrated (10 µl); 5, C. perfringens JP1011 mid‐log culture supernatant (10 µl); 6, C. perfringens JP1011 midleg culture supernatant, 10× concentrated (10 µl); 7, C. perfringens CP1‐1 overnight culture supernatant (10 µl). The data represent the mean ± SD of the results of three independent experiments. Nbs, nanobodies; PBS, phosphate‐buffered saline; VHH, Variable domain of the Heavy chain of Heavy chain
Article Snippet: Fresh C. perfringens (strain CL‐15, Type A, α and β2 toxins) challenge inoculum was prepared every day from a stock culture in fluid thioglycollate broth overnight at 35°C under anaerobic conditions by Microbial Research Inc. C. perfringens was administered by oral gavage at a target dose of approximately 1 × 10 8 CFUs/ml (1 ml/chick) on 17, and 18 days of age.
Techniques: Neutralization, Activity Assay, Recombinant, Control, Incubation, Positive Control, Negative Control, Purification, Western Blot, Binding Assay, Saline