- Indirect competition ELISA. 100μL aliquots of PBS contain-
ing 1μg/mL AHL-TG conjugates are dispensed in a polysty-
rene 96-well flat bottomed ELISA plate. The plate is incubated
at 37C for 1 h (or at 4C overnight). The plate is washed
twice with 250μL PBST and twice with 250μL PBS, blocked
with 200μL of MPBS per well, and incubated at 37C for 1 h.
Twofold sequential dilutions of free AHL compound solution
(free antigen) in PBS (125μL) are prepared in microfuge tubes
and mixed with an equal volume of a sub-saturating concen-
tration of the polyclonal sera purified instep 3as determined
from binding profiles. PBS is used in place of free antigen to
give a 100% signal (non-competitive binding). The reactions
are incubated for 1 h at room temperature. The wells are
washed twice with 250μL PBST and twice with 250μL PBS.
Then, 100μL of each sample containing free HSL compound
and polyclonal antiserum or the control corresponding to PBS
and polyclonal antiserum control is added to replicate wells and
incubated for 1 h at room temperature. The plate is washed
twice with 250μL PBST and twice with 250μL PBS. Then,
100 μL anti-sheep IgG antibody HRP conjugate diluted
1:1000 in PBS are added to the wells as the secondary antibody
and incubated for 1 h at room temperature. The wells are
washed as described instep 4and developed as described in
step 10. The absorbance value (A 450 ) obtained for polyclonal
sera incubated with PBS represents 100% binding. This value is
used to determine the % binding for polyclonal serum in the
presence of free antigen. The sensitivity of polyclonal sera
against free antigen can be represented as IC 50 and IC 20 values.
The IC 50 value is defined as the concentration of free antigen
required to reduce the binding of the antibody to HSL conju-
gate by 50%, and IC 20 as the free antigen concentration of free
antigen required to reduce the binding of the antibody to HSL
conjugate by 20%.
3.2 Anti-Virulence
Activity of the Anti-QS
Polyclonal Sera
3.2.1 Elastase Assay
Using Anti-QS Polyclonal
Sera
Elastase production can be measured using the published method
[10] with some modifications.- An overnight culture ofP. aeruginosaPA14 grown in LB broth
at 37C with shaking at 200 rpm is diluted to an absorbance at
600 nm wavelength (A 600 ) of 0.05 in two 15 mL tubes each
containing 2 mL of LB broth. Culture ofE. coliOP50 (non-
elastase producing strain) are prepared in a similar manner to
be used as a negative control. - Purified anti-AHL polyclonal sera prepared in Subheading3.1,
step 3, are added toP. aeruginosaandE. colicultures at final
IgG concentration of 600 nM. Pre-immune and nonspecific
antigen immunized polyclonal serum (600 nM) are added to
differentP. aeruginosaandE. colicultures as negative controls.
Monoclonal Antibodies Inactivating AHL Signal Molecules 335