Quorum Sensing

purified antibody fragments to the wells alongside double dilu-

tions of human whole IgG (кisotype) with starting concentra-

tion of 1.25μg/mL.

33. Incubate plates at room temperature for 1 h, wash as before,
    and add 100μL goat anti-human C-kappa light chain peroxi-
    dase conjugate diluted 1:1000 (Sigma). Incubate 1 h at room
    temperature, wash, and develop the plates as described in
    Subheading3.1,step 10.


34. scAb Binding ELISA. Coat 96-well flat bottomed Immulon 4
    plates with 100μLof1μg/mL AHL-conjugates (i.e., C 12 -
    BSA, 3OC 12 -BSA, 3OHC 12 -BSA) for 2 h at 37C or over-
    night at 4C. Include plates coated with 1μg/mL BSA as a
    control. Wash three times with 200μL of PBSTand three times
    with 200μL of PBS. Add 200μL of MPBS for blocking and
    incubate at 37C for 1 h. Repeat the washing step and add
    200 μL of purified antibody samples of known concentration
    (as determined by Capture ELISA,steps 31– 33 ) making dou-
    ble dilutions in PBS. Incubate at room temperature for 1 h.
    Wash plates as before and add 100μL of goat anti-HuCκ
    peroxidase conjugate diluted 1:1000 in PBS. Incubate 1 h at
    room temperature, wash, and develop the plates as described in
    Subheading3.1,step 10.


35. scAb Competition ELISA. Coat 96-well flat bottomed Immu-
    lon 4 plates with 1μg/mL AHL-BSA conjugates and block
    with MPBS as in the step above. Set up double dilutions of free
    AHL solutions (i.e., C 12 -HSL, 3OC 12 -HSL, 3OHC 12 -HSL,
    C 4 -HSL) in 125μL of PBS and mix with an equal volume of a
    sub-saturating concentration of AHL scAbs as determined by
    binding ELISA (Subheading3.7,step 34). For 100% binding
    control, use AHL scAb in PBS. The reactions are pre-incubated
    at room temperature for 1 h and 100μL of each reaction mix is
    added to replicate wells and incubated for a further 1 h at room
    temperature. Following normal washing steps, HRP-
    conjugated secondary antibody is added to the wells and incu-
    bated as described above. The plates are then washed and
    developed as described in Subheading3.1,step 10.

4 Notes

1. We routinely purchase customized AHL-compounds from Sal-
   ford Ultrafine Chemicals and Research Ltd., Manchester, UK
   (presently known as SAFC, a division of Sigma Aldrich
   Corporation).


2. In principle, the melon gel support bind to non-antibody
   serum proteins, such as albumin and transferrins, using

Monoclonal Antibodies Inactivating AHL Signal Molecules 351