- The plates containing young worms are washed with M9
buffer, transferred onto NGM agar plates containing fresh
lawns ofE. coliOP50, and incubated at 25C for the next
3 days. - GrowP. aeruginosaPA14 andE. coliOP50 cultures in 5 mL of
LB at 37C overnight, with shaking. Dilute theP. aeruginosa
PA14 overnight culture 1:100 in 5 mL of LB containing
600 nM anti-QS polyclonal sera or pre-immune and nonspe-
cific antigen immunized polyclonal sera (negative control) in
duplicate. Dilute theE. coliOP50 overnight culture 1:100 in
5 mL LB without any antibody. An additional negative control
consisting ofP. aeruginosaPA14 culture supplemented with
PBS can also be included. - Grow bacterial cultures by shaking at 37C until A 600 0.4 is
reached. 100μL of anti-QS polyclonal sera or control sera
made up to 600 nM in PBS are added to the center of 3.5 cm
NGM-EP plates in duplicates and allowed to dry. - Ten microliters ofP. aeruginosaPA14 orE. coliOP50 cultures
grown up to log phase are spotted onto NGM-EP plates and
incubated at 37C overnight. - The following day, another 100μL of 600 nM anti-QS poly-
clonal sera or control sera are added on top of PA14/OP50
cultures on NGM-EP plates and incubated at 37C for 8 h. - The process is repeated and the plates are incubated at 25C
overnight. - The plates were treated with antibodies as before and synchro-
nized adult worms suspended in a drop of M9 were added onto
the center of each of the PA14 plates containing anti-QS
polyclonal sera or control sera. As a positive control, worms
were added onto NGM-EP plates containingE. coliOP50. - The number of worms in each plate is counted and plates are
incubated at 25C for ~24 h. - Worm count is repeated every 12 h and dead worms are
removed with a wire pick. - The plates are treated with 600 nM polyclonal sera / control
antibodies at 12 h time intervals as before. Overall, the worms
are monitored for 80 h.
3.3 Preparation of
Monoclonal Antibodies
- To separate peripheral blood lymphocytes (PBLs) from sheep
blood, PBLs are prepared from sheep blood collected 4 days
after antigen-boost and processed within 18 h using Accuspin
system Histopaque 1077 columns (Sigma). About 250 mL of
production bleed in 3.2% (wt/vol) sodium citrate solution are
used for the procedure.
Monoclonal Antibodies Inactivating AHL Signal Molecules 337