Assay for Positive Colonies

1. Prepare serial dilutions of the "red eluate" in TBSG (see Note 6).

2. Mix 10 |L of diluted phage and 10 |L of K91Kan cells in NAP (Subheading 3.2.5.).

3. Rock for 10 min at room temperature.

4. Add 1 mL of LB containing 0.2 |g/mL tetracycline and shake at 37°C for 30 min.

5. Spread 200 |L on LB/Tet/Kan agar and grow overnight at 37°C (see Note 7).

6. Cut a nitrocellulose membrane (NCM) to fit a 90-mm Petri dish (see Note 8).

7. Put NCM onto LB/Tet/Kan agar and let it soak.

8. Using the same toothpicks transfer single colonies onto both a LB/Tet/Kan agar master plate and to a NCM on LB/Tet/Kan agar.

9. Incubate plates overnight at 37°C.

10. Put the master plates in a refrigerator (these can be stored for several weeks).

11. Remove the NCM from agar surface and transfer to another Petri dish with 10 mL of TBST. Using a piece of sponge, remove all the cells from the NCM.

12. Wash six to eight times with TBST. Block NCM for 30 min at room temperature in 3% low fat dried milk in TBST. Incubate for 1 h with MAbs (hybridoma supernatant, 1/50 in IB; see Note 9) at room temperature on rocker.

13. Wash five times with TBST. Incubate for 1 h with peroxidase-labeled rabbit anti-(mouse IgG) antibodies diluted 1/1000 in IB at room temperature on rocker.

14. Wash five times with TBST and two to three times with distilled water. Add 5 mL of DAB solution in substrate buffer supplemented with 0.012% hydrogen peroxide. Traces of the positive colonies should be bright-brown colored (see Notes 10 and 11).

15. Make replicas of positive clones with sterile toothpicks on another NCM placed on a LB/Tet/Kan agar surface so, that the replicas form columns that can be cut later into strips for incubation with individual MAbs from the mixture used for biopanning. Grow the cells overnight at 37°C. Repeat steps 11 to 14 with the exception that incubation with antibodies is performed individually with each MAb.

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