Protocol for Making Monoclonal Antibody
- Prewarm all culture media to 37oC. Thaw the parental myeloma cells (Sp2/0-Ag14 or X63-Ag8.653) from liquid nitrogen and culture in BiCell-mAb Medium MY for 3-5 days to fully recover cell growth to reach 1 x 106/ml cell density.
- Split cells at 1:5 into fresh BiCell-mAb Medium MY and let grow for 2-3 days until cell density reach 1 x 106/ml.
- Split cells again at 1:5 into fresh BiCell-mAb Medium MY and let grow for 2-3 days until cell density reach 1 x 106/ml. Passing 2 cycles will bring myeloma cells back to early-mid log phase prior to fusion.
- Centrifuge the parental myeloma cells in a 50 ml conical tube at 250 x g for 5 minutes at room temperature. Remove and discard the supernatant. Resuspend the cell pellet in 25 ml of BiCell-mAb Medium FU.
- Repeat the above step for two more times.
- Myeloma cells are now ready for fusion. Stain cells with Trypan Blue. The viability of the parental myeloma cells should be > 95%.
- Prewarm all culture media to 37oC. Dissect the spleen from immunized animal and place it in a sterile Petri dish containing 5 ml of BiCell-mAb Medium FU. Trim off fat and connective tissues.
- Transfer the spleen to a 70-μm mesh placed on top of a 50 ml conical tube, and use the plunger of a 3 ml syringe to grind the cells through the mesh. Rinse the screen with 25 ml of BiCell-mAb Medium FU until only the spleen membrane is left on the mesh.
- Centrifuge the splenocytes in a 50 ml conical tube at 250 x g for 5 minutes at room temperature. Remove and discard the supernatant. Resuspend the cell pellet in 5 ml of Red Blood Cell Lysis Buffer (155 mM NH4Cl, 10 mM HEPES, pH 7.2).
- Incubate the cell suspension at room temperature for 5 min. Stop the reaction by adding 20 ml of BiCell-mAb Medium FU.
- Centrifuge the splenocytes in a 50 ml conical tube at 250 x g for 5 minutes at room temperature. Remove and discard the supernatant. Resuspend the cell pellet in 25 ml of BiCell-mAb Medium FU.
- Repeat the above step for two more times.
- Splenocytes are now ready for fusion. Stain cells with Trypan Blue. The viability of the harvested splenocytes should be > 95%.
- Prewarm all culture media to 37oC. Mix 1 x 107 parental myeloma cells with 3 x 107 splenocytes in a 50 ml conical tube. The optimal ratio of myeloma to splenocyte fusion is from 1:1 to 1:5.
- Centrifuge the cell mixture in a 50 ml conical tube at 250 x g for 5 minutes at room temperature. Remove and discard the supernatant.
- Add 1 ml of BiCell-mAb Medium PEG to the cell pellet dropwise. Stir the cells gently with a pipette tip for 1 minute to fully dissolve the pellet.
- Add 4 ml of BiCell-mAb Medium FU to the fused cells dropwise. Stir the cells gently with a pipette tip for 1 minute to fully dissolve the pellet.
- Add 10 ml of BiCell-mAb Medium FU to the fused cells. Incubate the fused cells in a 37°C water bath for 10 minutes.
- Add 25 ml of BiCell-mAb Medium HAT to the fused cells. Centrifuge the cell mixture in a 50 ml conical tube at 250 x g for 5 minutes at room temperature. Remove and discard the supernatant.
- Resuspend the cell pellet in 10 ml of BiCell-mAb Medium HAT. Using a multi-channel pipettor, add 100 μl of cell suspension into each well of 10 x 96-well tissue culture plates. Each 100 μl of cell suspension contains 1 x 105 fused myeloma cells. BiCell-mAb Medium PEG can generate >1 stable hybridoma cell clone per 105 fused myeloma cells.
- Incubate plates at 37°C and 5% CO2 for 7-10 days until colonies appear.
- Prewarm all culture media to 37oC. Using a multi-channel pipettor, aspirate and discard BiCell-mAb Medium HAT medium from each well of the 96-well culture plates. Take care not to disrupt the hybridoma colonies.
- Add 100 μl of BiCell-mAb Medium HT to each well of the 96-well culture plates. Incubate plates at 37°C and 5% CO2 for 3-5 days until culture medium turns yellow. This step is essential to reduce the rate of false positives in screening experiments due to IgG carryover from the B cells that have died off during selection. Weaning hybridoma cells off aminopterin also facilitates the maturation process and promotes the stability of hybridoma clones.
- Using a multi-channel pipettor, aspirate BiCell-mAb Medium HT medium from each well of the 96-well culture plates and transfer into new 96-well culture plates for downstream screening experiments. Take care not to disrupt the hybridoma colonies.
- Add 100 μl of freezing medium (10% DMSO in FBS) to each well of the 96-well culture plates. Freeze the 96-well culture plates at -80oC. Hybridoma cells frozen at -80oC are stable for up to 6 months.
- Prewarm all culture media to 37oC. Thaw the 96-well culture plate that carries positive clones to room temperature. Using a multi-channel pipettor, aspirate and discard the freezing medium. Take care not to disrupt the hybridoma colonies.
- Add 100 μl of BiCell-mAb Medium HT to each well of the 96-well culture plates. Incubate plates at 37°C and 5% CO2 for 3-5 days until culture medium turns yellow.
- Using a single-channel pipettor, pipet the medium in each positive culture well up and down several times to dissociate the hybridoma cell colony. Transfer the dissociated cells to a 24-well plate containing 1 ml/well of BiCell-mAb Medium HT. Incubate plates at 37°C and 5% CO2 for 3-5 days until culture medium turns yellow.
- Transfer 500 μl of the cells in each well of the 24-well plate into a T-25cm2 flask containing 5 ml of BiCell-mAb Medium HT for cryopreservation. Transfer the remaining cells from each well of the 24-well plate into a T-25cm2 flask containing 5 ml of BiCell-mAb Medium SF for further expansion and antibody production. Antibody production in BiCell-mAb Medium SF can reach 100 μg/ml.
- Prewarm all culture media to 37oC. Thaw the hybridoma clones that need subcloning from liquid nitrogen and culture in BiCell-mAb Medium HT for 3-5 days to reach 1 x 106/ml cell density.
- Centrifuge the hybridoma cells in a 50 ml conical tube at 250 x g for 5 minutes at room temperature. Remove and discard the supernatant. Resuspend the cell pellet in 25 ml of BiCell-mAb Medium HT.
- Count the cells and dilute the hybridoma cells to 10 cells/ml in 10 ml of BiCell-mAb Medium HT.
- Using a multi-channel pipettor, add 100 μl of diluted hybridoma cell suspension into each well of a 96-well culture plate to plate at a limiting dilution cell density of 1 cell/well.
- Incubate plates at 37°C and 5% CO2 for 7-10 days until colonies appear.
- Harvest subcloned colonies, and then screen and expand colonies as described before.
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