overnight at 30 C with shaking. Centrifuge the plate at
700 gfor 10 min and transfer the supernatant containing
monoclonal phage to sterile 96-well plates.- Phage monoclonal binding ELISA. Coat 96-well Immulon 4
ELISA plates with 100μL per well of 1μg/mL AHL conju-
gates (i.e., C 12 -BSA, 3OC 12 -BSA, 3OHC 12 -BSA) and carrier
protein BSA for 2 h at 37C (or overnight at 4C). Wash and
block as explained in Subheading3.1,step 4. - Add 50μL of phage supernatant fromstep 8and 50μLof4%
MPBS and incubated at room temperature for 1 h. The follow-
ing steps are repeated as detailed in Subheading3.1,steps
7 – 10. Secondary antibody used here is 100μL of a 1 in 1000
dilution anti-M13 monoclonal HRP conjugate per well. - Monoclonal phage Competition ELISA. Monoclonal phage
clones specific for free AHLs are identified by Competition
ELISA. ELISA plates coated with HSL blocked with MPBS
and washed as described in Subheading3.1,step 4. - After washing, varying concentrations of free AHL solutions
(1μM–100 nM) were prepared in 4% MPBS and added to the
ELISA plate during incubation with the phage supernatant.
The rest of the assay is carried out as explained in Subheading
3.1,step 12, except the secondary antibody used is 100μLofa
1 in 1000 dilution anti-M13 monoclonal HRP conjugate per
well and the clones which showed reduced binding (greater
than 50% when compared with the control) against AHL-
conjugates in the presence of free AHLs, are selected. - Conversion of positive AHL binders into a single chain anti-
body format (scAb). Extract plasmid DNA from phage clones
recognizing free AHLs selected in the step above and sequence
using AH18REV and GIIIFOR primers. After sequence analy-
sis, the scFV region of unique positive clones is cloned into the
soluble antibody expression vector pIMS147 using NcoI and
NotI restriction sites following the general cloning procedures
described in Subheading3.4. Briefly, 40μg of pIMS147 DNA
and 2μg of each plasmid DNA extracted by the clones selected
in the step above are digested with 100 U of NcoI and 100 U
of NotI for 16 h at 37C. - The restriction products are separated by gel electrophoresis on
a 1% (wt/vol) agarose TAE gel and the correct sized DNA
band (~5.5 kbp for the vector and ~750 bp for the insert) are
purified by using a commercial kit. - Set up a ligation reaction using about a 1:7 (vector:insert)
molar ratio (i.e., for each clone, use 100 ng of insert, 100 ng
of vector, 200 U of T4 ligase and incubate at 16C overnight).
348 Soumya Palliyil