(c) MuVH1BACK-Sfi:5^0 -CAT GCC ATG ACT GCG GCC
CAG CCG GCC ATG GCC SAG GTS MAR CTG CAG
SAG TCW GG-3^0.
(d) Hu4aBACK-Sfi:5^0 -GTC CTC GCA ACT GCG GCC
CAG CCG GCC ATG GCC CAG GTG CAG CTG
CAG GAG TCG GG-3^0.
(e) OvJL1FOR-Not:5^0 -GAG TCA TTC TCG ACT TGC
GGC CGC ACC CAG GAC GGT CAG CCT GGT
CC-3^0.
(f) OvJL2FOR-Not:5^0 -GAG TCA TTC TCG ACT TGC
GGC CGC ACC CAG GAC GGT CAG YCK RGW
CC-3^0.
(g) OvJK1FOR-Not:5^0 -GAG TCA TTC TCG ACT TGC
GGC CGC CCG TTT GAT TTC CAC GTT GGT
CCC-3^0.
(h) OvJK2FOR-Not:5^0 -GAG TCA TTC TCG ACT TGC
GGC CGC CCT TTT GAT CTC TAG TTT GGT
TCC-3^0.
(i) OvJK3FOR-Not:5^0 -GAG TCA TTC TCG ACT TGC
GGC CGC CCT TTT GAT CTC TAC CTT GGT
TCC-3^0.3 Methods
3.1 Production and
ELISA Validation of
Anti-QS Polyclonal
Antisera
In order to construct an immunized antibody phage display library,
two Welsh breed sheep are hyper-immunized with AHLs conju-
gated to thyroglobulin (TG) (seeNote 1) as described below.- A mixture of three AHL-TG conjugates, i.e., C 12 -HSL-TG,
3OC 12 -HSL-TG, and 3OHC 12 -HSL-TG are injected into two
sheep. For primary immunization, 500μg of AHL-conjugate
mixture is made up to a 2 mL volume in PBS and administered
per sheep. For subsequent re-boosts (after 4, 8, 12, 16, 20, and
24 weeks from the primary immunization), 250μg of AHL-
conjugate mixture in 2 mL of PBS is used per sheep. - Serum samples are taken at weeks 6, 10, 14, 18, and 22 for
preliminary analyses, while the production bleed is collected
3–5 days after the final boost at week 24. - Purification of sheep polyclonal sera are performed by means of
commercial resins that allow removing nonrelevant proteins
often present in high abundance in the serum, according to
manufacturer’s instructions (seeNote 2). After purification,
antibodies are dialyzed 1:200 against PBS to remove any
remaining trace of purification buffer and stored at 4C until
needed.
Monoclonal Antibodies Inactivating AHL Signal Molecules 333