Cell Fusion

1. Mix the washed NS-0 cells harvested from the 2-T flasks with one quarter of the splenocytes from one spleen.

2. Pellet cells by centrifugation at 400g for 5 min.

3. Discard supernatant and slowly add 1 mL of PEG/RPMI mixture.

4. Gently resuspend the pellet by swirling.

5. Pellet cells by centrifugation at 250g for 5 min.

6. Slowly overlay cell/peg layer with 5 mL of RPMI 1640 with no FBS and then gently swirl to create a cell suspension.

7. Pellet cells by centrifugation at 400g for 5 min.

8. Discard PEG/RPMI mixture and replace with 10 mL of HAT medium. Do not disturb the cell pellet, then incubate the cell pellet for 5-10 min at 37°C.

9. Resuspend the cells by swirling and add 20 mL of HAT and 7.5 mL of allogeneic mixed thymocyte medium.

10. Dispense cells suspension into 96-well tissue culture plates (0.2 mL/ well; see Note 11).

12. After 7 d examine the wells for the presence of hybridomas and given an additional 0.1 mL of HAT medium.

13. Test wells exhibiting growth for the presence of monoclonal antibodies when the hybridoma growth covers approx 25% of the base of the cell well. The assay system used should mimic the final test format required. Commonly, Triple Antibody Sandwich (TAS) (5) enzyme-linked immunosorbent assay (ELISA) is use for screening hybridomas for specific antibody. It is important when testing hybri-

domas that a suitable negative is included as background antibody activity from unfused spleen cells may give apparently positive results. Up to 0.2 mL of medium can be harvested from each cell well, which can be split to assay against specific antigen and a suitable negative.

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