Editing Hybridoma production (section)

== Clone Testing ==

<br/>1. After 10-14 days when clones are visible by eye and medium in some wells just&nbsp;begins to turn yellowish, test all wells by ELISA.

2. Pick individual clones from ELISA positive wells

Transfer to round bottom, cell-treated 96 well plates, 1 clone/well

150ul medium/well

Use 30ul to pick clones

3. When 96-well clones are just visible by eye (usually 3 days later), test by ELISA.<br/>Test supernatants for IgG (or other fusion protein tag) and the protein of interest in parallel.

<br/>4. For clones that recognize the protein of interest and not the IgG or fusion protein tag, transfer to 24 well plates – 1ml/well

<br/>5. Transfer to 6well plates – 3-4ml/well. Collect the supernatants for additional testing depending on the protein: e.g. fusions for CD markers or surface proteins&nbsp;should be tested by flow cytometry. Monoclonal antibodies to an IgG isotype&nbsp;should be tested against various isotypes, etc to identify clones of highest interest.

<br/>6. When well-grown, fix an aliquot of cells for clonality testing by flow cytometry, freeze an aliquot, and collect supernatant for mouse isotype ELISA

<br/>7. After an additional 2 weeks in HAT (4 weeks total), wean cells into HT medium.

8. After 2 weeks in HT medium, wean cells into Hyb+10%FCS

9. Wean to Hyb-SFM for bulk culture for purification.