Selection of Phages Displaying Antibodies Reactive with Heparan Sulfate Using Biopanning

For a schematic outline of the selection procedure, see Fig. 1. 3.1.1. Growth of Antibody Phage Display Library

1. Inoculate 50 mL of 2XTY containing 100 ^g of ampicillin/mL and 1% (w/v) glucose with about 5 x 108 bacteria from a glycerol stock of the (semi)-synthetic scFv Library #1 (5).

Heparan Sulfate
Fig. 1. Schematic representation of the selection of phage display-derived anti-heparan sulfate antibodies by biopanning.

2. Grow the culture, while shaking, at 37°C until an absorbance at 600 nm of 0.5 is reached (see Note 5).

3. Pipet 10 mL of the culture into a sterile 50-mL tube and add about 4 x 1010 VCS-M13 helper phages (see Note 6).

4. Incubate in a water bath, without shaking, at 37°C for 30 min.

5. Spin the infected culture at 3000g for 10 min at room temperature. Decant the supernatant and resuspend the pellet in 30 mL of 2XTY containing 100 ^g of ampicillin and 25 ^g of kanamycin/mL.

6. Add the 30 mL of bacterial suspension to 270 mL of prewarmed (30°C) 2XTY containing 100 ^g of ampicillin and 25 ^g of kanamycin/mL (No glucose! see Note 7). Incubate, while shaking, at 30°C for 16-20 h to allow for large-scale (antibody-displaying) phage production.

7. Inoculate 5 mL of 2XTY with a single E. coli TG1 colony from a minimal medium plate and grow, while shaking, at 37°C for 16-20 h. This culture will be used in Subheading 3.1.3, step 16.

3.1.2. Isolation of Phages

1. Spin the culture from step 6 under Subheading 3.1.1 at 10,000g for 10 min at 4°C to remove bacteria. Decant the supernatant containing the phages into another sterile bucket.

2. Add 60 mL of ice-cold PEG/NaCl to the supernatant, mix well by inverting the bucket at least 30 times, and leave the bucket on ice for 1 h. In this step (and step 3) phages are precipitated (see Note 8).

3. Spin the phages at 10,000g for 30 min at 4°C. Resuspend the pellet in 40 mL of ice-cold, sterile milli-Q water. Transfer the suspension to a 50-mL tube and add 8 mL of ice-cold PEG/NaCl. Mix well (as in step 2) and leave for 30 min on ice.

4. Spin the mixture at 3000g for 30 min at 4°C. Decant the supernatant and remove the remains with a pipet. Respin briefly and remove residual PEG/NaCl. Invert the tube on a piece of paper tissue, and leave for 30 min to drain any remaining PEG/NaCl.

5. Resuspend the pellet in sterile PBS and spin at 3000g for 10 min at 4°C to remove any remaining bacterial debris. Decant the supernatant containing the phages into a sterile tube and store at 4°C until use.

3.1.3. Selection of Phages Binding to Heparan Sulfate

1. Add 2 mL of a 20-^g heparan sulfate/mL solution to an immunotube for 16 h, 4°C. Wash the immunotube 3 times with PBS and block the tube with PBS containing 2% (w/v) Marvel. Fill the tube to the brim, cover it with Parafilm, and incubate for at least 2 h at room temperature to avoid nonspecific binding of phages to the surface of the tube. This step should be performed early in the day, so the immunotube will be ready when the phages used for biopanning (step 5, Subheading 3.1.2.) are obtained.

2. Wash the blocked tube 3 times with PBS. Add 2 mL of PBS containing 4% (w/v) Marvel and 2 mL of phage supernatant (step 5, Subheading 3.1.2.) to the tube, cover with Parafilm, and incubate for 30 min on an under-and-over turntable (room temperature), followed by standing for 90 min (room temperature).

3. Discard the phage suspension and wash the tube 20 times with PBS containing 0.1% (v/v) Tween-20, followed by 20 washes with PBS.

4. Remove the last remains of PBS and elute the bound phages with 1 mL of 100 mM tri-ethylamine. Cover the tube with Parafilm and rotate for 10 min on an under-and-over turntable at room temperature.

5. Add the 1 mL of eluted phages to a 50-mL tube containing 0.5 mL of 1 MTris-HCl (pH 7.4) for pH neutralization. Also add 200 ^L of 1 M Tris-HCl (pH 7.4) to the remaining phages in the immunotube. At this point phages can be stored at 4°C for a short period of time (up to 2 d), or used the same day to infect E. coli TG1 cells. The latter is recommended.

6. Add 1 mL of the eluted phages from the 50-mL tube (Subheading 3.1.3., step 5) to 9 mL of exponentially growing E. coli TG1 cells in a 50-mL tube. Add 4 mL of the TG1 culture to the remaining phages in the immunotube (step 5). Incubate both cultures for 30 min at 37°C in a water bath, without shaking, to allow for infection. Exponentially growing TG1 culture is prepared as follows: Inoculate 50 mL of 2XTY with 0.5 mL of overnight culture from Subheading 3.1.1., step 7. Grow the culture, while shaking, at 37°C until an absorbance at 600 nm of 0.4-0.5 is reached. The culture obtained is ready for infection (see Note 5).

7. Pool both infected TG1 cultures. Take 100 ^L of the pooled culture and make 4 serial dilutions (1/102, 1/104, 1/106, and 1/108) in 2XTY containing 100 ^g of ampicillin/mL and 1%(w/v) glucose. Plate 100 ^l of these dilutions on 94/15 TYE plates containing 100 ^g of ampicillin/mL and 1% (w/v) glucose, and grow for 16-20 h at 37°C. Calculate the titer from these dilutions (see Note 9).

8. Spin the rest of the pooled culture at 3000g for 10 min at room temperature. Decant the supernatant and resuspend the pellet in 1 mL of 2XTY. Spread the cell suspension on a Nunclon TC plate with TYE containing 100 ^g of ampicillin/mL and 1% (w/v) glucose and grow for 16-20 h at 37°C.

9. Add 5 mL of ice-cold 2XTY containing 15% (v/v) glycerol to the Nunclon TC dish and scrape the bacterial cells from the plate with a glass spreader. Take 50 ^L of the bacterial suspension and use it for inoculation of 50 mL 2XTY containing 100 ^g of ampicillin/mL and 1% (w/v) glucose as in Subheading 3.1.1., step 1. Store the remaining bacteria at -70°C (see Note 10). Repeat the selection procedure for another 3-4 rounds (all steps Subheading 3.1.1.-3.1.3.).

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