Wells testing positive for specific antibody should be assayed more than once to ensure that activity continues over the course of a few days. The health of the cells should be checked daily to ensure that overcrowding does not occur and once the cells are confluent in the cell well they should be subcultured into 24-well plates. The cells must also be cloned by limiting dilution at this stage and plated out at 1 cell/well. It is useful to test the isotype of the secreted antibody when the cells are growing in the 24-well plates. This can be accomplished using a number of "dip stick" assays giving results in a matter of a few minutes. The isotype of the antibody is important for development of the antibody if purification and reagent development will be required. Antibodies of the sub classes G1, G2a, and G2b are the most desirable for further development as they are readily purified and are relatively stable. Antibodies with the G3 isotype, most commonly produced from bacterial antigens may be inherently unstable and liable to spontaneous aggregation.
Approximately 50% of hybridomas will secret antibody of the M subclass. IgM is a large molecule that does not readily purify but may be used for TAS ELISA. Some antigens will only give rise to IgM secreting hybridomas and are know as anamnestic antigens. These substances are frequently highly glycosylated and do not invoke a "memory" response in the immune system. The result of this is that B-lymphocytes never mature beyond the production of IgM regardless of the number of immunizations the animal is given.
3.5.1. Limiting Dilution Cloning
1. Agitate the contents of the cell well using a Pasteur pipet and then using a pipet with a sterile tip, draw up 2X 0.01-mL aliquots of cells.
Add one aliquot to 1 mL of warm RPMI 1640 medium with 15% FBS. Add the other aliquot to 0.01 mL of Trypan blue dye and count the cells using a Neubauer counting chamber.
2. Calculate the cell count in the 1 mL tube using the formula: (total cell count/number of grid squares counted) x 104 x 2 = cells/mL.
3. Calculate the volume of cell suspension from the 1-mL tube that would contain 60 cells.
4. Transfer the appropriate volume of the medium and cells from the 1-mL tube to the 9 mL of cloning medium and dispense 0.15 mL/well in 60 wells of the cloning plate.
5. Place the cloning plate into the tissue culture incubator 37°C/5% CO2 for 1 wk and then inspect for growth. Most cell wells should have single hybridoma colonies growing in them. It is important at this stage to record the wells containing single colonies. Give each of the cell wells 0.05-0.1 mL of cloning medium and reincubate until cells are between 30-50% confluent.
6. Test the wells that originally contained single colonies for specific activity, and record the numbers that are positive.
7. Subclone the cells using the previously mentioned method until 100% of the clones are positive for the specific antibody. Occasionally some cell lines will not achieve a cloning efficiency of 100% regardless of the number of subcloning attempts. In these cases a final clone should be selected, rapidly multiplied and cryogenically stored. Cells from these unstable stores should never be used to produce additional cell stocks without subcloning again.
8. Choose one of the clones from the final (100% efficiency) cloning as the master cell line, expand it in tissue culture and cryogenically preserve stocks of it as the master cell bank (at least 12 vials).
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