Protein Protocols Protein Protocols

Selection by Panning 1079
4. Notes
1. The protocols described were established using the phagemid vector pSEX81. A detailed
protocol regarding the cloning of an antibody repertoire into pSEX 81 with the title Production
of a Human Antibody Library in the Phage-Display Vector pSEX81 is provided in
Methods in Molecular Medicine, Vol. 13: Molecular Diagnosis of Infectious Diseases,
edited by U. Reischl, Humana Press, Totowa, NJ.
2. Both XL-1 Blue and TG1 bacteria are suitable hosts for phagemid libraries, whereby TG1
grows faster than XL-1 Blue. Plating transformed bacteria onto SOB-GA plates reduces
the risk of losing slower growing clones.
3. For performing phage rescue on a smaller or larger scale adjust the volume of the media
and the number of M13KO7 helper phages. It is important to use optimal growth conditions
for the bacteria at all times. It is therefore important to use large flasks for better aeration.
4. Phage rescue can also be started from an overnight culture or with bacteria from a glycerol
stock. In the latter case, the concentration of glycerol in the culture should not exceed 2%
to avoid inhibition of bacterial growth.
5. Do not handle the cells too vigorously before and during superinfection with helper
phage, as this may decrease their capacity for phage infection.
6. One milliliter of XL-1 Blue at A600 of 0.1 corresponds to 5 × 108 bacteria. To obtain a
multiplicity of infection of 20, add 1 × 10 10 phages to each milliliter of the culture. One
milliliter of TG1 at A600 of 0.1 corresponds to 1 × 108 bacteria, so 2 × 109 phages/mL are
required.
7. It is very important to use glucose-free medium at this step, as the presence of glucose will
inhibit expression of the antibody genes.
8. Extended incubation increases the number of rescued phages (infectious particles), but
decreases the ratio of functional recombinant antibody to phage particles. The binding
capacity of the rescued phage antibodies is lost mainly owing to protease activity in the
culture.
9. Determination of the number of infectious particles defined as colony-forming units (cfu)
can be performed according to standard methods (see ref. 25).
10. The number of infectious particles in the supernatant is usually about 1 × 10 10 to 1 × 1011 cfu/mL. If it is less than 1 × 109 cfu/mL, the rescue should be repeated.
11. Take care, as the pellet may be hardly visible.
12. Rinse that side of the tube where the pellet is to be expected with 1 mL of buffer several
times, pool in a polypropylene tube, and repeat this procedure 4×.
13. Panning should be performed as soon as possible following rescue, as some phage displayed
recombinant antibody preparations may be unstable. It is possible to freeze rescued
phage antibodies at –20°C, although it is presently unknown wether some antibodies
might possibly be denatured during freezing. In any case, repeated freezing and thawing
should be avoided.
14. The optimal conditions for coating may differ for each antigen. Small-scale trials should
therefore first be carried out. The present protocol is suitable for protein antigens. We use
200 µg of protein per tube if sufficient amounts are available, but the optimal amounts
may vary. Our suggestion is to make an empiricall estimation of the amount of antigen
necessary to coat a well of an ELISA plate. This amount can then be scaled up to the
volume of the Immunotubes (5 mL). The suitability of the plastic support for immobilization
of the particular antigen should also be determined prior to panning. If possible, an
immunoassay should be established prior to panning to ensure that under the conditions
used for coating the antigen binds to the tube wall.