Capítulo 1. Fenómeno de globalización, impulsor de los procesos
1.1 Contexto y evolución del proceso de globalización
display
2.12.1 Selection on immunotubes
In selection, scFv with high affinity against the targets (Table 2.3) were isolated from the display libraries by panning. Normally three rounds of selection is sufficient to isolate binders against each target.
An immunotube (Nunc) was coated overnight at 4˚C with 4 ml of a purified clostridial target protein at a concentration of 100 µg/ml in PBS. Next day, each tube was washed 3 times with PBS and filled to the brim with 2% MPBS as a blocking reagent. Tubes were left for 2 hours at room temperature to allow
blocking to take place. After 2 hours, the immunotubes were washed 3 times with PBS. Library phage (1012 to 1013) were mixed in 4 ml MPBS and added into each tube. Tubes were firstly incubated at room temperature for 1 hour and then rotated using an under and over turntable rotator for another hour, again at room temperature. The supernatant of each tube was thrown away and each tube was washed extensively with PBS-0.1% Tween 20: in the first round of selection, 10 washes were used; for selection rounds two and three, 20 washes were used. The excess PBS was removed by inverting each tube on a tissue for a few seconds.
The bound phages were eluted by adding 500 µl of trypsin-PBS (50 µl of 10 mg/ml trypsin stock in 450 µl PBS) and rotating at room temperature for 10 minutes. From this eluate, 250 µl was added into 1.75 ml of fresh E. coli TG1 (0.4) for static incubation at 37˚C for 30 minutes in a water bath. The remaining 250 µl of eluate was stored at 4˚C as reserve. Serial 10-fold dilutions were prepared from the infected E. coli and 10 µl aliquots were spotted on TYE plates containing 100µg/ml ampicilin and 1% glucose and incubated at 37˚C overnight. The number of colonies found the following day enabled estimates to be made of the number of phage recovered from the selection step.
The remaining infected bacteria were contrifuged at 11,600 x g for 5 minutes in a micro centrifuge. The cell pellet was resuspended in 50µl of 2xYT and plated onto TYE (100µg/ml ampicillin and 1% glucose) for overnight incubation at 37˚C. From the resulting colonies, phage were prepared to take forward further rounds of selection. To do this, the bacterial colonies were resuspended from the plate by adding 5 ml of 2xYT 15 % glycerol and loosening the cells with a sterile spreader. One ml of the resuspended cells were stored at -70˚C as a stock, 50 µl of were added into 50 ml of 2xYT (100 µg/ml ampicillin and 1% glucose) and grown at 37˚C until the absorbance reached 0.4. Ten ml were recovered and 5x1010 KM10 helper phage were added for static incubation at 37˚C in a water bath for 30 minutes. The mix was then centrifuged at 3000 x g for 10 minutes and the pellet was resuspended in 50 ml 2xYT (100µg/ml ampicillin, 50µg/ml kanamycin and 0.1%
glucose) and shaken overnight at 30˚C. The overnight culture was centrifuged at 3300 x g for 15 minutes and 10 ml of PEG/NaCl was added to 40 ml of the culture supernatant. To precipitate the phage, the suspension was mixed and placed on ice for 1 hour. After spinning at 3300 x g for 30 minutes, the supernatant was discarded and the pellet was spun again to remove any remaining PEG/NaCl. The viral pellet was resuspended in 2 ml PBS and again spun at 11,600 g for 10 min to remove bacterial debris. One ml of this phage was titred to determine the phage yield and then used for the next round of selection.
2.12.2 Screening phage by ELISA
ELISA using antibodies directed against the phage coat was used to monitor the specificity of reaction between scFv-pIII fusions and the target proteins. Mixed populations of phage recovered after each round of selection were tested in “polyclonal phage ELISA”; by picking single bacterial clones and preparing phage, virus were also tested in “monoclonal phage ELISA”.
2.12.2.1 Polyclonal phage ELISA
ELISA plates (96 well; Nunc) were coated with recombinant target proteins using the same concentration and buffer as applied in the selection process. For coating, 100 µl of protein was added to each well of the ELISA plate which was incubated overnight at 4˚C. Plates were then washed three times with PBS and blocked by addition of 200µl 2% MPBS per well for 2 hours at room temperature. After washing three times with PBS, 10 µl of PEG precipitated phages recovered from each round of selection were added to each well, diluted in 100µl 2% MPBS. Plates were incubated for 1 hour at room temperature to allow binding to take place. After this, plates were washed 3 times with PBS-0.1% Tween 20 and 100 µl of HRP-anti-M13 conjugate (GE Healthcare) was added to each well. The conjugate
was diluted 1/5000 in 2% MPBS prior to use. Plates were incubated for 1 hour and washed three times with PBS-0.1% Tween 20. As substrate for the colorometric reaction, 100µl of TMB (Promega) was added to each well and left at room temperature for 10 minutes to allow development of a blue colour reaction. The reaction was stopped by adding 50µl of 1M sulphuric acid and the intensity of the resulting yellow colour was measured at 650 nm in a plate reader.
2.12.2.2 Monoclonal Phage ELISA
Individual colonies were picked from titration plates after each round of selection and bacteria were added into 100µl 2xYT (100µg/ml ampicillin and 1% glucose) in 96 well plates. Plates were shaken at 250 rpm at 37˚C overnight. Two µl was sampled from each well and transferred into wells of a fresh plate, each containing 200µl of 2xYT (100µg/ml ampicillin and 1% glucose). Remaining overnight culture was mixed with glycerol to a final concentration of 15% and frozen to archive the strains.
Newly inoculated plates were shaken at 250 rpm at 37˚C for 2 hours and then 25µl 2xYT was added to each well, each aliquot supplemented with ampicillin and glucose as before, but also containing 109 KM10 helper phage. Plates were shaken for a further hour at 37˚C then centrifuged at 1800 x g for 10 minutes. Each bacterial pellet was resuspended in 200µl 2xYT (100µg/ml ampicillin and 50µg/ml kanamycin). After shaking overnight at 250 rpm, the plates were centrifuged at 1800 x g for 10 minutes and 50µl of the supernatant from each well was sampled and used in ELISA reaction as explained above.