Editing Phage Display Library Screening

== Library selection on immunotubes ==

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

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

Rinse&nbsp; Immunotubes with PBS

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

Rinse with PBS

Add >10<sup>12</sup> phage particles in 2% MPBS&nbsp; tothe immunotubes. 

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

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

Elute the bound phages&nbsp; by&nbsp;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&nbsp;TG1 ==

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

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

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

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

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

Incubate&nbsp;at 30°C overnight. 

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

Store the rescued bacteria at -80°C.

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

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

Add&nbsp; &nbsp;100 μl helper phage VCS-M13 (1x10<sup>12</sup> phage)&nbsp; to 10 ml (4 – 5 x 10<sup>8</sup> bacteria/ml)<br/>Incubate at 37°C for 40 min.&nbsp;<br/>Centrifuge&nbsp;the culture for 10 min at 3300 g and 4°C,

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

Incubate the culture &nbsp;at 30°C overnight. 

Centrifuge the overnight culture&nbsp;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&nbsp;each 40 ml of&nbsp;supernatant. 

Incubate the mixture on ice for 40 min

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

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

Incubate the mixture on ice for 40&nbsp;min 

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

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

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

Transfer superntant to a new tube and put on ice

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

Store&nbsp;the rest of phage at -20°C.

== ELISA screening ==

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

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

Add&nbsp;isopropyl-thiogalactopyranoside at a&nbsp;final concentration of 1 mM

Grow&nbsp;overnight at 30°C. 

Transfer&nbsp;supernatants to an ELISA plate.

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