2.5.1 Principle of the assay
In this method, proteins are separated electrophoretically on sodium dodecyl phosphate- polyacrylamide gels (SDS-PAGE), according to their molecular weight. Then these proteins are electrphoretically transferred into a nitrocellulose or PVDF membrane. After that, non-specific binding capacity on the membrane is blocked with bovine milk (skimmed) proteins. Thereafter, the target protein is probed with a specific primary antibody, which is then detected by chemiluminescence with an enzyme conjugated secondary antibody allowing it to react with its substrate.
2.5.2 Western Blotting of Pneumococcal CCS
Immunostaining of RrgA and RrgB proteins in pneumococcal CCS was performed following Western blotting protein transfer using rabbit anti-RrgA and anti-RrgB antisera (Novartis vaccine, Sienna, Italy) to confirm the presence of RrgA and RrgB
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pilus proteins in the TIGR4wt CCS and the absence of RrgA protein in RrgA-/- mutant strain and RrgB protein in RrgB-/- mutant strain.
2.5.2.1 Gel Electrophoresis
Samples were prepared in Laemmli reducing Buffer (1:2 dilutions) and then heated on a heat block at 100°C for 5 min. After that, 20μl of CCS (derived from TIGR4wt, RrgB-/- or RrgA-/- strain) and 5μl of precision plus protein kaleidoscope ladder (Bio- Rad, UK) were loaded into a 12% mini protean precast TGXTM gels (BioRad). The gel was run for 1 hr at a constant 250v in 50mA.
2.5.2.2 Protein Transfer by Western blotting
Western blotting transfer of protein was performed using a Transblot TurboTM transfer system (BioRad) into a 0.2μm nitrocellulose membrane (Transblot turbo transfer pack). After gel electrophoresis, the gel was carefully placed onto a nitrocellulose membrane and then placed on to the bottom ion reservoir (anode) stack. The top ion reservoir (cathode) stack was then laid over the gel, and run for 10 min at a constant 25v in 1000mA.
2.5.2.3 Detection of RrgA and RrgB proteins in pneumococcal CCS
Following Western blot transfer, the nitrocellulose membrane was blocked with 5% skimmed milk in TBS containing 0.05% Tween20 (TBS-T) (appendix I) for 2 hr at room temperature (RT). After washing 3 times in TBS-T, rabbit anti-RrgA and anti- RrgB antiserum, diluted 1:10,000 in blocking solution were added. The membrane was incubated in room temperature for 2 hr at RT. After, washing for 5 times in TBS-T, the membrane was incubated with secondary antibody (donkey anti-rabbit IgG-HRP) for 1 hr at RT. The secondary antibody was prepared by adding 2μl donkey anti-rabbit IgG-HRP (1:30,000) (Santa Cruz Biotechnology, Germany), to
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visualise the ladder 2μl Streptactin-HRP (1:30,000) (Bio-Rad) and to reduce background 30μl normal donkey serum (Sigma) into 60 ml blocking solution. The membrane was washed 5 times in TBS-T and then in PBS, before the addition of substrate to the membranes. Western Blot substrate was prepared by adding equal volume (1:1) of Immun-star WesternC chemiluminescence reagent A and B (BioRad). The membrane was incubated with the prepared substrate solution for 5 min in the dark. Finally, the membranes were imaged in the Chemi-DocXRS system (BioRad), after removal of excess substrate with PBS.
Figure 2.5.2.3: Western blot analysis of pneumococcal CCS (TIGR4wt, RrgB-/- and
RrgA-/-). (a) Nitrocellulose membrane blotted with pneumococcal CCS were immunostained with rabbit anti-RrgA antiserum showing that, RrgA band is present in TIGR4wt and RrgB-/- CCS but absent in RrgA-/- mutant CCS. RrgA band shows a higher molecular mass (∼120 kDa) than predicted from its sequence (92.7 kDa) (LeMieux et al. 2008). (b) Blotted with rabbit anti-RrgB antiserum showing that, RrgB band (~50 kDa) is present in TIGR4wt and RrgA-/- CCS but absent in RrgB-/- mutant CCS.
2.5.2.4 Western blotting of recombinant pilus proteins (rRrgA and rRrgB)
Western blotting and immunostaining of recombinant pilus (rRrgA and rRrgB) proteins (Gianfaldoni et al. 2007) were also performed with rabbit anti-RrgA and anti-RrgB antisera (Novartis vaccine, Sienna, Italy). This was later used as the standard control in the detection of pilus-specific antibodies in serum samples using Western blotting. Samples for this purpose was prepared by adding 5μl of
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recombinant proteins (rRrgA and rRrgB) to 95μl of Laemmli reducing Buffer (appendix-I), to make a 1:20 dilutions. After heating on a heat block at 100°C for 5 min, 5μl of these mixtures were loaded into the gel. The rest of the Western blot procedure was the same as that of the pneumococcal CCS, as described earlier.
Figure 2.5.2.4: Western blotting of recombinant RrgA (rRrgA) and RrgB (rRrgB)
proteins. (a) Probing with rabbit anti-RrgA antiserum showing the (~100 kDa) RrgA band.
(b) Probing with rabbit anti-RrgB antiserum showing the (~ 50 kDa) RrgB band.
2.5.2.5 Serum Western Blotting for detection of pilus-specific antibodies
Serum anti-RrgA and anti-RrgB antibodies were also analysed by Western blotting. Recombinant pilus proteins (rRrgA and rRrgB) were mixed with Laemmli reducing buffer (1:20) and heated at 100°C for 5 min. The mixtures were loaded into a mini protean TGX gel (BioRad, UK), and run for 1 hr at a constant 250v in 50mA. Transfer of protein to a 0.2μm nitrocellulose membrane was performed with a Transblot turbo transfer system (BioRad). The membrane was then blocked with 5% skimmed milk TBS-T for 2 hr. The membrane was then probed with serum samples (1:10,000 in blocking solution) for 2 hr. After washing, murine anti-human IgG-HRP (Sigma) and Streptactin-HRP (BioRad), both diluted (1:10,000) in blocking solution were added and incubated for 1 hr. The membrane was imaged on Chemi-DocXRS system (BioRad), 5 min after the addition of 1:1 mixture of Immun-star WesternC chemiluminescence reagent A and B (BioRad).
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Figure 2.5.2.5: Western blotting of recombinant RrgA and RrgB proteins with
patients’ sera. Figure shows the MW marker ladder in lane 1, positivity of anti-RrgA in
lane 2 and positivity of anti-RrgB in lane 3 in a representative serum sample.