Protocol for Making Monoclonal Antibody

Prewarm all culture media to 37^oC. 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 37^oC. 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 37^oC. 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 37^oC. 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 -80^oC. Hybridoma cells frozen at -80^oC are stable for up to 6 months.

Prewarm all culture media to 37^oC. 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 37^oC. 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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