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Monoclonal Antibody Production
Order 6 six week old Balb/C mice and let the ARC know they are coming. Have your antigen ready for when they arrive. Once they get there earmark the mice and perform a pre-bleed on them to be used as an ELISA control for monitoring the titer and screening of the hybridomas:
- Place the mouse in a mouse restrainer.
- Sterilize the tail with 70% ethanol.
- With a razor blade, nip off the last 2 mm of the tip of the tail.
- Using a milking motion, pull blood down and let drip off the end of the tail until you have collected ~200 µL. (You may have to pre-bleed twice, with a week or so between bleeds).
- Take the collected blood and place at 37 °C for 30 min. to remove complement.
- Place blood at 4 °C overnight to clot.
- Centrifuge samples at 10,000g 10 min.
- Pipet off the serum supernatant. Store at -20 °C. This is your pre-bleed control.
For one fusion you will need:
- 8 500 mL bottles of DMEM media
- 3 500 mL bottles of fetal calf serum (FCS)
- 2 50 mL bottles of 100X penicillin-streptomycin
- 10 mL of 100X HAT selection solution
- 10 mL of 100X HT solution
- 10 sterile flat-bottomed 96-well plates
Assaying for Positive Clones
Run an indirect ELISA using the antigen you want the MAb directed against. If you are raising the MAbs against a small hapten that you coupled to a carrier protein for immunization of the mice, then use the hapten coupled to a different carrier protein for the screen.
Expanding Positive Clones
- The day prior to any expansion, obtain feeder cells. For this first expansion from the 200 µL cultures in 96-well plates to 500 µL cultures in 24-well plates, add 200 µL of feeder cells/well in DMEM/20% FCS + HT to the 24-well plates the day before expansion. Let the feeder cells grow overnight in the CO2 incubator.
- For the initial expansion, resuspend the positive testing hybridoma cells and place all but the last 10 µL (200 µL) in the well of the 24-well plate with the feeder cells. Then bring to 500 µL with DMEM/20% FCS +HT.
- Add 200 µL of DMEM/20% FCS +HT to the little bit of cells left in the 96-well plate. This serves as a backup for your positive clones.
- Place all plates in the CO2 incubator.
- When the cells are beginning to acidify the media (gets orange-yellow), change the media and double the culture volume by pipetting off 400 µL, and replace with 900 µL fresh DMEM/20% FCS +HT.
- Harvest more feeder cells for your next expansion. Put 0.5 mL f feeder cells in DMEM/20% FCS (note the removal of the HT) in a T25 flask (one for each positive clone) for later use
- When the cells are ready for expansion out of the 24-well plate, resuspend them and pipet to the T25 flasks with feeder cells. Again leave a little bit in the 24-well plate and refill with DMEM/20 FCS as a backup. Volume to the T25 culture 5 mL with DMEM/20% FCS (add 3.5 mL) Place all cultures in the incubator.
Subcloning Positive Clones
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