Bacterial Strains and Reagents:
… Activated protective antigen was procured form List Biological Laboratories (USA). …
2.6. Binding to LF and Native-PAGE
For the confirmation of functional activity of the expressed PA, 2g of trypsin digested PA was incubated with 2g of LF at room temperature for 30 minutes. The samples were run on 4% native polyacrylamide gel at 4C and visualized by staining the gel with coomassie brilliant blue.
2.7. Animal Immunization
Six female Balb/c mice were procured from Institutional Animal Care Center, DRDE, and were provided with food and water ad libitum. Each mouse was immunized subcutaneously with 10g PA adsorbed on aluminium hydroxide adjuvant on days 0, 14, and 28. Mice receiving only PBS were taken as control. All mice were bled prior to first immunization on day 0 and on 35th day. Serum was separated and stored at 20C until further use.
2.8. Enzyme Linked Immunosorbent Assay
To test the reactivity of PA immunized sera, indirect ELISA was performed by coating 1gmL1 PA in ELISA plate overnight at 4C. After blocking with 5% skim milk for one hour, serial twofold dilution of PA immunized sera starting from 1:1000 was added and incubated for one hour at room temperature. …
2.9. Cell Culture
Mouse macrophage cell line J774.1 was procured from National centre for cell sciences, Pune, India and was maintained in high glucose Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS) and 100UmL1 penicillin and streptomycin as antibiotics. One day before the experiment 4 104 cells were seeded in a 96 well plate and incubated at 37C in presence of 5% humidified CO2. The next day spent media was replaced by a fresh medium containing gradient concentration of soluble PA and native PA of B. anthracis from List Biologicals (0.005gmL1 to 5gmL1) and was incubated at 37C along with 0.125gmL1 LF. After 4 hours, 20L of 5mgmL1 MTT (3-(4,5 dimethylthiazol-2 yl)-2,5 diphenyl tetrazolium bromide) was added to each well and further incubated for 30 minutes.