Micro titer elisa plate reader

Both pro (IFN-γ, IL-12, IL-1β, IL-6) and anti (IL-4, IL-10) inflammatory cytokines induced in ex vivo re-stimulated splenocyte culture supernatants (from various experimental and control groups) were estimated using OptEIA sandwich ELISA kits (BD Biosciences). Briefly, 100 μl of the purified capture antibodies were adsorbed overnight on polystyrene micro-titer plates (Maxisorp, Thermo Scientific) at 4°C in the kit recommended coating buffer. Plates were washed five times with PBST and blocked with 1% BSA. After washing, 100 μl of the supernatant (isolated from cultured splenocytes after 24 h) was dispensed in each well. After incubation for the time stipulated, the plates were thoroughly washed and incubated with respective biotinylated anti-mouse detection antibody. Afterward, the plates were washed three times with PBST. Subsequently, 100 μl of streptavidin-HRP conjugate was added to each well, and the plate was incubated for 30 min at room temperature (RT). The plates were again washed three times with PBST and finally a colored complex was developed with tetra methyl benzidine (TMB). The absorbance was read at 450 nm with a micro-titer ELISA plate reader (Bio-Rad).

Both Th1 and Th2 cytokines induced in splenocytes culture supernatants (belonging to various experimental groups) upon their co-culture in the presence of the respective recall antigens were estimated using BD OptEIA sandwich ELISA kits (BD Biosciences). Briefly, 100 µl of the purified capture antibodies were adsorbed overnight on polystyrene micro-titer plates (Maxisorp, Thermo Scientific) at 4 °C in carbonate buffer of pH 9.5. Plates were washed five times with PBST and blocked with 1% BSA. After the usual steps of washing, 100 µl of the supernatant (isolated from cultured splenocytes after 48 h) was dispensed in each well. After stipulated incubation time, the plates were thoroughly washed and incubated with biotinylated polyclonal goat anti-mouse detection antibody. Afterward, the plates were washed three times with PBST. Subsequently, 100 µl of streptavidin-HRP conjugate was added to each well and plate was incubated for 30 min at RT. The plates were again washed three times with PBST and finally colored complex was developed with tetra methyl benzidine (TMB). The absorbance was read at 450 nm with a micro-titer ELISA plate reader (Bio-Rad).

Mice were bled at various time intervals (post immunization and post challenge) and their sera were analyzed for the presence of Ag85B-specific antibodies. Subsequently, the antibodies were analyzed for their isotypes using kit purchased from BD Biosciences. Briefly, 96-well micro-titer plates were coated (in duplicate) with isotype specific monoclonal antibodies in carbonate bicarbonate buffer (0.05 M, pH 9.5) and incubated overnight at 4 °C. After washing and blocking steps, the plates were incubated with 100 µl test sera from each animal from each group and positive control (supplied with kit) at RT for 1 h. After excessive washing of the plates, 100 µl of (1:100 dilution of stock) rat anti-mouse IgG antibodies were added in each well and incubated for 1 h at RT. The plates were washed again followed by adding 100 µl of substrate solution (supplied with kit) and were finally incubated for 3–10 min at RT. The reaction was stopped by the addition of 50 µl of stop solution (1M phosphoric acid). On addition of stop solution, the absorbance of as-formed color complex was immediately read at 450 nm with a micro-titer ELISA plate reader (Bio-Rad, USA).