immunotube coated with AHL-conjugate as described insteps
11 – 17. Subsequent rounds of rescue and selection are carried
out and the stringency of panning is increased in each round by
increasing the number of washing steps and also by adding
1 mg/mL BSA, 1 mg/mL TG, 2% (wt/vol) skim milk, 0.5%
(vol/vol) Tween 20 along with PEG precipitated phage
(Fig.2).3.7 Production of
Anti-QS Antibodies
- Screening of antibody fragments binding to AHL-conjugates
by ELISA. A 96-well flat bottomed Immulon 4 ELISA plate
(Dynatech Laboratories Ltd.) is coated with 100μL per well of
10 μg/mL AHL-conjugates for 2 h at 37C. The plate is
washed three times with PBST and three times with MPBS
and incubated at 37C for 1 h. - The washing step is repeated and 50μL of PEG-precipitated
phage from pan 0 to pan 3 are added to distinct wells. 50μLof
4% MPBS are added to the same wells and incubated at room
temperature for 1 h. - After discarding the phage solution, the wells are washed with
PBST and PBS as instep 1, and 100μL of a 1:1000 dilution of
anti-M13 monoclonal HRP conjugate is added to the wells. - The plate is incubated for 1 h at room temperature, washed,
and the wells developed by adding 100μL per well of HRP
substrate solution. The reaction is stopped with 50μLof1M
H 2 SO 4 per well. The absorbance of the wells is measured at
450 nm using a microplate reader. - Individual bacterial colonies are picked from Pan 2 and Pan 3
serial dilution plates and inoculated into 100μLof2TY
containing 100μg/mL ampicillin and 1% (w/v) glucose in a
sterile 96-well tissue culture plate and incubated overnight at
37 C with shaking at 250 rpm. The following day, using a
sterile 96-well transfer device, a small inoculum is transferred
into a new 96-well plate containing 200μL per well of fresh
2 TY containing 100μg/mL ampicillin and 1% (w/v) glucose
(seeNote 3). - Incubate the refreshed 96-well plate at 37C with shaking for
2 h. Then add to each well 25μLof2TY containing 100μg/
mL ampicillin, 1% (wt/vol) glucose and helper phage
M13KO7 (10^9 PFU per well) obtained as described in Sub-
heading3.5,step 6. - The plate is incubated at 37C for 30 min without shaking
followed by 1 h shaking at 37C. Cells are pelleted by centrifu-
ging at 700gfor 10 min and supernatants are carefully
removed from the plate by pipetting. - Resuspend the pellets in 200μLof2TY containing 100μg/
mL ampicillin and 50 μg/mL kanamycin and incubate
Monoclonal Antibodies Inactivating AHL Signal Molecules 347