Phage Display Library Screening

Library selection on immunotubes

All selections were performed using recombinant or commercially available antigens with purity >90%.

Coat Immunotubes (Nunc; Wiesbaden, Germany)  with antigen at a concentration of 50 μg/ml in PBS over-night at room temperature.

Rinse  Immunotubes with PBS

Block with 2% MPBS for 2 hours at room temperature .

Rinse with PBS

Add >10¹² phage particles in 2% MPBS  tothe immunotubes.

Incubate the immunotubes  on a shaker for 30 minutes and then for 90 minutes standing upright at room temperature.

Wash unbound phage away by rinsing the immunotubes ten times with PBS 0.1% Tween 20 and ten times with PBS.

Elute the bound phages  by incubation of 1 ml 100 mM triethylamine for 5 minutes.

Add 0.5 ml 1 M Tris-HCl pH 7.4. to neutralize triethylamine

Infection of TG1

Add the eluted phage  to exponentially growing E.coli TG1, for 40 min at 37°C.

Dilute series of bacteria  and plate on small 2xYT agar plates, 100 μg/ml carb, 0.1% glucose (2xYT-Carb-Glu)

Incubate  at 30°C overnight to determine the titer of the eluted phage.

Centrifuge the remaining phage-infected bacteria for 10 min at 3300 g and 4°C.

Resuspend the pellet in 0.5 ml 2xYT, spread on a large 2xYT-Carb-Glu agar plate

Incubate at 30°C overnight.

Scape bacteria from the large plate using 5 ml 10% glycerol 2xYT using a sterile glass loop.

Store the rescued bacteria at -80°C.

Use the rescued bacteria to inoculate 50 ml 2xYT-Carb-Glu (initial OD600 nm between 0.05 and 0.1)

Grow the culture at 37°C and 200 rpm until OD600 nm 0.4 – 0.5.

Add   100 μl helper phage VCS-M13 (1x10¹² phage)  to 10 ml (4 – 5 x 10⁸ bacteria/ml)
Incubate at 37°C for 40 min. 
Centrifuge the culture for 10 min at 3300 g and 4°C,

Resuspend the pellet in 100 ml 2xYT-Carb-Glu including kanamycin 33 μg/ml (2xYT-Carb-Kan-Glu

Incubate the culture  at 30°C overnight.

Centrifuge the overnight culture for 30 min at 3300 g and 4°C.

Transfer the supernatant to new bottles

Add 10 ml 20% PEG/2.5 M NaCl to each 40 ml of supernatant.

Incubate the mixture on ice for 40 min

Centrifuge for 30 min at 3300 g and 4°C.

Resuspend the pellet in 40 ml sterile H2O and 10 ml PEG/NaCl.

Incubate the mixture on ice for 40 min

Centrifuge for 30 min at 3300 g and 4°C.

Resuspend the pellet  in 2 ml sterile PBS containing 15% glycerol.

Centrifuge  for 3 min at 15000 g and discard the pellet (cell debris)

Transfer superntant to a new tube and put on ice

Use the phage  on the same day for a further round of panning

Store the rest of phage at -20°C.

ELISA screening

Inoculate individual colonies  in 180 μl 2xYT, 100 μg\ml carb, 0.1 % glucose in 96-well plates 

Incubate the plates for 3 hours at 37°C in a shaker incubator.

Add isopropyl-thiogalactopyranoside at a final concentration of 1 mM

Grow overnight at 30°C.

Transfer supernatants to an ELISA plate.

ELISA assay using  anti-myc tag 9E10 mAb and anti-mouse horseradish peroxidase immunoglobulins.