Quorum Sensing

2.3 HSL/HS
Extraction of Cell
Pellets

1. Extraction solvent: Prepare 100 ml of anhydrous ethyl acetate
   and acidify the solution with 10μl glacial acetic acid.


2. EtOAc/ACN mixture: Prepare a 100 ml mixture of 50% (v/v)
   anhydrous ethyl acetate and 50% (v/v) acetonitrile (seeNote 4).

2.4 UHPLC-ESI-
QToF-MS Analysis

1. UHPLC Column: Use a Waters Acquity Ethylene Bridged
   Hybrid (BEH) C 18 column with the dimensions 1.0 
   150 mm and a particle size of 1.7μm or a column with similar
   separation properties.


2. Eluant A: Prepare a solution of purified deionized water with
   10% (v/v) acetonitrile and 0.1% (v/v) formic acid.


3. Eluant B: 100% Acetonitrile.

3 Methods

3.1 Sample
Preparation

In principle, liquid samples from any given habitat can be analyzed.

However, the matrix can lead to a considerable inhibition of the

assay and needs to be thoroughly tested. Especially rich bacterial

culture media, like nutrient broth (NB), are likely to cause a signifi-

cant matrix effect and should be avoided.

1. Separation of cell fraction and culture supernatant should
   be achieved by centrifugation or ultracentrifugation at 4C
   (seeNote 6).


2. Subsequently, the supernatant should be filtered through a
   0.22μm nitrocellulose membrane and frozen at 80 C for
   further extraction and analysis (seeNote 7).

3.2 ELISA The given amounts are calculated for three plates. Measure all
standards and samples at least in triplicates and take mean. Mix all
components in glass beakers and then pour in specifically labeled
plastic reservoirs for multi-pipettes. If used for one component
only, these plastic reservoirs can be reused after rinsing with tap
water. Plates should be covered with PCR foil (reusable if clean)
during every incubation step. It is advisable to process not more
than three plates in parallel and always finish one complete step with
one plate before repeating the step with the next one.

1. Coating: Prepare coating antigen solution by diluting 1μl
   stock solution of HSL-BSA in 34.5 ml of 50 mM carbonate
   buffer. Add 100μl of antigen solution per well into a Maxi Sorp
   microtiter plate and incubate overnight at 4 C without
   shaking.


2. Preparation: On the next day put all buffers out of the refrigera-
   tor to equilibrate to room temperature. If you are using an
   automated washer (seenext step) attach vacuum and buffer bot-
   tles, and perform a washing cycle with an empty 96-well plate.

Detection of Bacterial Quorum Sensing Molecules with ELISA and UHPLC-MS 65