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兔单抗亲和力和灵敏度更高
相对于鼠单抗（解离常数Kd在10-9-10-10M水平），兔单抗的亲和力可提高100-1000倍（Kd在10-10-10-12M水平）。实际使用时，兔单抗的工作浓度更低，灵敏度更高，因此产生的背景更低，大大降低了假阳性的发生。用作免疫组化等用途时，有时甚至可免去抗原修复的步骤。
兔单抗能识别更多表位
很多蛋白在人类和啮齿动物中同源性很高，这些保守表位在小鼠体内免疫原性很弱，不容易产生高质量抗体。而兔做为宿主，可以更好地识别这些表位，产生针对更多表位的抗体。当然，兔也适合生产针对鼠类蛋白的抗体。
Monoclonal Antibody Production
Mouse Immunization
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:
Bleeding Mice
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.
Hybridoma Fusion
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 ELISA.html">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.
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